Difference between revisions of "Team:Freiburg/Labbook/In Vivo"

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Lab Book: Expression

Short description of what has been done

Droplet cloning and characterization

04.06.19

- Made 1:10 dilutions of all primers

Primers		Cloning of Droplet forming proteins		TM °C	Ta °C

Name	Tube name	Sequence (overlap/spacer/ANNEAL)	Use
pBAD33_fwd	pBAD33_sfGFP_gibson_f	gctctacaaaTAAATCGATCGCGTTAGG	Gibson primer to join sfGFP and the start of the pBAD33 Backbone (	58.5	56.1
pBAD33_rev	pBAD_FUS_gibson_rev	tagacgccatGCTAGCATTATACCTAGG	Gibson primer to join FUS and the end of pBAD33 Backbone	55.1	56.1
FUS_fwd	FUS_pBAD33_gibson_fw	taatgctagcATGGCGTCTAATGACTACACTCAGC	Gibson primer to join FUS and the end of pBAD33 Backbone (reverse)	68.3	69.3
FUS_rev	FUS_sfGFP_gibson_rev	cggatcctccGTATGGGCGCTCGCGACG	Gibson primer to join FUS and the sfGFP with linker(reverse)	72.4	69.3
linker_spGFP_fwd	gsGFP_pBAD33_gibson_f	gcgcccatacGGAGGATCCGGAGGATCC	Joins the start of sfGFP to the end of fus	67.0	65.6
linker_spGFP_rev	spGFP_pBAD33_gibson_r	gatcgatttaTTTGTAGAGCTCATCCATGCC	Gibson primer to join sfGFP and the start of the pBAD33 Backbone	64.6	65.6

SPD5
pBAD33_fwd	pBAD_SPD5_fwd	gctctacaaaTAAATCGATCGCGTTAGG	Gibson primer to join stGFP and the start of the pBAD33 Backbone	58.5	56.1
pBAD33_rev	pBAD33_SPD5_gibson_rev	tatcttccatGCTAGCATTATACCTAGG	Gibson primer to join stGFP and the start of the pBAD33 Backbone (reverse)	55.1	56.1
SPD5_1_fwd	SPD5_pBAD33_gibson_fwd	taatgctagcATGGAAGATAACTCTGTCCTTAATGAAG	Gibson primer to join SPD5_1 at the end of pBAD33	63.5	64.5
SPD5_1_rev	SPD5_1_gibson_rev	gtgcaatagtGAAACGACGCGACGCTTC	Gibson primer to join SPD5_1 at the end of pBAD33 (reverse)	67.2	64.5
SPD5_2_fwd	SPD5_2_gibson_fw	gcgtcgtttcACTATTGCACCGGATGCTG	Gibson primer to join SPD5_2 at the end of SPD5_1	65.2	62.6
SPD5_2_rev	SPD5_2_sfGFP_gibson_rev	cggatcctccCTTCTTGCGAATTTCCTTTACG	Gibson primer to join SPD5_2 at the end of SPD5_1 (reverse)	61.6	62.6
linker_spGFP_fwd	sfGFP_SPD5_2_gibson_fw	tcgcaagaagGGAGGATCCGGAGGATCC	Forward primer for sfGFP (at the end of SPD5)	67.0	65.6
linker_spGFP_rev	sfGFP_pBAD33_gibson_rev	gatcgatttaTTTGTAGAGCTCATCCATGCC	Reverse primer GFP at the start of pBAD	64.6	65.6
mCherry
pBAD33_fwd	PBAD33_mCherry_gibson_fwd	cgagctgtacTAAATCGATCGCGTTAGG	Gibson primer to join mCHERRY and the start of the PBAD33 Backbone	58.5	56.1
pBAD33_rev	pBAD_EWSR1_gibson_rev	ttgaggccatGCTAGCATTATACCTAGG	Gibson primer to join mCHERRY and the start of the PBAD33 Backbone (reverse)	55.1	56.1
EWSR1_fwd	EWSR1_pBAD33_gibson_fwd	taatgctagcATGGCCTCAACTGACTACTC	Gibson primer to join EWSR1 and the end of the PBAD33 Backbone	63.7	64.7
EWSR1_rev	EWSR1_mCherry_gibson_rev	ccttgctcacggatcctccggatcctccATAGGGACGATCACGGCG	Gibson primer to join EWSR1 and the end of the PBAD33 Backbone (reverse)	67.1	64.7
GS_mCherry_fwd	mCherry_EWSR1_gibson_fwd	tcgtccctatggaggatccggaggatccGTGAGCAAGGGCGAGGAG	Gibson primer to join mCHERRY and the end of the EWSR1	68.8	66.9
GS_mCherry_rev	mCherry_pBAD33_gibson_rev	gatcgatttaGTACAGCTCGTCCATGCC	Gibson primer to join mCHERRY and the end of the EWSR1 (reverse)	65.9	66.9



NiCD
pBAD33_fwd	pBAD33_sfGFP_gibson_fwd	tgagctctacaaaTAAATCGATCGCGTTAGGC	Gibson primer to join sfGFP and the start of the PBAD33 Backbone	57.1	60.4
pBAD33_rev	pBAD33_NICD_gibson_rev	tgacgcattcatGCTAGCATTATACCTAGGACTG	Gibson primer to join sfGFP and the start of the PBAD33 Backbone (reverse)	56.9	60.4
NICD_fwd	NICD_pBAD33_gibson_fwd	gtataatgctagcATGAATGCGTCATGTGTAGGGG	Gibson primer to join NICD at the end of pBAD33	62.5	65.6
NICD_rev	NICD_sfGFP_rev	ctcctttgctcatggatcctccggatcctccGACCAGATGGCCGCGCAG	Gibson primer to join NICD at the end of pBAD33 (reverse)	65.7	65.6
sfGFP_fwd	sfGFP_NICD_fwd	ggccatctggtcggaggatccggaggatccATGAGCAAAGGAGAAGAACTTTTC	Gibson primer to join sfGFP at the end of NCID	59.1	60.8
sfGFP_rev	sfGFP_pBAD33_rev	gcgatcgatttaTTTGTAGAGCTCATCCATGC	Gibson primer to join sfGFP at the end of NCID (reverse)	57.3	60.8

Ordered gBlocks sequences:

EWSR1 (Ewing Sarcoma Breakpoint Region 1):

atggcctcaactgactactccacttactcgcaggcagcagcacagcaaggctacagcgcttatacagcccagccaacgcaaggatatgcccaaacaacgcaggcatacggtcagcagtcttacggcacttacggccagccaactgatgtctcttacactcaagctcagaccacagcgacttatggccagaccgcatatgccacctcttacggtcagcccccgaccgtggaaggaacgtcgactggctatacaactccaaccgctcctcaagcatacagccagccagtccaaggatatggaaccggtgcatacgatacaacaactgccacagtgacgactacacaggcttcttatgcagctcagagtgcatatgggactcaaccagcatacccggcatacggccagcagccagctgcaacagctccaacgcgtcctcaggatggcaacaagccaacagagacctcgcaaccccagtccagtacaggtggctacaatcagccctccttggggtatgggcagagtaactactcgtacccccaagtccccggttcttaccctatgcagcctgtcacggcgcccccgagctatccacctacctcctatagcagtacgcagcccacgtcatatgatcagtcctcatattcccagcaaaacacatatgggcaacctagcagttatggccagcagtcttcgtacgggcagcagtcatcgtatggacagcaacctccgacatcatatccaccacagaccggatcgtactctcaggcaccatcacaatattctcagcaatcatcgtcatacggtcagcagtcttcttttcgtcaggatcatccttcttcgatgggtgtatatggccaagaatcggggggattctctggaccgggtgagaaccgtagtatgtcaggcccagacaaccgtggccgtggtcgtggcggtttcgatcgtggtggcatgtcgcgcggtgggcgtggtgggggccgcgggggtatgggggcaggggagcgtggtgggtttaacaaaccaggtggaccgatggatgagggcccagatcttgacttgggtccaccagtcgatccggatgaggattcagacaatagtgcaatttacgtgcaggggttaaatgactccgtgacgctggatgatctggccgatttcttcaaacaatgcggcgtagtaaaaatgaataaacgcacaggtcaacccatgattcatatctacttggacaaagagactggcaaaccaaaaggagacgctacggtatcatatgaggaccctccaacagctaaagccgctgttgagtggtttgatggaaaagacttccagggaagcaaattaaaggtatcattagcgcgtaaaaaaccacctatgaactcaatgcgcggcggactgccgccccgcgaaggtcgtggtatgccaccgccacttcgtggaggacctggaggtccaggaggtccaggtggtcctatgggacgaatgggaggtcgtggcggcgaccgcgggggttttcctccgcgcgggccccgtggtagtcgtggtaacccttcagggggcggcaacgtgcaacatcgcgcaggcgattggcagtgtccaaacccgggttgcggtaatcagaacttcgcgtggcgtacagaatgtaatcagtgcaaagcgccaaaaccagaaggttttcttccacccccgtttccacctccaggcggagaccgtggccgtggagggccgggtggtatgcgcggcggacgcggaggtcttatggatcgcggtggtccgggcgggatgtttcgtgggggtcgcggcggggatcgtggaggtttccgcggaggtcgcggaatggatcgtggcggattcggaggaggacgtcgtggtggtccaggggggcctcctggtcctctgatggagcagatgggagggcgccgcggtggtcgtggcgggccaggcaagatggataaaggtgagcaccgtcaagagcgccgtgatcgtccctatggaggatccggaggatcc

FUS:

atggcgtctaatgactacactcagcaagctacccaatcttatggggcatatccgacccaacctggccaaggctattcccaacagtcatcacaaccttacgggcaacaaagttattccggttacagccagtcaacagatacttctggatatggtcagtcttcctattcgtcatacggtcagagtcagaatacgggttacgggacacaatcaaccccgcaggggtacggttccaccggcggctatggctccagccaatcgagtcaatcaagttatgggcaacaatcttcttatcccggctatggccaacagccggccccgagctccacatccggttcatatggttcttcaagccaatcaagctcgtatggtcagccgcagtcaggaagctattcacagcagccgtcctacgggggacaacaacagagttacggacagcaacagagttataacccaccgcaaggctacgggcagcagaatcagtacaattcctcttcaggtggtggtggtggcggtggtggtggagggaattacgggcaagatcaatctagtatgtcttcaggagggggcagcggtgggggatacggcaatcaagatcaaagtggtggaggtggatctggaggttatggacagcaggatcgtggaggtcgcggccgcggcggctctggtggtggtgggggaggaggaggtggtgggtacaatcgtagctccggaggatacgagccacgtgggcgtgggggcggtcgcggtgggcgcggtgggatgggtggctctgaccgcggcggttttaataagttcggtggtccccgtgatcagggttcacgccacgatagtgagcaagacaacagtgataacaatacgatcttcgttcaaggtcttggagagaacgttacaattgaatcggtagcagactatttcaaacagattggtattattaaaaccaacaagaagacaggccaaccgatgattaacttgtatactgaccgtgaaaccggtaaattaaaaggggaggctaccgtatcctttgacgaccctccaagcgcaaaagctgccattgactggtttgacggtaaagaattttctggcaacccgattaaggtgtccttcgcaacacgccgtgcggacttcaaccgtggcggggggaacggacgtggggggcgcggccgcggaggtccaatggggcgcggagggtatggaggaggaggcagtggtgggggtggacgtggtggtttccccagcggcggcggaggaggaggcggtcagcaacgtgccggtgattggaagtgtcctaatccaacctgcgaaaatatgaacttctcttggcgcaatgagtgtaaccagtgcaaggcaccgaaacctgatggcccaggtggcgggccagggggaagccacatgggcggaaattacggtgatgatcgtcgcggcggccgtgggggatacgaccgtggtggctatcgtggccgtggtggagatcgcggtggcttccgtggtgggcgcggaggaggtgaccgcggagggtttgggccggggaagatggacagccgtggcgaacaccgtcaagatcgtcgcgagcgcccatac

NICD (Nephrin Intracellular Domain)

atgaatgcgtcatgtgtagggggtgtgatttggcagcgccgtattcgccgtctggccgaaggaatttcggaaaaaacagaagccggttccgaggaggaccgtgttcgtaatgaatacgaagaatcgcaatggaccggtgagcgcgatacgcaatctagtaccgtatccacgacagaagcagagccttattatcgcagtatccgcgatgaaagtccacagcttcctcctacacaggaagaggtttcctatagccgtggcgaaactggggaggatgaggatatggcagagccgggtcacttgtacgatgaggtagagcgcacttacccacctagcggcgcatggggtcccctgtacgatgaagtccagatggggccctgggatctgcattggcctgaggacacttatcaagaccctcgcggaatctatgaccaggttgcgggcgatcttgacactctggagcctgattcgctgccgtttgagctgcgcggccatctggtcggaggatccggaggatcc

SPD5:

atggaagataactctgtccttaatgaagatagtaaccttgagcacgtcgaaggtcagcctcgtcgtagtatgtctcaaccagtactgaatgtcgaaggtgacaagcgcacttctagcacaagtgcaacacagcagcaagttctttccggtgcgttctcttccgctgatgtccgctcaattccgatcatccaaacgtgggaagaaaacaaggctttgaaaacgaagatcacaattcttcgcggtgagctgcaaatgtaccaacgccgctatagtgaagccaaggaggcaagccagaagcgtgtcaaagaggttatggatgactatgtggacttaaaattaggtcaagaaaatgtacaagagaagatggaacaatacaagttaatggaagaagacttattggcgatgcaatcccgtattgaaacgtcggaagataatttcgcgcgtcagatgaaggaatttgaagcccaaaaacatgccatggaagaacgtattaaggagttggaactttccgccaccgatgctaacaacactacagtaggtagttttcgcgggacgcttgacgatatcttgaagaagaacgaccccgactttaccttaacctccggatatgaagaacgtaagatcaatgacttagaagccaagttacttagcgaaatcgataaggtggcagaactggaggaccacatccagcagttgcgccaagaacttgatgatcaaagtgcgcgcttagctgattcagagaacgtgcgcgctcagctggaagcggctaccggacaaggaatcttgggagctgctgggaatgcaatggtcccaaattcaacgttcatgatcgggaacgggcgcgaatctcaaacgcgcgatcagttgaattacatcgatgatttggaaactaagcttgcagatgctaagaaggaaaatgacaaggcccgtcaggccttagtggagtatatgaataaatgttcaaagcttgaacacgagatccgcactatggtaaagaatagtacgtttgacagctcatcaatgttattgggcggtcagaccagcgacgaattaaagattcagattggaaaggtaaatggagaattaaacgtacttcgcgccgagaatcgcgagcttcgcattcgctgcgatcaacttactggaggggacggtaacctttctatcagtcttggccaaagtcgtctgatggctgggattgctacaaacgatgtggatagtattggccaagggaatgaaaccggcgggacgagtatgcgtatcttgccacgcgagtcgcagttagacgacttggaagaaagtaagttgcctttaatggatactagtagtgcggtccgcaaccaacaacagttcgccagcatgtgggaagattttgagtccgtgaaagactcactgcaaaacaaccataacgacacccttgagggctcgtttaatagtagtatgccacccccagggcgcgatgccacccagtcatttctttctcagaaatcttttaagaatagcccaattgtgatgcaaaagccgaagagcttacacttgcaccttaagtcacatcagagcgagggggcgggggagcagattcagaataatagtttctctactaagacggcgagtccgcatgtctcccagagccatatcccaatccttcacgacatgcaacaaattctggactcatcggcgatgtttttagaaggtcaacacgacgttgcagttaatgtggaacaaatgcaagaaaagatgtctcagattcgcgaggcccttgcgcgcttgttcgagcgcctgaaatcgagtgccgcattattcgaggaaattctggaacgcatgggcagttcagatccaaacgccgataagattaaaaaaatgaagttagctttcgagacgtcgatcaacgataaattaaacgtgtctgctattcttgaggcggccgagaaggatttacataacatgagcttaaacttttcaatcttagagaagagtatcgtaagccaagctgccgaagcgtcgcgtcgtttcactattgcaccggatgctgaagacgttgcatccagttctcttcttaatgcgagttactcgccgttgtttaagtttacttccaactccgatatcgttgaaaagcttcagaatgaagtctcagaacttaaaaacgagttagagatggcacgcacacgtgatatgcgcagcccccttaacggatcttccgggcgtcttagtgacgtgcagatcaacacaaaccgcatgtttgaagatcttgaggtatccgaggcaacgcttcagaaggccaaggaagagaactccactctgaaatcacagtttgctgagcttgaggcgaacttgcaccaggtgaattctaagttaggggaggtgcgctgcgagttaaatgaggccttggcccgtgtggacggagagcaagagactcgtgtaaaggctgagaacgcgttagaagaagctcgccaattgatttcttcccttaaacatgaggaaaatgagttaaagaagactattactgatatggggatgcgtcttaatgaagcaaaaaagtccgacgagttcctgaaatccgagctttctaccgctttggaggaggagaaaaaatcccaaaatttagcagacgagttgtcagaggaacttaacggttggcgtatgcgtacgaaggaagcggagaataaagtagaacatgcgtcctcggagaagagcgagatgctggaacgtatcgtgcatcttgaaacggaaatggagaagctgtcaacctctgaaattgcagcggactattgttctacgaagatgactgagcgcaaaaaggaaattgagttggcaaagtaccgcgaagattttgagaacgccgctattgtgggcctggaacgtatttcaaaagaaatctctgagttaactaagaagacactgaaggcaaagatcattccatcgaacatctcaagcatccagcttgtctgcgatgagctttgccgtcgtctgtcacgcgagcgcgagcaacaacacgaatacgccaaggttatgcgtgacgtcaatgagaagatcgaaaaattgcaattagaaaaagatgcgttggaacacgagttgaaaatgatgtcaagtaataatgaaaacgtgcctcctgtcgggacttcagttagcggcatgccgacaaagacgagtaatcaaaaatgcgcgcaaccacactacacgtcaccgactcgccaacttctgcatgagtcaaccatggcggtcgacgcgattgtccagaaattgaagaaaacacacaacatgagcgggatgggaccagagttgaaagaaacaattggtaacgtgatcaacgaatcacgtgtcttgcgcgactttcttcatcaaaagcttattttgtttaagggcattgatatgtctaattggaagaatgaaacggttgatcagctgatcaccgatttaggccagctgcaccaggacaatctgatgttggaagaacagatcaagaaatataaaaaggaacttaagcttaccaaaagtgcgatccctactcttggagtggaattccaggatcgtattaagaccgaaattgggaaaattgcgacggacatgggcggagccgtaaaggaaattcgcaagaag

Created Plasmids in first cloning phase
	Backbone	Insert	Insert	Promoter	Resistance
P1	pBAD33	EWSR1	mCherry	J23119	Chloramphenicol
P2	pBAD33	FUS	sfGFP	J23119	Chloramphenicol
P3	pBAD33	SPD5	sfGFP	J23119	Chloramphenicol
P4	pBAD33	NICD	sfGFP	J23119	Chloramphenicol

Plasmid 3 with split SPD5 gBlock due to its size. Linker for sfGFP is 2xGGS.

Plasmid 2 with Fus. Linker for the sfGFP is 2xGGS

Plasmid 1 with EWSR1. Linker for mCherry is also 2xGGS.

Plasmid 4 with NICD. Linker for sfGFP is 2xGGS

</p>

1 05.06.19
2 07.06.19
3 11.06.19
4 12.06.19
5 13.06.19
6 14.06.19
7 20.06.19
8 21.06.19
9 25.06.19
10 Subsection Title
11 Subsection Title #2
12 01.07.2019
13 02.07.2019
14
15 04.07.2019
16 05.07.2019
17 06.07.2019
18 08.07.2019
19 09.07.2019
20 10.07.2019
21 11.07.2019
22 12.07.2019
23 13.07.2019
24 15.07.2019
25 16.07.2019
26 16.07.2019
27 17.07.2019
28 18.07.2019
29 19.07.2019
30 24.07.2019
31 25.07.2019
32 26.07.2019
33 27.07.2019
34 28.07.2019
35 29.07.2019
36 30.07.2019
37 31.07.2019
38 01.08.2019
39 02.08.2019
40 04.08.2019
41 05.08.2019
42 11.08.2019
43 12.08.2019
44 14.08.2019
45 15.08.2019
46 16.08.2019
47 18.08.2019
48 20.08.2019
49 21.08.2019
50 22.08.2019
51 23.08.2019
52 25.08.2019
53 26.08.2019
54 27.08.2019
55 28.08.2019
56 29.08.2019
57 30.8.2019
58 31.8.2019
59 02.09.2019
60 03.09.2019
61 05.09.2019
62 06.09.2019
63 08.09.2019
64 11.09.2019
65 16.09.2019
66 17.09.2019
67 18.09.2019
68 19.09.2019
69 26.09.2019
70 27.09.2019
71 01.10.2019
72 02.10.2019
73 08.10.2019
74 09.10.2019
75 10.10.2019
76 11.10.2019
77 14.10.2019
78 15.10.2019
79 16.10.2019
80 17.10.2019
81
82 18.10.2019
83
84 20.10.2019
85 Primers and Constructs

05.06.19

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Labbook 05.06.2019

Performed amplifications of the Backbones (all Plasmids will be constructed with a pBAD33 backbone), sfGFP and mCherry genes from plasmids.

Following PCR constructs were made:

For FUS: (A) pBAD amplification with Primers 1&2* (not successful)

(B) sfGFP amplification with Primers 5&6*

For SPD5: (D) pBAD amplification with Primers 7&8* (not successful)

(E) sfGFP amplification with Primers 13&14* (not successful)

For EWSR1: (H) pBAD amplification with Primers 15&16*

(I) mCherry amplification with primers 19&20*

*For sequences of primers and roteins see Protocol from Day 04.06.2019

PCR tubes contained: 2,5 μl of each primer (diluted 1:10)

1 μl DNA

19 μl H2O

25 μl Phusion Flash Master Mix (Thermo Fisher Scientific)

Fragments B, H and I were successfully amplified, identified via Gel electrophoresis and purified.

PCR for DNA fragments A, D and E were set up again. To troubleshoot the PCRS, higher Backbone concentrations of about 2 ng were used. Also for fragment E, 3% of DMSO were added to the PCR mix. Also, two different Polymerases were used (Q5 and Phusion Flash).

Furthermore, constructs amplifying pBAD33 (A and D) were incubated overnight with 0,5μl DPN1 enzyme at 37°C to digest methylated bacterial DNA.

All amplifications and purifications were successful. Unfortunately, the PCR image has been lost because of technical issues with the GelViewer.

07.06.19

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NICD (Nephrin Intracellular Domain) was amplified, as well as sfGFP and pBAD33 with respective overlaps.

Following PCRS were set up:

(J) pBAD amplification with primers 1&22*

(K) sfGFP amplification with primers 25&6*

(L) NICD amplification with primers 23&24*

*For all sequences see table in protocol from 04.06.2019

All amplifications were successful, see picture below.

Afterwards, gibson cloning was set up. Considering sizes, amound of insert for 50 ng backbone was calculated.

NICD: 17.6 ng

sfGFP: 24 ng

The DNA was diluted with water until reaching 5 microliters of volume and mixed in a 1:1 ratio with a HiFi ligase/exoniuclease mastermix and incubated at 50°C for 15 minutes. Afterwards competent Top10 E. Coli were transformed with 2 microlitres of Gibson Assembly Mix and incubated overnight.

11.06.19

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After transformation of NICD was ineffective, proportions were calculated again and ligation was repeated. Incubation overnight follows.

Furthermore, gBlocks for EWSR1, FUS, SPD5_1 and SPD5_2 were amplified to create overlaps with following Primers:

EWSR1 with Primers 17 and 18*

FUS with Primers 3 and 4*

SPD5_1 and SPD5_2 with Primers 9&10 and 11&12 respectively*

*For primer and protein sequences see protocol from 04.06.2019.

Amplification was only successful for SPD5_2 in the first round:

PCR troubleshooting consisted of repeating the reaction with two different polymerases, being Q5 and Phusion Flash, as well as adding 3% DMSO to the DNA mix. In the case of EWSR1, DNA was increased to 2 ng.

In the second round, bands for each sequence could be seen, were cut and purified.

12.06.19

Transformation from the day before was unsuccessful, no colonies were on the plate. We found out we used Ampicillin plates, which is the wrong antibiotic.

Since all required sequences for the designed plasmids (for more information about plasmid constructs see protocol from day 04.06.2019) were now amplified, ligation for all plasmids could now start. The method used was Gibson Cloning. The gibson assemblies were then plated on Chloramphenicol plates and incubated overnight.

13.06.19

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Gibson assembly of the Plasmids containing NICD and SPD5, EWSR.

Following constructs were ligated with NEB HiFi Ligase:

Fus: A + B + C (Fus)

EWSR1: H, I, J (EWSR1)

SPD5: D, E, F(SPD5_1) and G (SPD5_2)

Together with the ligated NICD plasmid, all plasmids were transformed into competent E. Coli and plated on chloramphenicol plates.

Plates were incubated overnight at 37°C

14.06.19

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Plates containing FUS, SPD5 and NICD from 13.06.2019 had colonies. EWSR1 had no colonies.

Coloies were picked and LB medium with Chloramphenicol was inoculated, bacteria was grown in media for 5 hours and prepared for sequencing.

A second set of media were incoulated and prepared for microscopy. Samples were visualized with a Widefield microscope and analyzed with FiJi. Images attached.

SPD5 sample 2: Dispersed fluroescence (left). No indication for droplets found.

SPD5 sample 1. Lighter spots that can be an indication for droplets in the fluorescent image (Left) that are not visible on the right.

FUS sample 1. Low, dispersed fluorescence (left). No indications for droplets.

NICD sample 1. Low, dispersed fluorescence (left). No indications for droplets.

Problem with NICD: A cloning mistake has led to an excessive atg start codon at the begining of sfGFP, which may give misleading results.

Complementary primers for a site directed mutagenesis deleting the excessive three bases were ordered.

20.06.19

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Criteria for identifying if an accumulation of fluorescent proteins are dynamic droplets or dead aggregates:

1. They appear in a concentration dependent manner,

2. They fuse when they encounter another one of their kind,

3. Fluorescence regeneraties after photobleaching (FRAP)

In order to determine whether the results seen in the last protocol are significant, the constructed proteins have to be inducible to be seen under different concentrations.

Cloning steps were repeated so that the constructs could be positioned in front of inducible promoters. Additionally, EWSR1 will be inserted in a different backbone with

compatible origins of replications with the pBAD backbone, so that FUS and EWSR1 can be induced in the same bacteria without competition. The new backbone for

EWSR1 is pTrc99a (empty) with a prc promoter.

Following primers are now added to the list:

New primers
Number	Name	Tube name	Sequence	Description
31	pBAD33wt_SPD5_FWD	pBAD33wt_SPD5_FWD	TGGATGAGCTCTACAAATAAAAGCTTGGCTGTTTTGGCGG	Primer to amplify new pBAD33(induceable promoter) with right overlaps
32	pBAD33wt_SPD5_REV	pBAD33wt_SPD5_REV	AGGACAGAGTTATCTTCCATTCTAGAGGATCCCCGGGTAC	Primer to amplify new pBAD33(induceable promoter) with right overlaps
32_2	SPD5_1_mid_GFP_rev	SPD5_1_mid_GFP_rev	GTGCAATAGTGAAACGACGCGACGC
33	SPD5_sfGFP_pBAD33 FWD	SPD5_sfGFP_pBAD33 FWD	GTACCCGGGGATCCTCTAGAATGGAAGATAACTCTGTCCTTAA	Primer to amplify SPD5_1 with the right overlap for new backbone
34	SPD5_sfGFP_pBAD33 REV	SPD5_sfGFP_pBAD33 REV	CCGCCAAAACAGCCAAGCTTTTATTTGTAGAGCTCATCCATGC	Primer to amplify GFP with the right overlap for new backbone
35	EWSR1_mCherry_pBAD33 FWD	EWSR1_mCherry_pBAD33 FWD	CATGGAATTCGAGCTCGGTAATGGCCTCAACTGACTACTC	Primer to amplify EWSR1 with the right overlap for new backbone
36	EWSR1_mCherry_pBAD33 REV	EWSR1_mCherry_pBAD33 REV	GACTCTAGAGGATCCCCGGGTTAGTACAGCTCGTCCATGC	Primer to amplify mCherry with the right overlap for new backbone
37	pTrc99A FWD	pTrc99A FWD	GCATGGACGAGCTGTACTAACCCGGGGATCCTCTAGAGTC	Primer to amplify pTrc99A (induceable promoter) with right overlaps
38	pTrc99A REV	pTrc99A REV	GAGTAGTCAGTTGAGGCCATTACCGAGCTCGAATTCCATG	Primer to amplify pTrc99A (induceable promoter) with right overlaps
39	NICD_sfGFP_pBAD33_FWD	NICD_sfGFP_pBAD33_FWD	GTACCCGGGGATCCTCTAGAATGAATGCGTCATGTGTAGG	Primer to amplify NICD with the right overlap for new backbone
39_2	NICD_mid_sfGFP_REV	NICD_mid_sfGFP_REV	TCCTTTGCTCATGGATCCTCCGGATCCTC	Primer to amplify NICD with the right overlap for new backbone
40	NICD_sfGFP_pBAD33 REV	NICD_sfGFP_pBAD33 REV	CCGCCAAAACAGCCAAGCTTTTATTTGTAGAGCTCATCCATGC	Primer to amplify sfGFP with the right overlap for new backbone
41	pBAD33wt_NICD FWD	pBAD33wt_NICD FWD	TGGATGAGCTCTACAAATAAAAGCTTGGCTGTTTTGGCGG	Primer to amplify new pBAD33(induceable promoter) with right overlaps
42	pBAD33wt_NICD REV	pBAD33wt_NICD REV	CCTACACATGACGCATTCATTCTAGAGGATCCCCGGGTAC	Primer to amplify new pBAD33(induceable promoter) with right overlaps

	Primers for NICD atg deletion

	atg_del_fwd	GATCCGGAGGATCCAGCAAAGGAGAAGAAC
	atg_del_Rev	GTTCTTCTCCTTTGCTGGATCCTCCGGATC

Constructs cotnaining the backbone with inducible promoters will be the following:

FUS, containing the following fragments

XA	pBAD	w/primers 29&30	68°C
XB	GFP	w/ primers 5&28	63°C
XC	FUS	w/ primers 27&4	68°C

SPD5, containing the following fragments

XD	pBAD	w/primers 31&32	68°C
XE	GFP	w/primers 13&34	63°C
XF	SPD1	w/primers 33&10	60°C
G	SPD2

EWSR1, containing the following fragments:

XG	pBAD	w/primers 37&38	65°C
XH	EWSR1	w/primers 35&18	65°C
XI	mCherry	w/primers 19&36	65°C

NICD, containing the following fragments:

XJ	pBAD	w/primers 41&42	68°C
XK	sfGFP	w/primers 25&40	63°C
XL	NICD	w/primers 39&24	63°C

21.06.19

<!DOCTYPE html>

Amplifications for the gibson fragments with corrresponding sticky ends were started in order to achieve plasmids with the constructs in front of inducible promoters.

Image left and right: 1% agarose gel with results of amplification of gibson fragments.

All Fragments except EWSR1 gBlock could be successfully amplified. New primers were ordered as part of the troubleshooting procedure.

The new primers are:

43	pTrc99a_rev	pTrc99a_inducible_rev,	ttgaggccatTACCGAGCTCGAATTCCATG
44	EWSR1_fwd	EWSR1_inducible_fwd,	gagctcggtaATGGCCTCAACTGACTACTC
45	EWSR1_rev	EWSR1_inducible_rev	ccttgctcacggatcctccggatcctccATAGGGACGATCACGGCG
46	mCherry_fwd	mCherry_inducible_fwd	tcgtccctatggaggatccggaggatccGTGAGCAAGGGCGAGGAG
47	mCherry_rev	mCherry_inducible_rev,	gatccccgggGTACAGCTCGTCCATGCC
48	pTrc99a_fwd	pTrc99a_inducible_fwd	cgagctgtacCCCGGGGATCCTCTAGAG

25.06.19

<!DOCTYPE html>

Labbook

25/6/19

Repeat PCR for SPD5_1 and FUS and NICD with new primers and extract from agarose gel.

Transform into competent cells and grow overnight culture.

26/6/19

Planning of FRAP experiment

Widefield and Brightfield microscopy of samples

Gibson assembly of FUSinducible (XA, XB, XC)

50 ng of Backbone (1uL)

50 ng sfGFP (2,78 uL)

73 ng FUS (0.73 uL)

Retransformation of bacteria with correctly sequenced constructs (constitutive)

Repeat PCR for NICD and SPD5 fragments

27/6/19

Miniprep of assembled FUS

Repeat PCR of fragments XC, XF, XL, XH and HI (not successful except XC, XF)

Repeat PCR of fragments XH XI and XL with 3% DMSO

Gibson assembly of NICD and SPD5 with correct amplified fragments.

NICD: 50 ng Backbone (0,63 uL)

23,86 ng sfGFP (2,06 uL)

51,14 ng NICD (2,60 uL)

SPD5: 50 ng Backbone (2.5 uL)

sfGFP: 24,68 ng (1,1 uL)

SPD5_1: 68,18 (4,54 uL)

SPD5_2: 51,14 (1,17 uL)

2/7/19

PCR of EWSR1 with primers 17&18, 18&35 and 44&45 in order to amplify EWSR1 constitutive and EWSR1 inducible (twice)

PCR was not successful. Next step is a Touchdown PCR starting at 75°C and going down to 65°C (annealing temperature of the three constructs)

Induceable constructs were retransformed (NICD was made again by Gibson assembly)

Constitutive constructs were retransformed. All retranformations were plated on Chloramphenicol plates and placed overnight in the 37°C incubator.

3/7/19

Colonies were picked and new overnight cultures were prepared from the retransformation of the constructs (induceable and constitutive) for the FRAP experiment.

New Glycerol stocks from all constructs were frozen at -80°C

Glucose free TB medium was mixed, heated and autoclaved in order to prepare the induction and FRAP experiment.

New Chloramphenicol plates were made (LB medium + 400uL 34g/mol Chloramphenicol stock solution added at 50°C)

4/7/19

Microscopy of the working constructs (constitutive FUS, NICD and SPD5 as well as Arbinose-inducible FUS, NICD and SPD5) and the negative control IPTG-inducible

mini eYFP which is known to form aggregates at high concentrations (source: AG Di Ventura, BIOSS Freiburg).

Induction was carried out with the following concentrations:

No induction: E. coli carrying plasmids were grown overnight in TB, diluted to OD 0.01 in the morning, grown to OD 0.5 and further incubated for 2h at 37°C with vigorous shaking.

Induction: E. coli were grown in overnight culture in TB medium, diluted to OD 0.01 in the morning, grown to OD 0.5 and induced with the following concentrations of Arabinose:

LOW: 0.001% Arabinose

MID: 0.05% Arabinose

HIGH: 1% Arabinose

After 2h in the incubater at 37°C and vigorous shaking, miscroscopy was carried out.

At first, samples were analysed with brightfield and widefield GFP microscopy. Cells were checked for droplet like dots.

Most cells didn’t show droplet-like structures, only constitutive SPD5 (as already shown before). Induction seemed to not have worked, since the positive control

plasmid mini eYFP did not show the expected results. Furthermore, general fluorescence of mid and high induced cells was not to be distinguished from low induced cells in most cases.

The constitutive SPD5 probe containing droplet-like structures was analysed under the confocal microscope at 100x. Bleaching was done with 100% laser

intensity with an itinerary of 20. Single droplet like structures were bleached and the Region of Interest (ROI) was analysed, while other ROIs were only analised.

Results did not seem to be clear. Induceable constructs are analysed (sequenced) and microscopy will be repeated in a week.

FUS_const_04_07.jpg	NICD_cons_04_07.jpg	SPD5_const_04_07.jpg
FUS_const_04_07_2.jpg	NICD_const_04_07_2.jpg	SPD5_const_04_07_2.jpg
FUS_high_04_07.jpg	NICD_high 10_07.jpg	SPD5_high_04_07.jpg
FUS_high_04_07_2.jpg	NICD_high_04_07.jpg	SPD5_high_04_07_2.jpg
FUS_mid_04_07.jpg	NICD_mid_04_07.jpg	SPD5_mid_04_07_2.jpg
FUS_mid_04_07_2.jpg	NICD_mid_10_07.jpg	SPD5_mod_04_07.jpg
FUS_low_04_07.jpg	NICD_Low_04_07.jpg	SPD5_low_04_07.jpg
FUS_low_04_07_2.jpg	NICD_Low_04_07_1.jpg	SPD5_low_04_07_2.jpg

8/7/19

Touchdown PCR was started with the EWSR1 constructs, amplified with primer pairs 17&18(constitutive), 18&35 and 44&45 (with the araBAD promoter)

17&18(constitutive), 18&35 and 44&45 (with the araBAD promoter)

9/7/19

Ordered new gBlocks at twist. Ewsr in two parts. 999bp and 1002bp.

Made new TB buffer.

New cloning for EWSR1:

Kinase reaction on the insert with T4 polynucleotide kinase

Find restriction enzymes

pTrc99a empty: digest with sma1 (2h)
Inactivation wth heat (check temperature in NEB): 65°C 20 min
Column purification of the digested backbone: elute in 20uL of water
Measure concentration: take 30-50 ng of template
Take 150 ng of the gblock
Ligation overnight 4°C with ligase
Transform 50uL and plate
Colony PCR to verify (try 10 colonies at first)

Overnight culture in TB medium of constructs FUS3 ind (verified by sequencing), SPD5_1 ind (verified by sequencing) and NICD (not verified) and mini eYFP construct.

New constructs were sent for sequencing

10/7/19

Measurement of overnight cultures. All cultures are at an OD of 3,3. All cultures were diluted to an OD of 0.03 and incubated again until reaching OD 0.5

Retransformation of pTrc99a

Measurement of overnight cultures. All cultures are at an OD of 3,3. All cultures were diluted to an OD of 0.03 and incubated again until reaching OD 0.5

All cultures were split and induced with different levels of arabinose:

NICD: NICD uninduced, NICD low (0.001%), NICD mid (0.05 %) and high (1%)

FUS: FUS uninduced, FUS low (0.001%), FUS mid (0.05 %) and high (1%)

SPD5: SPD5 uninduced, SPD5 low (0.001%), SPD5 mid (0.05 %) and high (1%)

Control: mini eYFP uninduced and induced with 1 mM IPTG

None of the transformations were successful.

	10-7-FUS_constitutive (1).jpg		10-7-SPD5_constitutive-04.jpg
	10-7-FUS_constitutive (2).jpg
10-7-eYFP_high (1).jpg	7-10-FUS_high_induction widefield (1).jpg	10-7-NICD_high_10_07_2 (1).jpg
10-7-eYFP_high (2).jpg	7-10-FUS_high_induction widefield (2).jpg	10-7-NICD_high_10_07_2 (2).jpg
10-7-eYFP_uninduced (1).jpg	10-7-FUS_mid_induction-03 widefield (1).jpg	NICD_mid_10_07_2.jpg
10-7-eYFP_uninduced (2).jpg	10-7-FUS_mid_induction-03 widefield (2).jpg	NICD_mid_10_07.jpg
	10-7-FUS_low_induction-02 gfp (1).jpg	10-7-NICD_low_10_7_2 (1).jpg
	10-7-FUS_low_induction-02 gfp (2).jpg	10-7-NICD_low_10_7_2 (2).jpg
	10-7-FUS_no_induction-03 widefield (1).jpg	NICD_unicduced10_7-02.jpg
	10-7-FUS_no_induction-03 widefield (2).jpg	NICD_uninduced 10_7.jpg

11/7/19

Sequencing results show that promoter of the backbones used for the inducible constructs is not AraC but a modified construct. Empty pBad33 plasmid is now

sequenced and restriction site cloning prepared.

Also retransformation of pTrc99a was not successful and will be repeated in order to initialize restriction enzyme cloning of the EWSR1 cloning.

Retransformation ptrc and pbad33 empty as well as backbone for receptor

Pcr of fragments for essays were amplified

FRAP was ameliorated with the constitutive SPD5 construct that has been working since the beginning.

With a pinhole of 65 um, a laser intensity of 100% and 65 itinerations, droplets were bleached and recovery could be observed over time.

12/7/19

13.7.19

Blade light induction

O/N culture with 3 mL of terrific broth

Measurement of OD in the dark with red light lamp. ODs were all 2.0

Dilution of cutures to OD 0.1

Incubation for 1,5h hours to reach OD 0.4

(ODs were too high, between 0.55 and 0.78) -> dilute

Split cultures (1.5 mL), half of them in dark tubes (uninduced), half of them in light tubes (induced).

Transparent tubes were placed in front of blue LED plate (15,5 V) in 37°C shaker.

4h incubation/illumination

2mL on PBS+Agar plates for microscopy

New TB medium: 5g NaCl, 10g Bacto Tryptone and 1 mL of NaOH 1M. Autoclaved.

New overnight culture of FUS (ind), SPD5 (ind), NICD (ind) as well as cells with an empty pBAD (negative control) and a mini eYFP plasmid (positive control)

14. 7.19

Measurement of the 5 cultures’ OD at 8 am, dilution to OD 0.1.

1.5h incubation and measurement of OD. All ODs at 0.4

Split cultures:

NICD: NICD uninduced, NICD induced (4h light), NICD induced (1 % arabinose)

FUS: FUS uninduced, FUS induced (4h light), FUS induced (1 % arabinose)

SPD5: SPD5 uninduced, SPD5 induced (4h light), SPD5 induced (1% arabinose)

Empy pBAD: uninduced / 1% Arabinose

Mini eYFP: uninduced / 1% Arabinose

Incubation for 4 hours.

7-14 mini eYFP no arabinose (1).jpg	7-14-FUS_arabinose-02_2 (1).jpg	7-14-NICD_Arabinose_2 (1).jpg	7-14-pBAD negative control (1).jpg	7-14-SPD5_arabinose-02_2 (1).jpg
7-14 mini eYFP no arabinose (2).jpg	7-14-FUS_arabinose-02_2 (2).jpg	7-14-NICD_Arabinose_2 (2).jpg	7-14-pBAD negative control (2).jpg	7-14-SPD5_arabinose-02_2 (2).jpg
YFP_high-06.jpg	7-14-FUS_light_ind_2 (1).jpg	7-14-NICD_light induced_2 (1).jpg		7-14-SPD5_light_ind-02_2 (1).jpg
YFP_high-06_2.jpg	7-14-FUS_light_ind_2 (2).jpg	7-14-NICD_light induced_2 (2).jpg		7-14-SPD5_light_ind-02_2 (2).jpg
	7-14-FUS_uninduced-10 (1).jpg	7-14-NICD_uninducefd-07_2 (1).jpg		7-14SPD5_unind-05_2 (1).jpg
	7-14-FUS_uninduced-10 (2).jpg	7-14-NICD_uninducefd-07_2 (2).jpg		7-14SPD5_unind-05_2 (2).jpg

Text

                     </div>
                 </div>
             </div>

                     MCP and ms2 cloning and characterization

Subsection Title

Text

Subsection Title #2

Text

                     Intein Expression Project

01.07.2019

- PCR of the N-Intein (Npu DnaE, BBa_K1362100)

- Master Mix (50µL):

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19nifwd 10µM	2.5 µL	0.5 µM
Primer: oIG19nirev 10 µM	2.5 µL	0.5 µM
Template DNA 200-300 pg/µL	3 µL	600-900 pg
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	30.5 µL	-

- PCR Programm:

Step	Temp.	Time
Initial Denaturation	98°C	3 min
Denaturation Annealing 30x Extension	98°C 54°C 72°C	10 sec 25 sec 45 sec
Final Extension	72°C	2 min
Hold	4°C	-

02.07.2019

- PCR of the C-Intein (Npu DnaE, BBa_K136201)

Master Mix (50µL):

reagents	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19cifwd 10µM	2.5 µL	0.5 µM
Primer: oIG19cirev 10 µM	2.5 µL	0.5 µM
Template DNA 200-300 pg/µL	3 µL	600-900 pg
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	30.5 µL	-

PCR Programm:

Step	Temp.	Time
Initial Denaturation	98°C	3 min
Denaturation Annealing 30x Extension	98°C 56°C 72°C	10 sec 25 sec 42 sec
Final Extension	72°C	2 min
Hold	4°C	-

- Gelelectrophoresis (1% Agarose, 100 V, 50 min, with Midori Green (3 µL/100 mL):

H2O C-Int

H2O N-Int

...

1 kb ladder

...

C-Int

N-Int

 <img width="400" src="../../imgs/Intein_Project/190702_pcr_inteins.png" alt="PCR Gelelctrophoresis of Inteins">

- Gelextraction with NucleoSpin® Gel and PCR Clean-up from Machery-Nagel following the protocol (page 19) at:
https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf

04.07.2019

- PCR of pET302 for the C-Intein

Master Mix (50 µL):

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19bbcifwd 10µM	2.5 µL	0.5 µM
Primer: oIG19bbcirev 10 µM	2.5 µL	0.5 µM
Template DNA 86 ng/µL	1 µL	86 ng
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	32.5 µL	-

PCR Programm:

Step	Temp.	Time
Initial Denaturation	98°C	3 min
Denaturation Annealing 30x Extension	98°C 64°C 72°C	10 sec 30 sec 3 min
Final Extension	72°C	3 min
Hold	4°C	-

05.07.2019

- PCR of pET302 for N-Intein

Master Mix (50 µL):

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19bbnifwd 10µM	2.5 µL	0.5 µM
Primer: oIG19bbnirev 10 µM	2.5 µL	0.5 µM
Template DNA 86 ng/µL	1 µL	86 ng
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	32.5 µL	-

PCR Programm:

Step	Temp.	Time
Initial Denaturation	98°C	3 min
Denaturation Annealing 30x Extension	98°C 64°C 72°C	10 sec 30 sec 3 min
Final Extension	72°C	3 min
Hold	4°C	-

- Gelelektrophoresis (100 V, 90 min, 1% Agarose, Midori Green 3 µL/100 mL) showed no bands

- troubleshooting showed that i used too much DNA

- Did a gradient PCR for C-Int bb:

Master Mix 300 µL:

reagent	amount	concentration
5X Q5 Reaction Buffer	60 µL	1X
10 mM dNTPs	6 µL	200 µM
Primer: oIG19bbcifwd 10µM	15 µL	0.5 µM
Primer: oIG19bbcirev 10 µM	15 µL	0.5 µM
Template DNA 9.8 ng/µL	1 µL	1,96 ng
Q5 High-Fidelity DNA Polymerase	6 µL	0.02 U/µL
Nuclease-Free Water	200 µL	-

- took 50 µL of the mastermix away before adding the DNA to have a H2O control, put the rest in 5 tubes, annealed at the following temperatures:

tube 1	tube 2	tube 3	tube4	tube 5	H2O tube
55,1°C	56,8°C	59,5°C	61°C	63,7°C	63,7°C

PCR Programm:

Step	Temp.	Time
Initial Denaturation	98°C	3 min
Denaturation Annealing 30x Extension	98°C - °C 72°C	10 sec 30 sec 4,5 min
Final Extension	72°C	4.5 min
Hold	4°C	-

06.07.2019

- repeated the gradient-PCR with the pET302 for the N-Intein

08.07.2019

- Gelelektrophoresis (100V, 1% Agarose, 75 min) showed no bands

- found out that the touch-down function was activated

- did a Gradient PCR for the N-Intein Backbone:

Master Mix 350µL:

reagent	amount	concentration
5X Q5 Reaction Buffer	70 µL	1X
10 mM dNTPs	7 µL	200 µM
Primer: oIG19bbnifwd 10µM	17.5 µL	0.5 µM
Primer: oIG19bbnirev 10 µM	17.5 µL	0.5 µM
Template DNA 9.8 ng/µL (added after taking 50µL of the MM for H₂O control)	0.6 µL	1.96 ng
Q5 High-Fidelity DNA Polymerase	3.5 µL	0.02 U/µL
Nuclease-Free Water	233.9 µL	-

- took 50 µL of the mastermix away before adding the DNA to have a H2O control, put the rest in 6 tubes, annealed at the following temperatures:

tube 1	tube 2	tube 3	tube4	tube 5	tube 6	H2O tube
49,2°C	51,5°C	55,2°C	57,3°C	61,1°C	64,1°C	64,1°C

PCR Programm:

Step	Temp.	Time
Initial Denaturation	96°C	1 min
Denaturation Annealing 30x Extension	96°C - °C 72°C	30 sec 30 sec 4.5 min
Final Extension	72°C	10 min
Hold	4°C	-

09.07.2019

- Gelelektrophoresis of the N-Int. BB PCR (100V, 1% Agarose, 75 min, Midori Green (3 µL/100 mL)) showed:

 <img width="400" src="../../imgs/Intein_Project/190709 backbone n int.png"
 alt="Gelelekt. PCR NI BB">

-did a PCR extraction of the tubes of the bands 2,4,5 after the protocol of: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/General_Information/1/high-pure-pcr-product-purification-kit.pdf (page 7)

- repeated the PCR with the same conditions for the C-Intein BB

10.07.2019

- Gelelektrophoresis of the C-Int. BB PCR (100V, 1% Agarose, 75 min, Midori Green (3 µL/100 mL)) showed:

 <img width="400" src="../../imgs/Intein_Project/190710 backbone c int.png">

- did a PCR extraction of the tubes after the protocol of: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/General_Information/1/high-pure-pcr-product-purification-kit.pdf (page 7)

- did 2 Gibson assemblys 1 h, 50°C (protocol: https://international.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol) with the following amounts of DNA:

N-Intein (1464bp)	BB for N-Intein (5683bp)	C-Intein (1259bp)	BB for C-Intein (5682bp)
38.64ng	50ng	33.24ng	50ng

- did a transformation with competent TOP10 cells following the protocol of: https://international.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987

- platet the cells on Agar plates with Ampicillin and let grow overnight at 37°C

11.07.2019

- no colonies on the plates

- did a gelelectrophoresis (1% Agarose, 100 V, 75 min, Midori Green (3 µL/100 mL)) with the following DNA Products:

ladder

N-Int

BB for N-Int

Gibson BB+N-Int (40ng)

C-Int

BB for C-Int

Gibson BB+C-Int (40ng)

ladder

- had to do a post-staining with Gel-Red (30 min):

 <img width="400" src="../../imgs/Intein_Project/190711 gibson ints + bb.png">

- bands of coiled plasmids can be seen in the top area

- did another transformation with NEB 5-alpha competent cells following this protocol: https://international.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987

- plated on Agar plates with Ampicillin and let grow overnight at 37°C

12.07.2019

- colonies did grow overnight and did show red fluorescence as the should with a functioning biobrick inserted into the plasmid

- picked colonies and let grow overnight at 37°C in 2ml of LB-medium with Ampicillin (100 µg/ml) each

13.07.2019

- cultures did grow well, a miniprep was performed following this protocol: https://files.zymoresearch.com/protocols/_d4208t_d4209_d4210_d4211_d4212_zymopure_plasmid_miniprep.pdf (page 4 + 6)

- with the harvested plasmids a PCR was performed to insert a point mutation to delete a BsaI restriction site:

PCR Programm: BsaI point mutation :

Master Mix 1 (50µL) each for N-/C-Intein:

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19bbBsaI mut fwd 10µM	2.5 µL	0.5 µM
Primer: oIG19bbBsaI mut rev 10 µM	2.5 µL	0.5 µM
Template DNA 1.2 ng/µL	1 µL	1.2 ng
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	30.5 µL	-
DMSO	2 µL	4%

Master Mix 2 (50µL) each for N-/C-Intein:

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19bbBsaI mut fwd 10µM	2.5 µL	0.5 µM
Primer: oIG19bbBsaI mut rev 10 µM	2.5 µL	0.5 µM
Template DNA 1.2 ng/µL	1 µL	1.2 ng
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	32.5 µL	-

PCR Programm:

Step	Temp.	Time
Initial Denaturation	96°C	1.5 min
Denaturation Annealing 30x Extension	96°C 72°C 72°C	35 sec 30 sec 5 min 45 sec
Final Extension	72°C	10 min
Hold	4°C	-

15.07.2019

- did a gelelectrophoresis (1% Agarose, 100 V, 80 min, Midori Green (3 µL/100 mL)), no plasmid bands

- did a gradient PCR with the N-Intein + BB to get out the BsaI restriction site:

Master Mix (150µL for 10 tubes):

reagent	amount	concentration
5X Q5 Reaction Buffer	30 µL	1X
10 mM dNTPs	3 µL	200 µM
Primer: oIG19bbBsaI mut fwd 10µM	7.5 µL	0.5 µM
Primer: oIG19bbBsaI mut rev 10 µM	7.5 µL	0.5 µM
Template DNA 1.2 ng/µL	3 µL	1.2 ng/50 µL
Q5 High-Fidelity DNA Polymerase	1.5 µL	0.02 U/µL
Nuclease-Free Water	97.5 µL	-

Annealing Temperatures:

tube	1	2	3	4	5	6	7	8	9	H₂O
temp.	55°C	56.2°C	57.5°C	59.4°C	62°C	65°C	67.6°C	69.5°C	71.6°C	71.6°C

PCR Programm:

Step	Temp.	Time
Initial Denaturation	96°C	1.5 min
Denaturation Annealing 30x Extension	96°C - °C 72°C	35 sec 30 sec 5 min 45 sec
Final Extension	72°C	10 min
Hold	4°C	-

- Gelelectrophoresis showed no bands

16.07.2019

- Did a PCR with the N-Int. with 3, 5 and 10% DMSO whith the annealing temperatures = Tm - 0.6°C x 1% DMSO

- Gelelectrophoresis showed a band of the right size (on the right, same size as +ctrl on the left)

 <img width="400" src="../../imgs/Intein_Project/190716 N-Int bsa1 mut dmso (E).png ">

16.07.2019

- Did a DpnI Digestion of the N-Int 10% DMSO tube (0.5 µL DpnI, 1 h, 37°C)

- Did a PCR-Extraction after the protocol of: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Roche/Bulletin/1/hppcrpkrobul.pdf (page 7)

- Did 2 PCRs of the C-Intein, one with BsaI mut fwd and a rev Primer in the Amp. Resistance, and one with BsaI mut rev and a fwd primer in the Amp. Res.

17.07.2019

- Gelelctrophoresis showed bands of the right size for the two PCRs with the Primers inside of the Amp. Resistance, both constructs were isolated with Gel-Extraction and a Gibson Asembly was performed

- Did a Transformation with the N-Int construct (out of the 10% DMSO tube) and the C-Int construct (out of the Gibbson assembly) into NEB Alpha-5 competent cells

- plated on Agar plates with Amp and let incubate overnight at 37°C

18.07.2019

- colonies did grow for both constructs! picked 3 colonies each and put into 2 mL of LB medium with Amp, let grow overnight

19.07.2019

- did a mini-prep of each culture with Zymo-Miniprep kit, send the isolated plasmids to sequencing

24.07.2019

- Sequencing uncomplete because of wrong primers but point mutations were inserted succesful in both constructs

- repeated sequencing with other primers

25.07.2019

- Sequencing showed that the N-Int has no unwanted pointmutations and the insert is fully inserte

- but the C-Intein construct is not fully inserted, parts of the insert are missing, will repeat the point mutation PCR with 10% DMSO instead of the Gibson approach

26.07.2019

- Gelelectrophoresis showed no bands

- did a PCR (following https://international.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491) with the C-Intein + BB with the fwd and rev Primer in seperate tubes (16 cycles), then mixed together and did 14 cycles

27.07.2019

- Gelelectrophoresis showed a band that was approx. 500bp to small

- looking closer at the sequence showed ~75% GC content at one region

28.07.2019

- repeated the PCR of C-Int + BB with seperated Primer but added GC-Enhancer

Master Mix (50µL each)

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19BB bsaI mut fwd / rev	2.5 µL	0.5 µM
Template DNA (C-Int + BB) 1 ng/µL	1 µL	1 ng
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	25 µL	-
High GC-Enhancer	10 µL	1X

PCR Programm:

Step	Temp.	Time
Initial Denaturation	96°C	1 min
Denaturation Annealing 16x, then mix both tubes, 14x Extension	96°C 70°C 72°C	30 sec 30 sec 5 min
Final Extension	72°C	10 min
Hold	4°C	-

29.07.2019

- Gelelctrophoresis showed band of the right size (ladder, +ctrl, tube 1. tube 2, too short band without GC-Enhancer):

 <img width="400" src="../../imgs/Intein_Project/190729 C-Int bsaI mut.png ">

- did a PCR-Purification of the remaining samples

- did transformation of the mutated C-Int + BB construct with competent NEB 5-alpha cells, platet on Agarplates with Amp, incubated overnight

- picked colonies with the SUMO1-Plasmid (obtained from BIOSS Toolbox, University Freiburg) and cultured them in 3 mL LB-Medium with Amp. overnight

30.07.2019

- Did a Mini Prep of the SUMO1 cultures

- Did a PCR with TrxA (obtained from Di Ventura Lab, University Freiburg)(Primers: IG19BB TrxA fwd/rev) and with SUMO1 (Primers: IG19BB SUMO1 fwd/rev) , introduced the overhangs from RFC105 for the Golden Gate Assembly

- Gelelctrophoresis (100bp ladder, SUMO1, TrxA, 1kb ladder):

 <img width="400" src="../../imgs/Intein_Project/190730 PCR Products of SUMO1 and TRX.png ">

- both PCR products had the right size: PCR purification

- Picked colonies with the mutated C-Int + BB construct (from 29.07) and cultured them in 2 mL LB-Medium with Amp overnight

31.07.2019

- Did a Mini Prep of the cultures with the mutated C-Int + BB construct

- Send them to Sequencing

01.08.2019

- Sequencing succesful

- Inserted SUMO1 into N-Int + BB mut and TrxA into the C-Int. + BB mut constructs, both with Golden Gate Assembly:

Master Mix (each): 3:1 (Insert:Backbone(150ng)), 1.5 µL T4 DNA Ligase Buffer, 9 µL dH20 (filled up to 15 µL), 1µL BsaI, 1µL T4 DNA Ligase

Cycler Program:

Step	Temp.	Time
Restriction x25 Ligation	37°C 16°C	3 min 4 min
Final Restriction	50°C	5 min
Denaturation	80°C	5 min
Hold	4°C	-

- Did a Transformation with NEB 5-Alpha comp. cells with 2 or 4 µL of the Golden Gate product and plated the cells on Agar plates with Amp., incubated at 37°C o/n

02.08.2019

- colonies did grow! picked 2 of each construct and cultured in 2 mL LB-Medium with Amp.

- after 7 hours did a Mini Prep and sent all DNA to Sequencing

- reincubated the remaining cells in the tube in 4mL LB-Medium with Amp.

04.08.2019

- Did a Miniprep of the NI-SUMO1 and CI-Trx cultures, same cultures as sent to sequencing, did this to have enough DNA

05.08.2019

- Sequencing showed that Golden Gate Asembly was performed succesful

11.08.2019

- Cultured bacteria with NI-SUMO and CI-TRX in 2 mL LB-medium + Amp at 37°C for 6 hours

- measured OD and put the amount of bacteria into 4 mL of LB -medium + Amp to have OD 0.1, incubated 1 hour at 37°C

- after 1 h added IPTG with 1mM end concentration, incubated 2 more hours then pelletized and stored pellets o/n at -80°C

12.08.2019

- resuspended the pellets in 250 µL Promega FastBreak Lysis Buffer, added PMSF (1mM), sonicated the cells (3 x 6 cycles of 1 min, max volume)

- centrifuged the lysate 15 min at max speed at 4°C, took the supernatant and stored on ice

- prepared 4 tubes:

tube

1

2

3

4

ingredients

50 µL NI-SUMO1

5mM DTT

50 µL CI-TRX

5mM DTT

100 µL NI-SUMO1

100 µL CI-TRX

100 µL NI-SUMO1

100 µL CI-TRX

5mM DTT

- took 20 µL samples of each tube directly and then each hour (T0-T4), added 5 µL 5xSDS-Buffer and cooked 10 min at 95°C

- performed the following PCRs:

PCR-Programm: C-Int BB for Sec-Tag Insertion:

Master Mix (50µL)

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19BB C-Int BB T7 Term fwd	2.5 µL	0.5 µM
Primer: oIG19BB C-Int for sfGFP rev	2.5 µL	0.5 µM
Template DNA (C-Int + BB mut) 0.4ng/µL	2.5 µL	1 ng
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	21 µL	-
High GC-Enhancer	10 µL	1X

PCR Programm:

Step	Temp.	Time
Initial Denaturation	96°C	1 min
Denaturation Annealing x30 Extension	96°C 63°C 72°C	30 sec 30 sec 4 min
Final Extension	72°C	10 min
Hold	4°C	-

PCR-Programm: N-Int BB for Sec-Tag Insertion:

Master Mix (50µL)

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19BB N-Int for HlyA fwd	2.5 µL	0.5 µM
Primer: oIG19BB N-Int for HlyA rev	2.5 µL	0.5 µM
Template DNA (N-Int + BB mut) 0.4ng/µL	2.5 µL	1 ng
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	21 µL	-
High GC-Enhancer	10 µL	1X

PCR Programm:

Step	Temp.	Time
Initial Denaturation	96°C	1 min
Denaturation Annealing x30 Extension	96°C 68°C 72°C	30 sec 30 sec 5 min 30 sec
Final Extension	72°C	10 min
Hold	4°C	-

PCR-Programm: HlyA Insert (for N-Intein):

Master Mix (50µL):

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19BB HlyA fwd 10µM	2.5 µL	0.5 µM
Primer: oIG19BB HlyA rev 10 µM	2.5 µL	0.5 µM
Template DNA HlyA 0.3 ng/µL	1 µL	0.3 ng
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	32.5 µL	-

PCR Programm:

Step	Temp.	Time
Initial Denaturation	96°C	1 min
Denaturation Annealing 30x Extension	96°C 65°C 72°C	30 sec 30 sec 20 sec
Final Extension	72°C	3 min
Hold	4°C	-

PCR-Programm: sfGFP Insert (for C-Intein):

Master Mix (50µL):

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19BB sfGFP fwd 10µM	2.5 µL	0.5 µM
Primer: oIG19BB sfGFP rev 10 µM	2.5 µL	0.5 µM
Template DNA sfGFP 0.3 ng/µL	1 µL	0.3 ng
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	32.5 µL	-

PCR Programm:

Step	Temp.	Time
Initial Denaturation	96°C	1 min
Denaturation Annealing 30x Extension	96°C 66°C 72°C	30 sec 30 sec 40 sec
Final Extension	72°C	3 min
Hold	4°C	-

14.08.2019

- performed the following PCR:

PCR-Programm: C-Int for Sec-Tag Insertion:

Master Mix (50µL)

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: oIG19BB C-Int BB T7 Term rev	2.5 µL	0.5 µM
Primer: oIG19BB C-Int for sfGFP fwd	2.5 µL	0.5 µM
Template DNA (C-Int + BB mut) 0.4ng/µL	2.5 µL	1 ng
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µL
Nuclease-Free Water	21 µL	-
High GC-Enhancer	10 µL	1X

PCR Programm:

Step	Temp.	Time
Initial Denaturation	96°C	1 min
Denaturation Annealing x30 Extension	96°C 67°C 72°C	30 sec 30 sec 1 min
Final Extension	72°C	10 min
Hold	4°C	-

- performed an SDS-PAGE with the samples of the 12.08 (12% Gel, 1 h 20 min, 80 V):

ladder

Tube 2 - T0

Tube 1 - T0

Tube 3 - T0

Tube 4 - T0

Tube 4 - T1

Tube 4 - T2

Tube 4 - T4

Tube 3 -T4

- the Gel leaked at one side, so the ladder could not be used, after blotting and antibody staining (15.08) no bands could be identified and we found out that we used cells for the expression, that were not able to work with the T7 Promotor of our construct

- Gelelectrophoresis( ladder, HlyA, NI-BB, sfGFP, CI, CI-BB) 1 h, 1% Agarose, 120 V:

 <img width="400" src="../../imgs/Intein_Project/190814 PCR of Secretion Insertion Fragments.png ">

- all bands had the correct size besides NI-BB in lane 4 (no product)

15.08.2019

- repeated the PCR of NI-BB with a touchdown Program (72 - 57 °C), Gelelectrophoresis showed: did not work

- tranformed Rosetta cells with the NI-SUMO1 and the CI-TRX Plasmid and plated them, incubated at 37°C over night

16.08.2019

- repeated the PCR of NI-BB with a touchdown Program (62-57 degrees) and 10% DMSO, Gelelectrophoresis showed: did not work

- picked colonies from the transformed Rosetta cells, incubated in 2 mL LB-Medium + Amp overnight at 37°C

18.08.2019

- measured OD of the Rosetta cells and put the amount of bacteria into 4 mL of LB -medium to have OD 0.1, incubated 2 hours at 37°C

- after 2 h added IPTG with 1mM end concentration, incubated 3 more hours then pelletized and stored pellets over night at -80°C

20.08.2019

- prepared Reaction Buffer K: 100mM Tris (pH 7.5), 5mM DTT, 1mM EDTA

- resuspended pellets from yesterday in 300 µL Reaction Buffer K, sonicated the cells (4x 8 cycles of 1 min, max volume)

- centrifuged 15 min, 4°C, max speed

- prepared 4 tubes:

tube

1

2

3

4

ingredients

300 µL CI-TRX

300 µL NI-SUMO1

300 µL CI-TRX

300 µL NI-SUMO1

300 µL CI-TRX

(both in lysis buffer instead of Reaction Buffer K)

- took 20 µL samples of each tube directly and then each hour (T0-T4), added 5 µL 5xSDS-Buffer and cooked 10 min at 95°C

- did a gradient PCR of NI-BB with Q5, from 72 to 57°C

- did a touchdown PCR of NI-BB with Phusion 2xMM with and without 10% DMSO (66-61°C and 63-58°C)

21.08.2019

- Gelelctrophoresis of all PCR tubes from yesterday showed not one band :(

- Did a SDS-PAGE with the following samples (1 h 10 min, 100 V, 12% Gel):

Tube 1- T0

Tube 2- T0

Tube 3-T0

Tube 4-T0

ladder

Tube 3- T1

Tube 3- T2

Tube 3- T3

Tube 3- T4

Tube 4- T4

- Blotted 30 min, 1A, semi-dry

- Blocked 1 h with 5% BSA

- Incubated 1 h with 1. ABs (Rabbit anti SUMO1: 1/5000 and Mouse anti TRX1: 1/2000) at room temp.

- wash 3x in PBST

- Incubated 1 h with 2. ABs (Goat anti Rabbit-Cy5: 1/2000 and Goat anti Mouse Alexa Flour 488: 1/2000)

- wash 3x in PBST

- Took flouressence pictures, TRX:

 <img width="400" src="../../imgs/Intein_Project/190821-Western Blot stained against TRX.png ">

- lowest band is at 17kDa and should show CI-TRX, the band of a splice product(SUMO1-TRX) should be at 24kDa and cannot be seen

22.08.2019

- Did a gradient PCR of NI-BB with Phusion (52-62°C), gelelectrophorsis showed no bands, orderd new primers with shorter overhangs, bringein in the wanted overhangs in 2 PCRs in a row

23.08.2019

- Did a SDS-PAGE with the samples from 20.08 (1 h 10 min, 100 V, 12% gel):

ladder

tube 1-T0

tube 2-T0

tube 3-T0

tube3-T4

- stained 30min with FastBlue Comassie staining:

 <img width="400" src="../../imgs/Intein_Project/190823 comassie stain of NI SUMO and CI TRX.png ">

- The C-Int-TRX can be seen in tube 1 and 3 at 17kDa, but the N-Int-SUMO cannot be seen at 24kDa and has not been overexpressed

- picked colonies of Rosetta cells with CI-TRX and NI-SUMO and cultured them in 2 mL LB with Amp overnight at 37°C

25.08.2019

- did a PCR-Purification of HlyA(46 ng/uL), sfGFP(41 ng/uL), CI(37 ng/uL) and CI-BB(30 ng/uL) from 14.08.

- did a Gibson assembly (following: https://international.neb.com/protocols/2012/09/25/gibson-assembly-master-mix-assembly) with CI-BB, CI and sfGFP

- did a Transformation with TOP10 cells, plated them on agar plates with Amp. and incubated them at 37°C overnight

- took the cultures with CI-TRX and NI-SUMO fom yesterday and put the amount of culture into 6x 5ml LB medium to have an OD600 of 0.1 in each, incubated them
all 2 h at 37°C, then induced them in different ways:

tube	1	2	3	4	5	6
construct	NI-SUMO	NI-SUMO	NI-SUMO	NI-SUMO	CI-TRX	CI-TRX
induction/incubation	no IPTG/ 25°C o.n.	1mM IMPTG/ 25°C o.n.	no IPTG/ 18°C o.n.	1mM IMPTG/ 18°C o.n.	no IPTG/ 25°C o.n.	1mM IMPTG/ 25°C o.n.

26.08.2019

- pelletized each culture: 2x 2 mL: pellet into the freezer (-80°C) and 1 mL: resuspended in 100 uL Reaction Buffer K

- lysed the resuspended pellets via sonication: 4 times 6 cycles of 1 min with, max volume

- centrifuged them 15 min at 4°C with max rpm

- took 20 uL of the supernatant and denatured them with 4x SDS Buffer and at 95°C for 10 min

- loaded the samples on a 12% SDS-Gel in the following order: ladder, tube1-6

- let run gel 1 h 10 min at 100 V, stained 30 min with InstantBlue™ Protein Stain

- took picture:

 <img width="400" src="../../imgs/Intein_Project/190826 vers sumo expr.png ">

- can see no difference between the induced and uninduced samples at the expected size, maybe NI-SUMO builds inclusion bodys

- picked 3 colonies of the TOP10 cells with the sfGFP-CI construct from yesterday, incubated them in 2 mL LB + Amp o/n

27.08.2019

- took a 2 mL pellet of tube 1 and 2 (from 25.08, to check if mutation occur after induction) and the 3 sfGFP-CI cultures from yesterday, performed a mini prep and send DNA to sequencing

28.08.2019

- took a 2 mL pellet of tube 1 and 2 (from 25.08), resuspended them in 200 uL Reaction Buffer K, sonicated the cells with 4x 6 cycles of 1 min at max volume

- took a 20 uL sample of the whole lysate of tube 2 (ind. NI-SUMO1) and denatured it with Laemmli-Buffer and at 95°C for 10 min

- centrifuged the lysates 15 min, max rpm, 4°C

- took 20 uL samples of the supernatant and the resuspended debris of lysate of tube 2 and a 20 uLsample of the resuspended debris of the lysate of tube 1 and denatured them with Laemmli-Buffer and at 95°C for 10 min

- loaded samples on a 12% SDS-Gel: ladder / tube 2 lysate / tube 2 SN / tube 2 debris / tube 1 debris / ladder / tube 2 lysate / tube 2 SN / tube 2 debris / tube 1 debris

- let run 1 h, 100 V

- cut the gel in the middle, blotted halves separately on PVDF membranes (30 min, 1 A, semi dry) then blocked both membranes with 5% BSA in TBST o/n at 4°C

- sequencing showed that sfGFP-CI was assembled successfully, inserted TRX into sfGFP-CI with Golden Gate Assembly:

Master Mix (each): 3:1 (Insert:Backbone(150ng)), 1.5 µL T4 DNA Ligase Buffer, 9.2 µL dH20 (filled up to 15 µL), 1µL BsaI, 1µL T4 DNA Ligase

Cycler Program:

Step	Temp.	Time
Restriction x25 Ligation	37°C 16°C	3 min 4 min
Final Restriction	50°C	5 min
Denaturation	80°C	5 min
Hold	4°C	-

- tried PCR of NI-BB for Sec-Tag with new Primers: also failed

29.08.2019

- the 2 PVDF were washed 1x 5 min with TBST, then the 1. antibodies were added: 1. Rabbit anti SUMO1 (1:5000) / 2. Mouse anti His-Tag (1:1000) both in TBST with 5% BSA, incubated 2h at RT

- 3x washed with TBST

- 2. antibodies were added: 1. Goat anti Rabbit Cy5 (1:2000) / 2. Goat anti Mouse 488 (1:2000) both in TBST with 5% BSA, incubated 2h at RT

- washed 3x with TBST, took picture with Amersham™ Typhoon™Biomolecular Imager:

 <img width="400" src="../../imgs/Intein_Project/190829 SUMO1 Western Blot Inclusion bodies.PNG ">

- lane 2 and 3 show in both stainings that the main amount of SUMO1 (band at 24kDa) stayed in the debris and possibly is building inclusion bodies, decided to use Maltose binding Protein instead (MBP)

- transformed competent TOP10 cells with the sfGFP-CI-TRX construct from the Golden Gate Assembly from yesterday, plated on Agar + Amp, incubated at 37°C o/n

30.8.2019

- Picked 3 colonies (C-int 1, C-int 2, C-int 3)

- Inoculated them in LB+Amp (4 mL), incubated at 37°C, 220 rpm for 8 hours

- Picked a Colony from MBP (pMal) plate (obtained from BIOSS Toolbox, University Freiburg) and incubated in 4 mL LB+Amp for 7 hrs at 37°C, 220 rpm

- Did a mini prep of 1 mL of cultures

- Eluted in 30 uL Elution Buffer

- Sent 20 uL of eluates to sequencing

31.8.2019

- PCR of MBP fragment

Reagent	Amount
2X Q5 Mastermix	25 uL
oIG19BB MBP forward primer	2.5 uL
oIG19BB MBP reverse primer	2.5 uL
Template DNA MBP	1 uL
dH2O	19 uL

- did a gel electrophoresis (bands at 1.2kb as expected)

 <img width="200" src="../../imgs/Intein_Project/190831first mbp PCR.png ">

- PCR purification with the rest in the PCR-tube

02.09.2019

- Inserted MBP into N-Int + BB mut with Golden Gate Assembly:

Master Mix (each): 3:1 molarity (Insert:Backbone(150ng)), 1.5 µL T4 DNA Ligase Buffer, 0.2 µL dH20 (filled up to 15 µL), 1µL BsaI, 1µL T4 DNA Ligase

Cycler Program:

Step	Temp.	Time
Restriction x25 Ligation	37°C 16°C	3 min 4 min
Final Restriction	50°C	5 min
Denaturation	80°C	5 min
Hold	4°C	-

- transformed competent TOP10 cells with 3/6 µL of the Golden Gate Product, plated, let grow o/n at 37°C

- repeated PCR of NI-BB mut for Sec-Tag with Phusion Flash Polymerase, gelelectrophoresis: no band

- Sequencing of sfGFP-CI-TRX showed that the construct was assembled correctly

 <img width="800" src="../../imgs/Intein_Project/190902 sfGFP_CI_TRX Sequencing.PNG">

- transformed competent BL21(DE3) cells with the sfGFP-CI-TRX construct, plated them on Agar plates + Amp, incubated o/n at 37°C

03.09.2019

- barely colonies grown from the with NI-MBP transformed cells, picked 3 and cultured in 4 mL LB + Amp, Mini prep after 8 h, send to Sequencing

- no BL21(DE3) cells with sfGFP-CI-TRX did grow, realized that competent cells have been defrozen several times before, repeated transformation with new competent BL21(DE3) cells

- tried gradient PCRs with DMSO, with GC-Enhancer, under normal conditions of the NI + BB mut for Sec-Tag construct, all failed

05.09.2019

- colonies of BL21(DE3) cells with sfGFP-CI-TRX did grow, picked one and cultured in 8 mL LB + Amp o/n, 37°C, 200 rpm

- sequencing of the NI-MBP constructs show that MBP was not inserted, found a BsaI restriction site in the MBP sequence, ordered phosphorylated primers to insert a point mutation

06.09.2019

- prewarmed 500 mL of LB-medium + Amp.

- added the BL21(DE3) culture with the sfGFP-CI-TRX construct to have 500 mL culture with OD 0.1, incubated at 37°C, 200 rpm until OD 0.5

- took 15x 20 mL samples, centrifuged them 10 min at 3000 g, discard SN, resuspend pellet in 20 mL LB+Amp (12x) or in Auto-Induction Medium+Amp (3x) and put them in 100 mL erlenmayer flasks

- induced the cultures with different concentrations of IPTG and then let them incubate at different temperatures, at 200 rpm and for different period of times:

flask	medium	induction	temperature	induction time
1	LB	1mM IPTG	37°C	16 h
2	LB	1mM IPTG	30°C	16 h
3	LB	1mM IPTG	25°C	24 h
4	LB	0.5mM IPTG	37°C	16 h
5	LB	0.5mM IPTG	30°C	16 h
6	LB	0.5mM IPTG	25°C	24 h
7	LB	0.1mM IPTG	37°C	16 h
8	LB	0.1mM IPTG	30°C	16 h
9	LB	0.1mM IPTG	25°C	24 h
10	LB	no IPTG	37°C	16 h
11	LB	no IPTG	30°C	16 h
12	LB	no IPTG	25°C	24 h
13	auto induction	-	37°C	16 h
14	auto induction	-	30°C	24 h
15	auto induction	-	25°C	48 h

- after induction time, measure OD of each flask, take 1 mL samples, centrifuge them 1 min at max speed, take supernatant and resuspend pellet in LB+Amp, measure flourescence of each samples SN and pellet with 2x 200 µL in the platereader at 495 nm excitation and 515 nm emission

- also take 40 µL of each supernatant and denature it with 4x Laemmli Buffer and at 95°C for 10 min

08.09.2019

- results of the sfGFP experiment:

 <img width="400" src="../../imgs/Intein_Project/190908 1 fluorescence medium.png ">

 <img width="400" src="../../imgs/Intein_Project/190908 2 fluorescence pellet.png ">

BBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDDDDDDDD

- LB-medium, 37°C, 0.1 mM IPTG, 16 h looks the most promising

11.09.2019

- to verify that not just sfGFP was in the mdium, but also the CI-TRX construct linked to it, performed SDS-Page and Western Blot with the denatured samples in the following lanes:

LB-medium / SN 37°C unind. / pellet 37°C 1mM (1:20) / SN 37°C 1mM / SN 37°C 0.5 mM / ladder / SN 37°C 0.1 mM / SN 37°C auto-ind. / SN 30°C auto-ind. / SN 25°C auto-ind.

- let run on 12% SDS-Gel for 1 h 10 min, 100 V

- blotted on PVDF membrane (30 min, 1 A), blocked with 5% BSA (2 h), washed 1x with TBST, added 1. antibody (Mouse anti TRX 1:1000) in 10 mL TBST with 5% BSA (1 h), washed 3x with TBST, added 2. antibody (Goat anti Mouse 488 1:2000) in 10 mL TBST with 5% BSA (1 h), washed 3x with TBST, took picture with Amersham™ Typhoon™Biomolecular Imager at 488 nm:

BBBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDD

- sfGFP-CI-TRX should be seen at 44 kDa and all fluorescent samples show the right band, indicating that if sfGFP gets secreted it can carry a protein fused to it

16.09.2019

- did a PCR of MBP in pMal plasmid to insert a point mutation in MBP to get rid of the BsaI restriction site, used Primers: oIG19BB MBP mut P fwd and rev, both with a phosphorylation at the 5' end, tried different temperatures from 54 to 64°C and tried it with DMSO, GC-Enhancer and under normal conditions

- Gelelctrophoresis:

BBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDDDD

- lane 5 (62.6°C with GC-Enahncer), 6 (64.1°C with GC-Enhancer), 10 (62.6°C with 5% DMSO) and 11 (64.1°C with 5% DMSO) showed bands of the right size (5.7 kb)

- took the rest of the samples, added them together, did a PCR purification, added 10x T4 Ligase Buffer and 1 µL T4 Ligase, let incubate 1 h at room temp.

- then added 0.5 µL DpnI and let incubate 1 h at 37°C

- 5 min 80°C to denaturate enzymes

- used this sample to do a PCR of MBP with Primers: oIG19BB MBP forward and reverse

17.09.2019

- Gelelectrophoresis:

BBBBBBBBBBBBBBBbbIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDD

- PCR product has the right size (1.2 kb), did a PCR purification of the rest of the sample

- realized that our Npu DnaE Inteins won't splice together MBP and TRX because we have a serine at the extein +1 position. Threrefor we decided to order the Biobricks BBa_K1362160 and BBa_K1362161 (containing gp41.1 N-Intein and C-Intein) as G-Blocks

- picked a BL21(DE3) colony of sfGFP-CI-TRX and cultured it in 8 mL LB + Amp at 37°C, 200 rpm, o/n for an experiment testing the sfGFP secretion over time, compared to OD development, to figure out if the sfGFP gets into the medium through secretion or lysis of the cells

18.09.2019

- prewarmed 200 mL of LB-medium + Amp.

- added the BL21(DE3) culture with the sfGFP-CI-TRX construct to have 200 mL culture with OD 0.1, incubated at 37°C, 180 rpm until OD 0.5

- took 2x 50 mL samples, centrifuged them 10 min at 3000 g, discard SN, resuspend pellet in 50 mL LB+Amp and put in 200 mL erlenmayer flask

- induced one culture with 0.1 mM IPTG and the other not, then incubated them both at 37°C, 180 rpm, 16 h

- every 2 hours measure OD600 of each flask, take 1 mL samples, centrifuge them 1 min at max speed, take supernatant and resuspend pellet in LB+Amp, measure flourescence of each samples SN and pellet with 3x 200 µL in the platereader at 495 nm excitation and 515 nm emission after all samples have been taken

19.09.2019

- results from the sfGFP secretion experiment:

BBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDd

BBBBBBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDD

BBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDD

- fluorescence in the medium already rises at 8 h after induction, strongly indicating secretion of sfGFP-CI-TRX

26.09.2019

- repeated the sfGFP-CI-TRX secretion experiment from 18.09 under same conditions

27.09.2019

- results:

BBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDdd

BBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDd

BBBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDD

01.10.2019

- repeated the sfGFP-CI-TRX secretion experiment from 18.09 under same conditions but this time also two cultures of BL21(DE3) with the CI-TRX construct are added, one induced with 0.1 mM IPTG and one uninduced

- also this time not just fluorescence was measured but also 40 µL of each supernatant was taken and denatured with 4x Laemmli Buffer and at 95°C for 10 min, stored at -20°C

02.10.2019

- results of fluorescence measurement:

BBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDd

BBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDDDDDDD

BBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDDD

08.10.2019

- did a SDS-PAGE with the denatured samples from 02.10, all samples were adjusted to an OD of 4

Gel 1:

sfGFP-CI-TRX unind. / CI-TRX unind. / sfGFP-CI-TRX ind. Pellet T16 (1:20) / CI-TRX ind. Pellet T16 (1:20) / ladder / sfGFP-CI-TRX ind. T4 / CI-TRX ind. T4 / sfGFP-CI-TRX ind. T6 / CI-TRX ind. T6 / sfGFP-CI-TRX ind. T8

Gel 2:

sfGFP-CI-TRX unind. / CI-TRX unind. / sfGFP-CI-TRX ind. Pellet T16 (1:20) / CI-TRX ind. Pellet T16 (1:20) / CI-TRX ind. T8 / ladder / sfGFP-CI-TRX ind. T10 / CI-TRX ind. T10 / sfGFP-CI-TRX ind. T12 / CI-TRX ind. T12

- let run 12% SDS-gel 1 h 10 min, 100V, then blotted 30 min, 1A, semi dry on 2 PVDF membranes

- blocked o/n in 5% BSA in TBST at 4°C

09.10.2019

- washed membranes 1x in TBST

- added 1. antibody: mouse anti TRX (1:1000) in 10 mL TBST with 5% BSA to both membranes, let incubate 1 h

- washed 3x in TBST

- added 2. antibody: goat anti mouse 488 (1:2000) in mL TBST with 5% BSA to both membranes let incubate 1 h

- washed 3x in TBST

- took picture with Amersham™ Typhoon™Biomolecular Imager at 488 nm:

BBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDD

- the Western Blot clearly shows a big difference in the amount of protein that were in the medium, at each timepoint the sfGFP-CI-TRX bands (44 kDa) are way more intense than the CI-TRX bands (17kDa), indicating that the fusionprotein enters the medium mainly via secretion instead of lysis

10.10.2019

- G-Blocks with the gp41.1 Inteins and the Primers arrived

- did a PCR of pET302 to open it up and introduce overhangs to insert the G-Blocks with Gibson assembly:

PCR of pET302 for GBlocks (gp41.1):

Master Mix (50 µL)

reagent	amount	concentration
5X Q5 Reaction Buffer	10 µL	1X
10 mM dNTPs	1 µL	200 µM
Primer: pET302 for GBlock fwd	2.5 µL	0.5 µM
Primer: pET302 for GBlock rev	2.5 µL	0.5 µM
Template DNA: CI-TRX in pET302 mut	1 µL	-
Q5 High-Fidelity DNA Polymerase	0.5 µL	0.02 U/µl
Nuclease-Free Water	(22.5) 32.5 µL	-
High GC-Enhancer	(10) µL	1X

PCR Programm:

Step	Temp.	Time
Initial Denaturation	96°C	1 min
Denaturation Extension x30 Annealing	96°C 62°C 72°C	30 sec 30 sec 3 min 30sec
Final Extension	72°C	5 min
Hold	4°C	-

- Gelelectrophoresis:

BBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDD

- the left band, with GC-Enhancer, has the perfect size (5.6 kbp) and the rest of sample was purified by PCR purification

- put both G-Blocks into pET302 for G-Blocks with Gibson Assembly, following: https://international.neb.com/protocols/2014/11/26/nebuilder-hifi-dna-assembly-reaction-protocol

- transformed competent TOP10 cells with 4 µLof the gibson products and plated them on agar + amp, incubated at 37°C o/n

11.10.2019

- picked colonies, cultured each in 4 mL LB+Amp at 37°C, 200 rpm, 8 h

- mini prep of each culture and send to sequencing

14.10.2019

- sequencing showed that G-Blocks were inserted successfully

- Inserted MBP mut into gp41.1 N-Int in pET302 mut with Golden Gate Assembly:

Master Mix (each): 3:1 molarity (Insert:Backbone(150 ng)), 1.5 µL T4 DNA Ligase Buffer, 8.3 µL dH20 (filled up to 15 µL), 1µL BsaI, 1µL T4 DNA Ligase

Cycler Program:

Step	Temp.	Time
Restriction x25 Ligation	37°C 16°C	3 min 4 min
Final Restriction	50°C	5 min
Denaturation	80°C	5 min
Hold	4°C	-

- Inserted TRX into gp41.1 C-Int in pET302 mut with Golden Gate Assembly:

Master Mix (each): 3:1 molarity (Insert:Backbone(150 ng)), 1.5 µL T4 DNA Ligase Buffer, 9.1 µL dH20 (filled up to 15 µL), 1µL BsaI, 1µL T4 DNA Ligase

Cycler Program:

Step	Temp.	Time
Restriction x25 Ligation	37°C 16°C	3 min 4 min
Final Restriction	50°C	5 min
Denaturation	80°C	5 min
Hold	4°C	-

- transformed competent TOP10 cells with 4 µL of the Golden Gate products, plated on agar + amp, incubated at 37°C o/n

15.10.2019

- picked colonies, cultured each in 4 mL LB+Amp at 37°C, 200 rpm, 8 h

- mini prep of each culture and send to sequencing

16.10.2019

- Sequencing showed that TRX and MBP mut were both inserted successfully

17.10.2019

Retransformation in competent Rosetta cells:

2 µL DNA (4 samples: gp4.1 N-Int (BBa_K1362160); gp41.1 C-Int (BBa_K1362161); gp41.1 N-Int with MBP mut; gp41.1 C-Int with TRX)

50 µL competent cells

plate on agar plates with ampicillin

18.10.2019

Measure OD₆₀₀ by diluting 1:10

1. N-gp41.1-Intein w/o MBP: 0.35

2. N-gp41.1-Intein w/o MBP: 0.37

1. C-gp41.1-Intein w/o TRX: 0.36

2. C-gp41.1-Intein w/o TRX: 0.40

1. C-gp41.1-Intein-TRX : 0.38

2. C-gp41.1-Intein-TRX: 0.38

1. N-gp41.1-Intein-MBP: 0.35

2. N-gp41.1-Intein-MBP: 0.37

Dilute all cultures to an OD₆₀₀ of 0.1. Add to 4 mL LB+Ampicillin

1. N-gp41.1-Intein w/o MBP: 114 µL

2. N-gp41.1-Intein w/o MBP: 108 µL

1. C-gp41.1-Intein w/o TRX: 111 µL

2. C-gp41.1-Intein w/o TRX: 100 µL

1. C-gp41.1-Intein-TRX: 105 µL

2. C-gp41.1-Intein-TRX: 105 µL

1. N-gp41.1-Intein-MBP: 114 µL

2. N-gp41.1-Intein-MBP: 108 µL

Incubate for 2 hours at 37°C.

Induce with 5 mM IPTG 2 µL each culture (4 mL).

Incubate for 3 hours at 37°C and 200 rpm.

Transferre samples in 2 mL microtubes (2x2 mL tubes for each culture) and spin down for 1 minute at maximum speed.

Discard supernatant and store C-gp41.1-Intein w/o TRX and N-gp41.1-Intein w/o MBP and one sample from N-gp41.1-Intein MBP and one sample from C-gp41.1-Intein TRX (6 samples in total) at -80°C.

Resuspend the pellet from C-gp41.1-Intein TRX w/o secretion tag and N-gp41.1-Intein w/o secretion tag in 260 µL Reaction Buffer K (2 samples in total)

Add 1 pipet tip PMSF (protease inhibitor) to each sample

Lyse the cell via sonication (5x6 cycles each 30 seconds and pause 30 seconds) until the suspension is clear

Centrifuge for 15 minutes at 4°C and maxium speed.

Transferre the supernatant (250 µL) into a 1.5 mL microtube

Prepare following samples from this supernatant.

1. C-gp41.1-Intein-TRX	2. N-gp41.1-Intein-MBP	3. N-gp41.1-Intein-MBP + C-Intein-TRX	4. N-gp41.1-Intein-MBP + C-gp41.1-Intein-TRX + DTT (4 mM)
50 µL	50 µL	100 µL from each (200 µL in total)	100 µL from each (200 µL in total)

Take 20 µL from samples 1.-3. and add 20 µL 2xLämmLi-Buffer and incubate it for 10 minutes at 95°C. Store them at -20°C

Incubate samples 3. and 4. at 42°C and 350 rpm overnight.

Take 20 µL samples after 1, 2, 3 and 16 hours of incubation and add 20 µL 2xLämmLi Buffer and incubate them for 10 minutes at 95°C. Store them at -20°C.

20.10.2019

Resuspend C-gp41.1-Intein w/o MBP and N-gp41.1-Intein w/o TRX pellet from yesterday in 260 µL Reaction Buffer K.

Add 1 pipet tip PMSF (protease inhibitor) to each sample

Lyse the cell via sonication (4x6 cycles each 30 seconds and pause 30 seconds) until the suspension is clear

Centrifuge for 15 minutes at 4°C and maxium speed.

Transferre 20 µL supernatant into a 1.5 mL microtube and add 20 µL 2xLämmLi Buffer.

Denaturate for 10 minutes at 95°C.

Expression test

Load a 12% acrylamide gel with 18 µL of each sample:

C-gp41.1-Intein w/o MBP

C-gp41.1-Intein-MBP T=0

ladder

N-gp41.1-Intein w/o TRX

N-gp41.1-Intein-TRX T=0

Run the gel at 100 V for 1 hour 20 minutes

Wash gel short with ddH₂O

Stain the gel with Instant Blue for 30 minutes.

BBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDd

Splicing test

Load a 12% acrylamide gel with 18 µL of each sample:

C-gp41.1-Intein-MBP T=0

N-gp41.1-Intein-TRX T=0

N-gp41.1-I-TRX+C-gp41.1-I-MBP T=0

Positive control (MBP-TRX)

Ladder

N-gp41.1-I-MBP + C-gp41.1-TRX + DTT T=1

N-gp41.1-I-MBP + C-gp41.1-TRX T=1

N-gp41.1-I-MBP + C-gp41.1-TRX + DTT T=3

N-gp41.1-I-MBP + C-gp41.1-TRX T=3

N-gp41.1-I-MBP + C-gp41.1-TRX + DTT T=16

Run the gel at 100 V for 1 hour 20 minutes.

Perform a Western Blot with the gel (30 minutes)

Block membrane with 15 mL of 5% BSA in TBST for 2 hours.

Wash 1x5 minutes with TBST.

Incubate for 1 hour with primary antibody (4 µL mouse anti TRX in 10 mL TBST with 5% BSA)

Wash 3x5 minutes with TBST.

Incubate for 1 hour with secondary antibody (5 µL goat anti mouse 488 in 10 mL TBST with 5% BSA).

Visualize with Typhoon laser scanner at 488 nm:

BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDDDDd

Primers and Constructs

Primer	Sequence
oIG19nifwd	AATAATTTTGTTTAACTTTAAGAAG
oIG19nirev	TTTATTACTAATGGTGATGGTG
oIG19cifwd	AATAATTTTGTTTAACTTTAAGAAGG
oIG19cirev	TTATTATGAGACCTATAAACGC
oIG19bbcifwd	CTGCGTTTATAGGTCTCATAATAAGTATAATCGGATCCGGCTGCTAAC
oIG19bbcirev	CTCCTTCTTAAAGTTAAACAAAATTATTAACAAAATTATTTCTAGAGGGGAATTGTTATC
oIG19bbnifwd	CATCACCATCACCATTAGTAATAAAGTATAATCGGATCCGGCTGCTAAC
oIG19bbnirev	CTCCTTCTTAAAGTTAAACAAAATTATTAACAAAATTATTTCTAGAGGGGAATTGTTATC
oIG19bbBsaI mut fwd	GTGAGCGTGGATCTCGCGGTATC
oIG19bbBsaI mut rev	GATACCGCGAGATCCACGCTCAC
fwd primer in the Amp. Res.	GGAGTCAGGCAACTATGGA
rev Primer in the Amp. Res.	CGTTCATCCATAGTTGCCTGAC
IG19BB TrxA fwd	GGTCTCGCAACAGCGATAAAATTATTCACCTG
IG19BB TrxA rev	GGTCTCTATTAGGCCAGGTTAGCG
IG19BB SUMO1 fwd	GGTCTCAGATGGGATCCTCTGACCAG
IG19BB SUMO1 rev	GGTCTCCAGCAGCCGGATCCAACTGTTGAATGACCC
oIG19BB C-Int BB T7 Term fwd	TTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGAATCCGGCTGCTAACAAAGCC
oIG19BB C-Int for sfGFP rev	GCTCTTCGCCTTTACGCATCTAGTATCTCCTTCTTAAAGTTAAACAAAATTATTAAC
oIG19BB N-Int for HlyA fwd	TAGTAATCAGACTGACATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGAAGTATAATCGGATCCGGCTGC
oIG19BB N-Int for HlyA rev	GAATTTGAGTTCGAATGGTGATGGTGATGGTGATGATGACCAC
oIG19BB HlyA fwd	CACCATTCGAACTCAAATTCGTTAGCCTATGGAAGTCAGGGTG
oIG19BB HlyA rev	GGTTATGTCAGTCTGATTACTATTATTATGCTGATGTGGTCAGGG
oIG19BB sfGFP fwd	ATGCGTAAAGGCGAAGAGC
oIG19BB sfGFP rev	GATGCGAATTTGAGTTCGATTTGTACAGTTCATCCATACCATGC
oIG19BB C-Int BB T7 Term rev	CCCGTTTAGAGGCCCCAAGGGGTTATCCGATTATACTTATTATGAGACCTATAAACGCAG
oIG19BB C-Int for sfGFP fwd	TCGAACTCAAATTCGCATCACCATCATCACCATCACCACGG
oIG19BB MBP forward	GGTCTCAGATGATGAAAATCGAAGAAGGTAAACTGG
oIG19BB MBP reverse	GGTCTCCAGCAGCCGGATCCCCCGAGGTTGTTGTTATTGTTATTG
oIG19BB MBP mut P fwd	CCTCGCTGAAGTCGGTAAG
oIG19BB MBP mut P rev	CCGTTATAGCCTTTATCGCC
pET302 for GBlock fwd	TAACAAAGCCCGAAAGGAAG
pET302 for GBlock rev	AACAAAATTATTTCTAGAGGGGAATTG



Construct	Sequence
N-Int Npu DnaE (BBa_K1362100)	aataattttgtttaactttaagaaggagatactagatgagagacccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacatactagagaaagaggagaaatactagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataacgctgatagtgctagtgtagatcgctactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttataggtctcatgcttaagctatgaaacggaaatattgacagtagaatatggattattaccgattggtaaaattgtagaaaagcgcatcgaatgtactgtttatagcgttgataataatggaaatatttatacacaacctgtagcacaatggcacgatcgcggagaacaagaggtgtttgagtattgtttggaagatggttcattgattcgggcaacaaaagaccataagtttatgactgttgatggtcaaatgttgccaattgatgaaatatttgaacgtgaattggatttgatgcgggttgataatttgccgaatggtggtggtcatcatcaccatcaccattagtaataaa




C-Int Npu DnaE (BBa_K1362101)	aataattttgtttaactttaagaaggagatactagatgcatcatcaccatcaccacggtggtggaatcaaaatagccacacgtaaatatttaggcaaacaaaatgtctatgacattggagttgagcgcgaccataattttgcactcaaaaatggcttcatagcttccaacagagacccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacatactagagaaagaggagaaatactagatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataacgctgatagtgctagtgtagatcgctactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttataggtctcataataa




pET302	GATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAAACTTTAAGAAGGAGATATACATATGCATCATCATCATCATCACGTGAATTCGCTCGAGATCGATGATATTCGAGCCTAGGTATAATCGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGA




N-Int in pET302 (NI+BB)	GATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTAATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGAGAGACCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGTCTCATGCTTAAGCTATGAAACGGAAATATTGACAGTAGAATATGGATTATTACCGATTGGTAAAATTGTAGAAAAGCGCATCGAATGTACTGTTTATAGCGTTGATAATAATGGAAATATTTATACACAACCTGTAGCACAATGGCACGATCGCGGAGAACAAGAGGTGTTTGAGTATTGTTTGGAAGATGGTTCATTGATTCGGGCAACAAAAGACCATAAGTTTATGACTGTTGATGGTCAAATGTTGCCAATTGATGAAATATTTGAACGTGAATTGGATTTGATGCGGGTTGATAATTTGCCGAATGGTGGTGGTCATCATCACCATCACCATTAGTAATAAAGTATAATCGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGA




C-Int in pET302 (CI+BB)	GATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTAATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGCATCATCACCATCACCACGGTGGTGGAATCAAAATAGCCACACGTAAATATTTAGGCAAACAAAATGTCTATGACATTGGAGTTGAGCGCGACCATAATTTTGCACTCAAAAATGGCTTCATAGCTTCCAACAGAGACCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGTCTCATAATAAGTATAATCGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGA




N-Int in pET302 with point mutation in BsaI Restriction site in the Amp Resistance (NI + BB mut)	GATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTAATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGAGAGACCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGTCTCATGCTTAAGCTATGAAACGGAAATATTGACAGTAGAATATGGATTATTACCGATTGGTAAAATTGTAGAAAAGCGCATCGAATGTACTGTTTATAGCGTTGATAATAATGGAAATATTTATACACAACCTGTAGCACAATGGCACGATCGCGGAGAACAAGAGGTGTTTGAGTATTGTTTGGAAGATGGTTCATTGATTCGGGCAACAAAAGACCATAAGTTTATGACTGTTGATGGTCAAATGTTGCCAATTGATGAAATATTTGAACGTGAATTGGATTTGATGCGGGTTGATAATTTGCCGAATGGTGGTGGTCATCATCACCATCACCATTAGTAATAAAGTATAATCGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGATCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGA




C-Int in pET302 with point mutation in BsaI Restriction site in the Amp Resistance (CI + BB mut)	GATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTAATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGCATCATCACCATCACCACGGTGGTGGAATCAAAATAGCCACACGTAAATATTTAGGCAAACAAAATGTCTATGACATTGGAGTTGAGCGCGACCATAATTTTGCACTCAAAAATGGCTTCATAGCTTCCAACAGAGACCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGTCTCATAATAAGTATAATCGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGATCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGA




SUMO1	GGATCCTCTGACCAGGAGGCAAAACCTTCAACTGAGGACTTGGGGGATAAGAAGGAAGGTGAATATATTAAACTCAAAGTCATTGGACAGGATAGCAGTGAGATTCACTTCAAAGTGAAAATGACAACACATCTCAAGAAACTCAAAGAATCATACTGTCAAAGACAGGGTGTTCCAATGAATTCACTCAGGTTTCTCTTTGAGGGTCAGAGAATTGCTGATAATCATACTCCAAAAGAACTGGGAATGGAGGAAGAAGATGTGATTGAAGTTTATCAGGAACAAACGGGGGGTCATTCAACAGTTTAG




TrxA (TRX)	ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCC




N-Int + SUMO1 in pET302 mut (NI-SUMO1)	GATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTAATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGGGATCCTCTGACCAGGAGGCAAAACCTTCAACTGAGGACTTGGGGGATAAGAAGGAAGGTGAATATATTAAACTCAAAGTCATTGGACAGGATAGCAGTGAGATTCACTTCAAAGTGAAAATGACAACACATCTCAAGAAACTCAAAGAATCATACTGTCAAAGACAGGGTGTTCCAATGAATTCACTCAGGTTTCTCTTTGAGGGTCAGAGAATTGCTGATAATCATACTCCAAAAGAACTGGGAATGGAGGAAGAAGATGTGATTGAAGTTTATCAGGAACAAACGGGGGGTCATTCAACAGTTGGATCCGGCTGCTTAAGCTATGAAACGGAAATATTGACAGTAGAATATGGATTATTACCGATTGGTAAAATTGTAGAAAAGCGCATCGAATGTACTGTTTATAGCGTTGATAATAATGGAAATATTTATACACAACCTGTAGCACAATGGCACGATCGCGGAGAACAAGAGGTGTTTGAGTATTGTTTGGAAGATGGTTCATTGATTCGGGCAACAAAAGACCATAAGTTTATGACTGTTGATGGTCAAATGTTGCCAATTGATGAAATATTTGAACGTGAATTGGATTTGATGCGGGTTGATAATTTGCCGAATGGTGGTGGTCATCATCACCATCACCATTAGTAATAAAGTATAATCGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGATCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGA




C-Int + TrxA in pET302 mut (CI-TRX)	GATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTAATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGCATCATCACCATCACCACGGTGGTGGAATCAAAATAGCCACACGTAAATATTTAGGCAAACAAAATGTCTATGACATTGGAGTTGAGCGCGACCATAATTTTGCACTCAAAAATGGCTTCATAGCTTCCAACAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCTAATAAGTATAATCGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGATCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGA




C-Int backbone for Insertion of sfGFP ( C-Int BB for Sec-Tag Insertion)	TTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGAATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGATCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTAATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGCGTAAAGGCGAAGAGC




N-Int backbone for Insertion of HlyA ( N-Int BB for Sec-Tag Insertion)	TAGTAATCAGACTGACATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGAAGTATAATCGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTAGCTTATCGATGATAAGCTGTCAAACATGAGAATTAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGATCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGCCAGGACCCAACGCTGCCCGAGATGCGCCGCGTGCGGCTGCTGGAGATGGCGGACGCGATGGATATGTTCTGCCAAGGGTTGGTTTGCGCATTCACAGTTCTCCGCAAGAATTGATTGGCTCCAATTCTTGGAGTGGTGAATCCGTTAGCGAGGTGCCGCCGGCTTCCATTCAGGTCGAGGTGGCCCGGCTCCATGCACCGCGACGCAACGCGGGGAGGCAGACAAGGTATAGGGCGGCGCCTACAATCCATGCCAACCCGTTCCATGTGCTCGCCGAGGCGGCATAAATCGCCGTGACGATCAGCGGTCCAATGATCGAAGTTAGGCTGGTAAGAGCCGCGAGCGATCCTTGAAGCTGTCCCTGATGGTCGTCATCTACCTGCCTGGACAGCATGGCCTGCAACGCGGGCATCCCGATGCCGCCGGAAGCGAGAAGAATCATAATGGGGAAGGCCATCCAGCCTCGCGTCGCGAACGCCAGCAAGACGTAGCCCAGCGCGTCGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTAATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGAGAGACCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGTCTCATGCTTAAGCTATGAAACGGAAATATTGACAGTAGAATATGGATTATTACCGATTGGTAAAATTGTAGAAAAGCGCATCGAATGTACTGTTTATAGCGTTGATAATAATGGAAATATTTATACACAACCTGTAGCACAATGGCACGATCGCGGAGAACAAGAGGTGTTTGAGTATTGTTTGGAAGATGGTTCATTGATTCGGGCAACAAAAGACCATAAGTTTATGACTGTTGATGGTCAAATGTTGCCAATTGATGAAATATTTGAACGTGAATTGGATTTGATGCGGGTTGATAATTTGCCGAATGGTGGTGGTCATCATCACCATCACCATCACCATTCGAACTCAAATTC




C-Int for sfGFP Insertion (C-Int for Sec-Tag Insertion)	TCGAACTCAAATTCGCATCACCATCATCACCATCACCACGGTGGTGGAATCAAAATAGCCACACGTAAATATTTAGGCAAACAAAATGTCTATGACATTGGAGTTGAGCGCGACCATAATTTTGCACTCAAAAATGGCTTCATAGCTTCCAACAGAGACCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGTCTCATAATAAGTATAATCGGATAACCCCTTGGGGCCTCTAAACGGG




sfGFP (BBa_I746916)	ATGCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTACCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAA




HlyA (BBa_K208006)	TTAGCCTATGGAAGTCAGGGTGATCTTAATCCATTAATTAATGAAATCAGCAAAATCATTTCAGCAGCAGGTAGCTTCGATGTTAAAGAGGAAAGAACCGCAGCTTCTTTATTGCAGTTGTCCGGTAATGCCAGTGATTTTTCATATGGACGGAACTCAATAACCCTGACCACATCAGCATAATAA




sfGFP-CI	ATGCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTACCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATCGAACTCAAATTCGCATCACCATCATCACCATCACCACGGTGGTGGAATCAAAATAGCCACACGTAAATATTTAGGCAAACAAAATGTCTATGACATTGGAGTTGAGCGCGACCATAATTTTGCACTCAAAAATGGCTTCATAGCTTCCAACAGAGACCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGTCTCATAATAA




sfGFP-CI-TRX	ATGCGTAAAGGCGAAGAGCTGTTCACTGGTGTCGTCCCTATTCTGGTGGAACTGGATGGTGATGTCAACGGTCATAAGTTTTCCGTGCGTGGCGAGGGTGAAGGTGACGCAACTAATGGTAAACTGACGCTGAAGTTCATCTGTACTACTGGTAAACTGCCGGTACCTTGGCCGACTCTGGTAACGACGCTGACTTATGGTGTTCAGTGCTTTGCTCGTTATCCGGACCATATGAAGCAGCATGACTTCTTCAAGTCCGCCATGCCGGAAGGCTATGTGCAGGAACGCACGATTTCCTTTAAGGATGACGGCACGTACAAAACGCGTGCGGAAGTGAAATTTGAAGGCGATACCCTGGTAAACCGCATTGAGCTGAAAGGCATTGACTTTAAAGAAGACGGCAATATCCTGGGCCATAAGCTGGAATACAATTTTAACAGCCACAATGTTTACATCACCGCCGATAAACAAAAAAATGGCATTAAAGCGAATTTTAAAATTCGCCACAACGTGGAGGATGGCAGCGTGCAGCTGGCTGATCACTACCAGCAAAACACTCCAATCGGTGATGGTCCTGTTCTGCTGCCAGACAATCACTATCTGAGCACGCAAAGCGTTCTGTCTAAAGATCCGAACGAGAAACGCGATCATATGGTTCTGCTGGAGTTCGTAACCGCAGCGGGCATCACGCATGGTATGGATGAACTGTACAAATCGAACTCAAATTCGCATCACCATCATCACCATCACCACGGTGGTGGAATCAAAATAGCCACACGTAAATATTTAGGCAAACAAAATGTCTATGACATTGGAGTTGAGCGCGACCATAATTTTGCACTCAAAAATGGCTTCATAGCTTCCAACAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCTAATAA




MBP	GGTCTCAGATGATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGTGAAAGATCCGCGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAACAACCTCGGGGGATCCGGCTGCTGGAGACC




MBP mut	GGTCTCAGATGATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGATCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGTGAAAGATCCGCGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAACAACCTCGGGGGATCCGGCTGCTGGAGACC




gp41.1 N-Intein (BBa_K1362160)	AATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGAGAGACCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGTCTCATGCTTGGATCTGAAAACCCAGGTTCAGACCCCGCAGGGTATGAAGGAAATTTCCAACATCCAGGTCGGTGATCTGGTACTGAGCAACACGGGTTACAACGAAGTTCTGAACGTCTTCCCGAAATCTAAAAAAAAGTCTTACAAAATCACCCTGGAAGATGGCAAGGAAATCATCTGTTCCGAAGAACACCTGTTTCCGACGCAGACTGGTGAAATGAACATCTCCGGTGGTCTGAAAGAAGGTATGTGTCTGTATGTTAAAGAAGGTGGTGGTCATCATCACCATCACCATTAGTAATAAA




gp41.1 C-Intein (BBa_K1362161)	AATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGCATCATCACCATCACCACGGTGGTGGTATGATGCTGAAAAAAATCCTGAAAATCGAAGAGCTGGATGAACGTGAACTGATCGATATTGAGGTGTCCGGTAACCACCTGTTTTACGCTAACGATATTCTGACCCACAACAGAGACCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACATACTAGAGAAAGAGGAGAAATACTAGATGGCTTCCTCCGAAGACGTTATCAAAGAGTTCATGCGTTTCAAAGTTCGTATGGAAGGTTCCGTTAACGGTCACGAGTTCGAAATCGAAGGTGAAGGTGAAGGTCGTCCGTACGAAGGTACCCAGACCGCTAAACTGAAAGTTACCAAAGGTGGTCCGCTGCCGTTCGCTTGGGACATCCTGTCCCCGCAGTTCCAGTACGGTTCCAAAGCTTACGTTAAACACCCGGCTGACATCCCGGACTACCTGAAACTGTCCTTCCCGGAAGGTTTCAAATGGGAACGTGTTATGAACTTCGAAGACGGTGGTGTTGTTACCGTTACCCAGGACTCCTCCCTGCAAGACGGTGAGTTCATCTACAAAGTTAAACTGCGTGGTACCAACTTCCCGTCCGACGGTCCGGTTATGCAGAAAAAAACCATGGGTTGGGAAGCTTCCACCGAACGTATGTACCCGGAAGACGGTGCTCTGAAAGGTGAAATCAAAATGCGTCTGAAACTGAAAGACGGTGGTCACTACGACGCTGAAGTTAAAACCACCTACATGGCTAAAAAACCGGTTCAGCTGCCGGGTGCTTACAAAACCGACATCAAACTGGACATCACCTCCCACAACGAAGACTACACCATCGTTGAACAGTACGAACGTGCTGAAGGTCGTCACTCCACCGGTGCTTAATAACGCTGATAGTGCTAGTGTAGATCGCTACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGTCTCATAATAA




gp41.1 NI-MBP mut	AATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGATCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGTGAAAGATCCGCGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAACAACCTCGGGGGATCCGGCTGCTTGGATCTGAAAACCCAGGTTCAGACCCCGCAGGGTATGAAGGAAATTTCCAACATCCAGGTCGGTGATCTGGTACTGAGCAACACGGGTTACAACGAAGTTCTGAACGTCTTCCCGAAATCTAAAAAAAAGTCTTACAAAATCACCCTGGAAGATGGCAAGGAAATCATCTGTTCCGAAGAACACCTGTTTCCGACGCAGACTGGTGAAATGAACATCTCCGGTGGTCTGAAAGAAGGTATGTGTCTGTATGTTAAAGAAGGTGGTGGTCATCATCACCATCACCATTAGTAATAAA




gp41.1 CI-TRX	AATAATTTTGTTTAACTTTAAGAAGGAGATACTAGATGCATCATCACCATCACCACGGTGGTGGTATGATGCTGAAAAAAATCCTGAAAATCGAAGAGCTGGATGAACGTGAACTGATCGATATTGAGGTGTCCGGTAACCACCTGTTTTACGCTAACGATATTCTGACCCACAACAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCTAATAA

           </div>

                   <a target="_blank" rel="noopener noreferrer" href="https://www.abcam.com/"><img class="gold" src=""/></a>
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                   <a target="_blank" rel="noopener noreferrer" href="https://www.bioss.uni-freiburg.de/"><img class="platin" src=""/></a>
                   <a target="_blank" rel="noopener noreferrer" href="https://www.cibss.uni-freiburg.de/"><img class="platin" src=""/></a>
                   <a target="_blank" rel="noopener noreferrer" href="http://maria-ladenburger-stiftung.de/"><img class="platin" src=""/></a>
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                   <a target="_blank" rel="noopener noreferrer" href="https://www.molekularmedizin.uni-freiburg.de/index_html"><img class="platin" style="width: 10%" src=""/></a>
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                   <a target="_blank" rel="noopener noreferrer" href="https://www.freunde.uni-freiburg.de/"><img class="gold" src=""/></a>

       </div>
   </body>

@@ Line 7: / Line 7: @@
                  <div> Lab Book: Expression </div>
              </div>
-            <div class="labbook">
+                      <div class="labbook">
                <p style="color: black; margin: 0 10%; font-size: 1.6em; padding-bottom: 50px;"> Short description of what has been done </p>
                <div class="labbookentry">
@@ Line 16: / Line 16: @@
                        <div class="wrapper">
                            <h2> 04.06.19 </h2>
-        <p><span style="font-size:14px;">- Made 1:10 dilutions of all primers&nbsp;</span></p>
+<p><span style="font-size:14px;">- Made 1:10 dilutions of all primers&nbsp;</span></p>
-    <table style="border-collapse:collapse;  border:none">
+<table style="border-collapse:collapse; width:1306pt; border:none" >
+	<colgroup>
+		<col style="width:110pt" width="147" />
+		<col style="width:205pt" width="273" />
+		<col style="width:389pt" width="518" />
+		<col style="width:436pt" width="582" />
+		<col style="width:86pt" width="115" />
+		<col style="width:80pt" width="107" />
+	</colgroup>
 	<tbody>
 		<tr height="24" style="height:18.0pt">
@@ Line 320: / Line 327: @@
 <p>EWSR1 (Ewing Sarcoma Breakpoint Region 1):</p>
-<span class="wordbreak"><p>atggcctcaactgactactccacttactcgcaggcagcagcacagcaaggctacagcgcttatacagcccagccaacgcaaggatatgcccaaacaacgcaggcatacggtcagcagtcttacggcacttacggccagccaactgatgtctcttacactcaagctcagaccacagcgacttatggccagaccgcatatgccacctcttacggtcagcccccgaccgtggaaggaacgtcgactggctatacaactccaaccgctcctcaagcatacagccagccagtccaaggatatggaaccggtgcatacgatacaacaactgccacagtgacgactacacaggcttcttatgcagctcagagtgcatatgggactcaaccagcatacccggcatacggccagcagccagctgcaacagctccaacgcgtcctcaggatggcaacaagccaacagagacctcgcaaccccagtccagtacaggtggctacaatcagccctccttggggtatgggcagagtaactactcgtacccccaagtccccggttcttaccctatgcagcctgtcacggcgcccccgagctatccacctacctcctatagcagtacgcagcccacgtcatatgatcagtcctcatattcccagcaaaacacatatgggcaacctagcagttatggccagcagtcttcgtacgggcagcagtcatcgtatggacagcaacctccgacatcatatccaccacagaccggatcgtactctcaggcaccatcacaatattctcagcaatcatcgtcatacggtcagcagtcttcttttcgtcaggatcatccttcttcgatgggtgtatatggccaagaatcggggggattctctggaccgggtgagaaccgtagtatgtcaggcccagacaaccgtggccgtggtcgtggcggtttcgatcgtggtggcatgtcgcgcggtgggcgtggtgggggccgcgggggtatgggggcaggggagcgtggtgggtttaacaaaccaggtggaccgatggatgagggcccagatcttgacttgggtccaccagtcgatccggatgaggattcagacaatagtgcaatttacgtgcaggggttaaatgactccgtgacgctggatgatctggccgatttcttcaaacaatgcggcgtagtaaaaatgaataaacgcacaggtcaacccatgattcatatctacttggacaaagagactggcaaaccaaaaggagacgctacggtatcatatgaggaccctccaacagctaaagccgctgttgagtggtttgatggaaaagacttccagggaagcaaattaaaggtatcattagcgcgtaaaaaaccacctatgaactcaatgcgcggcggactgccgccccgcgaaggtcgtggtatgccaccgccacttcgtggaggacctggaggtccaggaggtccaggtggtcctatgggacgaatgggaggtcgtggcggcgaccgcgggggttttcctccgcgcgggccccgtggtagtcgtggtaacccttcagggggcggcaacgtgcaacatcgcgcaggcgattggcagtgtccaaacccgggttgcggtaatcagaacttcgcgtggcgtacagaatgtaatcagtgcaaagcgccaaaaccagaaggttttcttccacccccgtttccacctccaggcggagaccgtggccgtggagggccgggtggtatgcgcggcggacgcggaggtcttatggatcgcggtggtccgggcgggatgtttcgtgggggtcgcggcggggatcgtggaggtttccgcggaggtcgcggaatggatcgtggcggattcggaggaggacgtcgtggtggtccaggggggcctcctggtcctctgatggagcagatgggagggcgccgcggtggtcgtggcgggccaggcaagatggataaaggtgagcaccgtcaagagcgccgtgatcgtccctatggaggatccggaggatcc</p></span>
+<p><span class="wordbreak">&nbsp;atggcctcaactgactactccacttactcgcaggcagcagcacagcaaggctacagcgcttatacagcccagccaacgcaaggatatgcccaaacaacgcaggcatacggtcagcagtcttacggcacttacggccagccaactgatgtctcttacactcaagctcagaccacagcgacttatggccagaccgcatatgccacctcttacggtcagcccccgaccgtggaaggaacgtcgactggctatacaactccaaccgctcctcaagcatacagccagccagtccaaggatatggaaccggtgcatacgatacaacaactgccacagtgacgactacacaggcttcttatgcagctcagagtgcatatgggactcaaccagcatacccggcatacggccagcagccagctgcaacagctccaacgcgtcctcaggatggcaacaagccaacagagacctcgcaaccccagtccagtacaggtggctacaatcagccctccttggggtatgggcagagtaactactcgtacccccaagtccccggttcttaccctatgcagcctgtcacggcgcccccgagctatccacctacctcctatagcagtacgcagcccacgtcatatgatcagtcctcatattcccagcaaaacacatatgggcaacctagcagttatggccagcagtcttcgtacgggcagcagtcatcgtatggacagcaacctccgacatcatatccaccacagaccggatcgtactctcaggcaccatcacaatattctcagcaatcatcgtcatacggtcagcagtcttcttttcgtcaggatcatccttcttcgatgggtgtatatggccaagaatcggggggattctctggaccgggtgagaaccgtagtatgtcaggcccagacaaccgtggccgtggtcgtggcggtttcgatcgtggtggcatgtcgcgcggtgggcgtggtgggggccgcgggggtatgggggcaggggagcgtggtgggtttaacaaaccaggtggaccgatggatgagggcccagatcttgacttgggtccaccagtcgatccggatgaggattcagacaatagtgcaatttacgtgcaggggttaaatgactccgtgacgctggatgatctggccgatttcttcaaacaatgcggcgtagtaaaaatgaataaacgcacaggtcaacccatgattcatatctacttggacaaagagactggcaaaccaaaaggagacgctacggtatcatatgaggaccctccaacagctaaagccgctgttgagtggtttgatggaaaagacttccagggaagcaaattaaaggtatcattagcgcgtaaaaaaccacctatgaactcaatgcgcggcggactgccgccccgcgaaggtcgtggtatgccaccgccacttcgtggaggacctggaggtccaggaggtccaggtggtcctatgggacgaatgggaggtcgtggcggcgaccgcgggggttttcctccgcgcgggccccgtggtagtcgtggtaacccttcagggggcggcaacgtgcaacatcgcgcaggcgattggcagtgtccaaacccgggttgcggtaatcagaacttcgcgtggcgtacagaatgtaatcagtgcaaagcgccaaaaccagaaggttttcttccacccccgtttccacctccaggcggagaccgtggccgtggagggccgggtggtatgcgcggcggacgcggaggtcttatggatcgcggtggtccgggcgggatgtttcgtgggggtcgcggcggggatcgtggaggtttccgcggaggtcgcggaatggatcgtggcggattcggaggaggacgtcgtggtggtccaggggggcctcctggtcctctgatggagcagatgggagggcgccgcggtggtcgtggcgggccaggcaagatggataaaggtgagcaccgtcaagagcgccgtgatcgtccctatggaggatccggaggatcc</span></p>
 <p>FUS:</p>
-<p class="wordbreak">atggcgtctaatgactacactcagcaagctacccaatcttatggggcatatccgacccaacctggccaaggctattcccaacagtcatcacaaccttacgggcaacaaagttattccggttacagccagtcaacagatacttctggatatggtcagtcttcctattcgtcatacggtcagagtcagaatacgggttacgggacacaatcaaccccgcaggggtacggttccaccggcggctatggctccagccaatcgagtcaatcaagttatgggcaacaatcttcttatcccggctatggccaacagccggccccgagctccacatccggttcatatggttcttcaagccaatcaagctcgtatggtcagccgcagtcaggaagctattcacagcagccgtcctacgggggacaacaacagagttacggacagcaacagagttataacccaccgcaaggctacgggcagcagaatcagtacaattcctcttcaggtggtggtggtggcggtggtggtggagggaattacgggcaagatcaatctagtatgtcttcaggagggggcagcggtgggggatacggcaatcaagatcaaagtggtggaggtggatctggaggttatggacagcaggatcgtggaggtcgcggccgcggcggctctggtggtggtgggggaggaggaggtggtgggtacaatcgtagctccggaggatacgagccacgtgggcgtgggggcggtcgcggtgggcgcggtgggatgggtggctctgaccgcggcggttttaataagttcggtggtccccgtgatcagggttcacgccacgatagtgagcaagacaacagtgataacaatacgatcttcgttcaaggtcttggagagaacgttacaattgaatcggtagcagactatttcaaacagattggtattattaaaaccaacaagaagacaggccaaccgatgattaacttgtatactgaccgtgaaaccggtaaattaaaaggggaggctaccgtatcctttgacgaccctccaagcgcaaaagctgccattgactggtttgacggtaaagaattttctggcaacccgattaaggtgtccttcgcaacacgccgtgcggacttcaaccgtggcggggggaacggacgtggggggcgcggccgcggaggtccaatggggcgcggagggtatggaggaggaggcagtggtgggggtggacgtggtggtttccccagcggcggcggaggaggaggcggtcagcaacgtgccggtgattggaagtgtcctaatccaacctgcgaaaatatgaacttctcttggcgcaatgagtgtaaccagtgcaaggcaccgaaacctgatggcccaggtggcgggccagggggaagccacatgggcggaaattacggtgatgatcgtcgcggcggccgtgggggatacgaccgtggtggctatcgtggccgtggtggagatcgcggtggcttccgtggtgggcgcggaggaggtgaccgcggagggtttgggccggggaagatggacagccgtggcgaacaccgtcaagatcgtcgcgagcgcccatac</p>
+<p><span class="wordbreak">atggcgtctaatgactacactcagcaagctacccaatcttatggggcatatccgacccaacctggccaaggctattcccaacagtcatcacaaccttacgggcaacaaagttattccggttacagccagtcaacagatacttctggatatggtcagtcttcctattcgtcatacggtcagagtcagaatacgggttacgggacacaatcaaccccgcaggggtacggttccaccggcggctatggctccagccaatcgagtcaatcaagttatgggcaacaatcttcttatcccggctatggccaacagccggccccgagctccacatccggttcatatggttcttcaagccaatcaagctcgtatggtcagccgcagtcaggaagctattcacagcagccgtcctacgggggacaacaacagagttacggacagcaacagagttataacccaccgcaaggctacgggcagcagaatcagtacaattcctcttcaggtggtggtggtggcggtggtggtggagggaattacgggcaagatcaatctagtatgtcttcaggagggggcagcggtgggggatacggcaatcaagatcaaagtggtggaggtggatctggaggttatggacagcaggatcgtggaggtcgcggccgcggcggctctggtggtggtgggggaggaggaggtggtgggtacaatcgtagctccggaggatacgagccacgtgggcgtgggggcggtcgcggtgggcgcggtgggatgggtggctctgaccgcggcggttttaataagttcggtggtccccgtgatcagggttcacgccacgatagtgagcaagacaacagtgataacaatacgatcttcgttcaaggtcttggagagaacgttacaattgaatcggtagcagactatttcaaacagattggtattattaaaaccaacaagaagacaggccaaccgatgattaacttgtatactgaccgtgaaaccggtaaattaaaaggggaggctaccgtatcctttgacgaccctccaagcgcaaaagctgccattgactggtttgacggtaaagaattttctggcaacccgattaaggtgtccttcgcaacacgccgtgcggacttcaaccgtggcggggggaacggacgtggggggcgcggccgcggaggtccaatggggcgcggagggtatggaggaggaggcagtggtgggggtggacgtggtggtttccccagcggcggcggaggaggaggcggtcagcaacgtgccggtgattggaagtgtcctaatccaacctgcgaaaatatgaacttctcttggcgcaatgagtgtaaccagtgcaaggcaccgaaacctgatggcccaggtggcgggccagggggaagccacatgggcggaaattacggtgatgatcgtcgcggcggccgtgggggatacgaccgtggtggctatcgtggccgtggtggagatcgcggtggcttccgtggtgggcgcggaggaggtgaccgcggagggtttgggccggggaagatggacagccgtggcgaacaccgtcaagatcgtcgcgagcgcccatac</span></p>
 <p>NICD (Nephrin Intracellular Domain)</p>
-<p class="wordbreak">atgaatgcgtcatgtgtagggggtgtgatttggcagcgccgtattcgccgtctggccgaaggaatttcggaaaaaacagaagccggttccgaggaggaccgtgttcgtaatgaatacgaagaatcgcaatggaccggtgagcgcgatacgcaatctagtaccgtatccacgacagaagcagagccttattatcgcagtatccgcgatgaaagtccacagcttcctcctacacaggaagaggtttcctatagccgtggcgaaactggggaggatgaggatatggcagagccgggtcacttgtacgatgaggtagagcgcacttacccacctagcggcgcatggggtcccctgtacgatgaagtccagatggggccctgggatctgcattggcctgaggacacttatcaagaccctcgcggaatctatgaccaggttgcgggcgatcttgacactctggagcctgattcgctgccgtttgagctgcgcggccatctggtcggaggatccggaggatcc</p>
+<p><span class="wordbreak">atgaatgcgtcatgtgtagggggtgtgatttggcagcgccgtattcgccgtctggccgaaggaatttcggaaaaaacagaagccggttccgaggaggaccgtgttcgtaatgaatacgaagaatcgcaatggaccggtgagcgcgatacgcaatctagtaccgtatccacgacagaagcagagccttattatcgcagtatccgcgatgaaagtccacagcttcctcctacacaggaagaggtttcctatagccgtggcgaaactggggaggatgaggatatggcagagccgggtcacttgtacgatgaggtagagcgcacttacccacctagcggcgcatggggtcccctgtacgatgaagtccagatggggccctgggatctgcattggcctgaggacacttatcaagaccctcgcggaatctatgaccaggttgcgggcgatcttgacactctggagcctgattcgctgccgtttgagctgcgcggccatctggtcggaggatccggaggatcc</span></p>
 <p>SPD5:</p>
-<p class="wordbreak"> atggaagataactctgtccttaatgaagatagtaaccttgagcacgtcgaaggtcagcctcgtcgtagtatgtctcaaccagtactgaatgtcgaaggtgacaagcgcacttctagcacaagtgcaacacagcagcaagttctttccggtgcgttctcttccgctgatgtccgctcaattccgatcatccaaacgtgggaagaaaacaaggctttgaaaacgaagatcacaattcttcgcggtgagctgcaaatgtaccaacgccgctatagtgaagccaaggaggcaagccagaagcgtgtcaaagaggttatggatgactatgtggacttaaaattaggtcaagaaaatgtacaagagaagatggaacaatacaagttaatggaagaagacttattggcgatgcaatcccgtattgaaacgtcggaagataatttcgcgcgtcagatgaaggaatttgaagcccaaaaacatgccatggaagaacgtattaaggagttggaactttccgccaccgatgctaacaacactacagtaggtagttttcgcgggacgcttgacgatatcttgaagaagaacgaccccgactttaccttaacctccggatatgaagaacgtaagatcaatgacttagaagccaagttacttagcgaaatcgataaggtggcagaactggaggaccacatccagcagttgcgccaagaacttgatgatcaaagtgcgcgcttagctgattcagagaacgtgcgcgctcagctggaagcggctaccggacaaggaatcttgggagctgctgggaatgcaatggtcccaaattcaacgttcatgatcgggaacgggcgcgaatctcaaacgcgcgatcagttgaattacatcgatgatttggaaactaagcttgcagatgctaagaaggaaaatgacaaggcccgtcaggccttagtggagtatatgaataaatgttcaaagcttgaacacgagatccgcactatggtaaagaatagtacgtttgacagctcatcaatgttattgggcggtcagaccagcgacgaattaaagattcagattggaaaggtaaatggagaattaaacgtacttcgcgccgagaatcgcgagcttcgcattcgctgcgatcaacttactggaggggacggtaacctttctatcagtcttggccaaagtcgtctgatggctgggattgctacaaacgatgtggatagtattggccaagggaatgaaaccggcgggacgagtatgcgtatcttgccacgcgagtcgcagttagacgacttggaagaaagtaagttgcctttaatggatactagtagtgcggtccgcaaccaacaacagttcgccagcatgtgggaagattttgagtccgtgaaagactcactgcaaaacaaccataacgacacccttgagggctcgtttaatagtagtatgccacccccagggcgcgatgccacccagtcatttctttctcagaaatcttttaagaatagcccaattgtgatgcaaaagccgaagagcttacacttgcaccttaagtcacatcagagcgagggggcgggggagcagattcagaataatagtttctctactaagacggcgagtccgcatgtctcccagagccatatcccaatccttcacgacatgcaacaaattctggactcatcggcgatgtttttagaaggtcaacacgacgttgcagttaatgtggaacaaatgcaagaaaagatgtctcagattcgcgaggcccttgcgcgcttgttcgagcgcctgaaatcgagtgccgcattattcgaggaaattctggaacgcatgggcagttcagatccaaacgccgataagattaaaaaaatgaagttagctttcgagacgtcgatcaacgataaattaaacgtgtctgctattcttgaggcggccgagaaggatttacataacatgagcttaaacttttcaatcttagagaagagtatcgtaagccaagctgccgaagcgtcgcgtcgtttcactattgcaccggatgctgaagacgttgcatccagttctcttcttaatgcgagttactcgccgttgtttaagtttacttccaactccgatatcgttgaaaagcttcagaatgaagtctcagaacttaaaaacgagttagagatggcacgcacacgtgatatgcgcagcccccttaacggatcttccgggcgtcttagtgacgtgcagatcaacacaaaccgcatgtttgaagatcttgaggtatccgaggcaacgcttcagaaggccaaggaagagaactccactctgaaatcacagtttgctgagcttgaggcgaacttgcaccaggtgaattctaagttaggggaggtgcgctgcgagttaaatgaggccttggcccgtgtggacggagagcaagagactcgtgtaaaggctgagaacgcgttagaagaagctcgccaattgatttcttcccttaaacatgaggaaaatgagttaaagaagactattactgatatggggatgcgtcttaatgaagcaaaaaagtccgacgagttcctgaaatccgagctttctaccgctttggaggaggagaaaaaatcccaaaatttagcagacgagttgtcagaggaacttaacggttggcgtatgcgtacgaaggaagcggagaataaagtagaacatgcgtcctcggagaagagcgagatgctggaacgtatcgtgcatcttgaaacggaaatggagaagctgtcaacctctgaaattgcagcggactattgttctacgaagatgactgagcgcaaaaaggaaattgagttggcaaagtaccgcgaagattttgagaacgccgctattgtgggcctggaacgtatttcaaaagaaatctctgagttaactaagaagacactgaaggcaaagatcattccatcgaacatctcaagcatccagcttgtctgcgatgagctttgccgtcgtctgtcacgcgagcgcgagcaacaacacgaatacgccaaggttatgcgtgacgtcaatgagaagatcgaaaaattgcaattagaaaaagatgcgttggaacacgagttgaaaatgatgtcaagtaataatgaaaacgtgcctcctgtcgggacttcagttagcggcatgccgacaaagacgagtaatcaaaaatgcgcgcaaccacactacacgtcaccgactcgccaacttctgcatgagtcaaccatggcggtcgacgcgattgtccagaaattgaagaaaacacacaacatgagcgggatgggaccagagttgaaagaaacaattggtaacgtgatcaacgaatcacgtgtcttgcgcgactttcttcatcaaaagcttattttgtttaagggcattgatatgtctaattggaagaatgaaacggttgatcagctgatcaccgatttaggccagctgcaccaggacaatctgatgttggaagaacagatcaagaaatataaaaaggaacttaagcttaccaaaagtgcgatccctactcttggagtggaattccaggatcgtattaagaccgaaattgggaaaattgcgacggacatgggcggagccgtaaaggaaattcgcaagaag
+<p><span class="wordbreak">atggaagataactctgtccttaatgaagatagtaaccttgagcacgtcgaaggtcagcctcgtcgtagtatgtctcaaccagtactgaatgtcgaaggtgacaagcgcacttctagcacaagtgcaacacagcagcaagttctttccggtgcgttctcttccgctgatgtccgctcaattccgatcatccaaacgtgggaagaaaacaaggctttgaaaacgaagatcacaattcttcgcggtgagctgcaaatgtaccaacgccgctatagtgaagccaaggaggcaagccagaagcgtgtcaaagaggttatggatgactatgtggacttaaaattaggtcaagaaaatgtacaagagaagatggaacaatacaagttaatggaagaagacttattggcgatgcaatcccgtattgaaacgtcggaagataatttcgcgcgtcagatgaaggaatttgaagcccaaaaacatgccatggaagaacgtattaaggagttggaactttccgccaccgatgctaacaacactacagtaggtagttttcgcgggacgcttgacgatatcttgaagaagaacgaccccgactttaccttaacctccggatatgaagaacgtaagatcaatgacttagaagccaagttacttagcgaaatcgataaggtggcagaactggaggaccacatccagcagttgcgccaagaacttgatgatcaaagtgcgcgcttagctgattcagagaacgtgcgcgctcagctggaagcggctaccggacaaggaatcttgggagctgctgggaatgcaatggtcccaaattcaacgttcatgatcgggaacgggcgcgaatctcaaacgcgcgatcagttgaattacatcgatgatttggaaactaagcttgcagatgctaagaaggaaaatgacaaggcccgtcaggccttagtggagtatatgaataaatgttcaaagcttgaacacgagatccgcactatggtaaagaatagtacgtttgacagctcatcaatgttattgggcggtcagaccagcgacgaattaaagattcagattggaaaggtaaatggagaattaaacgtacttcgcgccgagaatcgcgagcttcgcattcgctgcgatcaacttactggaggggacggtaacctttctatcagtcttggccaaagtcgtctgatggctgggattgctacaaacgatgtggatagtattggccaagggaatgaaaccggcgggacgagtatgcgtatcttgccacgcgagtcgcagttagacgacttggaagaaagtaagttgcctttaatggatactagtagtgcggtccgcaaccaacaacagttcgccagcatgtgggaagattttgagtccgtgaaagactcactgcaaaacaaccataacgacacccttgagggctcgtttaatagtagtatgccacccccagggcgcgatgccacccagtcatttctttctcagaaatcttttaagaatagcccaattgtgatgcaaaagccgaagagcttacacttgcaccttaagtcacatcagagcgagggggcgggggagcagattcagaataatagtttctctactaagacggcgagtccgcatgtctcccagagccatatcccaatccttcacgacatgcaacaaattctggactcatcggcgatgtttttagaaggtcaacacgacgttgcagttaatgtggaacaaatgcaagaaaagatgtctcagattcgcgaggcccttgcgcgcttgttcgagcgcctgaaatcgagtgccgcattattcgaggaaattctggaacgcatgggcagttcagatccaaacgccgataagattaaaaaaatgaagttagctttcgagacgtcgatcaacgataaattaaacgtgtctgctattcttgaggcggccgagaaggatttacataacatgagcttaaacttttcaatcttagagaagagtatcgtaagccaagctgccgaagcgtcgcgtcgtttcactattgcaccggatgctgaagacgttgcatccagttctcttcttaatgcgagttactcgccgttgtttaagtttacttccaactccgatatcgttgaaaagcttcagaatgaagtctcagaacttaaaaacgagttagagatggcacgcacacgtgatatgcgcagcccccttaacggatcttccgggcgtcttagtgacgtgcagatcaacacaaaccgcatgtttgaagatcttgaggtatccgaggcaacgcttcagaaggccaaggaagagaactccactctgaaatcacagtttgctgagcttgaggcgaacttgcaccaggtgaattctaagttaggggaggtgcgctgcgagttaaatgaggccttggcccgtgtggacggagagcaagagactcgtgtaaaggctgagaacgcgttagaagaagctcgccaattgatttcttcccttaaacatgaggaaaatgagttaaagaagactattactgatatggggatgcgtcttaatgaagcaaaaaagtccgacgagttcctgaaatccgagctttctaccgctttggaggaggagaaaaaatcccaaaatttagcagacgagttgtcagaggaacttaacggttggcgtatgcgtacgaaggaagcggagaataaagtagaacatgcgtcctcggagaagagcgagatgctggaacgtatcgtgcatcttgaaacggaaatggagaagctgtcaacctctgaaattgcagcggactattgttctacgaagatgactgagcgcaaaaaggaaattgagttggcaaagtaccgcgaagattttgagaacgccgctattgtgggcctggaacgtatttcaaaagaaatctctgagttaactaagaagacactgaaggcaaagatcattccatcgaacatctcaagcatccagcttgtctgcgatgagctttgccgtcgtctgtcacgcgagcgcgagcaacaacacgaatacgccaaggttatgcgtgacgtcaatgagaagatcgaaaaattgcaattagaaaaagatgcgttggaacacgagttgaaaatgatgtcaagtaataatgaaaacgtgcctcctgtcgggacttcagttagcggcatgccgacaaagacgagtaatcaaaaatgcgcgcaaccacactacacgtcaccgactcgccaacttctgcatgagtcaaccatggcggtcgacgcgattgtccagaaattgaagaaaacacacaacatgagcgggatgggaccagagttgaaagaaacaattggtaacgtgatcaacgaatcacgtgtcttgcgcgactttcttcatcaaaagcttattttgtttaagggcattgatatgtctaattggaagaatgaaacggttgatcagctgatcaccgatttaggccagctgcaccaggacaatctgatgttggaagaacagatcaagaaatataaaaaggaacttaagcttaccaaaagtgcgatccctactcttggagtggaattccaggatcgtattaagaccgaaattgggaaaattgcgacggacatgggcggagccgtaaaggaaattcgcaagaag</span></p>
-</p>
+<table style="border-collapse:collapse; width:379pt; border:none" width="505">
+	<colgroup>
-<table >
+		<col span="5" style="width:60pt" width="80" />
+		<col style="width:79pt" width="105" />
+	</colgroup>
 	<tbody>
 		<tr height="19" style="height:14.25pt">
@@ Line 387: / Line 396: @@
 </table>
+<img id="spd1" style="width:50%" src="../../imgs/In-Vivo/SPD5.JPG"/>
-<img id="spd1" src="/imgs/In-Vivo/SPD5.JPG"/>
 <figcaption>Plasmid 3 with split SPD5 gBlock due to its size. Linker for sfGFP is 2xGGS.</figcaption>
 </figure>
-<img id="fus" src="/imgs/In-Vivo/FUS.JPG"/>
+<img id="FUS" style="width:50%" src="../../imgs/In-Vivo/FUS.JPG"/>
 <figcaption>Plasmid 2 with Fus. Linker for the sfGFP is 2xGGS</figcaption>
 </figure>
-<img id="ewsr" src="/imgs/In-Vivo/EWSR1.JPG"/>
+<img id="spd1" style="width:50%" src="../../imgs/In-Vivo/EWSR1.JPG"/>
 <figcaption>Plasmid 1 with EWSR1. Linker for mCherry is also 2xGGS.</figcaption>
 </figure>
-<img id="nicd" src="/imgs/In-Vivo/NICD.JPG"/>
+<img id="nicd" style="width:50%" src="../../imgs/In-Vivo/NICD.JPG"/>
 <figcaption>Plasmid 4 with NICD. Linker for sfGFP is 2xGGS</figcaption>
 </figure>
@@ Line 407: / Line 415: @@
 <p>&nbsp;</p>
- </p>
+</body>
-                           <h2> Subsection Title #2 </h2>
+</html> </p>
+                           <h2> 05.06.19 </h2>
+                          <p> <!DOCTYPE html>
+<html>
+<head><meta charset="utf-8">
+	<title></title>
+</head>
+<body>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Labbook 05.06.2019</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Performed amplifications of the Backbones (all Plasmids will be constructed with a pBAD33 backbone), sfGFP and mCherry genes from plasmids.</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Following PCR constructs were made:</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">For FUS: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; (A) pBAD amplification with Primers 1&amp;2* (not successful)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;(B) sfGFP amplification with Primers 5&amp;6*</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">For SPD5: &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(D) pBAD amplification with Primers 7&amp;8* (not successful)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; (E) sfGFP amplification with Primers 13&amp;14* (not successful)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">For EWSR1:&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; (H) pBAD amplification with Primers 15&amp;16*</span></span></span></p>
+<p style="margin-left:35.4pt; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; (I) mCherry amplification with primers 19&amp;20*</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">*For sequences of primers and roteins see<a href="...%2004.06.19">&nbsp;Protocol from Day 04.06.2019</a></span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">PCR tubes contained: &nbsp;&nbsp; 2,5 <span style="background:white"><span style="color:#222222">&mu;</span></span>l of each primer (diluted 1:10)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 <span style="background:white"><span style="color:#222222">&mu;</span></span>l DNA </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 19 <span style="background:white"><span style="color:#222222">&mu;</span></span>l H2O</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 25 <span style="background:white"><span style="color:#222222">&mu;</span></span><span lang="EN-GB" style="background:white"><span style="color:#222222">l Phusion Flash Master Mix (Thermo Fisher Scientific) </span></span></span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Fragments B, H and I were successfully amplified, identified via Gel electrophoresis and purified. </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><img alt="Pic1" height="616" src="../../imgs/In-Vivo/Unbenannt.JPG" width="573" /></span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><img alt="Pic2" height="272" src="../../imgs/In-Vivo/Unbenannt2.JPG" width="572" /></span></span></span></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">PCR for DNA fragments A, D and E were set up again. To troubleshoot the PCRS, higher Backbone concentrations of about 2 ng were used. Also for fragment E, 3% of DMSO were added to the PCR mix. Also, two different Polymerases were used (Q5 and Phusion Flash). </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Furthermore, constructs amplifying pBAD33 (A and D) were incubated overnight with 0,5<span style="background:white"><span style="color:#222222">&mu;</span></span><span lang="EN-GB" style="background:white"><span style="color:#222222">l DPN1 enzyme at 37&deg;C to digest methylated bacterial DNA. </span></span></span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">All amplifications and purifications were successful. Unfortunately, the PCR image has been lost because of technical issues with the GelViewer. </span></span></span></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+</body>
+</html> </p>
+                          <h2> 07.06.19 </h2>
+                          <p> <!DOCTYPE html>
+<html>
+<head><meta charset="utf-8">
+	<title></title>
+</head>
+<body>
+<p>NICD (Nephrin Intracellular Domain) was amplified, as well as sfGFP and pBAD33 with respective overlaps.&nbsp;</p>
+<p>Following PCRS were set up:&nbsp;</p>
+<p>(J) pBAD amplification with primers 1&amp;22*</p>
+<p>(K) sfGFP amplification with primers 25&amp;6*</p>
+<p>(L) NICD amplification with primers 23&amp;24*</p>
+<p>*For all sequences see table in <a href="...%2004.06.19">protocol from 04.06.2019</a></p>
+<p>All amplifications were successful, see picture below.</p>
+<p><img alt="" height="303" src="../../imgs/In-Vivo/Unbenannt3.JPG" width="411" /></p>
+<p>Afterwards, gibson cloning was set up. Considering sizes, amound of insert for 50 ng backbone was calculated.</p>
+<p>NICD: 17.6 ng</p>
+<p>sfGFP: 24 ng</p>
+<p>The DNA was diluted with water until reaching 5 microliters of volume and mixed in a 1:1 ratio with a&nbsp;HiFi ligase/exoniuclease&nbsp;mastermix and incubated at 50&deg;C for 15 minutes. Afterwards competent Top10 E. Coli were transformed with 2 microlitres of Gibson Assembly Mix and incubated overnight.&nbsp;</p>
+</body>
+</html> </p>
+                          <h2>11.06.19</h2>
+                          <p><!DOCTYPE html>
+<html>
+<head><meta charset="utf-8">
+	<title></title>
+</head>
+<body>
+<p>After transformation of NICD was ineffective, proportions were calculated again and ligation was repeated. Incubation overnight follows.</p>
+<p>Furthermore, gBlocks for EWSR1, FUS, SPD5_1 and SPD5_2 were amplified&nbsp;to create overlaps with following Primers:</p>
+<p>EWSR1 with Primers 17 and 18*</p>
+<p>FUS with Primers 3 and 4*</p>
+<p>SPD5_1 and SPD5_2 with Primers 9&amp;10 and 11&amp;12 respectively*</p>
+<p>*For primer and protein sequences see <a href="...%2004.06.19">protocol from 04.06.2019.</a></p>
+<p>Amplification was only successful for SPD5_2 in the first round:</p>
+<p><img alt="" src="./11.06.019%202.JPG" /><img alt="" height="329" src="../../imgs/In-Vivo/11.06.019%202.JPG" width="438" /></p>
+<p>PCR troubleshooting consisted of repeating the reaction with two different polymerases, being Q5 and Phusion Flash, as well as adding 3% DMSO to the DNA mix. In the case of EWSR1, DNA was increased to 2 ng.</p>
+<p>In the second round, bands for each sequence could be seen, were cut and purified.&nbsp;</p>
+<p><img alt="" height="327" src="../../imgs/In-Vivo/11.06.2019.JPG" width="438" /></p>
+</body>
+</html> </p>
+                          <h2> 12.06.19 </h2>
+                          <p> Transformation from the day before was unsuccessful, no colonies were on the plate. We found out we used Ampicillin plates, which is the wrong antibiotic. </p>
+<p>Since all required sequences for the designed plasmids (for more information about plasmid constructs see protocol from day 04.06.2019) were now amplified, ligation for all plasmids could now start. The method used was Gibson Cloning. The gibson assemblies were then plated on Chloramphenicol plates and incubated overnight.</p>
+                          <h2> 13.06.19 </h2>
+                          <p> <!DOCTYPE html>
+<html>
+<head><meta charset="utf-8">
+	<title></title>
+</head>
+<body>
+<p>Gibson assembly of the Plasmids containing&nbsp;NICD and SPD5, EWSR.</p>
+<p>Following constructs were ligated with NEB HiFi Ligase:</p>
+<p>Fus:&nbsp; A + B + C (Fus)</p>
+<p>EWSR1: H, I, J (EWSR1)</p>
+<p>SPD5: D, E, F(SPD5_1) and G (SPD5_2)</p>
+<p>Together with the ligated NICD plasmid, all plasmids were transformed into competent E. Coli and plated on chloramphenicol plates.</p>
+<p>Plates were incubated overnight at 37&deg;C</p>
+</body>
+</html></p>
+                          <h2> 14.06.19 </h2>
+                          <p> <!DOCTYPE html>
+<html>
+<head><meta charset="utf-8">
+	<title></title>
+</head>
+<body>
+<p>Plates containing FUS, SPD5 and NICD from 13.06.2019 had colonies. EWSR1 had no colonies.</p>
+<p>Coloies were picked and LB medium with Chloramphenicol was inoculated, bacteria was grown in media for 5 hours and prepared for sequencing.&nbsp;</p>
+<p>A second set of media were incoulated and prepared for microscopy. Samples were visualized with a Widefield microscope and analyzed with FiJi. Images attached.</p>
+<p>&nbsp;</p>
+<p>SPD5 sample 2: Dispersed fluroescence (left). No indication for droplets found.</p>
+<p><img alt="" height="345" src="../../imgs/In-Vivo/SPD5_1-04.jpg" width="430" /><img alt="" height="344" src="../../imgs/In-Vivo/SPD5_1-04_02.jpg" width="430" /></p>
+<p>SPD5 sample 1. Lighter spots that can be an indication for droplets in the fluorescent image (Left) that are not visible on the right.</p>
+<p><img alt="" height="343" src="../../imgs/In-Vivo/SPD5_1-02.jpg" width="427" /><img alt="" height="342" src="../../imgs/In-Vivo/SPD5_1-02_02.jpg" width="426" /></p>
+<p>FUS sample 1. Low, dispersed fluorescence (left). No indications for droplets.</p>
+<p><img alt="" height="343" src="../../imgs/In-Vivo/FUS_2-03.jpg" width="427" /><img alt="" height="343" src="../../imgs/In-Vivo/FUS_2-03_02.jpg" width="428" /></p>
+<p>NICD sample 1.&nbsp;Low, dispersed fluorescence (left). No indications for droplets.</p>
+<p><img alt="" height="342" src="../../imgs/In-Vivo/NICD_2-06.jpg" width="426" /><img alt="" height="341" src="../../imgs/In-Vivo/NICD_2-06_02.jpg" width="425" /></p>
+<p>Problem with NICD: A cloning mistake has led to an excessive atg start codon at the begining of sfGFP, which may give misleading results.</p>
+<p>Complementary primers for a site directed mutagenesis deleting the excessive three bases were ordered.&nbsp;</p>
+<p><img alt="" height="318" src="../../imgs/In-Vivo/atg.JPG" width="321" /></p>
+</body>
+</html> </p>
+                          <h2> 20.06.19 </h2>
+                          <p> <!DOCTYPE html>
+<html>
+<head><meta charset="utf-8">
+	<title></title>
+</head>
+<body>
+<p>Criteria for identifying if an accumulation of fluorescent proteins are dynamic droplets or dead aggregates:</p>
+<p>1. They appear in a concentration dependent manner,</p>
+<p>2. They fuse when they encounter another one of their kind,</p>
+<p>3. Fluorescence regeneraties after photobleaching (FRAP)</p>
+<p>In order to determine whether the results seen in the last protocol are significant, the constructed proteins have to be inducible to be seen under different concentrations.</p>
+<p>Cloning steps were repeated so that the constructs could be positioned in front of inducible promoters. Additionally, EWSR1 will be inserted in a different backbone with</p>
+<p>compatible origins of replications with the pBAD backbone, so that FUS and EWSR1 can be induced in the same bacteria without competition. The new backbone for&nbsp;</p>
+<p>EWSR1 is pTrc99a (empty) with a prc promoter.</p>
+<p>Following primers are now added to the list:</p>
+<table style="border-collapse:collapse; width:1041pt; border:none" width="1389">
+	<colgroup>
+		<col style="width:60pt" width="80" />
+		<col style="width:136pt" width="182" />
+		<col style="width:204pt" width="272" />
+		<col style="width:311pt" width="415" />
+		<col style="width:330pt" width="440" />
+	</colgroup>
+	<tbody>
+		<tr height="19" style="height:14.25pt">
+			<td height="19" style="border:none; height:14.25pt; width:60pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap" width="80"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">New primers</span></span></span></span></span></span></td>
+			<td style="border:none; width:136pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap" width="182">&nbsp;</td>
+			<td style="border:none; width:204pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap" width="272">&nbsp;</td>
+			<td style="border:none; width:311pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap" width="415">&nbsp;</td>
+			<td style="border:none; width:330pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap" width="440">&nbsp;</td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td height="19" style="border:none; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Number</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Name</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Tube name</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Sequence</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Description</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl66" height="19" style="border:none; background:#e2efda; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">31</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pBAD33wt_SPD5_FWD</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pBAD33wt_SPD5_FWD</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">TGGATGAGCTCTACAAATAA<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">AAGCTTGGCTGTTTTGGCGG</span></span></span></span></span></span></font><font><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">&nbsp;</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify new pBAD33(induceable promoter) with right overlaps</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl66" height="19" style="border:none; background:#e2efda; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">32</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pBAD33wt_SPD5_REV</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pBAD33wt_SPD5_REV</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">AGGACAGAGTTATCTTCCAT<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">TCTAGAGGATCCCCGGGTAC</span></span></span></span></span></span></font><font><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">&nbsp;</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify new pBAD33(induceable promoter) with right overlaps</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td class="xl69" height="19" style="border:none; background:#ffe699; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">32_2</span></span></span></span></span></span></td>
+			<td class="xl70" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#344043"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">SPD5_1_mid_GFP_rev</span></span></span></span></span></span></td>
+			<td class="xl70" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#344043"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">SPD5_1_mid_GFP_rev</span></span></span></span></span></span></td>
+			<td class="xl72" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">GTGCAATAGT<font><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">GAAACGACGCGACGC</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl69" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">&nbsp;</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl69" height="19" style="border:none; background:#ffe699; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">33</span></span></span></span></span></span></td>
+			<td class="xl71" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">SPD5_sfGFP_pBAD33 FWD</span></span></span></span></span></span></td>
+			<td class="xl71" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">SPD5_sfGFP_pBAD33 FWD</span></span></span></span></span></span></td>
+			<td class="xl73" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">GTACCCGGGGATCCTCTAGA<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">ATGGAAGATAACTCTGTCCTTAA</span></span></span></span></span></span></font><font><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">&nbsp;</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl69" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify SPD5_1 with the right overlap for new backbone</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl66" height="19" style="border:none; background:#e2efda; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">34</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">SPD5_sfGFP_pBAD33 REV</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">SPD5_sfGFP_pBAD33 REV</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">CCGCCAAAACAGCCAAGCTT<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">TTATTTGTAGAGCTCATCCATGC</span></span></span></span></span></span></font><font><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">&nbsp;</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify GFP with the right overlap for new backbone</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl66" height="19" style="border:none; background:#e2efda; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">35</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">EWSR1_mCherry_pBAD33 FWD</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">EWSR1_mCherry_pBAD33 FWD</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">CATGGAATTCGAGCTCGGTA<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">ATGGCCTCAACTGACTACTC</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify EWSR1 with the right overlap for new backbone</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl66" height="19" style="border:none; background:#e2efda; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">36</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">EWSR1_mCherry_pBAD33 REV</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">EWSR1_mCherry_pBAD33 REV</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">GACTCTAGAGGATCCCCGGG<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">TTAGTACAGCTCGTCCATGC</span></span></span></span></span></span></font><font><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">&nbsp;</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify mCherry with the right overlap for new backbone</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl66" height="19" style="border:none; background:#e2efda; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">37</span></span></span></span></span></span></td>
+			<td class="xl74" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-weight:700"><span style="font-family:Arial,sans-serif"><span style="font-style:normal"><span style="text-decoration:none">&nbsp;<font><span style="font-size:10pt"><span style="color:#333333"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">pTrc99A FWD</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl74" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-weight:700"><span style="font-family:Arial,sans-serif"><span style="font-style:normal"><span style="text-decoration:none">&nbsp;<font><span style="font-size:10pt"><span style="color:#333333"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">pTrc99A FWD</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">GCATGGACGAGCTGTACTAA<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">CCCGGGGATCCTCTAGAGTC</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify pTrc99A (induceable promoter) with right overlaps</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl66" height="19" style="border:none; background:#e2efda; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">38</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pTrc99A REV</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pTrc99A REV</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">GAGTAGTCAGTTGAGGCCAT<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">TACCGAGCTCGAATTCCATG</span></span></span></span></span></span></font><font><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">&nbsp;</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify pTrc99A (induceable promoter) with right overlaps</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl69" height="19" style="border:none; background:#ffe699; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">39</span></span></span></span></span></span></td>
+			<td class="xl71" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">NICD_sfGFP_pBAD33_FWD</span></span></span></span></span></span></td>
+			<td class="xl71" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">NICD_sfGFP_pBAD33_FWD</span></span></span></span></span></span></td>
+			<td class="xl73" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">GTACCCGGGGATCCTCTAGA<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">ATGAATGCGTCATGTGTAGG</span></span></span></span></span></span></font><font><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">&nbsp;</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl69" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify NICD with the right overlap for new backbone</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td class="xl69" height="19" style="border:none; background:#ffe699; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">39_2</span></span></span></span></span></span></td>
+			<td class="xl75" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:#344043"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">NICD_mid_sfGFP_REV</span></span></span></span></span></span></td>
+			<td class="xl70" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#344043"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">NICD_mid_sfGFP_REV</span></span></span></span></span></span></td>
+			<td class="xl72" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">TCCTTTGCTCAT<font><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">GGATCCTCCGGATCCTC</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl69" style="border:none; background:#ffe699; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify NICD with the right overlap for new backbone</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl66" height="19" style="border:none; background:#e2efda; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">40</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">NICD_sfGFP_pBAD33 REV</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">NICD_sfGFP_pBAD33 REV</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">CCGCCAAAACAGCCAAGCTT<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">TTATTTGTAGAGCTCATCCATGC</span></span></span></span></span></span></font><font><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">&nbsp;</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify sfGFP with the right overlap for new backbone</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl66" height="19" style="border:none; background:#e2efda; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">41</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pBAD33wt_NICD FWD</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pBAD33wt_NICD FWD</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">TGGATGAGCTCTACAAATAA<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">AAGCTTGGCTGTTTTGGCGG</span></span></span></span></span></span></font><font><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">&nbsp;</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify new pBAD33(induceable promoter) with right overlaps</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl66" height="19" style="border:none; background:#e2efda; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">42</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pBAD33wt_NICD REV</span></span></span></span></span></span></td>
+			<td class="xl67" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pBAD33wt_NICD REV</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">CCTACACATGACGCATTCAT<font><span style="font-size:10pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">TCTAGAGGATCCCCGGGTAC</span></span></span></span></span></span></font><font><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Arial,sans-serif">&nbsp;</span></span></span></span></span></span></font></span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; background:#e2efda; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primer to amplify new pBAD33(induceable promoter) with right overlaps</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td height="19" style="border:none; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+		</tr>
+		<tr height="21" style="height:15.75pt">
+			<td height="21" style="border:none; height:15.75pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td class="xl63" colspan="2" style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:12pt"><span style="color:#333333"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">Primers for NICD atg deletion</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td height="19" style="border:none; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td height="19" style="border:none; height:14.25pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">atg_del_fwd</span></span></span></span></span></span></td>
+			<td class="xl64" style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">GATCCGGAGGATCCAGCAAAGGAGAAGAAC</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+		</tr>
+		<tr height="21" style="height:15.75pt">
+			<td height="21" style="border:none; height:15.75pt; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">atg_del_Rev</span></span></span></span></span></span></td>
+			<td class="xl65" style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:12pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">GTTCTTCTCCTTTGCTGGATCCTCCGGATC&nbsp;</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap">&nbsp;</td>
+		</tr>
+	</tbody>
+</table>
+<p>&nbsp;</p>
+<p>Constructs cotnaining the backbone with inducible promoters will be the following:</p>
+<p>FUS, containing the following fragments</p>
+<table border="1" cellpadding="1" cellspacing="1" style="width: 960px;">
+	<tbody>
+		<tr>
+			<td>XA</td>
+			<td>pBAD</td>
+			<td>w/primers 29&amp;30</td>
+			<td>68&deg;C</td>
+		</tr>
+		<tr>
+			<td>XB</td>
+			<td>GFP</td>
+			<td>w/ primers 5&amp;28</td>
+			<td>63&deg;C</td>
+		</tr>
+		<tr>
+			<td>XC</td>
+			<td>FUS</td>
+			<td>w/ primers 27&amp;4</td>
+			<td>68&deg;C</td>
+		</tr>
+	</tbody>
+</table>
+<p>&nbsp;</p>
+<p>SPD5, containing the following fragments</p>
+<table border="1" cellpadding="1" cellspacing="1" style="width: 960px;">
+	<tbody>
+		<tr>
+			<td>XD</td>
+			<td>pBAD</td>
+			<td>w/primers 31&amp;32</td>
+			<td>68&deg;C</td>
+		</tr>
+		<tr>
+			<td>XE</td>
+			<td>GFP</td>
+			<td>w/primers 13&amp;34</td>
+			<td>63&deg;C</td>
+		</tr>
+		<tr>
+			<td>XF</td>
+			<td>SPD1</td>
+			<td>w/primers 33&amp;10</td>
+			<td>60&deg;C</td>
+		</tr>
+		<tr>
+			<td>G</td>
+			<td>SPD2</td>
+			<td>&nbsp;</td>
+			<td>&nbsp;</td>
+		</tr>
+	</tbody>
+</table>
+<p>EWSR1, containing the following fragments:</p>
+<table border="1" cellpadding="1" cellspacing="1" style="width: 960px;">
+	<tbody>
+		<tr>
+			<td>XG</td>
+			<td>pBAD</td>
+			<td>w/primers 37&amp;38</td>
+			<td>65&deg;C</td>
+		</tr>
+		<tr>
+			<td>XH</td>
+			<td>EWSR1</td>
+			<td>w/primers 35&amp;18</td>
+			<td>65&deg;C</td>
+		</tr>
+		<tr>
+			<td>XI</td>
+			<td>mCherry</td>
+			<td>w/primers 19&amp;36</td>
+			<td>65&deg;C</td>
+		</tr>
+	</tbody>
+</table>
+<p>NICD, containing the following fragments:</p>
+<table border="1" cellpadding="1" cellspacing="1" style="width: 960px;">
+	<tbody>
+		<tr>
+			<td>XJ</td>
+			<td>pBAD</td>
+			<td>w/primers 41&amp;42</td>
+			<td>68&deg;C</td>
+		</tr>
+		<tr>
+			<td>XK</td>
+			<td>sfGFP</td>
+			<td>w/primers 25&amp;40</td>
+			<td>63&deg;C</td>
+		</tr>
+		<tr>
+			<td>XL</td>
+			<td>NICD</td>
+			<td>w/primers 39&amp;24</td>
+			<td>63&deg;C</td>
+		</tr>
+	</tbody>
+</table>
+<p>&nbsp;</p>
+</body>
+</html> </p>
+                          <h2> 21.06.19 </h2>
+                          <p> <!DOCTYPE html>
+<html>
+<head><meta charset="utf-8">
+	<title></title>
+</head>
+<body>
+<p>Amplifications for the gibson fragments with corrresponding sticky ends were started in order to achieve plasmids with the constructs in front of inducible promoters.</p>
+<p><img alt="" height="338" src="../../imgs/In-Vivo/PCR.JPG" width="516" /><img alt="" height="334" src="../../imgs/In-Vivo/PCRteil2.JPG" width="561" /></p>
+<p>Image left and right: 1% agarose gel with results of amplification of gibson fragments.&nbsp;</p>
+<p>All Fragments except EWSR1 gBlock could be successfully amplified. New primers were ordered as part of the troubleshooting procedure.&nbsp;</p>
+<p>The new primers are:</p>
+<table style="border-collapse:collapse; width:777pt; border:none" width="1036">
+	<colgroup>
+		<col style="width:60pt" width="80" />
+		<col style="width:158pt" width="211" />
+		<col style="width:216pt" width="288" />
+		<col style="width:343pt" width="457" />
+	</colgroup>
+	<tbody>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl63" height="19" style="border:none; border-bottom:none; height:14.25pt; width:60pt; border-top:1pt solid windowtext; border-right:none; border-left:1pt solid windowtext; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap" width="80"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">43</span></span></span></span></span></span></td>
+			<td class="xl64" style="border:none; border-bottom:none; width:158pt; border-top:1pt solid windowtext; border-right:none; border-left:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap" width="211"><span style="font-size:10pt"><span style="color:#333333"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">pTrc99a_rev</span></span></span></span></span></span></td>
+			<td class="xl65" style="border:none; border-bottom:none; width:216pt; border-top:1pt solid windowtext; border-right:none; border-left:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap" width="288"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">pTrc99a_inducible_rev,&nbsp;</span></span></span></span></span></span></td>
+			<td class="xl66" style="border:none; border-bottom:none; width:343pt; border-top:1pt solid windowtext; border-right:1pt solid windowtext; border-left:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap" width="457"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">ttgaggccatTACCGAGCTCGAATTCCATG</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl67" height="19" style="border:none; border-bottom:none; height:14.25pt; border-top:none; border-right:none; border-left:1pt solid windowtext; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">44</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">EWSR1_fwd</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">EWSR1_inducible_fwd,</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; border-bottom:none; border-top:none; border-right:1pt solid windowtext; border-left:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">gagctcggtaATGGCCTCAACTGACTACTC</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl67" height="19" style="border:none; border-bottom:none; height:14.25pt; border-top:none; border-right:none; border-left:1pt solid windowtext; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">45</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">EWSR1_rev</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">EWSR1_inducible_rev</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; border-bottom:none; border-top:none; border-right:1pt solid windowtext; border-left:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">ccttgctcacggatcctccggatcctccATAGGGACGATCACGGCG</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl67" height="19" style="border:none; border-bottom:none; height:14.25pt; border-top:none; border-right:none; border-left:1pt solid windowtext; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">46</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">mCherry_fwd</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">mCherry_inducible_fwd</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; border-bottom:none; border-top:none; border-right:1pt solid windowtext; border-left:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">tcgtccctatggaggatccggaggatccGTGAGCAAGGGCGAGGAG</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="19" style="height:14.25pt">
+			<td align="right" class="xl67" height="19" style="border:none; border-bottom:none; height:14.25pt; border-top:none; border-right:none; border-left:1pt solid windowtext; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">47</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">mCherry_rev</span></span></span></span></span></span></td>
+			<td style="border:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">mCherry_inducible_rev,</span></span></span></span></span></span></td>
+			<td class="xl68" style="border:none; border-bottom:none; border-top:none; border-right:1pt solid windowtext; border-left:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">gatccccgggGTACAGCTCGTCCATGCC</span></span></span></span></span></span></td>
+		</tr>
+		<tr height="20" style="height:14.65pt">
+			<td align="right" class="xl69" height="20" style="border:none; border-bottom:1pt solid windowtext; height:14.65pt; border-top:none; border-right:none; border-left:1pt solid windowtext; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">48</span></span></span></span></span></span></td>
+			<td class="xl70" style="border:none; border-bottom:1pt solid windowtext; border-top:none; border-right:none; border-left:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">pTrc99a_fwd</span></span></span></span></span></span></td>
+			<td class="xl70" style="border:none; border-bottom:1pt solid windowtext; border-top:none; border-right:none; border-left:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:11pt"><span style="color:black"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none"><span style="font-family:Calibri,sans-serif">pTrc99a_inducible_fwd</span></span></span></span></span></span></td>
+			<td class="xl71" style="border:none; border-bottom:1pt solid windowtext; border-top:none; border-right:1pt solid windowtext; border-left:none; padding-top:1px; padding-right:1px; padding-left:1px; vertical-align:bottom; white-space:nowrap"><span style="font-size:10pt"><span style="color:#a2b9be"><span style="font-family:Arial,sans-serif"><span style="font-weight:400"><span style="font-style:normal"><span style="text-decoration:none">cgagctgtacCCCGGGGATCCTCTAGAG</span></span></span></span></span></span></td>
+		</tr>
+	</tbody>
+</table>
+<p>&nbsp;</p>
+<p>&nbsp;</p>
+</body>
+</html> </p>
+                          <h2> 25.06.19 </h2>
+                          <p> <!DOCTYPE html>
+<html>
+<head><meta charset="utf-8">
+	<title></title>
+</head>
+<body>
+<h3 style="margin: 0cm 0cm 8pt;"><strong><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Labbook </span></span></span></strong></h3>
+<h3 style="margin: 0cm 0cm 8pt;"><strong><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">25/6/19</span></span></span></strong></h3>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Repeat PCR for SPD5_1 and FUS and NICD with new primers and extract from agarose gel. </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Transform into competent cells and grow overnight culture.</span></span></span></p>
+<h2 style="margin: 0cm 0cm 8pt;"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">26/6/19</span></span></span></h2>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Planning of FRAP experiment</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Widefield and Brightfield microscopy of samples </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Gibson assembly of&nbsp; FUSinducible (XA, XB, XC) </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">50 ng of Backbone (1uL) </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">50 ng sfGFP (2,78 uL)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">73 ng FUS (0.73 uL)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Retransformation of bacteria with correctly sequenced constructs (constitutive)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Repeat PCR for NICD and SPD5 fragments</span></span></span></p>
+<h2 style="margin: 0cm 0cm 8pt;"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">27/6/19</span></span></span></h2>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Miniprep of assembled FUS </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Repeat PCR of fragments XC, XF, XL, XH and HI (not successful except XC, XF)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Repeat PCR of fragments XH XI and XL with 3% DMSO</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Gibson assembly of NICD and &nbsp;SPD5 with correct amplified fragments.</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">NICD: 50 ng Backbone (0,63 uL)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">23,86 ng sfGFP (2,06 uL)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">51,14 ng NICD (2,60 uL)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">SPD5: 50 ng Backbone (2.5 uL)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">sfGFP: 24,68 ng (1,1 uL)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">SPD5_1: 68,18 (4,54 uL)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">SPD5_2: 51,14 (1,17 uL)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<h2 style="margin: 0cm 0cm 8pt;"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">2/7/19</span></span></span></h2>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">PCR of EWSR1 with primers 17&amp;18, 18&amp;35 and 44&amp;45 in order to amplify EWSR1 constitutive and EWSR1 inducible (twice) </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">PCR was not successful. Next step is a Touchdown PCR starting at 75&deg;C and going down to 65&deg;C (annealing temperature of the three constructs)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Induceable constructs were retransformed (NICD was made again by Gibson assembly)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Constitutive constructs were retransformed. All retranformations were plated on Chloramphenicol plates and placed overnight in the 37&deg;C incubator. </span></span></span></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<h2 style="margin: 0cm 0cm 8pt;"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">3/7/19</span></span></span></h2>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Colonies were picked and new overnight cultures were prepared from the retransformation of the constructs (induceable and constitutive) for the FRAP experiment.</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">New Glycerol stocks from all constructs were frozen at -80&deg;C</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Glucose free TB medium was mixed, heated and autoclaved in order to prepare the induction and FRAP experiment.</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">New Chloramphenicol plates were made (LB medium + 400uL 34g/mol Chloramphenicol stock solution added at 50&deg;C)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<h2 style="margin: 0cm 0cm 8pt;"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">4/7/19</span></span></span></h2>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Microscopy of the working constructs (constitutive FUS, NICD and SPD5 as well as Arbinose-inducible FUS, NICD and SPD5) and the negative control IPTG-inducible</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">mini eYFP which is known to form aggregates at high concentrations (source: AG Di Ventura, BIOSS Freiburg).</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Induction was carried out with the following concentrations:</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">No induction: E. coli carrying plasmids were grown overnight in TB, diluted to OD 0.01 in the morning, grown to OD 0.5 and further incubated for 2h at 37&deg;C with vigorous shaking.</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Induction: E. coli were grown in overnight culture in TB medium, diluted to OD 0.01 in the morning, grown to OD 0.5 and induced with the following concentrations of Arabinose:</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">LOW: 0.001% Arabinose</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">MID: 0.05% Arabinose</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">HIGH: 1% Arabinose</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">After 2h in the incubater at 37&deg;C and vigorous shaking, miscroscopy was carried out.</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">At first, samples were analysed with brightfield and widefield GFP microscopy. Cells were checked for droplet like dots. </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Most cells didn&rsquo;t show droplet-like structures, only constitutive SPD5 (as already shown before). Induction seemed to not have worked, since the positive control </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">plasmid mini eYFP did not show the expected results. Furthermore, general fluorescence of mid and high induced cells was not to be distinguished from low induced cells in most cases. </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">The constitutive SPD5 probe containing droplet-like structures was analysed under the confocal microscope at 100x. Bleaching was done with 100% laser </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">intensity with an itinerary of 20. Single droplet like structures were bleached and the Region of Interest (ROI) was analysed, while other ROIs were only analised. </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Results did not seem to be clear. Induceable constructs are analysed (sequenced) and microscopy will be repeated in a week. </span></span></span></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<table border="1" cellpadding="1" cellspacing="1" style="width: 960px;">
+	<tbody>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385136/FUS_const_04_07.jpg" onclick="window.location.href = '1385136?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">FUS_const_04_07.jpg</a>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385191/NICD_cons_04_07.jpg" onclick="window.location.href = '1385191?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">NICD_cons_04_07.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385231/SPD5_const_04_07.jpg" onclick="window.location.href = '1385231?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">SPD5_const_04_07.jpg</a></td>
+		</tr>
+		<tr>
+			<td>
+			<p style="margin:0cm 0cm 8pt"><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385141/FUS_const_04_07_2.jpg" onclick="window.location.href = '1385141?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">FUS_const_04_07_2.jpg</a>&nbsp; &nbsp; &nbsp;</p>
+			</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385196/NICD_const_04_07_2.jpg" onclick="window.location.href = '1385196?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">NICD_const_04_07_2.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385236/SPD5_const_04_07_2.jpg" onclick="window.location.href = '1385236?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">SPD5_const_04_07_2.jpg</a></td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385146/FUS_high_04_07.jpg" onclick="window.location.href = '1385146?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">FUS_high_04_07.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385201/NICD_high%2010_07.jpg" onclick="window.location.href = '1385201?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">NICD_high 10_07.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385241/SPD5_high_04_07.jpg" onclick="window.location.href = '1385241?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">SPD5_high_04_07.jpg</a></td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385151/FUS_high_04_07_2.jpg" onclick="window.location.href = '1385151?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">FUS_high_04_07_2.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385206/NICD_high_04_07.jpg" onclick="window.location.href = '1385206?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">NICD_high_04_07.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385246/SPD5_high_04_07_2.jpg" onclick="window.location.href = '1385246?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">SPD5_high_04_07_2.jpg</a></td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385166/FUS_mid_04_07.jpg" onclick="window.location.href = '1385166?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">FUS_mid_04_07.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385226/NICD_mid_04_07.jpg" onclick="window.location.href = '1385226?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">NICD_mid_04_07.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385261/SPD5_mid_04_07_2.jpg" onclick="window.location.href = '1385261?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">SPD5_mid_04_07_2.jpg</a></td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385171/FUS_mid_04_07_2.jpg" onclick="window.location.href = '1385171?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">FUS_mid_04_07_2.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385106/NICD_mid_10_07.jpg" onclick="window.location.href = '1385106?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">NICD_mid_10_07.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385266/SPD5_mod_04_07.jpg" onclick="window.location.href = '1385266?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">SPD5_mod_04_07.jpg</a></td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385156/FUS_low_04_07.jpg" onclick="window.location.href = '1385156?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">FUS_low_04_07.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385216/NICD_Low_04_07.jpg" onclick="window.location.href = '1385216?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">NICD_Low_04_07.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385251/SPD5_low_04_07.jpg" onclick="window.location.href = '1385251?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">SPD5_low_04_07.jpg</a></td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385161/FUS_low_04_07_2.jpg" onclick="window.location.href = '1385161?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">FUS_low_04_07_2.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385221/NICD_Low_04_07_1.jpg" onclick="window.location.href = '1385221?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">NICD_Low_04_07_1.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385256/SPD5_low_04_07_2.jpg" onclick="window.location.href = '1385256?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">SPD5_low_04_07_2.jpg</a></td>
+		</tr>
+	</tbody>
+</table>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="margin:0cm 0cm 8pt"><u><strong><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">8/7/19</span></span></span></strong></u></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Touchdown PCR was started with the EWSR1 constructs, amplified with primer pairs 17&amp;18(constitutive), 18&amp;35 and 44&amp;45 (with the araBAD promoter)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">17&amp;18(constitutive), 18&amp;35 and 44&amp;45 (with the araBAD promoter)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="margin:0cm 0cm 8pt"><strong><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">9/7/19 </span></span></span></strong></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Ordered new gBlocks at twist. Ewsr in two parts. 999bp and 1002bp.</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Made new TB buffer.</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">New cloning for EWSR1:</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><b>Kinase reaction on the insert with T4 polynucleotide kinase </b></span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><b>Find restriction enzymes</b></span></span></span></p>
+<ol>
+	<li style="margin:0cm 0cm 0.0001pt 36pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><b>pTrc99a empty: digest with sma1 (2h)</b></span></span></span></li>
+	<li style="margin:0cm 0cm 0.0001pt 36pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><b>Inactivation wth heat (check temperature in NEB): 65&deg;C 20 min</b></span></span></span></li>
+	<li style="margin:0cm 0cm 0.0001pt 36pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><b>Column purification of the digested backbone: elute in 20uL of water</b></span></span></span></li>
+	<li style="margin:0cm 0cm 0.0001pt 36pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><b>Measure concentration: take 30-50 ng of template</b></span></span></span></li>
+	<li style="margin:0cm 0cm 0.0001pt 36pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><b>Take 150 ng of the gblock</b></span></span></span></li>
+	<li style="margin:0cm 0cm 0.0001pt 36pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><b>Ligation overnight 4&deg;C with ligase </b></span></span></span></li>
+	<li style="margin:0cm 0cm 0.0001pt 36pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><b>Transform 50uL and plate</b></span></span></span></li>
+	<li style="margin:0cm 0cm 8pt 36pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><b>Colony PCR to verify (try 10 colonies at first)</b></span></span></span></li>
+</ol>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Overnight culture in TB medium of constructs FUS3 ind (verified by sequencing), SPD5_1 ind (verified by sequencing) and NICD (not verified) and mini eYFP construct.</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">New constructs were sent for sequencing</span></span></span></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="margin:0cm 0cm 8pt"><strong><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">10/7/19</span></span></span></strong></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Measurement of overnight cultures. All cultures are at an OD of 3,3. All cultures were diluted to an OD of 0.03 and incubated again until reaching OD 0.5</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Retransformation of pTrc99a </span></span></span></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Measurement of overnight cultures. All cultures are at an OD of 3,3. All cultures were diluted to an OD of 0.03 and incubated again until reaching OD 0.5</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">All cultures were split and induced with different levels of arabinose:</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">NICD: NICD uninduced, NICD low (0.001%), NICD mid (0.05 %) and high (1%)</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">FUS: FUS uninduced, FUS low (0.001%), FUS mid (0.05 %) and high (1%)</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">SPD5: SPD5 uninduced, SPD5 low (0.001%), SPD5 mid (0.05 %) and high (1%)</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Control: mini eYFP uninduced and induced with 1 mM IPTG</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span lang="EN-GB" style="font-size:11.0pt"><span style="line-height:107%"><span style="font-family:&quot;Calibri&quot;,sans-serif">None of the transformations were successful.</span></span></span></p>
+<table border="1" cellpadding="1" cellspacing="1" style="width: 960px;">
+	<tbody>
+		<tr>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385026/10-7-FUS_constitutive%20(1).jpg" onclick="window.location.href = '1385026?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-FUS_constitutive (1).jpg</a></td>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385101/10-7-SPD5_constitutive-04.jpg" onclick="window.location.href = '1385101?op=preview&amp;back_url=1366648'; return false">10-7-SPD5_constitutive-04.jpg</a></td>
+		</tr>
+		<tr>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385031/10-7-FUS_constitutive%20(2).jpg" onclick="window.location.href = '1385031?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-FUS_constitutive (2).jpg</a></td>
+			<td>&nbsp;</td>
+			<td>&nbsp;</td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385006/10-7-eYFP_high%20(1).jpg" onclick="window.location.href = '1385006?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-eYFP_high (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384996/7-10-FUS_high_induction%20widefield%20(1).jpg" onclick="window.location.href = '1384996?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">7-10-FUS_high_induction widefield (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385071/10-7-NICD_high_10_07_2%20(1).jpg" onclick="window.location.href = '1385071?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-NICD_high_10_07_2 (1).jpg</a></td>
+			<td>&nbsp;</td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385011/10-7-eYFP_high%20(2).jpg" onclick="window.location.href = '1385011?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-eYFP_high (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385001/7-10-FUS_high_induction%20widefield%20(2).jpg" onclick="window.location.href = '1385001?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">7-10-FUS_high_induction widefield (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385076/10-7-NICD_high_10_07_2%20(2).jpg" onclick="window.location.href = '1385076?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-NICD_high_10_07_2 (2).jpg</a></td>
+			<td>&nbsp;</td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385016/10-7-eYFP_uninduced%20(1).jpg" onclick="window.location.href = '1385016?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-eYFP_uninduced (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385051/10-7-FUS_mid_induction-03%20widefield%20(1).jpg" onclick="window.location.href = '1385051?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-FUS_mid_induction-03 widefield (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385116/NICD_mid_10_07_2.jpg" onclick="window.location.href = '1385116?op=preview&amp;back_url=1366648'; return false">NICD_mid_10_07_2.jpg</a></td>
+			<td>&nbsp;</td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385021/10-7-eYFP_uninduced%20(2).jpg" onclick="window.location.href = '1385021?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-eYFP_uninduced (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385056/10-7-FUS_mid_induction-03%20widefield%20(2).jpg" onclick="window.location.href = '1385056?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-FUS_mid_induction-03 widefield (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385106/NICD_mid_10_07.jpg" onclick="window.location.href = '1385106?op=preview&amp;back_url=1366648'; return false">NICD_mid_10_07.jpg</a></td>
+			<td>&nbsp;</td>
+		</tr>
+		<tr>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385041/10-7-FUS_low_induction-02%20gfp%20(1).jpg" onclick="window.location.href = '1385041?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-FUS_low_induction-02 gfp (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385081/10-7-NICD_low_10_7_2%20(1).jpg" onclick="window.location.href = '1385081?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-NICD_low_10_7_2 (1).jpg</a></td>
+			<td>&nbsp;</td>
+		</tr>
+		<tr>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385046/10-7-FUS_low_induction-02%20gfp%20(2).jpg" onclick="window.location.href = '1385046?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-FUS_low_induction-02 gfp (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385086/10-7-NICD_low_10_7_2%20(2).jpg" onclick="window.location.href = '1385086?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-NICD_low_10_7_2 (2).jpg</a></td>
+			<td>&nbsp;</td>
+		</tr>
+		<tr>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385061/10-7-FUS_no_induction-03%20widefield%20(1).jpg" onclick="window.location.href = '1385061?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-FUS_no_induction-03 widefield (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384986/NICD_unicduced10_7-02.jpg" onclick="window.location.href = '1384986?op=preview&amp;back_url=1366648'; return false">NICD_unicduced10_7-02.jpg</a></td>
+			<td>&nbsp;</td>
+		</tr>
+		<tr>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385066/10-7-FUS_no_induction-03%20widefield%20(2).jpg" onclick="window.location.href = '1385066?op=preview&amp;back_url=1366648%3fclient_size%3d544x710'; return false">10-7-FUS_no_induction-03 widefield (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384991/NICD_uninduced%2010_7.jpg" onclick="window.location.href = '1384991?op=preview&amp;back_url=1366648'; return false">NICD_uninduced 10_7.jpg</a></td>
+			<td>&nbsp;</td>
+		</tr>
+	</tbody>
+</table>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="margin:0cm 0cm 8pt"><strong><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">11/7/19</span></span></span></strong></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Sequencing results show that promoter of the backbones used for the inducible constructs is not AraC but a modified construct. Empty pBad33 plasmid is now </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">sequenced and restriction site cloning prepared.</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Also retransformation of pTrc99a was not successful and will be repeated in order to initialize restriction enzyme cloning of the EWSR1 cloning. </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Retransformation ptrc and pbad33 empty as well as backbone for receptor</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Pcr of fragments for essays were amplified</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">FRAP was ameliorated with the constitutive SPD5 construct that has been working since the beginning.&nbsp;</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">With a pinhole of 65 um, a laser intensity of 100% and 65 itinerations, droplets were bleached and recovery could be observed over time.&nbsp;</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><strong>12/7/19</strong></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">13.7.19</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Blade light induction</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">O/N culture with 3 mL of terrific broth</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Measurement of OD in the dark with red light lamp. ODs were all 2.0</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Dilution of cutures to OD 0.1</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Incubation for 1,5h hours to reach OD 0.4</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">(ODs were too high, between 0.55 and 0.78) -&gt; dilute</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Split cultures (1.5 mL), half of them in dark tubes (uninduced), half of them in light tubes (induced). </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Transparent tubes were placed in front of blue LED plate (15,5 V) in 37&deg;C shaker. </span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">4h incubation/illumination</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">2mL on PBS+Agar plates for microscopy</span></span></span></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">New TB medium: 5g NaCl, 10g Bacto Tryptone and 1 mL of NaOH 1M. Autoclaved.</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">New overnight culture of FUS (ind), SPD5 (ind), NICD (ind) as well as cells with an empty pBAD (negative control) and a mini eYFP plasmid (positive control)</span></span></span></p>
+<p style="margin:0cm 0cm 8pt"><strong><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif"><span lang="EN-GB" style="color:#385723">14. 7.19</span></span></span></span></strong></p>
+<p style="margin:0cm 0cm 8pt">&nbsp;</p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Measurement of the 5 cultures&rsquo; OD at 8 am, dilution to OD 0.1.</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">1.5h incubation and measurement of OD. All ODs at 0.4</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Split cultures: </span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">NICD: NICD uninduced, NICD induced (4h light), NICD induced (1 % arabinose)</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">FUS: FUS uninduced, FUS induced (4h light), FUS induced (1 % arabinose)</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">SPD5: SPD5 uninduced, SPD5 induced (4h light), SPD5 induced (1% arabinose)</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Empy pBAD: uninduced / 1% Arabinose</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Mini eYFP: uninduced / 1% Arabinose</span></span></span></p>
+<p style="text-align:justify; margin:0cm 0cm 8pt"><span style="font-size:11pt"><span style="line-height:107%"><span style="font-family:Calibri,sans-serif">Incubation for 4 hours. </span></span></span></p>
+<table border="1" cellpadding="1" cellspacing="1" style="width: 960px;">
+	<tbody>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384876/7-14%20mini%20eYFP%20no%20arabinose%20(1).jpg" onclick="window.location.href = '1384876?op=preview&amp;back_url=1366648'; return false">7-14 mini eYFP no arabinose (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384886/7-14-FUS_arabinose-02_2%20(1).jpg" onclick="window.location.href = '1384886?op=preview&amp;back_url=1366648'; return false">7-14-FUS_arabinose-02_2 (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384926/7-14-NICD_Arabinose_2%20(1).jpg" onclick="window.location.href = '1384926?op=preview&amp;back_url=1366648'; return false">7-14-NICD_Arabinose_2 (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384956/7-14-pBAD%20negative%20control%20(1).jpg" onclick="window.location.href = '1384956?op=preview&amp;back_url=1366648'; return false">7-14-pBAD negative control (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384966/7-14-SPD5_arabinose-02_2%20(1).jpg" onclick="window.location.href = '1384966?op=preview&amp;back_url=1366648'; return false">7-14-SPD5_arabinose-02_2 (1).jpg</a></td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384881/7-14%20mini%20eYFP%20no%20arabinose%20(2).jpg" onclick="window.location.href = '1384881?op=preview&amp;back_url=1366648'; return false">7-14 mini eYFP no arabinose (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384891/7-14-FUS_arabinose-02_2%20(2).jpg" onclick="window.location.href = '1384891?op=preview&amp;back_url=1366648'; return false">7-14-FUS_arabinose-02_2 (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384931/7-14-NICD_Arabinose_2%20(2).jpg" onclick="window.location.href = '1384931?op=preview&amp;back_url=1366648'; return false">7-14-NICD_Arabinose_2 (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384961/7-14-pBAD%20negative%20control%20(2).jpg" onclick="window.location.href = '1384961?op=preview&amp;back_url=1366648'; return false">7-14-pBAD negative control (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384851/7-14-SPD5_arabinose-02_2%20(2).jpg" onclick="window.location.href = '1384851?op=preview&amp;back_url=1366648'; return false">7-14-SPD5_arabinose-02_2 (2).jpg</a></td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385126/YFP_high-06.jpg" onclick="window.location.href = '1385126?op=preview&amp;back_url=1366648'; return false">YFP_high-06.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384896/7-14-FUS_light_ind_2%20(1).jpg" onclick="window.location.href = '1384896?op=preview&amp;back_url=1366648'; return false">7-14-FUS_light_ind_2 (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384936/7-14-NICD_light%20induced_2%20(1).jpg" onclick="window.location.href = '1384936?op=preview&amp;back_url=1366648'; return false">7-14-NICD_light induced_2 (1).jpg</a></td>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384856/7-14-SPD5_light_ind-02_2%20(1).jpg" onclick="window.location.href = '1384856?op=preview&amp;back_url=1366648'; return false">7-14-SPD5_light_ind-02_2 (1).jpg</a></td>
+		</tr>
+		<tr>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1385131/YFP_high-06_2.jpg" onclick="window.location.href = '1385131?op=preview&amp;back_url=1366648'; return false">YFP_high-06_2.jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384901/7-14-FUS_light_ind_2%20(2).jpg" onclick="window.location.href = '1384901?op=preview&amp;back_url=1366648'; return false">7-14-FUS_light_ind_2 (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384941/7-14-NICD_light%20induced_2%20(2).jpg" onclick="window.location.href = '1384941?op=preview&amp;back_url=1366648'; return false">7-14-NICD_light induced_2 (2).jpg</a></td>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384861/7-14-SPD5_light_ind-02_2%20(2).jpg" onclick="window.location.href = '1384861?op=preview&amp;back_url=1366648'; return false">7-14-SPD5_light_ind-02_2 (2).jpg</a></td>
+		</tr>
+		<tr>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384906/7-14-FUS_uninduced-10%20(1).jpg" onclick="window.location.href = '1384906?op=preview&amp;back_url=1366648'; return false">7-14-FUS_uninduced-10 (1).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384946/7-14-NICD_uninducefd-07_2%20(1).jpg" onclick="window.location.href = '1384946?op=preview&amp;back_url=1366648'; return false">7-14-NICD_uninducefd-07_2 (1).jpg</a></td>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384866/7-14SPD5_unind-05_2%20(1).jpg" onclick="window.location.href = '1384866?op=preview&amp;back_url=1366648'; return false">7-14SPD5_unind-05_2 (1).jpg</a></td>
+		</tr>
+		<tr>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384911/7-14-FUS_uninduced-10%20(2).jpg" onclick="window.location.href = '1384911?op=preview&amp;back_url=1366648'; return false">7-14-FUS_uninduced-10 (2).jpg</a></td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384951/7-14-NICD_uninducefd-07_2%20(2).jpg" onclick="window.location.href = '1384951?op=preview&amp;back_url=1366648'; return false">7-14-NICD_uninducefd-07_2 (2).jpg</a></td>
+			<td>&nbsp;</td>
+			<td><a href="https://groups.uni-freiburg.de/bscw/bscw.cgi/d1384871/7-14SPD5_unind-05_2%20(2).jpg" onclick="window.location.href = '1384871?op=preview&amp;back_url=1366648'; return false">7-14SPD5_unind-05_2 (2).jpg</a></td>
+		</tr>
+	</tbody>
+</table>
+<p>&nbsp;</p>
+</body>
+</html> </p>
                            <p> Text </p>
                        </div>
@@ Line 432: / Line 1,405: @@
                    <div class="content">
                        <div class="wrapper">
                          <h2>01.07.2019</h2>
@@ Line 661: / Line 1,632: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190702_pcr_inteins.png" alt="PCR Gelelctrophoresis of Inteins">
+   <img width="400" src="../../imgs/Intein_Project/190702_pcr_inteins.png" alt="PCR Gelelctrophoresis of Inteins">
 </div>
@@ Line 1,160: / Line 2,131: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190709_backbone_n_int.png"
+   <img width="400" src="../../imgs/Intein_Project/190709 backbone n int.png"
    alt="Gelelekt. PCR NI BB">
@@ Line 1,180: / Line 2,151: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190710_backbone_c_int.png">
+   <img width="400" src="../../imgs/Intein_Project/190710 backbone c int.png">
 </div>
@@ Line 1,238: / Line 2,209: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190711_gibson_ints_%2B_bb.png">
+   <img width="400" src="../../imgs/Intein_Project/190711 gibson ints + bb.png">
 </div>
@@ Line 1,576: / Line 2,547: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190716_N-Int_bsa1_mut_dmso_(E).png">
+   <img width="400" src="../../imgs/Intein_Project/190716 N-Int bsa1 mut dmso (E).png ">
 </div>
@@ Line 1,785: / Line 2,756: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190729_C-Int_bsaI_mut.png">
+   <img width="400" src="../../imgs/Intein_Project/190729 C-Int bsaI mut.png ">
 </div>
@@ Line 1,810: / Line 2,781: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190730_PCR_Products_of_SUMO1_and_TRX.png">
+   <img width="400" src="../../imgs/Intein_Project/190730 PCR Products of SUMO1 and TRX.png ">
 </div>
@@ Line 2,544: / Line 3,515: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190814_PCR_of_Secretion_Insertion_Fragments.png">
+   <img width="400" src="../../imgs/Intein_Project/190814 PCR of Secretion Insertion Fragments.png ">
 </div>
@@ Line 2,679: / Line 3,650: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190821-Western_Blot_stained_against_TRX.png">
+   <img width="400" src="../../imgs/Intein_Project/190821-Western Blot stained against TRX.png ">
 </div>
@@ Line 2,719: / Line 3,690: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190823_comassie_stain_of_NI_SUMO_and_CI_TRX.png">
+   <img width="400" src="../../imgs/Intein_Project/190823 comassie stain of NI SUMO and CI TRX.png ">
 </div>
@@ Line 2,798: / Line 3,769: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190826_vers_sumo_expr.png">
+   <img width="400" src="../../imgs/Intein_Project/190826 vers sumo expr.png ">
 </div>
@@ Line 2,911: / Line 3,882: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190829_SUMO1_Western_Blot_Inclusion_bodies.PNG">
+   <img width="400" src="../../imgs/Intein_Project/190829 SUMO1 Western Blot Inclusion bodies.PNG ">
 </div>
@@ Line 2,990: / Line 3,961: @@
 <div class="centerimgs">
-   <img width="200" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190831first_mbp_PCR.png">
+   <img width="200" src="../../imgs/Intein_Project/190831first mbp PCR.png ">
 </div>
@@ Line 3,065: / Line 4,036: @@
 <div class="centerimgs">
-   <img width="800" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190902_sfGFP_CI_TRX_Sequencing.PNG">
+   <img width="800" src="../../imgs/Intein_Project/190902 sfGFP_CI_TRX Sequencing.PNG">
 </div>
@@ Line 3,244: / Line 4,215: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190908_1_fluorescence_medium.png">
+   <img width="400" src="../../imgs/Intein_Project/190908 1 fluorescence medium.png ">
 </div>
@@ Line 3,252: / Line 4,223: @@
 <div class="centerimgs">
-   <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190908_2_fluorescence_pellet.png">
+   <img width="400" src="../../imgs/Intein_Project/190908 2 fluorescence pellet.png ">
 </div>
@@ Line 3,259: / Line 4,230: @@
 <p>&nbsp;</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDDDDDDDD</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190908_3_OD_whole_culture.png">
-</div>
 <p>&nbsp;</p>
@@ Line 3,283: / Line 4,251: @@
 <p>- blotted on PVDF membrane (30 min, 1 A), blocked with 5% BSA (2 h), washed 1x with TBST, added 1. antibody (Mouse anti TRX 1:1000) in 10 mL TBST with 5% BSA (1 h), washed 3x with TBST, added 2. antibody (Goat anti Mouse 488 1:2000) in 10 mL TBST with 5% BSA (1 h), washed 3x with TBST, took picture with Amersham&trade; Typhoon&trade;Biomolecular Imager at 488 nm:</p>
-div class="centerimgs">
+<p>BBBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDD</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190911_anti-TRX_for_sfGFP_Secretion_488.png">
-</div>
 <p>- sfGFP-CI-TRX should be seen at 44 kDa and all fluorescent samples show the right band, indicating that if sfGFP gets secreted it can carry a protein fused to it</p>
@@ Line 3,302: / Line 4,266: @@
 <p>- Gelelctrophoresis:</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDDDD</p>
-  <img width="400" src=" https://2019.igem.org/File:T--Freiburg---Intein_Project-190916_MBP_mut_plasmid_PCR.png">
-</div>
 <p>- lane 5 (62.6&deg;C with GC-Enahncer), 6 (64.1&deg;C with GC-Enhancer), 10 (62.6&deg;C with 5% DMSO) and 11 (64.1&deg;C with 5% DMSO) showed bands of the right size (5.7 kb)</p>
@@ Line 3,327: / Line 4,287: @@
 <p>- Gelelectrophoresis:</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBBBbbIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDD</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190917_MBP_mut_product.png">
-</div>
 <p>- PCR product has the right size (1.2 kb), did a PCR purification of the rest of the sample</p>
@@ Line 3,364: / Line 4,321: @@
 <p>- results from the sfGFP secretion experiment:</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDd</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190919_1_fluorescence_SN.png">
-</div>
+<p>BBBBBBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDD</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDD</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190919_2_fluoresence_pellet.png">
-</div>
-<div class="centerimgs">
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190919_3_OD600_of_culture.png">
-</div>
 <p>- fluorescence in the medium already rises at 8 h after induction, strongly indicating secretion of sfGFP-CI-TRX</p>
@@ Line 3,402: / Line 4,347: @@
 <p>- results:</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDdd</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190927_1_fluorescence_sn.png">
-</div>
-<div class="centerimgs">
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190927_2_fluorescence_pellet.png">
-</div>
-<div class="centerimgs">
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-190927_3_OD_culture.png">
-</div>
+<p>BBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDd</p>
+<p>BBBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDD</p>
 <p>&nbsp;</p>
@@ Line 3,441: / Line 4,373: @@
 <p>- results of fluorescence measurement:</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDd</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-191002_1_fluorescence_SN.png">
-</div>
+<p>BBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDDDDDDD</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDDD</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-191002_2_fluorescence_pellet.png">
-</div>
-<div class="centerimgs">
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-191002_3_OD600.png">
-</div>
 <p>&nbsp;</p>
@@ Line 3,497: / Line 4,419: @@
 <p>- took picture with Amersham&trade; Typhoon&trade;Biomolecular Imager at 488 nm:</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDD</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-191009_sfGFP_secretion_western_blot.png">
-</div>
 <p>- the Western Blot clearly shows a big difference in the amount of protein that were in the medium, at each timepoint the sfGFP-CI-TRX bands (44 kDa) are way more intense than the CI-TRX bands (17kDa), indicating that the fusionprotein enters the medium mainly via secretion instead of lysis</p>
@@ Line 3,624: / Line 4,542: @@
 <p>- Gelelectrophoresis:</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDD</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-191010_pcr_pET302_for_GBlock.png">
-</div>
 <p>- the left band, with GC-Enhancer, has the perfect size (5.6 kbp) and the rest of sample was purified by PCR purification</p>
@@ Line 3,952: / Line 4,866: @@
 <p>Stain the gel with Instant Blue for 30 minutes.</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDd</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-191020_1_comassie_expression.png">
-</div>
+<p><u>Splicing test</u></p>
 <p>Load a 12% acrylamide gel with 18 &micro;L of each sample:</p>
@@ Line 3,993: / Line 4,905: @@
 <p>Visualize with Typhoon laser scanner at 488 nm:</p>
-<div class="centerimgs">
+<p>BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIILLLLLLLLLLLLLLLLLLLLLLLLLLDDDDDDDDDDDDDDDDDDDDDDDDDd</p>
-  <img width="400" src="https://2019.igem.org/File:T--Freiburg---Intein_Project-191020_2_Western_Blot_Splicing.png">
-</div>
 <p>&nbsp;</p>
@@ Line 4,178: / Line 5,087: @@
   <tr><td>&nbsp;</td><td></td></tr>
 </tbody></table>
                        </div>
@@ Line 4,184: / Line 5,092: @@
                </div>
              </div>
              <div class="sponsors">
                  <div class="sponsorsrow">

Difference between revisions of "Team:Freiburg/Labbook/In Vivo"

Revision as of 16:47, 28 November 2019

04.06.19

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Subsection Title

Subsection Title #2

01.07.2019

02.07.2019

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