Difference between revisions of "Team:HBUT-China/Notebook"

 
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         border-left: #67c23a solid 10px;
 
         border-left: #67c23a solid 10px;
 
         padding-left: 10px;
 
         padding-left: 10px;
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        font-variant: small-caps;
 
 
         }
 
         }
 
         table
 
         table
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     </style>
 
     </style>
 
  <div id="notebookapp">
 
  <div id="notebookapp">
−
         <div style="width: 60%;margin:100px auto 0">
+
         <div style="width: 65%;margin:100px auto 0">
 
             <div id="Method-Protocol1">
 
             <div id="Method-Protocol1">
 
                 <div id="Method-Protocol" style="position: relative;top:-120px"></div>
 
                 <div id="Method-Protocol" style="position: relative;top:-120px"></div>
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                             href="https://2019.igem.org/wiki/images/2/2d/T--HBUT-China--Yeast--Transformation--Protocol.pdf">Yeast
 
                             href="https://2019.igem.org/wiki/images/2/2d/T--HBUT-China--Yeast--Transformation--Protocol.pdf">Yeast
 
                             Transformation—Protocol</a>
 
                             Transformation—Protocol</a>
 +
                    </li>
 +
                    <li>
 +
                        <a style="color:rgb(30, 63, 12);font-size: 18px;"
 +
                            href="https://2019.igem.org/wiki/images/5/57/T--HBUT-China--Seamless_Cloning.pdf">Seamless Cloning</a>
 
                     </li>
 
                     </li>
 
                 </ol>
 
                 </ol>
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                     <p>Verified pictures of pSB1C3-pTEF1/tCYC1 digested by <em>EcoR</em>1 and <em>Pst</em>1:</p>
 
                     <p>Verified pictures of pSB1C3-pTEF1/tCYC1 digested by <em>EcoR</em>1 and <em>Pst</em>1:</p>
 
                     <div style="text-align:center">
 
                     <div style="text-align:center">
−
                         <img src="https://2019.igem.org/wiki/images/0/06/T--HBUT-China--_BBa-k3126001.png" width="400">
+
                         <img src="https://2019.igem.org/wiki/images/e/e7/T--HBUT-China--photoBBa-k3126001.png" width="400">
 
                     </div>
 
                     </div>
 
                     <p>Verified pictures of pSB1C3-pGPM1/tTDH1 digested by <em>EcoR</em>1 and <em>Pst</em>1:</p>
 
                     <p>Verified pictures of pSB1C3-pGPM1/tTDH1 digested by <em>EcoR</em>1 and <em>Pst</em>1:</p>
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                 </el-collapse>
 
                 </el-collapse>
 
                 <p style="word-wrap:break-word">
 
                 <p style="word-wrap:break-word">
−
                     The resistance gene(kanR) was linked to the target gene(hexa-His, nixA, TgMTP1t2) to screen the engineering yeast and the original yeast.
+
                     The resistance gene(kanR) was linked to the target gene(hexa-His, nixA, TgMTP1t2) to screen the engineered yeast and the original yeast.
 
                 </p>
 
                 </p>
 
                 <p>pSB1C3-BBa_K3126018 (pSB1C3-hexa-His/ kanR):</p>
 
                 <p>pSB1C3-BBa_K3126018 (pSB1C3-hexa-His/ kanR):</p>
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             </div>
 
             </div>
 +
 +
          <div id="888">
 +
                    <div id="date8" style="position: relative;top:-120px"></div>
 +
                    <h1 class="greenp">2019.8.25 ~ 9.10</h1>
 +
                    <h4>Adsorption of Nickel ion by <em>S. cerevisiae</em>
 +
                    </h4>
 +
                    <p>1.Culture yeast  for a period of time, measure its OD600</p>
 +
                    <p>2. Convert the required dry weight volume according to OD600</p>
 +
                    <p>3.Add the corresponding volume of yeast solution into 50mL centrifuge tube, centrifugate at 8000 rpm for 5 min, discard the supernatant and enrich the cells</p>
 +
                    <p>4.Prepare 21 bottles of 50mL 15mg / L nickel ion solution and 21 bottles of 50mL 200mg / L nickel ion solution, put the put the six kinds of engineered yeast and original yeast into the solution respectively, and culture them in a shaker at 30℃</p>
 +
                    <p>5.Take the engineered yeast solution every interval to measure the nickel ion concentration</p>
 +
                    <p>6.After about half an hour of adsorption, the equilibrium is reached</p>
 +
                    <p>7.Draw the curve about the enrichment of Ni<sup>2+</sup> by yeast.</p>
 +
                    <div style="text-align: center">
 +
                        <img src="https://2019.igem.org/wiki/images/e/e1/T--HBUT-China--notebook-new1.png" width="80%">
 +
                    </div>
 +
                    <div style="text-align: center">
 +
                        <img src="https://2019.igem.org/wiki/images/1/1c/T--HBUT-China--notebook-new2.png" width="80%">
 +
                    </div>
 +
   
 +
                </div>
 +
 +
 
             <div id="Improvement">
 
             <div id="Improvement">
 
                 <div id="Improvement1" style="position: relative;top:-120px"></div>
 
                 <div id="Improvement1" style="position: relative;top:-120px"></div>
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                 <p><strong>[Protocols]</strong></p>
 
                 <p><strong>[Protocols]</strong></p>
 
                 <p>1.Digested the pSB1C3-BBa_K2652001 circular plasmid with <em>Bam</em>HI.</p>
 
                 <p>1.Digested the pSB1C3-BBa_K2652001 circular plasmid with <em>Bam</em>HI.</p>
−
                 <p>2.Amplified our target gene BBa_K3126026 <em>（His-Tag-nikABCDE- His-Tag）</em> using PCR technology.</p>
+
                 <p>2.Amplified our target gene BBa_K3126026 <em>(His-Tag-nikABCDE- His-Tag)</em> using PCR technology.</p>
−
                 <p>3.Recovered the digested part and inserted BBa_K3126026 <em>His-Tag-nikABCDE-His-Tag</em> into pSB1C3-BBa_K2652001 by
+
                 <p>3.Recovered the digested part and inserted BBa_K3126026 <em>(His-Tag-nikABCDE-His-Tag)</em> into pSB1C3-BBa_K2652001 by
 
                     seamless cloning, and the mixed system was reacted at 37℃ for 30 minutes.</p>
 
                     seamless cloning, and the mixed system was reacted at 37℃ for 30 minutes.</p>
 
                 <p>4.converted all of the products of the above seamless cloning system to DH5α.</p>
 
                 <p>4.converted all of the products of the above seamless cloning system to DH5α.</p>
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                 <h1 class="greenp"  style="margin-top:30px">Characterization</h1>
 
                 <h1 class="greenp"  style="margin-top:30px">Characterization</h1>
 
                 <br/>
 
                 <br/>
−
                 <h1 class="greenp">2019.7.1 ~ 2019.7.10</h1>
+
                 <h1 class="greenp">2019.9.10 ~ 2019.9.20</h1>
 
                 <p>We used the pSB1C3-BBa_K2652001 from last year and digested with <em>Bam</em>H I enzyme.</p>
 
                 <p>We used the pSB1C3-BBa_K2652001 from last year and digested with <em>Bam</em>H I enzyme.</p>
 
                 <p>The pSB1C3 digestion system we used:</p>
 
                 <p>The pSB1C3 digestion system we used:</p>
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                         <ul style="list-style: none;padding:0 10px 10px;margin: 0">
 
                         <ul style="list-style: none;padding:0 10px 10px;margin: 0">
 
                             <li style="margin: 5px ;">
 
                             <li style="margin: 5px ;">
−
                                 <el-link @click="scrollEle('Characterization1')"><strong style="font-size: 14px;color:#67c23a">2019.7.1 ~ 2019.7.10</strong></el-link>
+
                                 <el-link @click="scrollEle('Characterization1')"><strong style="font-size: 14px;color:#67c23a">2019.9.10 ~ 2019.9.20</strong></el-link>
 
                             </li>
 
                             </li>
 
                         </ul>
 
                         </ul>

Latest revision as of 01:38, 22 October 2019

2019.5

We synthesized:

BBa_K3126001 (promoter TEF1)

BBa_K3126002 (promoter GPM1)

BBa_K3126003 (promoter TDH3)

BBa_K3126004 (promoter TEF2)

BBa_K3126005 (hexa-His gene)

BBa_K3126006 (nixA gene)

BBa_K3126007 (TgMTP1t2 gene)

BBa_K3126008 (kanR gene)

BBa_K3126010 (terminator CYC1)

BBa_K3126011 (terminator TDH1)

BBa_K3126012 (terminator PGK1)

BBa_K3126013 (terminator ENO2)

GAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGGATCCTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGCCTTACTAGTAGCGGCCG

GAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCCTTACTAGTAGCGGCCGCTGCAG

GAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGGATCCATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTAGGCCTTACTAGTAGCGGCCGCTGCAG

GAATTCGCGGCCGCTTCTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCAG

GGTCTCAATCTAAGTTTTAATTACAAAGGATCCATGAGATTCCCTAGTATCTTCACCGCCGTCTTGTTCGCTGCTAGTTCTGCTTTGGCTGCTCCAGTCAACACTACCACCGAAGACGAAACCGCTCAGATCCCAGCTGAAGCCGTCATCGGTTACCTTGACCTTGAGGGTGACTTCGATGTCGCCGTCCTTCCATTCTCTAACAGTACCAACAACGGTCTTTTGTTCATTAACACCACCATTGCTTCTATCGCCGCTAAGGAGGAGGGTGTCTCTCTTGACAAGAGACATCACCACCACCATCACAGTGCTAAGTCTAGTTTCATCTCTACCACCACTACCGACCTTACCTCTATCAACACCAGTGCCTACAGTACCGGTTCTATCTCTACCGTCGAAACCGGAAACAGAACCACCTCTGAGGTCATCTCTCATGTCGTCACCACCTCTACCAAGCTTAGTCCTACCGCCACCACCAGTCTTACCATCGCTCAAACTTCTATTTATAGTACCGACTCTAATATTACCGTCGGTACCGACATCCACACCACCAGTGAAGTCATCTCTGACGTCGAAACCATCTCTAGGGAAACCGCCTCTACCGTTGTTGCCGCCCCAACTTCTACCACTGGTTGGACCGGTGCCATGAACACTTACATCTCTCAATTCACTTCTTCTAGTTTTGCTACTATTAACTCTACCCCTATCATCTCTTCTAGTGCCGTCTTCGAAACCAGTGACGCCTCTATCGTCAACGTCCATACCGAGAACATCACCAACACTGCTGCCGTTCCATCTGAGGAGCCTACCTTCGTCAACGCCACTAGAAACTCTCTTAATTCTTTTTGTTCTTCTAAACAACCATCTTCTCCTAGTTCTTATACTTCTTCTCCTTTGGTTTCTTCTTTGTCTGTTTCTAAGACCTTGTTGTCTACCTCTTTCACCCCTTCTGTCCCAACTTCTAATACTTATATTAAGACCAAGAACACTGGTTACTTCGAGCACACTGCCTTGACCACCTCTAGTGTTGGATTGAATAGTTTCTCTGAAACCGCCGTCTCTAGTCAAGGTACCAAGATTGACACCTTTTTGGTTTCTAGTTTGATCGCCTACCCATCTTCTGCCTCTGGTTCTCAGCTTAGTGGTATCCAGCAAAATTTTACCTCTACTTCTTTGATGATCAGTACCTACGAGGGTAAGGCCAGTATCTTCTTCTCTGCCGAGTTGGGTTCTATTATTTTTTTGTTGCTTAGTTATTTGTTGTTTTAAAACGGGTACTGTACAGTTAGTACATTGAGTCGAAATATACGAAATTATTGTTCATAATTTTCATCCTGGCTCTTTTTTTCTTCAACCATAGTTAAATGGACAGTTCATATCTTAACTCTAATAATACTTTTCTAGTTCTTATCCTTTTCCGTCTCACCGCAGATTTTATCATAGTATTAAATTTATATTTTGTTCGTAAAAAGAAAAATTTGTGAGCGTTACCGCTCGTTTCATTACCCGAAGGCTGTTTCAGTAGACCACTGATTAAGTAAGTAGATGAAAAAATTTCATCACCATGAAAGAGTTCGATGAGAGCTACTTTTTCAAATGCTTAACAGCTAACCGCCATTCAATAATGTTACGCTCTCTTCATTCTGCGGCTACGTTATCTAACAAGAGGTTTTACTCTCTCATATCTCATTCAAATAGAAAGAACATAATCAAAAGGATCCTCATGTAATTAGTTATGTCACGAGACC

GGTCTCACAAACACACATATTACAATAGGATCCATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTATCCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAAGGATCCATAAAGCAATCTTGATGAGGAGAGACC

GGTCTCAAACACACATAAACAAACAAAGGATCCATGGAGAGTTCTTCTCATATTATTGAAGTCAACGGAGGTAGATCTGACGAGGAAAGAAGGGCTGTCGCTAGTAAGGTCTGCGGAGAAGCTCCTTGCGGTTTCAGTGACGCTAAGAACGCTTCTGGTGACGCCCAAGAAAGGAACGCCTCTATGAGGAAATTGTGCATCGCCGTCGTCTTGTGCCTTGTCTTCATGTCTGTCGAGGTCGTTGGTGGTGTCAAGGCCAACAGTTTGGCCATCATGACCGATGCCGCTCATCTTTTGAGTGACGTTGCCGCTTTCGCCATCTCTTTGTTCAGTCTTTGGGCTGCCGGTTGGGAGGCTACCCCAAGGCACACCTACGGATTCTTCAGAATCGAAATTCTTGGAGCTCTTGTTTCTATCCAATTGATTTGGTTGCTTACTGGTATCCTTGTTTACGAGGCCATCATTAGACTTTTGACCGAAACCTCTGAGGTCAACGGTTTCTTGATGTTCGCTGTCGCCACTTTCGGTTTGCTTGTCAACATTATCATGGCTGTCATGCTTGGTCACGACCATGACAATGGTCACGGTCATAGTCACGGTGAGGACAAACACGATGAAGCCCATGGTGACGTCACTGAACAGCTTCTTGAAAAGCCAAAACAGCAGAAGGAAAAAAAGAAGAGGAACATCAACGCCCAAGGTGCCTATTTGCACGTCTTGGGTGACTCTATCCAGTCTGTCGGAGTCATGATCGGTGGTGCTGCTATCTGGTACAATCCAAAGTGGAAGATCATTGACTTGATCTGCACCTTGGTCTTCTCTGTCATCGTCTTGGGAACCACCATCAATATGATCAGAAGTATTCTTGAGGTCTTGATGGAGAGTACCCCAAGAGAAATCGACGCCACCAAGCTTGAGAAGGGTCTTCTTGAGATGGAGGAGGTCGTCGCTGTTCACGAATTGCACATCTGGGCCTCTACCGTTGGTAAGGTCTTGCTTGCTTGCCATGTCAACGCCAGACCAGATGCCGATGCCGATATGGTCCTTAACAAAGTCGTTGACTATATTAGGAGAGAATATAACATTTCTCATGTTACTATCCAAATTGAAAGATAAGGATCCATTGAATTGAATTGAAATCGAGAGACC

GGTCTCACGAACTATAATTAACTAAACGGATCCATAACTTCGTATAATGTATGCTATACGAAGTTATATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAATAACTTCGTATAATGTATGCTATACGAAGTTATGGATCCAGTGCTTTTAACTAAGAATTAGAGACC

2019.6.1 ~ 6.10

Construction plasmids：

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGGATCCTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGCCTTACTAGTAGCGGCCGCTGCA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCCTTACTAGTAGCGGCCGCTGCA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGGATCCATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTAGGCCTTACTAGTAGCGGCCGCTGCA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

We insert promoter TEF1 and terminator CYC1, promoter GPM1 and terminator TDH1, promoter TDH3 and terminator PGK1, promoter TEF2 and terminator ENO2 into vector pSB1C3 using restriction endonuclease and T4 DNA ligase, respectively.

The pSB1C3 digestion system we used:

pSB1C3 15 μL
EcoRI 3 μL
PstI 3 μL
Cutsmart Buffer 5 μL
ddH2O 24 μL


The enzyme system we use:

pSB1C3 2 μL
fragment 6 μL
T4 DNA Ligase 0.5 μL
T4 Buffer 1 μL
ddH2O 0.5 μL

Verified pictures of pSB1C3-pTEF1/tCYC1 digested by EcoR1 and Pst1:

Verified pictures of pSB1C3-pGPM1/tTDH1 digested by EcoR1 and Pst1:

Verified pictures of pSB1C3-pTDH3/tPGK1 digested by EcoR1 and Pst1:

Verified pictures of pSB1C3-pTEF2/tENO2 digested by EcoR1 and Pst1:

2019.6.10 ~ 6.20

Construction plasmids：

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGGATCCATGAGATTCCCTAGTATCTTCACCGCCGTCTTGTTCGCTGCTAGTTCTGCTTTGGCTGCTCCAGTCAACACTACCACCGAAGACGAAACCGCTCAGATCCCAGCTGAAGCCGTCATCGGTTACCTTGACCTTGAGGGTGACTTCGATGTCGCCGTCCTTCCATTCTCTAACAGTACCAACAACGGTCTTTTGTTCATTAACACCACCATTGCTTCTATCGCCGCTAAGGAGGAGGGTGTCTCTCTTGACAAGAGACATCACCACCACCATCACAGTGCTAAGTCTAGTTTCATCTCTACCACCACTACCGACCTTACCTCTATCAACACCAGTGCCTACAGTACCGGTTCTATCTCTACCGTCGAAACCGGAAACAGAACCACCTCTGAGGTCATCTCTCATGTCGTCACCACCTCTACCAAGCTTAGTCCTACCGCCACCACCAGTCTTACCATCGCTCAAACTTCTATTTATAGTACCGACTCTAATATTACCGTCGGTACCGACATCCACACCACCAGTGAAGTCATCTCTGACGTCGAAACCATCTCTAGGGAAACCGCCTCTACCGTTGTTGCCGCCCCAACTTCTACCACTGGTTGGACCGGTGCCATGAACACTTACATCTCTCAATTCACTTCTTCTAGTTTTGCTACTATTAACTCTACCCCTATCATCTCTTCTAGTGCCGTCTTCGAAACCAGTGACGCCTCTATCGTCAACGTCCATACCGAGAACATCACCAACACTGCTGCCGTTCCATCTGAGGAGCCTACCTTCGTCAACGCCACTAGAAACTCTCTTAATTCTTTTTGTTCTTCTAAACAACCATCTTCTCCTAGTTCTTATACTTCTTCTCCTTTGGTTTCTTCTTTGTCTGTTTCTAAGACCTTGTTGTCTACCTCTTTCACCCCTTCTGTCCCAACTTCTAATACTTATATTAAGACCAAGAACACTGGTTACTTCGAGCACACTGCCTTGACCACCTCTAGTGTTGGATTGAATAGTTTCTCTGAAACCGCCGTCTCTAGTCAAGGTACCAAGATTGACACCTTTTTGGTTTCTAGTTTGATCGCCTACCCATCTTCTGCCTCTGGTTCTCAGCTTAGTGGTATCCAGCAAAATTTTACCTCTACTTCTTTGATGATCAGTACCTACGAGGGTAAGGCCAGTATCTTCTTCTCTGCCGAGTTGGGTTCTATTATTTTTTTGTTGCTTAGTTATTTGTTGTTTTAAAACGGGTACTGTACAGTTAGTACATTGAGTCGAAATATACGAAATTATTGTTCATAATTTTCATCCTGGCTCTTTTTTTCTTCAACCATAGTTAAATGGACAGTTCATATCTTAACTCTAATAATACTTTTCTAGTTCTTATCCTTTTCCGTCTCACCGCAGATTTTATCATAGTATTAAATTTATATTTTGTTCGTAAAAAGAAAAATTTGTGAGCGTTACCGCTCGTTTCATTACCCGAAGGCTGTTTCAGTAGACCACTGATTAAGTAAGTAGATGAAAAAATTTCATCACCATGAAAGAGTTCGATGAGAGCTACTTTTTCAAATGCTTAACAGCTAACCGCCATTCAATAATGTTACGCTCTCTTCATTCTGCGGCTACGTTATCTAACAAGAGGTTTTACTCTCTCATATCTCATTCAAATAGAAAGAACATAATCAAAAGGATCCTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGCCTTACTAGTAGCGGCCGCTGCA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTATCCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCCTTACTAGTAGCGGCCGCTGCA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGGATCCATGGAGAGTTCTTCTCATATTATTGAAGTCAACGGAGGTAGATCTGACGAGGAAAGAAGGGCTGTCGCTAGTAAGGTCTGCGGAGAAGCTCCTTGCGGTTTCAGTGACGCTAAGAACGCTTCTGGTGACGCCCAAGAAAGGAACGCCTCTATGAGGAAATTGTGCATCGCCGTCGTCTTGTGCCTTGTCTTCATGTCTGTCGAGGTCGTTGGTGGTGTCAAGGCCAACAGTTTGGCCATCATGACCGATGCCGCTCATCTTTTGAGTGACGTTGCCGCTTTCGCCATCTCTTTGTTCAGTCTTTGGGCTGCCGGTTGGGAGGCTACCCCAAGGCACACCTACGGATTCTTCAGAATCGAAATTCTTGGAGCTCTTGTTTCTATCCAATTGATTTGGTTGCTTACTGGTATCCTTGTTTACGAGGCCATCATTAGACTTTTGACCGAAACCTCTGAGGTCAACGGTTTCTTGATGTTCGCTGTCGCCACTTTCGGTTTGCTTGTCAACATTATCATGGCTGTCATGCTTGGTCACGACCATGACAATGGTCACGGTCATAGTCACGGTGAGGACAAACACGATGAAGCCCATGGTGACGTCACTGAACAGCTTCTTGAAAAGCCAAAACAGCAGAAGGAAAAAAAGAAGAGGAACATCAACGCCCAAGGTGCCTATTTGCACGTCTTGGGTGACTCTATCCAGTCTGTCGGAGTCATGATCGGTGGTGCTGCTATCTGGTACAATCCAAAGTGGAAGATCATTGACTTGATCTGCACCTTGGTCTTCTCTGTCATCGTCTTGGGAACCACCATCAATATGATCAGAAGTATTCTTGAGGTCTTGATGGAGAGTACCCCAAGAGAAATCGACGCCACCAAGCTTGAGAAGGGTCTTCTTGAGATGGAGGAGGTCGTCGCTGTTCACGAATTGCACATCTGGGCCTCTACCGTTGGTAAGGTCTTGCTTGCTTGCCATGTCAACGCCAGACCAGATGCCGATGCCGATATGGTCCTTAACAAAGTCGTTGACTATATTAGGAGAGAATATAACATTTCTCATGTTACTATCCAAATTGAAAGATAAGGATCCATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTAGGCCTTACTAGTAGCGGCCGCTGCA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

Insertion of gene into plasmid carrying promoter and terminator.

pSB1C3-BBa_K3126014 (pSB1C3-pTEF1/ hexa His/ tCYC1):

Enzyme digestion system:

pSB1C3-pTEF1/tCYC120 μL
BamHI 3 μL
buffer5 μL
ddH2O22 μL

Seamless cloning system:

pSB1C3-pTEF1/tCYC1 2 μL
hexa-His4 μL
5×CEⅡ Buffer2 μL
Exnase Ⅱ1 μL
ddH2O1 μL

pSB1C3-BBa_K3126015 (pSB1C3-pGPM1/ nixA/tTDH1):

Enzyme digestion system:

pSB1C3-pGPM1/tTDH120 μL
BamHI 3 μL
buffer5 μL
ddH2O22 μL

Seamless cloning system:

pSB1C3-pGPM1/tTDH1 2 μL
nixA4 μL
5×CEⅡ Buffer2 μL
Exnase Ⅱ1 μL
ddH2O1 μL

pSB1C3-BBa_K3126016 (pSB1C3-pTDH3/TgMTP1t2/tPGK1):

Enzyme digestion system:

pSB1C3-pTDH3/ tPGK120 μL
BamHI 3 μL
buffer5 μL
ddH2O22 μL

Seamless cloning system:

pSB1C3-pTDH3/ tPGK1 2 μL
TgMTP1t24 μL
5×CEⅡ Buffer2 μL
Exnase Ⅱ1 μL
ddH2O1 μL

pSB1C3-BBa_K3126017 (pSB1C3-pTEF2/kanR/tENO2):

Enzyme digestion system:

pSB1C3-pTEF2/tENO220 μL
BamHI 3 μL
buffer5 μL
ddH2O22 μL

Seamless cloning system:

pSB1C3-pTEF2/tENO2 2 μL
kanR4 μL
5×CEⅡ Buffer2 μL
Exnase Ⅱ1 μL
ddH2O1 μL

2019.6.20 ~ 6.30

Construction of plasmids for the translation and expression of genes in S. cerevisiae:

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGGATCCATGAGATTCCCTAGTATCTTCACCGCCGTCTTGTTCGCTGCTAGTTCTGCTTTGGCTGCTCCAGTCAACACTACCACCGAAGACGAAACCGCTCAGATCCCAGCTGAAGCCGTCATCGGTTACCTTGACCTTGAGGGTGACTTCGATGTCGCCGTCCTTCCATTCTCTAACAGTACCAACAACGGTCTTTTGTTCATTAACACCACCATTGCTTCTATCGCCGCTAAGGAGGAGGGTGTCTCTCTTGACAAGAGACATCACCACCACCATCACAGTGCTAAGTCTAGTTTCATCTCTACCACCACTACCGACCTTACCTCTATCAACACCAGTGCCTACAGTACCGGTTCTATCTCTACCGTCGAAACCGGAAACAGAACCACCTCTGAGGTCATCTCTCATGTCGTCACCACCTCTACCAAGCTTAGTCCTACCGCCACCACCAGTCTTACCATCGCTCAAACTTCTATTTATAGTACCGACTCTAATATTACCGTCGGTACCGACATCCACACCACCAGTGAAGTCATCTCTGACGTCGAAACCATCTCTAGGGAAACCGCCTCTACCGTTGTTGCCGCCCCAACTTCTACCACTGGTTGGACCGGTGCCATGAACACTTACATCTCTCAATTCACTTCTTCTAGTTTTGCTACTATTAACTCTACCCCTATCATCTCTTCTAGTGCCGTCTTCGAAACCAGTGACGCCTCTATCGTCAACGTCCATACCGAGAACATCACCAACACTGCTGCCGTTCCATCTGAGGAGCCTACCTTCGTCAACGCCACTAGAAACTCTCTTAATTCTTTTTGTTCTTCTAAACAACCATCTTCTCCTAGTTCTTATACTTCTTCTCCTTTGGTTTCTTCTTTGTCTGTTTCTAAGACCTTGTTGTCTACCTCTTTCACCCCTTCTGTCCCAACTTCTAATACTTATATTAAGACCAAGAACACTGGTTACTTCGAGCACACTGCCTTGACCACCTCTAGTGTTGGATTGAATAGTTTCTCTGAAACCGCCGTCTCTAGTCAAGGTACCAAGATTGACACCTTTTTGGTTTCTAGTTTGATCGCCTACCCATCTTCTGCCTCTGGTTCTCAGCTTAGTGGTATCCAGCAAAATTTTACCTCTACTTCTTTGATGATCAGTACCTACGAGGGTAAGGCCAGTATCTTCTTCTCTGCCGAGTTGGGTTCTATTATTTTTTTGTTGCTTAGTTATTTGTTGTTTTAAAACGGGTACTGTACAGTTAGTACATTGAGTCGAAATATACGAAATTATTGTTCATAATTTTCATCCTGGCTCTTTTTTTCTTCAACCATAGTTAAATGGACAGTTCATATCTTAACTCTAATAATACTTTTCTAGTTCTTATCCTTTTCCGTCTCACCGCAGATTTTATCATAGTATTAAATTTATATTTTGTTCGTAAAAAGAAAAATTTGTGAGCGTTACCGCTCGTTTCATTACCCGAAGGCTGTTTCAGTAGACCACTGATTAAGTAAGTAGATGAAAAAATTTCATCACCATGAAAGAGTTCGATGAGAGCTACTTTTTCAAATGCTTAACAGCTAACCGCCATTCAATAATGTTACGCTCTCTTCATTCTGCGGCTACGTTATCTAACAAGAGGTTTTACTCTCTCATATCTCATTCAAATAGAAAGAACATAATCAAAAGGATCCTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGCCTTACTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTATCCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCCTTACTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGGATCCATGGAGAGTTCTTCTCATATTATTGAAGTCAACGGAGGTAGATCTGACGAGGAAAGAAGGGCTGTCGCTAGTAAGGTCTGCGGAGAAGCTCCTTGCGGTTTCAGTGACGCTAAGAACGCTTCTGGTGACGCCCAAGAAAGGAACGCCTCTATGAGGAAATTGTGCATCGCCGTCGTCTTGTGCCTTGTCTTCATGTCTGTCGAGGTCGTTGGTGGTGTCAAGGCCAACAGTTTGGCCATCATGACCGATGCCGCTCATCTTTTGAGTGACGTTGCCGCTTTCGCCATCTCTTTGTTCAGTCTTTGGGCTGCCGGTTGGGAGGCTACCCCAAGGCACACCTACGGATTCTTCAGAATCGAAATTCTTGGAGCTCTTGTTTCTATCCAATTGATTTGGTTGCTTACTGGTATCCTTGTTTACGAGGCCATCATTAGACTTTTGACCGAAACCTCTGAGGTCAACGGTTTCTTGATGTTCGCTGTCGCCACTTTCGGTTTGCTTGTCAACATTATCATGGCTGTCATGCTTGGTCACGACCATGACAATGGTCACGGTCATAGTCACGGTGAGGACAAACACGATGAAGCCCATGGTGACGTCACTGAACAGCTTCTTGAAAAGCCAAAACAGCAGAAGGAAAAAAAGAAGAGGAACATCAACGCCCAAGGTGCCTATTTGCACGTCTTGGGTGACTCTATCCAGTCTGTCGGAGTCATGATCGGTGGTGCTGCTATCTGGTACAATCCAAAGTGGAAGATCATTGACTTGATCTGCACCTTGGTCTTCTCTGTCATCGTCTTGGGAACCACCATCAATATGATCAGAAGTATTCTTGAGGTCTTGATGGAGAGTACCCCAAGAGAAATCGACGCCACCAAGCTTGAGAAGGGTCTTCTTGAGATGGAGGAGGTCGTCGCTGTTCACGAATTGCACATCTGGGCCTCTACCGTTGGTAAGGTCTTGCTTGCTTGCCATGTCAACGCCAGACCAGATGCCGATGCCGATATGGTCCTTAACAAAGTCGTTGACTATATTAGGAGAGAATATAACATTTCTCATGTTACTATCCAAATTGAAAGATAAGGATCCATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTAGGCCTTACTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

The resistance gene(kanR) was linked to the target gene(hexa-His, nixA, TgMTP1t2) to screen the engineered yeast and the original yeast.

pSB1C3-BBa_K3126018 (pSB1C3-hexa-His/ kanR):

Enzyme digestion system:

pSB1C3-BBa_K312601425 μL
SpeI 3 μL
PstI 3 μL
Cutsmart Buffer5 μL
ddH2O14 μL

pSB1C3-BBa_K312601725 μL
XbaI 3 μL
PstI 3 μL
Cutsmart Buffer5 μL
ddH2O14 μL

The enzyme system we use:

BBa_K3126014 linear plasmid 2 μL
kanR part6 μL
T4 DNA Ligase0.5 μL
T4Buffer1 μL
ddH2O0.5 μL

pSB1C3-BBa_K3126019 (pSB1C3-nixA/ kanR)

Enzyme digestion system:

pSB1C3-BBa_K312601525 μL
SpeI 3 μL
PstI 3 μL
Cutsmart Buffer5 μL
ddH2O14 μL

pSB1C3-BBa_K312601725 μL
XbaI 3 μL
PstI 3 μL
Cutsmart Buffer5 μL
ddH2O14 μL

The enzyme system we use:

pSB1C3-BBa_K3126015 linear plasmid 2 μL
kanR part4 μL
T4 DNA Ligase0.5 μL
T4Buffer1 μL
ddH2O0.5 μL

pSB1C3-BBa_K3126020 (pSB1C3-TgMTP1t2/ kanR)

Enzyme digestion system:

pSB1C3-BBa_K312601625 μL
SpeI 3 μL
PstI 3 μL
Cutsmart Buffer5 μL
ddH2O14 μL

pSB1C3-BBa_K312601725 μL
XbaI 3 μL
PstI 3 μL
Cutsmart Buffer5 μL
ddH2O14 μL

The enzyme system we use:

pSB1C3-BBa_K3126016 linear plasmid 2 μL
kanR part6 μL
T4 DNA Ligase0.5 μL
T4Buffer1 μL
ddH2O0.5 μL

2019.7.1 ~ 7.10

Construction of plasmids for the translation and expression of genes in S. cerevisiae:

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGGATCCATGAGATTCCCTAGTATCTTCACCGCCGTCTTGTTCGCTGCTAGTTCTGCTTTGGCTGCTCCAGTCAACACTACCACCGAAGACGAAACCGCTCAGATCCCAGCTGAAGCCGTCATCGGTTACCTTGACCTTGAGGGTGACTTCGATGTCGCCGTCCTTCCATTCTCTAACAGTACCAACAACGGTCTTTTGTTCATTAACACCACCATTGCTTCTATCGCCGCTAAGGAGGAGGGTGTCTCTCTTGACAAGAGACATCACCACCACCATCACAGTGCTAAGTCTAGTTTCATCTCTACCACCACTACCGACCTTACCTCTATCAACACCAGTGCCTACAGTACCGGTTCTATCTCTACCGTCGAAACCGGAAACAGAACCACCTCTGAGGTCATCTCTCATGTCGTCACCACCTCTACCAAGCTTAGTCCTACCGCCACCACCAGTCTTACCATCGCTCAAACTTCTATTTATAGTACCGACTCTAATATTACCGTCGGTACCGACATCCACACCACCAGTGAAGTCATCTCTGACGTCGAAACCATCTCTAGGGAAACCGCCTCTACCGTTGTTGCCGCCCCAACTTCTACCACTGGTTGGACCGGTGCCATGAACACTTACATCTCTCAATTCACTTCTTCTAGTTTTGCTACTATTAACTCTACCCCTATCATCTCTTCTAGTGCCGTCTTCGAAACCAGTGACGCCTCTATCGTCAACGTCCATACCGAGAACATCACCAACACTGCTGCCGTTCCATCTGAGGAGCCTACCTTCGTCAACGCCACTAGAAACTCTCTTAATTCTTTTTGTTCTTCTAAACAACCATCTTCTCCTAGTTCTTATACTTCTTCTCCTTTGGTTTCTTCTTTGTCTGTTTCTAAGACCTTGTTGTCTACCTCTTTCACCCCTTCTGTCCCAACTTCTAATACTTATATTAAGACCAAGAACACTGGTTACTTCGAGCACACTGCCTTGACCACCTCTAGTGTTGGATTGAATAGTTTCTCTGAAACCGCCGTCTCTAGTCAAGGTACCAAGATTGACACCTTTTTGGTTTCTAGTTTGATCGCCTACCCATCTTCTGCCTCTGGTTCTCAGCTTAGTGGTATCCAGCAAAATTTTACCTCTACTTCTTTGATGATCAGTACCTACGAGGGTAAGGCCAGTATCTTCTTCTCTGCCGAGTTGGGTTCTATTATTTTTTTGTTGCTTAGTTATTTGTTGTTTTAAAACGGGTACTGTACAGTTAGTACATTGAGTCGAAATATACGAAATTATTGTTCATAATTTTCATCCTGGCTCTTTTTTTCTTCAACCATAGTTAAATGGACAGTTCATATCTTAACTCTAATAATACTTTTCTAGTTCTTATCCTTTTCCGTCTCACCGCAGATTTTATCATAGTATTAAATTTATATTTTGTTCGTAAAAAGAAAAATTTGTGAGCGTTACCGCTCGTTTCATTACCCGAAGGCTGTTTCAGTAGACCACTGATTAAGTAAGTAGATGAAAAAATTTCATCACCATGAAAGAGTTCGATGAGAGCTACTTTTTCAAATGCTTAACAGCTAACCGCCATTCAATAATGTTACGCTCTCTTCATTCTGCGGCTACGTTATCTAACAAGAGGTTTTACTCTCTCATATCTCATTCAAATAGAAAGAACATAATCAAAAGGATCCTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTATCCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCCTTACTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTATCCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGGATCCATGGAGAGTTCTTCTCATATTATTGAAGTCAACGGAGGTAGATCTGACGAGGAAAGAAGGGCTGTCGCTAGTAAGGTCTGCGGAGAAGCTCCTTGCGGTTTCAGTGACGCTAAGAACGCTTCTGGTGACGCCCAAGAAAGGAACGCCTCTATGAGGAAATTGTGCATCGCCGTCGTCTTGTGCCTTGTCTTCATGTCTGTCGAGGTCGTTGGTGGTGTCAAGGCCAACAGTTTGGCCATCATGACCGATGCCGCTCATCTTTTGAGTGACGTTGCCGCTTTCGCCATCTCTTTGTTCAGTCTTTGGGCTGCCGGTTGGGAGGCTACCCCAAGGCACACCTACGGATTCTTCAGAATCGAAATTCTTGGAGCTCTTGTTTCTATCCAATTGATTTGGTTGCTTACTGGTATCCTTGTTTACGAGGCCATCATTAGACTTTTGACCGAAACCTCTGAGGTCAACGGTTTCTTGATGTTCGCTGTCGCCACTTTCGGTTTGCTTGTCAACATTATCATGGCTGTCATGCTTGGTCACGACCATGACAATGGTCACGGTCATAGTCACGGTGAGGACAAACACGATGAAGCCCATGGTGACGTCACTGAACAGCTTCTTGAAAAGCCAAAACAGCAGAAGGAAAAAAAGAAGAGGAACATCAACGCCCAAGGTGCCTATTTGCACGTCTTGGGTGACTCTATCCAGTCTGTCGGAGTCATGATCGGTGGTGCTGCTATCTGGTACAATCCAAAGTGGAAGATCATTGACTTGATCTGCACCTTGGTCTTCTCTGTCATCGTCTTGGGAACCACCATCAATATGATCAGAAGTATTCTTGAGGTCTTGATGGAGAGTACCCCAAGAGAAATCGACGCCACCAAGCTTGAGAAGGGTCTTCTTGAGATGGAGGAGGTCGTCGCTGTTCACGAATTGCACATCTGGGCCTCTACCGTTGGTAAGGTCTTGCTTGCTTGCCATGTCAACGCCAGACCAGATGCCGATGCCGATATGGTCCTTAACAAAGTCGTTGACTATATTAGGAGAGAATATAACATTTCTCATGTTACTATCCAAATTGAAAGATAAGGATCCATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTAGGCCTTACTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATAACTTCGTATAATGTATGCTATACGAAGTTATATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAATAACTTCGTATAATGTATGCTATACGAAGTTATGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGGATCCATGAGATTCCCTAGTATCTTCACCGCCGTCTTGTTCGCTGCTAGTTCTGCTTTGGCTGCTCCAGTCAACACTACCACCGAAGACGAAACCGCTCAGATCCCAGCTGAAGCCGTCATCGGTTACCTTGACCTTGAGGGTGACTTCGATGTCGCCGTCCTTCCATTCTCTAACAGTACCAACAACGGTCTTTTGTTCATTAACACCACCATTGCTTCTATCGCCGCTAAGGAGGAGGGTGTCTCTCTTGACAAGAGACATCACCACCACCATCACAGTGCTAAGTCTAGTTTCATCTCTACCACCACTACCGACCTTACCTCTATCAACACCAGTGCCTACAGTACCGGTTCTATCTCTACCGTCGAAACCGGAAACAGAACCACCTCTGAGGTCATCTCTCATGTCGTCACCACCTCTACCAAGCTTAGTCCTACCGCCACCACCAGTCTTACCATCGCTCAAACTTCTATTTATAGTACCGACTCTAATATTACCGTCGGTACCGACATCCACACCACCAGTGAAGTCATCTCTGACGTCGAAACCATCTCTAGGGAAACCGCCTCTACCGTTGTTGCCGCCCCAACTTCTACCACTGGTTGGACCGGTGCCATGAACACTTACATCTCTCAATTCACTTCTTCTAGTTTTGCTACTATTAACTCTACCCCTATCATCTCTTCTAGTGCCGTCTTCGAAACCAGTGACGCCTCTATCGTCAACGTCCATACCGAGAACATCACCAACACTGCTGCCGTTCCATCTGAGGAGCCTACCTTCGTCAACGCCACTAGAAACTCTCTTAATTCTTTTTGTTCTTCTAAACAACCATCTTCTCCTAGTTCTTATACTTCTTCTCCTTTGGTTTCTTCTTTGTCTGTTTCTAAGACCTTGTTGTCTACCTCTTTCACCCCTTCTGTCCCAACTTCTAATACTTATATTAAGACCAAGAACACTGGTTACTTCGAGCACACTGCCTTGACCACCTCTAGTGTTGGATTGAATAGTTTCTCTGAAACCGCCGTCTCTAGTCAAGGTACCAAGATTGACACCTTTTTGGTTTCTAGTTTGATCGCCTACCCATCTTCTGCCTCTGGTTCTCAGCTTAGTGGTATCCAGCAAAATTTTACCTCTACTTCTTTGATGATCAGTACCTACGAGGGTAAGGCCAGTATCTTCTTCTCTGCCGAGTTGGGTTCTATTATTTTTTTGTTGCTTAGTTATTTGTTGTTTTAAAACGGGTACTGTACAGTTAGTACATTGAGTCGAAATATACGAAATTATTGTTCATAATTTTCATCCTGGCTCTTTTTTTCTTCAACCATAGTTAAATGGACAGTTCATATCTTAACTCTAATAATACTTTTCTAGTTCTTATCCTTTTCCGTCTCACCGCAGATTTTATCATAGTATTAAATTTATATTTTGTTCGTAAAAAGAAAAATTTGTGAGCGTTACCGCTCGTTTCATTACCCGAAGGCTGTTTCAGTAGACCACTGATTAAGTAAGTAGATGAAAAAATTTCATCACCATGAAAGAGTTCGATGAGAGCTACTTTTTCAAATGCTTAACAGCTAACCGCCATTCAATAATGTTACGCTCTCTTCATTCTGCGGCTACGTTATCTAACAAGAGGTTTTACTCTCTCATATCTCATTCAAATAGAAAGAACATAATCAAAAGGATCCTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTATCCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGGATCCATGGAGAGTTCTTCTCATATTATTGAAGTCAACGGAGGTAGATCTGACGAGGAAAGAAGGGCTGTCGCTAGTAAGGTCTGCGGAGAAGCTCCTTGCGGTTTCAGTGACGCTAAGAACGCTTCTGGTGACGCCCAAGAAAGGAACGCCTCTATGAGGAAATTGTGCATCGCCGTCGTCTTGTGCCTTGTCTTCATGTCTGTCGAGGTCGTTGGTGGTGTCAAGGCCAACAGTTTGGCCATCATGACCGATGCCGCTCATCTTTTGAGTGACGTTGCCGCTTTCGCCATCTCTTTGTTCAGTCTTTGGGCTGCCGGTTGGGAGGCTACCCCAAGGCACACCTACGGATTCTTCAGAATCGAAATTCTTGGAGCTCTTGTTTCTATCCAATTGATTTGGTTGCTTACTGGTATCCTTGTTTACGAGGCCATCATTAGACTTTTGACCGAAACCTCTGAGGTCAACGGTTTCTTGATGTTCGCTGTCGCCACTTTCGGTTTGCTTGTCAACATTATCATGGCTGTCATGCTTGGTCACGACCATGACAATGGTCACGGTCATAGTCACGGTGAGGACAAACACGATGAAGCCCATGGTGACGTCACTGAACAGCTTCTTGAAAAGCCAAAACAGCAGAAGGAAAAAAAGAAGAGGAACATCAACGCCCAAGGTGCCTATTTGCACGTCTTGGGTGACTCTATCCAGTCTGTCGGAGTCATGATCGGTGGTGCTGCTATCTGGTACAATCCAAAGTGGAAGATCATTGACTTGATCTGCACCTTGGTCTTCTCTGTCATCGTCTTGGGAACCACCATCAATATGATCAGAAGTATTCTTGAGGTCTTGATGGAGAGTACCCCAAGAGAAATCGACGCCACCAAGCTTGAGAAGGGTCTTCTTGAGATGGAGGAGGTCGTCGCTGTTCACGAATTGCACATCTGGGCCTCTACCGTTGGTAAGGTCTTGCTTGCTTGCCATGTCAACGCCAGACCAGATGCCGATGCCGATATGGTCCTTAACAAAGTCGTTGACTATATTAGGAGAGAATATAACATTTCTCATGTTACTATCCAAATTGAAAGATAAGGATCCATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTAGGCCTTACTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATAACTTCGTATAATGTATGCTATACGAAGTTATATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAATAACTTCGTATAATGTATGCTATACGAAGTTATGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

We obtained the fragments to be inserted by PCR, then combine several target genes by seamless cloning.

pSB1C3-BBa_K3126021 (pSB1C3- hexa-His/ nixA/ kanR):

The PCR system of pGPM1-nixA-tTDH1:

(P-nixA-T_F

CGAAGGCTTTAATTTGCAGGGCCAAACTTTTCGGTTAACA

P-nixA-T_R

ACCTATCAACTCTAGTAAGGCCTCGTTCAGGGTAATATATTTT)

pSB1C3-BBa_K3126015/SpeⅠ PstⅠ1 μL
P-nixA-T_F 2 μL
P-nixA-T_R2 μL
2×PCR mix20 μL
ddH2O15 μL

Enzyme digestion system:

pSB1C3-BBa_K312601815 μL
StuI 3 μL
buffer 5 μL
ddH2O27 μL

Seamless cloning system:

pSB1C3-BBa_K3126018/StuⅠ 2 μL
pGPM1-nixA-tTDH14 μL
5×CEⅡ Buffer2 μL
Exnase Ⅱ1 μL
ddH2O1 μL

pSB1C3-BBa_K3126022 (pSB1C3- nixA/ TgMTP1t2/ kanR)

The PCR system of pTDH3-TgMTP1t2-tPGK1

pSB1C3-BBa_K3126016/SpeⅠ PstⅠ1 μL
P-TgMTP1t2-T_F2 μL
P-TgMTP1t2-T_R2 μL
2×PCR mix20 μL
ddH2O15 μL

Enzyme digestion system:

pSB1C3-BBa_K312602115 μL
StuI 3 μL
buffer 5 μL
ddH2O27 μL

Seamless cloning system:

pSB1C3-BBa_K3126021/StuⅠ 2 μL
pTDH3-TgMTP1t2-tPGK14 μL
5×CEⅡ Buffer2 μL
Exnase Ⅱ1 μL
ddH2O1 μL

pSB1C3-BBa_K3126023 (pSB1C3- hexa-His/ nixA/ TgMTP1t2/ kanR)

Enzyme digestion system:

pSB1C3-BBa_K312602115 μL
StuI 3 μL
buffer5 μL
ddH2O27 μL

Seamless cloning system:

pSB1C3-BBa_K3126021/StuⅠ2 μL
pTDH3-TgMTP1t2-tPGK1 4 μL
5×CEⅡ Buffer2 μL
Exnase Ⅱ1 μL
ddH2O1 μL

2019.7.20 ~ 7.25

The plasmid is digested with Bsa1 to get the fragment to be integrated.

pSB1C3- hexa-His- kanR/ BsaⅠ

Enzyme digestion system:

pSB1C3-BBa_K312601825μL
BsaI 3 μL
buffer5 μL
ddH2O17 μL

pSB1C3-nixA- kanR/ BsaⅠ

Enzyme digestion system:

pSB1C3-BBa_K312601925μL
BsaI 3 μL
buffer5 μL
ddH2O17 μL

pSB1C3-TgMTP1t2- kanR/ BsaⅠ

Enzyme digestion system:

pSB1C3-BBa_K312602025μL
BsaI 3 μL
buffer5 μL
ddH2O17 μL

pSB1C3-hexa-His-nixA- kanR/ BsaⅠ

Enzyme digestion system:

pSB1C3-BBa_K312602125μL
BsaI 3 μL
buffer5 μL
ddH2O17 μL

pSB1C3-nixA-TgMTP1t2- kanR/ BsaⅠ

Enzyme digestion system:

pSB1C3-BBa_K312602225μL
BsaI 3 μL
buffer5 μL
ddH2O17 μL

pSB1C3-hexa-His-nixA-TgMTP1t2- kanR/ BsaⅠ

Enzyme digestion system:

pSB1C3-BBa_K312602325μL
BsaI 3 μL
buffer5 μL
ddH2O17 μL

2019.7.25 ~ 8.5

The fragments were transformed into S. cerevisiae to express the corresponding protein.

2019.8.25 ~ 9.10

Adsorption of Nickel ion by S. cerevisiae

1.Culture yeast for a period of time, measure its OD600

2. Convert the required dry weight volume according to OD600

3.Add the corresponding volume of yeast solution into 50mL centrifuge tube, centrifugate at 8000 rpm for 5 min, discard the supernatant and enrich the cells

4.Prepare 21 bottles of 50mL 15mg / L nickel ion solution and 21 bottles of 50mL 200mg / L nickel ion solution, put the put the six kinds of engineered yeast and original yeast into the solution respectively, and culture them in a shaker at 30℃

5.Take the engineered yeast solution every interval to measure the nickel ion concentration

6.After about half an hour of adsorption, the equilibrium is reached

7.Draw the curve about the enrichment of Ni2+ by yeast.

Improvement


2019.9.20 ~ 2019.9.30

[Protocols]

1.Digested the pSB1C3-BBa_K2652001 circular plasmid with BamHI.

2.Amplified our target gene BBa_K3126026 (His-Tag-nikABCDE- His-Tag) using PCR technology.

3.Recovered the digested part and inserted BBa_K3126026 (His-Tag-nikABCDE-His-Tag) into pSB1C3-BBa_K2652001 by seamless cloning, and the mixed system was reacted at 37℃ for 30 minutes.

4.converted all of the products of the above seamless cloning system to DH5α.

5.Applied DH5α to the plasmid to a plate containing chloramphenicol resistance.

[Enzyme Digestion System]

pSB1C3-BBa_K2652001 circular plasmid 20 μL
BamHI 3 μL
Buffer 5 μL
ddH2O 27 μL

[PCR System]

Template 1 μL
Primer-F 2 μL
Primer-R 2 μL
2×Rapid Taq Master Mix 20 μL
ddH2O 27 μL

[PCR System]

1 95℃ 30s
2 95℃ 8s
3 60℃ 30s
4 72℃ 2min30s
5 72℃ 5min
6 4℃ ∞

（2、3、4-thirty cycles）


[Seamless cloning system]

pSB1C3-BBa_K2652001 circular plasmid/ BamHⅠ 3 μL
His-Tag-nikABCDE- His-Tag 4 μL
Buffer 2 μL
ExnaseⅡ 1 μL

Enzyme Digestion picture:

[Adsorption Experiment of E.coli]

1. Culture E.coli for a period of time, measure its OD600

2. Convert the required dry weight volume according to OD600

3.Add the corresponding volume of bacterial solution into 50mL centrifuge tube, centrifugate at 8000 rpm for 12 min, discard the supernatant and enrich the cells (more than 30mL is divided into two centrifuges);

4.Prepare 6 bottles of 50mL 30mg / L nickel ion solution, put the E.coli into the solution respectively, and culture them in a shaker at 37℃.

5.Take 1.5mL solution every 2 min in the centrifuge tube, take 5 times, and then take 3 times every 5min;

6.Using dimethylglyoxime spectrophotometry to measure the nickel ion concentration of the above samples;

7. Draw the relation curve about time and nickel ion concentration.


GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCAGGAAGAAAC

CATCATCATCATCATCACATGCTCTCCACACTCCGCCGCACTCTATTTGCGCTGCTGGCTTGTGCGTCTTTTATCGTCCATGCCGCTGCACCAGATGAAATCACCACCGCCTGGCCGGTGAATGTCGGGCCACTAAACCCGCACCTTTACACGCCTAACCAGATGTTCGCCCAGAGCATGGTTTATGAACCATTGGTGAAATATCAGGCAGACGGTTCGGTGATCCCGTGGCTGGCAAAAAGCTGGACTCATTCAGAAGATGGTAAAACCTGGACCTTCACCCTGCGTGATGACGTGAAATTCTCCAACGGTGAACCGTTCGATGCCGAGGCGGCGGCAGAAAACTTCCGCGCAGTGCTCGATAACCGTCAACGTCACGCCTGGCTGGAGCTGGCAAACCAGATTGTTGATGTTAAAGCACTCAGTAAAACAGAGCTGCAAATTACCCTGAAAAGCGCCTACTATCCTTTCCTGCAAGAACTGGCCCTGCCCCGTCCTTTCCGTTTTATCGCTCCCTCGCAGTTTAAAAACCATGAAACCATGAACGGAATTAAAGCGCCGATTGGCACCGGACCGTGGATTTTGCAGGAATCGAAACTGAATCAGTACGATGTCTTCGTCCGTAACGAAAACTACTGGGGCGAAAAGCCAGCGATTAAAAAGATCACCTTTAACGTCATCCCGGACCCGACTACCCGCGCGGTGGCGTTTGAAACTGGCGATATCGACCTGCTGTACGGAAACGAAGGGTTATTACCGCTCGATACCTTCGCCCGCTTTAGCCAGAATCCGGCTTACCACACCCAACTGTCACAGCCGATCGAAACCGTGATGCTGGCGCTCAATACCGCCAAAGCCCCCACCAACGAGCTGGCAGTACGTGAAGCTCTTAATTACGCGGTAAACAAAAAATCGCTGATTGATAACGCGTTGTATGGCACCCAGCAGGTCGCCGACACCCTGTTTGCCCCTTCTGTGCCCTACGCCAACCTCGGCCTGAAACCGAGCCAGTACGATCCGCAAAAAGCGAAAGCGTTGCTGGAAAAAGCCGGTTGGACGCTGCCTGCGGGCAAAGACATCCGCGAGAAAAATGGTCAGCCGCTGCGCATTGAACTTTCGTTCATCGGCACCGATGCGTTAAGCAAATCGATGGCGGAAATCATTCAGGCTGATATGCGCCAGATTGGCGCAGATGTCTCGCTGATTGGCGAAGAAGAGAGCAGTATCTATGCTCGTCAGCGCGACGGTCGTTTTGGCATGATTTTCCACCGCACCTGGGGCGCGCCATATGATCCACACGCCTTCCTCAGTTCAATGCGCGTACCGTCACACGCTGACTTCCAGGCACAGCAAGGATTAGCCGACAAACCGCTGATTGATAAAGAGATCGGCGAAGTGCTGGCGACCCATGACGAAACGCAACGTCAGGCGCTGTATCGCGACATTCTGACCCGTCTGCATGACGAGGCGGTTTATCTGCCTATCAGTTACATCTCAATGATGGTGGTATCAAAACCGGAGCTGGGTAACATCCCCTACGCGCCGATCGCCACCGAAATTCCGTTCGAACAGATTAAACCGGTGAAACCTTAAATGTTGCGTTACGTATTACGCCGCTTTCTGCTGCTGATCCCGATGGTGCTCGCCGCCTCGGTGATCATTTTTCTGATGCTGCGCCTCGGTACCGGCGACCCGGCGCTCGATTATTTGCGTCTGTCTAACCTGCCGCCGACGCCGGAGATGCTGGCCTCTACCCGCACCATGCTGGGACTGGATCAGCCGCTGTACGTCCAGTACGGCACCTGGTTGTGGAAGGCGCTGCATCTTGACTTTGGTATCTCATTCGCCAGCCAACGCCCGGTACTGGACGATATGCTGAACTTCCTGCCCGCCACGCTGGAACTTGCAGGTGCGGCGCTGGTATTAATTCTGCTCACTTCCGTACCGCTCGGTATCTGGGCGGCGCGCCATCGCGACCGTCTGCCGGATTTCGCCGTACGTTTCATCGCGTTTCTTGGCGTGTCGATGCCTAACTTCTGGCTGGCGTTTTTACTGGTGATGGCGTTTTCGGTGTATCTGCAATGGCTACCCGCGATGGGTTACGGCGGCTGGCAGCACATCATTTTGCCTGCGGTTTCCATTGCCTTTATGTCGCTGGCGATTAACGCGCGTTTACTGCGCGCCAGTATGCTGGACGTCGCCGGTCAGCGTCACGTCACCTGGGCGCGTCTGCGCGGCCTGAACGACAAACAGACCGAACGTCGCCACATCCTGCGCAATGCCTCGCTGCCGATGATCACCGCCGTGGGGATGCATATCGGCGAACTGATTGGCGGGACGATGATTATCGAAAACATCTTTGCCTGGCCGGGCGTCGGGCGCTATGCGGTGTCGGCGATTTTTAACCGTGACTATCCGGTGATCCAGTGCTTTACGCTGATGATGGTGGTGGTTTTTGTGGTCTGTAATTTGATTGTCGATTTGCTCAACGCCGCGCTGGACCCGCGCATTCGTCGTCATGAAGGAGCGCACGCGTGAGTGAACTTTTTCCTCTCTTCCCGCTGGTCGGTACGCCTGGCGCTGATCATTATCGCCCTGCTGGCGCTGATTGCGCTCACCAGCCAGTGGTGGCTGCCGTATGACCCACAGGCGATTGATTTGCCGTCGCGCCTGCTTTCGCCGGATGCGCAGCACTGGCTGGGCACCGATCACTTAGGTCGCGATATTTTCTCGCGGCTGATGGCAGCGACCCGCGTGTCGCTCGGTTCGGTAATGGCCTGCCTGCTGCTGGTGCTGACATTAGGGCTGGTTATTGGCGGCAGCGCCGGGTTGATTGGCGGGCGCGTTGATCAGGCCACCATGCGCGTCGCCGATATGTTTATGACCTTCCCGACCTCGATTCTGTCGTTCTTTATGGTTGGCGTGCTCGGCACCGGGCTGACCAACGTAATTATCGCCATCGCCCTGTCGCACTGGGCGTGGTATGCACGCATGGTGCGCAGCCTGGTGATTTCACTACGCCAACGCGAGTTTGTGCTGGCGTCACGGCTTTCCGGTGCGGGCCATGTGCGGGTGTTTGTCGATCATCTGGCAGGCGCGGTGATCCCTTCGCTGCTGGTGCTGGCAACGCTGGATATCGGCCATATGATGCTGCACGTCGCGGGGATGTCTTTCCTTGGCCTCGGTGTGACCGCGCCGACCGCCGAATGGGGCGTGATGATTAACGACGCGCGCCAGTATATCTGGACCCAGCCGCTGCAAATGTTCTGGCCGGGGCTGGCGCTGTTTATCAGCGTGATGGCCTTTAACCTGGTGGGTGACGCACTGCGCGATCATCTGGACCCTCATCTGGTGACGGAGCACGCACACTAAATGCCGCAACAGATTGAACTACGTAATATCGCGCTACAGGCCGCGCAGCCGCTGGTACACGGTGTATCGTTAACCCTGCAACGCGGGCGCGTGCTGGCGTTAGTCGGCGGTAGCGGCAGCGGGAAATCATTAACCTGCGCCGCAACGCTGGGCATTTTGCCCGCTGGCGTTCGCCAGACGGCGGGGGAAATTTTAGCCGATGGCAAACCGGTTTCGCCTTGCGCCCTGCGCGGCATCAAAATTGCCACCATCATGCAGAACCCGCGCAGCGCCTTTAATCCACTGCACACCATGCACACCCACGCGCGGGAAACCTGCCTGGCGTTAGGGAAACCCGCCGATGACGCTACGCTTACCGCTGCCATAGAAGCGGTGGGGCTGGAAAACGCCGCGCGCGTGCTGAAGCTGTACCCGTTCGAGATGAGCGGCGGCATGTTGCAGCGCATGATGATTGCGATGGCGGTGCTGTGTGAATCACCGTTTATCATCGCCGATGAACCGACCACCGACCTTGACGTGGTAGCACAGGCGCGCATCCTCGATCTGCTGGAAAGCATTATGCAAAAACAAGCGCCGGGAATGCTGCTGGTGACCCATGATATGGGCGTTGTGGCGCGTCTGGCGGATGACGTGGCGGTGATGTCTGACGGTAAGATTGTCGAACAGGGCGATGTAGAAACGCTGTTTAACGCCCCCAAACATACAGTGACGCGCAGCCTGGTTTCCGCTCATCTCGCCCTCTACGGTATGGAGCTGGCATCATGAATGACTTTACTTAACATCTCCGGCCTTTCCCATCACTATGCGCACGGTGGATTTAACGGAAAACATCAACATCAGGCGGTGCTGAATAACGTTTCCCTGACCCTGAAAAGCGGCGAAACCGTCGCCCTGCTGGGGCGCAGCGGCTGCGGGAAAAGTACCCTCGCGCGGTTGCTGGTGGGTTTAGAATCGCCCGCGCAGGGGAATATTAGCTGGCGTGGCGAACCGCTGGCGAAACTCAATCGCGCCCAGCGTAAAGCGTTCCGCCGCGATATTCAGATGGTATTTCAGGACTCCATCAGCGCCGTGAATCCGCGCAAAACCGTGCGCGAGATCCTGCGTGAACCGATGCGCCACCTGCTATCACTGAAAAAATCCGAACAACTGGCGCGCGCCAGCGAAATGCTGAAGGCGGTCGATCTCGATGACAGCGTTCTCGACAAACGCCCACCGCAGTTAAGCGGCGGCCAGCTCCAGCGCGTCTGCCTGGCTCGCGCGCTGGCGGTCGAACCGAAACTACTGATTCTGGATGAAGCCGTTTCTAACCTTGATCTCGTGTTACAGGCGGGTGTCATTCGCCTGCTGAAAAAGCTACAACAACAGTTTGGCACCGCCTGCCTGTTCATCACCCACGACTTACGCCTGGTAGAACGCTTTTGCCAGCGGGTAATGGTTATGGACAACGGACAAATCGTCGAAACCCAGGTGGTGGGAGAGAAATTAACCTTTTCCTCTGACGCCGGACGTGTGCTACAAAACGCGGTATTACCCGCATTCCCCGTGCGCCGTCGCACCACAGAAAAGGTTCACCACCACCACCACCACTAA

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCCATCATCATCATCATCACATGCTCTCCACACTCCGCCGCACTCTATTTGCGCTGCTGGCTTGTGCGTCTTTTATCGTCCATGCCGCTGCACCAGATGAAATCACCACCGCCTGGCCGGTGAATGTCGGGCCACTAAACCCGCACCTTTACACGCCTAACCAGATGTTCGCCCAGAGCATGGTTTATGAACCATTGGTGAAATATCAGGCAGACGGTTCGGTGATCCCGTGGCTGGCAAAAAGCTGGACTCATTCAGAAGATGGTAAAACCTGGACCTTCACCCTGCGTGATGACGTGAAATTCTCCAACGGTGAACCGTTCGATGCCGAGGCGGCGGCAGAAAACTTCCGCGCAGTGCTCGATAACCGTCAACGTCACGCCTGGCTGGAGCTGGCAAACCAGATTGTTGATGTTAAAGCACTCAGTAAAACAGAGCTGCAAATTACCCTGAAAAGCGCCTACTATCCTTTCCTGCAAGAACTGGCCCTGCCCCGTCCTTTCCGTTTTATCGCTCCCTCGCAGTTTAAAAACCATGAAACCATGAACGGAATTAAAGCGCCGATTGGCACCGGACCGTGGATTTTGCAGGAATCGAAACTGAATCAGTACGATGTCTTCGTCCGTAACGAAAACTACTGGGGCGAAAAGCCAGCGATTAAAAAGATCACCTTTAACGTCATCCCGGACCCGACTACCCGCGCGGTGGCGTTTGAAACTGGCGATATCGACCTGCTGTACGGAAACGAAGGGTTATTACCGCTCGATACCTTCGCCCGCTTTAGCCAGAATCCGGCTTACCACACCCAACTGTCACAGCCGATCGAAACCGTGATGCTGGCGCTCAATACCGCCAAAGCCCCCACCAACGAGCTGGCAGTACGTGAAGCTCTTAATTACGCGGTAAACAAAAAATCGCTGATTGATAACGCGTTGTATGGCACCCAGCAGGTCGCCGACACCCTGTTTGCCCCTTCTGTGCCCTACGCCAACCTCGGCCTGAAACCGAGCCAGTACGATCCGCAAAAAGCGAAAGCGTTGCTGGAAAAAGCCGGTTGGACGCTGCCTGCGGGCAAAGACATCCGCGAGAAAAATGGTCAGCCGCTGCGCATTGAACTTTCGTTCATCGGCACCGATGCGTTAAGCAAATCGATGGCGGAAATCATTCAGGCTGATATGCGCCAGATTGGCGCAGATGTCTCGCTGATTGGCGAAGAAGAGAGCAGTATCTATGCTCGTCAGCGCGACGGTCGTTTTGGCATGATTTTCCACCGCACCTGGGGCGCGCCATATGATCCACACGCCTTCCTCAGTTCAATGCGCGTACCGTCACACGCTGACTTCCAGGCACAGCAAGGATTAGCCGACAAACCGCTGATTGATAAAGAGATCGGCGAAGTGCTGGCGACCCATGACGAAACGCAACGTCAGGCGCTGTATCGCGACATTCTGACCCGTCTGCATGACGAGGCGGTTTATCTGCCTATCAGTTACATCTCAATGATGGTGGTATCAAAACCGGAGCTGGGTAACATCCCCTACGCGCCGATCGCCACCGAAATTCCGTTCGAACAGATTAAACCGGTGAAACCTTAAATGTTGCGTTACGTATTACGCCGCTTTCTGCTGCTGATCCCGATGGTGCTCGCCGCCTCGGTGATCATTTTTCTGATGCTGCGCCTCGGTACCGGCGACCCGGCGCTCGATTATTTGCGTCTGTCTAACCTGCCGCCGACGCCGGAGATGCTGGCCTCTACCCGCACCATGCTGGGACTGGATCAGCCGCTGTACGTCCAGTACGGCACCTGGTTGTGGAAGGCGCTGCATCTTGACTTTGGTATCTCATTCGCCAGCCAACGCCCGGTACTGGACGATATGCTGAACTTCCTGCCCGCCACGCTGGAACTTGCAGGTGCGGCGCTGGTATTAATTCTGCTCACTTCCGTACCGCTCGGTATCTGGGCGGCGCGCCATCGCGACCGTCTGCCGGATTTCGCCGTACGTTTCATCGCGTTTCTTGGCGTGTCGATGCCTAACTTCTGGCTGGCGTTTTTACTGGTGATGGCGTTTTCGGTGTATCTGCAATGGCTACCCGCGATGGGTTACGGCGGCTGGCAGCACATCATTTTGCCTGCGGTTTCCATTGCCTTTATGTCGCTGGCGATTAACGCGCGTTTACTGCGCGCCAGTATGCTGGACGTCGCCGGTCAGCGTCACGTCACCTGGGCGCGTCTGCGCGGCCTGAACGACAAACAGACCGAACGTCGCCACATCCTGCGCAATGCCTCGCTGCCGATGATCACCGCCGTGGGGATGCATATCGGCGAACTGATTGGCGGGACGATGATTATCGAAAACATCTTTGCCTGGCCGGGCGTCGGGCGCTATGCGGTGTCGGCGATTTTTAACCGTGACTATCCGGTGATCCAGTGCTTTACGCTGATGATGGTGGTGGTTTTTGTGGTCTGTAATTTGATTGTCGATTTGCTCAACGCCGCGCTGGACCCGCGCATTCGTCGTCATGAAGGAGCGCACGCGTGAGTGAACTTTTTCCTCTCTTCCCGCTGGTCGGTACGCCTGGCGCTGATCATTATCGCCCTGCTGGCGCTGATTGCGCTCACCAGCCAGTGGTGGCTGCCGTATGACCCACAGGCGATTGATTTGCCGTCGCGCCTGCTTTCGCCGGATGCGCAGCACTGGCTGGGCACCGATCACTTAGGTCGCGATATTTTCTCGCGGCTGATGGCAGCGACCCGCGTGTCGCTCGGTTCGGTAATGGCCTGCCTGCTGCTGGTGCTGACATTAGGGCTGGTTATTGGCGGCAGCGCCGGGTTGATTGGCGGGCGCGTTGATCAGGCCACCATGCGCGTCGCCGATATGTTTATGACCTTCCCGACCTCGATTCTGTCGTTCTTTATGGTTGGCGTGCTCGGCACCGGGCTGACCAACGTAATTATCGCCATCGCCCTGTCGCACTGGGCGTGGTATGCACGCATGGTGCGCAGCCTGGTGATTTCACTACGCCAACGCGAGTTTGTGCTGGCGTCACGGCTTTCCGGTGCGGGCCATGTGCGGGTGTTTGTCGATCATCTGGCAGGCGCGGTGATCCCTTCGCTGCTGGTGCTGGCAACGCTGGATATCGGCCATATGATGCTGCACGTCGCGGGGATGTCTTTCCTTGGCCTCGGTGTGACCGCGCCGACCGCCGAATGGGGCGTGATGATTAACGACGCGCGCCAGTATATCTGGACCCAGCCGCTGCAAATGTTCTGGCCGGGGCTGGCGCTGTTTATCAGCGTGATGGCCTTTAACCTGGTGGGTGACGCACTGCGCGATCATCTGGACCCTCATCTGGTGACGGAGCACGCACACTAAATGCCGCAACAGATTGAACTACGTAATATCGCGCTACAGGCCGCGCAGCCGCTGGTACACGGTGTATCGTTAACCCTGCAACGCGGGCGCGTGCTGGCGTTAGTCGGCGGTAGCGGCAGCGGGAAATCATTAACCTGCGCCGCAACGCTGGGCATTTTGCCCGCTGGCGTTCGCCAGACGGCGGGGGAAATTTTAGCCGATGGCAAACCGGTTTCGCCTTGCGCCCTGCGCGGCATCAAAATTGCCACCATCATGCAGAACCCGCGCAGCGCCTTTAATCCACTGCACACCATGCACACCCACGCGCGGGAAACCTGCCTGGCGTTAGGGAAACCCGCCGATGACGCTACGCTTACCGCTGCCATAGAAGCGGTGGGGCTGGAAAACGCCGCGCGCGTGCTGAAGCTGTACCCGTTCGAGATGAGCGGCGGCATGTTGCAGCGCATGATGATTGCGATGGCGGTGCTGTGTGAATCACCGTTTATCATCGCCGATGAACCGACCACCGACCTTGACGTGGTAGCACAGGCGCGCATCCTCGATCTGCTGGAAAGCATTATGCAAAAACAAGCGCCGGGAATGCTGCTGGTGACCCATGATATGGGCGTTGTGGCGCGTCTGGCGGATGACGTGGCGGTGATGTCTGACGGTAAGATTGTCGAACAGGGCGATGTAGAAACGCTGTTTAACGCCCCCAAACATACAGTGACGCGCAGCCTGGTTTCCGCTCATCTCGCCCTCTACGGTATGGAGCTGGCATCATGAATGACTTTACTTAACATCTCCGGCCTTTCCCATCACTATGCGCACGGTGGATTTAACGGAAAACATCAACATCAGGCGGTGCTGAATAACGTTTCCCTGACCCTGAAAAGCGGCGAAACCGTCGCCCTGCTGGGGCGCAGCGGCTGCGGGAAAAGTACCCTCGCGCGGTTGCTGGTGGGTTTAGAATCGCCCGCGCAGGGGAATATTAGCTGGCGTGGCGAACCGCTGGCGAAACTCAATCGCGCCCAGCGTAAAGCGTTCCGCCGCGATATTCAGATGGTATTTCAGGACTCCATCAGCGCCGTGAATCCGCGCAAAACCGTGCGCGAGATCCTGCGTGAACCGATGCGCCACCTGCTATCACTGAAAAAATCCGAACAACTGGCGCGCGCCAGCGAAATGCTGAAGGCGGTCGATCTCGATGACAGCGTTCTCGACAAACGCCCACCGCAGTTAAGCGGCGGCCAGCTCCAGCGCGTCTGCCTGGCTCGCGCGCTGGCGGTCGAACCGAAACTACTGATTCTGGATGAAGCCGTTTCTAACCTTGATCTCGTGTTACAGGCGGGTGTCATTCGCCTGCTGAAAAAGCTACAACAACAGTTTGGCACCGCCTGCCTGTTCATCACCCACGACTTACGCCTGGTAGAACGCTTTTGCCAGCGGGTAATGGTTATGGACAACGGACAAATCGTCGAAACCCAGGTGGTGGGAGAGAAATTAACCTTTTCCTCTGACGCCGGACGTGTGCTACAAAACGCGGTATTACCCGCATTCCCCGTGCGCCGTCGCACCACAGAAAAGGTTCACCACCACCACCACCACTAAGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCA

Characterization


2019.9.10 ~ 2019.9.20

We used the pSB1C3-BBa_K2652001 from last year and digested with BamH I enzyme.

The pSB1C3 digestion system we used:

pSB1C3-BBa_K2652001 15μL
BamHI 3 μL
Cutsmart Buffer 5 μL
ddH2O 27 μL

pSB1C3-BBa_K3126028(pSB1C3-BBa_K2652001/ BBa_E0032)

pSB1C3-BBa_K3126029(pSB1C3-BBa_K2652001/ BBa_E0034)

pSB1C3-BBa_K3126030(pSB1C3-BBa_K2652001/GFP)


We obtained the fragments to be inserted by PCR, then combine several target genes by seamless cloning.

The PCR system of BBa_E0032 and BBa_E0034:

BBa_E0032_F

AGGGAGCTTTAAATCGGATCCatggtgagcaagggcgagga

BBa_E0032_R

TGCCTGGCTCTAGTAGGATCCTTATTAAGCTACTAAAGCGT


pSB1C3- BBa_E0032/ BamHI 1μL
BBa_E0032_F 2 μL
BBa_E0032_R 2 μL
2×PCR mix 20 μL
ddH2O 27 μL

BBa_E0034_F

AGGGAGCTTTAAATCGGATCCatggtgagcaagggcgagga

BBa_E0034_R

TGCCTGGCTCTAGTAGGATCCTTATTAAACAGCAGCAGCGT


pSB1C3- BBa_E0034/ BamHI 1μL
BBa_E0034_F 2 μL
BBa_E0034_R 2 μL
2×PCR mix 20 μL
ddH2O 15 μL

pSB1C3-GFP 1μL
GFP_F 2 μL
GFP_R 2 μL
2×PCR mix 20 μL
ddH2O 15 μL

Seamless cloning system:


pSB1C3-BBa_K2652001/BamHI 2μL
BBa_E0032 4 μL
5×CEⅡ Buffer 2 μL
Exnase Ⅱ 1 μL
ddH2O 1 μL

pSB1C3-BBa_K2652001/BamHI 2μL
BBa_E0034 4 μL
5×CEⅡ Buffer 2 μL
Exnase Ⅱ 1 μL
ddH2O 1 μL

pSB1C3-BBa_K2652001/BamHI 2μL
GFP 4 μL
5×CEⅡ Buffer 2 μL
Exnase Ⅱ 1 μL
ddH2O 1 μL

GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCAGGAAGAAAC

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctttagtagcttaataa

TTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctttagtagcttaataaGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcg

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctttagtagcttaataaGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCA

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctgctgctgtttaataa

TTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctgctgctgtttaataaGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcg

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctgctgctgtttaataaGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCA

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

TTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcg

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCC ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA GGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCA

option date

  • Method-Protocol
  • Notebook
    • {{item.data}}
  • Improvement-notebook
    • 2019.9.20 ~ 2019.9.30
  • Characterization-notebook
    • 2019.9.10 ~ 2019.9.20