Team:Freiburg/Labbook/In Vivo
Click on an item for a detailed description
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 |
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.
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.
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
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
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.
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 |
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 |
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.
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.
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
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.
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
.jpg){kind=link}
Text
07/06/19
PCR to amplify sfGFP, MCP, mMBT1
10/06/19
PCR (2x)
sfGFP, mMBT1
2. Annealing Temp: 66.0°C, M64°C
3. Annealing Temp: 67.0°C, M60°C
- Gelelectrophoresis
- PCR purification
13/06/19
→ Primer: CmR,pKD46
PCR-Protocol
| Compound [µl] | Purpose |
|---|---|
| 25 | Q5 polymerase |
| 20 | Primer FW/RV |
| 1 | DNA template |
| up to 50 | ddH2O |
- PCR purification
- DNA digestion
- Transformation of ligated plasmids
14/06/19
Colonies observed on plates
test colonies using colony PCR, with primers: mRrev, PKUGfwd
18/06/19
05/08/19
Transformation of E. coli C321.A with WT sfGFP and E. coli with sfGFP(Y66→Amber)
06/08/19
Inoculated E.coli C21.A with WT sfGFP and E. coli with sfGFP(Y66→Amber), induction did not work as no fluorescence could be observed.
Retransformation of BLADE mutations in MG1655 and C321.A
Picked colonies from Amber mutation plater and make new overnight liquid culture
07/08/19
Sequencing: AraC - constructs, check for mutations (pBAD-FP); Blade - constructs, check for mutations and BLADE itself
Retransformation: BLADE constructs in C321.A and MG1655 → overday liquid culture with MG1655
Induction: mGFP*P-C → weird, mGFP-M → good, mGFP-C → nothing
Sequencing: All GFP constructs without loops have BLADE!
08/08/19
Overnight culture of mGFP-M, m-GFP-C, mGFP*C → induction Phase 1 in TB-medium (LB w/o yeast) Chloramphinicol (25ng/µl) → dilution 1:1000, 1:300
OD measured → diluted to OD600 0.1 → Grow to OD600=0.4-06 (~2-3h)
Induce with 0.1 Arabinose (2 ml cultures) → split to 1 ml add 10 µl of 10% Arabinose Stock
Grow for 2 h
prepare slides for microscope
Sequencing results: 1. AraC: pBAD-FP confirmed mutations: 05 → loops,03 → loops,01 → no loops,04 → not ready yet, 02 - stop codon, but in wrong position, missing codon → loops, P1-3 → correct, C1-3 → correct
1. pBAD-FP (mutations) O1,O2,O3 (incorrect)
2.1UF (GFP Seqence), P1 contains BLADE, O3 contains BLADE, WT3 unsequenced
→ check for ms2 loops, only O3 contains 3 loops?
→Microscopy of new induction AraC mGFP+C, mGFP+M, MGFP*n+C (goal signals
→DCR for BLADE A and O mutations + o/n Ligation
→o/n liquid cultures of BLADE mGFP and mGFP*P in (and M grown in dark)
09/08/19
Transformations: mGFP O+C, mGFP A+C (single) → chloramphenicol; mGFP+C, mGFP A+M, GFP A+C, mGFP O+C → Kanamycin (double)
Sequencing: O1 has random GFP insert in the beginning, O2 missing codon because overlab of AAA and TT, O5 → missing codon, try to express, O3 → correct, may be used,
All constructs have 3 loops exp. come C: C1 → 2x ms2 loops, C2 → 2x ms2 loops, C3 → 2x ms2 loops
Liquid cultures: mGFP+C, mGFP+M, mGFP*P+C in 8 ml LB; then use TB for dilution (better growth + stirring)
Microscopy: Possibly BLADE? constructs from yesterday; imaged GFP-M (WT): lower, but good signal; GFP*P: very bad signal
10/08/19
Liquid cultures: 4 ml AraC inducable: dilute with TB media! → diluted @ 11am, induced at 2.30pm
Transformations: Plates checked → groth on all, though C321.A grow work as expected; talenout @5pm
Liquid culture of new plates o/n in LB → only Phase II constructs; (mGFP A+C/O+C an O5)
Cloning: Purify SPD-fragments (Sequencing); Amplify backbone (pUltra) and fragment with overhangs (Primers, plasmid map)
11/08/19
Liquid cultures: OD of o/n cultures of Phase II measured at 9.30am, diluted and incubated at 10.30am; Measured (0.4-0.6) and induced @ 2pm prepare microscope slides of: mGFP*P+C, mGFP A+M, mGFP A+C, mGFP O+P in pUltra all samples with and without D-Phe (1 mM)
Cloning: PCR for SPD + pUltra (with GFP and without); Amplify Backbone pU1/2 → without GFP; pU5/6 → with GFP; Amplify SPD5: pU3/4 → without GFP; pU7/8 → with GFP
Tryed with Phusion flash, elongation time 15s/kb; Q5 Hifi 20-30/kb,
| Final construct | Primer | Temperature | Primer | Temperature |
|---|---|---|---|---|
| pUltra without GFP | SPD (pU1)fwd | 60.9°C | pU3 | 63.2°C |
| pUltra without GFP | SPD (pU1)rev | 63.4°C | pU4 | 85.9°C |
| pUltra with sfGFP | pU5 | 63.9°C | pU7 | 63.2°C |
| pUltra with sfGFP | pU6 | 65.4°C | pU8 | 67.8°C |
12/08/19
Microscopy: mGFP A+U, mGFP A+C, mGFP O+C in pUltra controls: MG1655, C321.A samples were treated as explained above.
Cloning: Gel of PCR products from BK 1/2 & 5/6 and Insert SPD 3/4 & 7/8: BK without GFP 1/2: ~6.1 kb BK with GFP 5/6: 6 kb; SPD without GFP 3/4: ~3.6 kb SPD with GFP 7/8: ~4.2 kb
Ligation of pUltra + SPD5 without GFP: 1/2 BK + 3/4 inster & pUltra + SPD5 with GFP: 5/6 + 7/8 instert; BK:insert ratio 1:3 → Transformation on Kanamycin plates
13/08/19
Microscopy: mGFP A+U, mGFP A+C, mGFP O+C in pUltra controls: MG1655, C321.A samples were treated as explained above.
Cloning: overday cultures of pUlt-SPD5 clonings, both plates (1 and 5) look good but colonies did't grow fast enough → colonie PCR was negative
o/n cultures for tomorrow: C321.AdeltaA, pUlt+MCP (with and without 8µM IPTG), pUlt (with and without 8µM IPTG), A+C +D-AA (with and without 40µl Arabinose)
14/08/19
Liquid cultures: old pU+SPD5 cultures grew overnight, new cultures (4x with, 4x without sfGFP) grow @ 37°C 220 RPM, miniprep and sequencing of (4x without and 2x with GFP) → MCR-rev, SPD-fwd, pUlt-Seq
o/n cultures for microscopy measured & diluted @ 9.00 am, measured induced @ 12 am
Microscopy:
- C321.A → no fluorescence
- pUlt + MCP → no fluorescence
- pUlt → no fluorescence
- A + C + N-AS
→ Prepared o/n cultures for phase III attempt 3:
- A+ M + pUlt
- A + C + pUlt
- O + C + pUlt
→ Miniprp of pUlt-SPD liquid cultures without GFP (1.1-1.4), with GFP (5.1-5.4) → colony PCR with S/9 and 6R (MCP rev), 25x, min extention, Q5, 63°C
15/08/19
Liquid cultures: o/n cultures of phase III: A+C, A+M, O+C + pUlt → measured & cultured @ 10am → measured at 2 pm
Cloning: Gel of colony PCR with S19 and MCP-rev, expected size: 2kb
Transformations:
- sfGFP A+C
- sfGFP A+M + pUlt
- sfGFP O+C
- C321.A: empty pBAD, empty pET, empty pET + pBAD
- mGFP05 + pUlt MCP
Microscopy: Better pictures! → repeat w same conditions, (OD600=0.5-0.6) new IPTG stock (500 mM, 8 µl), Arabinose 10% (40 µl), D-AA (3x 40 µl/ 80 µl)
Liquid cultures with pUltra but not D-Phe! ( A+C I+II; A+M I+II+pUlt; O+C I+II, no D-AA)
16/08/19
Transformation: Plates of "no loops/no MCP" TF
Successful: A+C, A+M (+pUlt), O+C,(P+C),mGFP 05 + pUlt +MCP
To Do: Empty pBAD/pET in C321.A and MG1655
Liquid cultures: o/n
m GFP , A+C I+II; A+M I+II; O+ I+ II → pUlt-MCP, no D-AA
17/08/19
Liquid culture: OD measured/ diluted in morning (mGFP,pUMCP,no D-AA)
Sequencing: Results for pU-SPD cloning: sent sequences S19, 12,13,14,S5,S4 Send primer: 10R -pUltra fwd; GFC2-MCP rev; S19-SPD1/2; S20-SP1/2 binding
Results:
| 12 | S19 | empty |
| S20 | Aligns SPD1 fragment | |
| 13 | S19 | Aligns SPD5 fragment |
| S20 | Aligns SPD1 fragment | |
| S5 | S19 | Aligns SPD5 fragment but mutations |
| S20 | Aligns SPD1 fragmentbut mutations |
| 12 (GF) | no sequencing/ alignent |
| 12 (10R) | missing |
| 13 (GF) | no sequencing/ alignment |
| 10 F | Aligns LacI |
| S5 (6F) | Aligns MCR/ RS half |
| 10R | Aligns LacI |
| 11-10R | Aligns short sequence after (10DF13) |
| 6F | empty |
| S19 | Align SPD! |
| 14 - S20 | empty |
| S19 | empty |
| 10R | empty |
| 6F | empty |
| S2 - S20 | empty |
| S19 | empty |
| 10R | empty |
| 6F | empty |
Microscopy plan
| 17/08 | 18/08 | 19/08 | 20/08 | 21/08 |
| mGFP* + pU-MCP | GFP* + pU | GFP + pU | empty pBAD/ pEvol | GFP* + pU |
| no D-AA | + D-AA | + D-AA | control | No D-AA |
c
A+C, A+M, O+C 2x with and without D-Phe + pUlt-MCP
OD600 + D-AS: ~ 10.15 am; no AS: ~ 11.30 am
pUlt-SPD: ~ 11:45 regrowing cultures 12,13,51,53,54
Microscopy: Repetition of no D-AS due to inconclusive results
| D-Phe | without D-AA |
|---|---|
|
A+C I |
A+C I |
| A+C II | A+C II |
| A+M I | A+M I |
| A+M II | A+M II |
| O+C I | O+C I |
| O+C II | O+C II |
Miniprep + Sequencing: pUlt+MCP+SPD (with and without GFP)
Sequence fusio sites to check if cloning is correct!
→ LacR for LAc-SPD fusion
→ GFP-fwd for GFP-pUlt fusion
→ Find primer for construct without OFP
→ Check sequencing , if correct retransform
→ check date/ picutes pf drop forming etc.
20/08/19
Liquid culture: Repetition of transformation only A+M GFP + pUltra → minicells
Microscopy: cells incredibly small → contamination?
Transformation:
- A+C GFP + pU
- A+M GFP + pU
- O5+C
- retransformation: C1, C2, O5,O3; pET → TOP10; pUlt + MCP → TOP10
Sequencing: Eurofins (samples resend, reordered)
PCR? → design plasmids and primers
21/08/19
Sequencing: no results
Liquid culture: (checked plates)
in 8 ml LB + D-AA (80 µl) + Chloramphinicol + Kanamycin
- GFP + pU A+C
- GFP + pU A+M
- GFP + pU O3+C
- GFP + pU O5+C
- GFP + pU A+C
only plasmid for purification: in 5 ml LB + respective antibiotics
- pEVOL (Kan)
- pU + MCP (Kan)
- C1 (Chl)
- C2 (Chl)
- C3 (Chl)
- C5 (Chl)
22/08/19
FACS: Analyze results
Microscopy:
- GFP + pU + A+C 3x
- GFP + pU + O+C 3x
- GFP + pU + A+M 3x
→ slides bad, repeat tomorrow
Sequencing: Re-sequence o/n cultures of A+M, mGFP O+C + pU(MCP), Aa+C
Retransformation of...
- O3 (outside normal)
- O5 (outside w/o codon)
- C1 inside chrophore
- C2 inside chrophore
- pET 2x for Kanamycin resistance
- pUlt-MCP for plasmid
23/08/19
Liquid cultures: diluted and induced
- A+C GFP + pU
- A+M GFP + pU
- O+C GFP + pU
sequencing: SPD + pU fusion sites
| without GFP | 12 | LacR | empty |
| 12 | S23 | empty | |
| confirmed fragment by colonie | 13 | LacR | promoter |
| 13 | S23 | empty | |
| 51 | LacR | empty | |
| 51 | S23 | empty | |
| with GFP | 53 | LacR |
Lac promoter |
| ... | 53 | S23 | aligns a bit between SPD/GS-linker |
| 54 | LacR | empty | |
| 54 | S23 | empty |
p8.32 (pUltra-MCP-retrafo) → 6F, 10R fine
p8.31 (pUltra-MCP-retrafo) → 6F, 10R fine
Microscopy: Difference between induced/ uninduced visikle D-AA or not?
24/08/19
Liquid cultures:
- A+M II
- A+C II
- O+C II
→ Miniprep, sequencing on Monday
26/08/19
→ Transformation
→ Retransformation
27/08/19
Transformation: pU8.3-MCP-SPD5 + pBAD
12(19.08) C1(inside)
12(21.08)O3(outside)
Liquid cultures for FACS:
NEW p8.3 (+MCP) +pBAD transformations:
induced/ uninduced with D-AA/without D-AA ~40 samples
- A+M
- A+C
- O+C
- C321 ctrl (Chl/Kan)
- MG1655 (Chl/Kan)
- pU + sfGFP
→ prepare liquid cultures o/n of pU8.3 (+MCP) + pBAD, if FACS results are good
02/09/19
Microscopy:
- O+C I + II
- A+C I + II
- sfGFP I + II
- pUltra enzel I + II
04/09 FACS
→ O51, A+M, O+C, pU solo, pos ctrl., neg. ctrl, A+C, SPD5, +/-, +D-AA/ -D-AA
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 | C-Int | C-Int | N-Int | N-Int |
- 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:
-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:
- 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):
- 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)
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):
- 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):
- 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:
- 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 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:
- 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:
- 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:
- 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:
- 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)
- 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
- 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:
- 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:
- 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:
- 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:
- 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:
- 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:
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:
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:
- 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:
- 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.
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:
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 |
