iGEM 2019 — Team HBUT-China

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# Team:HBUT-China/Notebook

[![](https://static.igem.org/mediawiki/2019/0/07/T--HBUT-China--weblogo.png)](https://2019.igem.org/Team%3AHBUT-China)

* [Home](https://2019.igem.org/Team%3AHBUT-China)
* Project
  + [Inspiration & Description](https://2019.igem.org/Team%3AHBUT-China/Description)
  + [Design](https://2019.igem.org/Team%3AHBUT-China/Design)
  + [Improvement](https://2019.igem.org/Team%3AHBUT-China/Improvement)
  + [Characterization](https://2019.igem.org/Team%3AHBUT-China/Characterization)
  + [Future plan](https://2019.igem.org/Team%3AHBUT-China/Future_Plan)
* Lab
  + [Notebook](https://2019.igem.org/Team%3AHBUT-China/Notebook)
  + [Results](https://2019.igem.org/Team%3AHBUT-China/Result)
  + [Parts](https://2019.igem.org/Team%3AHBUT-China/Parts)
  + [Safety](https://2019.igem.org/Team%3AHBUT-China/Safety)
* [Model](https://2019.igem.org/Team%3AHBUT-China/Model)
* Human Practices
  + [Overview](https://2019.igem.org/Team%3AHBUT-China/Human_Practices#Overview)
  + [Integrated Hunan Practices](https://2019.igem.org/Team%3AHBUT-China/Human_Practices#Integrated_Hunan_Practices)
  + [Education & Public Engagement](https://2019.igem.org/Team%3AHBUT-China/Human_Practices#Education_&_Public_Engagement)
* Team
  + [Team Member](https://2019.igem.org/Team%3AHBUT-China/TeamMember)
  + [Attributions](https://2019.igem.org/Team%3AHBUT-China/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3AHBUT-China/Collaborations)
* Device
  + [Mini Factory](https://2019.igem.org/Team%3AHBUT-China/Mini_Factory)
  + [Demonstrate](https://2019.igem.org/Team%3AHBUT-China/Demonstrate)
  + [3D Printing Technology](https://2019.igem.org/Team%3AHBUT-China/3DPrintingTechnology)
  + [Automatic Control System](https://2019.igem.org/Team%3AHBUT-China/AutomaticControlSystem)
* Awards
  + [Medals](https://2019.igem.org/Team%3AHBUT-China/Medals)
  + [Judging Form](https://igem.org/2019_Judging_Form?id=3126)

# Method-Protocol

1. [Characterization](https://static.igem.org/mediawiki/2019/a/a1/T--HBUT-China--Characterization.pdf)
2. [Cloning
   Protocol E.coli—Transformation](https://static.igem.org/mediawiki/2019/a/ac/T--HBUT-China--Cloning--Protocol--E.coli--Transformation.pdf)
3. [Cytotoxicity
   of Yeast to Ni2+](https://static.igem.org/mediawiki/2019/b/b4/T--HBUT-China--Cytotoxicity--of--Yeast--to--Ni2%2B.pdf)
4. [Fabrication
   Method of Ni2+](https://static.igem.org/mediawiki/2019/5/52/T--HBUT-China--Fabrication--Method--of--Ni2%2B.pdf)
5. [Molecular
   Cloning Plasmid extraction DNA electrophoresis DNA agarose gel—extraction](https://static.igem.org/mediawiki/2019/5/5a/T--HBUT-China--Molecular--Cloning--Plasmid--extraction--DNA--electrophoresis--DNA--agarose--gel--extraction.pdf)
6. [Molecular
   Cloning Protocol DNA—Recombination](https://static.igem.org/mediawiki/2019/7/71/T--HBUT-China--Molecular--Cloning--Protocol--DNA--Recombination.pdf)
7. [Standard
   Curves of OD600 and Cell Dry—Weight](https://static.igem.org/mediawiki/2019/f/f9/T--HBUT-China--Standard--Curves--of--OD600--and--Cell--Dry--Weight.pdf)
8. [Yeast
   Cell—Immobilization](https://static.igem.org/mediawiki/2019/9/99/T--HBUT-China--Yeast--Cell--Immobilization.pdf)
9. [Yeast
   Transformation—Protocol](https://static.igem.org/mediawiki/2019/2/2d/T--HBUT-China--Yeast--Transformation--Protocol.pdf)
10. [Seamless Cloning](https://static.igem.org/mediawiki/2019/5/57/T--HBUT-China--Seamless_Cloning.pdf)

# 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)

pTEF1-tCYC1

GAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCC**CCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAA**GGATCC**TCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGC**AGGCCTTACTAGTAGCGGCCG

pGPM1-tTDH1

GAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCC**GCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATA**GGATCC**ATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACG**AGGCCTTACTAGTAGCGGCCGCTGCAG

pTDH3-tPGK1

GAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCC**CAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAA**GGATCC**ATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGT**AGGCCTTACTAGTAGCGGCCGCTGCAG

pTEF2-tENO2

GAATTCGCGGCCGCTTCTAGAG**TTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAAC**GGATCC**AGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAAT**AAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCAG

BBa\_K3126005（hexa-His）

GGTCTCAATCTAAGTTTTAATTACAAAGGATCC**ATGAGATTCCCTAGTATCTTCACCGCCGTCTTGTTCGCTGCTAGTTCTGCTTTGGCTGCTCCAGTCAACACTACCACCGAAGACGAAACCGCTCAGATCCCAGCTGAAGCCGTCATCGGTTACCTTGACCTTGAGGGTGACTTCGATGTCGCCGTCCTTCCATTCTCTAACAGTACCAACAACGGTCTTTTGTTCATTAACACCACCATTGCTTCTATCGCCGCTAAGGAGGAGGGTGTCTCTCTTGACAAGAGACATCACCACCACCATCACAGTGCTAAGTCTAGTTTCATCTCTACCACCACTACCGACCTTACCTCTATCAACACCAGTGCCTACAGTACCGGTTCTATCTCTACCGTCGAAACCGGAAACAGAACCACCTCTGAGGTCATCTCTCATGTCGTCACCACCTCTACCAAGCTTAGTCCTACCGCCACCACCAGTCTTACCATCGCTCAAACTTCTATTTATAGTACCGACTCTAATATTACCGTCGGTACCGACATCCACACCACCAGTGAAGTCATCTCTGACGTCGAAACCATCTCTAGGGAAACCGCCTCTACCGTTGTTGCCGCCCCAACTTCTACCACTGGTTGGACCGGTGCCATGAACACTTACATCTCTCAATTCACTTCTTCTAGTTTTGCTACTATTAACTCTACCCCTATCATCTCTTCTAGTGCCGTCTTCGAAACCAGTGACGCCTCTATCGTCAACGTCCATACCGAGAACATCACCAACACTGCTGCCGTTCCATCTGAGGAGCCTACCTTCGTCAACGCCACTAGAAACTCTCTTAATTCTTTTTGTTCTTCTAAACAACCATCTTCTCCTAGTTCTTATACTTCTTCTCCTTTGGTTTCTTCTTTGTCTGTTTCTAAGACCTTGTTGTCTACCTCTTTCACCCCTTCTGTCCCAACTTCTAATACTTATATTAAGACCAAGAACACTGGTTACTTCGAGCACACTGCCTTGACCACCTCTAGTGTTGGATTGAATAGTTTCTCTGAAACCGCCGTCTCTAGTCAAGGTACCAAGATTGACACCTTTTTGGTTTCTAGTTTGATCGCCTACCCATCTTCTGCCTCTGGTTCTCAGCTTAGTGGTATCCAGCAAAATTTTACCTCTACTTCTTTGATGATCAGTACCTACGAGGGTAAGGCCAGTATCTTCTTCTCTGCCGAGTTGGGTTCTATTATTTTTTTGTTGCTTAGTTATTTGTTGTTTTAAAACGGGTACTGTACAGTTAGTACATTGAGTCGAAATATACGAAATTATTGTTCATAATTTTCATCCTGGCTCTTTTTTTCTTCAACCATAGTTAAATGGACAGTTCATATCTTAACTCTAATAATACTTTTCTAGTTCTTATCCTTTTCCGTCTCACCGCAGATTTTATCATAGTATTAAATTTATATTTTGTTCGTAAAAAGAAAAATTTGTGAGCGTTACCGCTCGTTTCATTACCCGAAGGCTGTTTCAGTAGACCACTGATTAAGTAAGTAGATGAAAAAATTTCATCACCATGAAAGAGTTCGATGAGAGCTACTTTTTCAAATGCTTAACAGCTAACCGCCATTCAATAATGTTACGCTCTCTTCATTCTGCGGCTACGTTATCTAACAAGAGGTTTTACTCTCTCATATCTCATTCAAATAGAAAGAACATAATCAAAA**GGATCCTCATGTAATTAGTTATGTCACGAGACC

BBa\_K3126006（nixA）

GGTCTCACAAACACACATATTACAATAGGATCCATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTAT**CCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAA**GGATCCATAAAGCAATCTTGATGAGGAGAGACC

BBa\_K3126007（TgMTP1t2）

GGTCTCAAACACACATAAACAAACAAAGGATCC**ATGGAGAGTTCTTCTCATATTATTGAAGTCAACGGAGGTAGATCTGACGAGGAAAGAAGGGCTGTCGCTAGTAAGGTCTGCGGAGAAGCTCCTTGCGGTTTCAGTGACGCTAAGAACGCTTCTGGTGACGCCCAAGAAAGGAACGCCTCTATGAGGAAATTGTGCATCGCCGTCGTCTTGTGCCTTGTCTTCATGTCTGTCGAGGTCGTTGGTGGTGTCAAGGCCAACAGTTTGGCCATCATGACCGATGCCGCTCATCTTTTGAGTGACGTTGCCGCTTTCGCCATCTCTTTGTTCAGTCTTTGGGCTGCCGGTTGGGAGGCTACCCCAAGGCACACCTACGGATTCTTCAGAATCGAAATTCTTGGAGCTCTTGTTTCTATCCAATTGATTTGGTTGCTTACTGGTATCCTTGTTTACGAGGCCATCATTAGACTTTTGACCGAAACCTCTGAGGTCAACGGTTTCTTGATGTTCGCTGTCGCCACTTTCGGTTTGCTTGTCAACATTATCATGGCTGTCATGCTTGGTCACGACCATGACAATGGTCACGGTCATAGTCACGGTGAGGACAAACACGATGAAGCCCATGGTGACGTCACTGAACAGCTTCTTGAAAAGCCAAAACAGCAGAAGGAAAAAAAGAAGAGGAACATCAACGCCCAAGGTGCCTATTTGCACGTCTTGGGTGACTCTATCCAGTCTGTCGGAGTCATGATCGGTGGTGCTGCTATCTGGTACAATCCAAAGTGGAAGATCATTGACTTGATCTGCACCTTGGTCTTCTCTGTCATCGTCTTGGGAACCACCATCAATATGATCAGAAGTATTCTTGAGGTCTTGATGGAGAGTACCCCAAGAGAAATCGACGCCACCAAGCTTGAGAAGGGTCTTCTTGAGATGGAGGAGGTCGTCGCTGTTCACGAATTGCACATCTGGGCCTCTACCGTTGGTAAGGTCTTGCTTGCTTGCCATGTCAACGCCAGACCAGATGCCGATGCCGATATGGTCCTTAACAAAGTCGTTGACTATATTAGGAGAGAATATAACATTTCTCATGTTACTATCCAAATTGAAAGATAA**GGATCCATTGAATTGAATTGAAATCGAGAGACC

BBa\_K3126008（kanR）

GGTCTCACGAACTATAATTAACTAAACGGATCCATAACTTCGTATAATGTATGCTATACGAAGTTAT**ATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAA**ATAACTTCGTATAATGTATGCTATACGAAGTTATGGATCCAGTGCTTTTAACTAAGAATTAGAGACC

# 2019.6.1 ~ 6.10

#### Construction plasmids：

#### pSB1C3-pTEF1/tCYC1

The sequence of pSB1C3-pTEF1/tCYC1

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGGATCCTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGCCTTACTAGTAGCGGCCGCTGCA

#### pSB1C3-pGPM1/tTDH1

The sequence of pSB1C3-pGPM1/tTDH1

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCCTTACTAGTAGCGGCCGCTGCA

#### pSB1C3-pTDH3/tPGK1

The sequence of pSB1C3-pTDH3/tPGK1

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGGATCCATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTAGGCCTTACTAGTAGCGGCCGCTGCA

#### pSB1C3-pTEF2/tENO2

The sequence of pSB1C3-pTEF2/tENO2

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 |
| *EcoR*I | 3 μL |
| *Pst*I | 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 *EcoR*1 and *Pst*1:

![](https://static.igem.org/mediawiki/2019/e/e7/T--HBUT-China--photoBBa-k3126001.png)

Verified pictures of pSB1C3-pGPM1/tTDH1 digested by *EcoR*1 and *Pst*1:

![](https://static.igem.org/mediawiki/2019/8/8b/T--HBUT-China--photoBBa-k3126002.png)

Verified pictures of pSB1C3-pTDH3/tPGK1 digested by *EcoR*1 and *Pst*1:

![](https://static.igem.org/mediawiki/2019/c/cf/T--HBUT-China--photoBBa-k3126003.png)

Verified pictures of pSB1C3-pTEF2/tENO2 digested by *EcoR*1 and *Pst*1:

![](https://static.igem.org/mediawiki/2019/4/48/T--HBUT-China--photoBBa-k3126004.png)

![](https://static.igem.org/mediawiki/2019/a/a8/T--HBUT-China--photoPlasmid--figure--BBa-k3126001.png)
![](https://static.igem.org/mediawiki/2019/4/43/T--HBUT-China--photoPlasmid--figure--BBa-k3126002.png)

![](https://static.igem.org/mediawiki/2019/8/88/T--HBUT-China--photoPlasmid--figure--BBa-k3126003.png)
![](https://static.igem.org/mediawiki/2019/4/48/T--HBUT-China--photoPlasmid--figure--BBa-k3126004.png)

# 2019.6.10 ~ 6.20

#### Construction plasmids：

#### pSB1C3-BBa\_K3126014 (pSB1C3-pTEF1/ hexa-His/ tCYC1)

The sequence of pSB1C3-BBa\_K3126014

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGGATCC**ATGAGATTCCCTAGTATCTTCACCGCCGTCTTGTTCGCTGCTAGTTCTGCTTTGGCTGCTCCAGTCAACACTACCACCGAAGACGAAACCGCTCAGATCCCAGCTGAAGCCGTCATCGGTTACCTTGACCTTGAGGGTGACTTCGATGTCGCCGTCCTTCCATTCTCTAACAGTACCAACAACGGTCTTTTGTTCATTAACACCACCATTGCTTCTATCGCCGCTAAGGAGGAGGGTGTCTCTCTTGACAAGAGACATCACCACCACCATCACAGTGCTAAGTCTAGTTTCATCTCTACCACCACTACCGACCTTACCTCTATCAACACCAGTGCCTACAGTACCGGTTCTATCTCTACCGTCGAAACCGGAAACAGAACCACCTCTGAGGTCATCTCTCATGTCGTCACCACCTCTACCAAGCTTAGTCCTACCGCCACCACCAGTCTTACCATCGCTCAAACTTCTATTTATAGTACCGACTCTAATATTACCGTCGGTACCGACATCCACACCACCAGTGAAGTCATCTCTGACGTCGAAACCATCTCTAGGGAAACCGCCTCTACCGTTGTTGCCGCCCCAACTTCTACCACTGGTTGGACCGGTGCCATGAACACTTACATCTCTCAATTCACTTCTTCTAGTTTTGCTACTATTAACTCTACCCCTATCATCTCTTCTAGTGCCGTCTTCGAAACCAGTGACGCCTCTATCGTCAACGTCCATACCGAGAACATCACCAACACTGCTGCCGTTCCATCTGAGGAGCCTACCTTCGTCAACGCCACTAGAAACTCTCTTAATTCTTTTTGTTCTTCTAAACAACCATCTTCTCCTAGTTCTTATACTTCTTCTCCTTTGGTTTCTTCTTTGTCTGTTTCTAAGACCTTGTTGTCTACCTCTTTCACCCCTTCTGTCCCAACTTCTAATACTTATATTAAGACCAAGAACACTGGTTACTTCGAGCACACTGCCTTGACCACCTCTAGTGTTGGATTGAATAGTTTCTCTGAAACCGCCGTCTCTAGTCAAGGTACCAAGATTGACACCTTTTTGGTTTCTAGTTTGATCGCCTACCCATCTTCTGCCTCTGGTTCTCAGCTTAGTGGTATCCAGCAAAATTTTACCTCTACTTCTTTGATGATCAGTACCTACGAGGGTAAGGCCAGTATCTTCTTCTCTGCCGAGTTGGGTTCTATTATTTTTTTGTTGCTTAGTTATTTGTTGTTTTAAAACGGGTACTGTACAGTTAGTACATTGAGTCGAAATATACGAAATTATTGTTCATAATTTTCATCCTGGCTCTTTTTTTCTTCAACCATAGTTAAATGGACAGTTCATATCTTAACTCTAATAATACTTTTCTAGTTCTTATCCTTTTCCGTCTCACCGCAGATTTTATCATAGTATTAAATTTATATTTTGTTCGTAAAAAGAAAAATTTGTGAGCGTTACCGCTCGTTTCATTACCCGAAGGCTGTTTCAGTAGACCACTGATTAAGTAAGTAGATGAAAAAATTTCATCACCATGAAAGAGTTCGATGAGAGCTACTTTTTCAAATGCTTAACAGCTAACCGCCATTCAATAATGTTACGCTCTCTTCATTCTGCGGCTACGTTATCTAACAAGAGGTTTTACTCTCTCATATCTCATTCAAATAGAAAGAACATAATCAAAA**GGATCCTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGCCTTACTAGTAGCGGCCGCTGCA

#### pSB1C3-BBa\_K3126015(pSB1C3-pGPM1/nixA/tTDH1/)

The sequence of pSB1C3-BBa\_K3126015

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCC**ATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTATCCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAA**GGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCCTTACTAGTAGCGGCCGCTGCA

#### pSB1C3-BBa\_K3126016(pSB1C3-pTDH3/TgMTP1t2/tPGK1)

The sequence of pSB1C3-BBa\_K3126016

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGGATCC**ATGGAGAGTTCTTCTCATATTATTGAAGTCAACGGAGGTAGATCTGACGAGGAAAGAAGGGCTGTCGCTAGTAAGGTCTGCGGAGAAGCTCCTTGCGGTTTCAGTGACGCTAAGAACGCTTCTGGTGACGCCCAAGAAAGGAACGCCTCTATGAGGAAATTGTGCATCGCCGTCGTCTTGTGCCTTGTCTTCATGTCTGTCGAGGTCGTTGGTGGTGTCAAGGCCAACAGTTTGGCCATCATGACCGATGCCGCTCATCTTTTGAGTGACGTTGCCGCTTTCGCCATCTCTTTGTTCAGTCTTTGGGCTGCCGGTTGGGAGGCTACCCCAAGGCACACCTACGGATTCTTCAGAATCGAAATTCTTGGAGCTCTTGTTTCTATCCAATTGATTTGGTTGCTTACTGGTATCCTTGTTTACGAGGCCATCATTAGACTTTTGACCGAAACCTCTGAGGTCAACGGTTTCTTGATGTTCGCTGTCGCCACTTTCGGTTTGCTTGTCAACATTATCATGGCTGTCATGCTTGGTCACGACCATGACAATGGTCACGGTCATAGTCACGGTGAGGACAAACACGATGAAGCCCATGGTGACGTCACTGAACAGCTTCTTGAAAAGCCAAAACAGCAGAAGGAAAAAAAGAAGAGGAACATCAACGCCCAAGGTGCCTATTTGCACGTCTTGGGTGACTCTATCCAGTCTGTCGGAGTCATGATCGGTGGTGCTGCTATCTGGTACAATCCAAAGTGGAAGATCATTGACTTGATCTGCACCTTGGTCTTCTCTGTCATCGTCTTGGGAACCACCATCAATATGATCAGAAGTATTCTTGAGGTCTTGATGGAGAGTACCCCAAGAGAAATCGACGCCACCAAGCTTGAGAAGGGTCTTCTTGAGATGGAGGAGGTCGTCGCTGTTCACGAATTGCACATCTGGGCCTCTACCGTTGGTAAGGTCTTGCTTGCTTGCCATGTCAACGCCAGACCAGATGCCGATGCCGATATGGTCCTTAACAAAGTCGTTGACTATATTAGGAGAGAATATAACATTTCTCATGTTACTATCCAAATTGAAAGATAA**GGATCCATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTAGGCCTTACTAGTAGCGGCCGCTGCA

#### pSB1C3-BBa\_K3126017 (pSB1C3-pTEF2/kanR/tENO2)

The sequence of pSB1C3-BBa\_K3126017

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCC**ATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAA**GGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

Insertion of gene into plasmid carrying promoter and terminator.

pSB1C3-BBa\_K3126014 (pSB1C3-pTEF1/ hexa His/ tCYC1):

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-pTEF1/tCYC1 | 20 μL |
| *Bam*HI | 3 μL |
| buffer | 5 μL |
| ddH2O | 22 μL |

Seamless cloning system:

|  |  |
| --- | --- |
| pSB1C3-pTEF1/tCYC1 | 2 μL |
| hexa-His | 4 μL |
| 5×CEⅡ Buffer | 2 μL |
| Exnase Ⅱ | 1 μL |
| ddH2O | 1 μL |

pSB1C3-BBa\_K3126015 (pSB1C3-pGPM1/ nixA/tTDH1):

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-pGPM1/tTDH1 | 20 μL |
| *Bam*HI | 3 μL |
| buffer | 5 μL |
| ddH2O | 22 μL |

Seamless cloning system:

|  |  |
| --- | --- |
| pSB1C3-pGPM1/tTDH1 | 2 μL |
| nixA | 4 μL |
| 5×CEⅡ Buffer | 2 μL |
| Exnase Ⅱ | 1 μL |
| ddH2O | 1 μL |

pSB1C3-BBa\_K3126016 (pSB1C3-pTDH3/TgMTP1t2/tPGK1):

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-pTDH3/ tPGK1 | 20 μL |
| *Bam*HI | 3 μL |
| buffer | 5 μL |
| ddH2O | 22 μL |

Seamless cloning system:

|  |  |
| --- | --- |
| pSB1C3-pTDH3/ tPGK1 | 2 μL |
| TgMTP1t2 | 4 μL |
| 5×CEⅡ Buffer | 2 μL |
| Exnase Ⅱ | 1 μL |
| ddH2O | 1 μL |

pSB1C3-BBa\_K3126017 (pSB1C3-pTEF2/kanR/tENO2):

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-pTEF2/tENO2 | 20 μL |
| *Bam*HI | 3 μL |
| buffer | 5 μL |
| ddH2O | 22 μL |

Seamless cloning system:

|  |  |
| --- | --- |
| pSB1C3-pTEF2/tENO2 | 2 μL |
| kanR | 4 μL |
| 5×CEⅡ Buffer | 2 μL |
| Exnase Ⅱ | 1 μL |
| ddH2O | 1 μL |

![](https://static.igem.org/mediawiki/2019/f/f0/T--HBUT-China--photoPlasmid--figure--BBa-k3126005.png)
![](https://static.igem.org/mediawiki/2019/1/15/T--HBUT-China--photoPlasmid--figure--BBa-k3126006.png)

![](https://static.igem.org/mediawiki/2019/6/65/T--HBUT-China--photoPlasmid--figure--BBa-k3126007.png)
![](https://static.igem.org/mediawiki/2019/c/cb/T--HBUT-China--photoPlasmid--figure--BBa-k3126008.png)

# 2019.6.20 ~ 6.30

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

#### pSB1C3-BBa\_K3126018 (pSB1C3- hexa-His/ kanR)

The sequence of pSB1C3-BBa\_K3126018

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGGATCCATGAGATTCCCTAGTATCTTCACCGCCGTCTTGTTCGCTGCTAGTTCTGCTTTGGCTGCTCCAGTCAACACTACCACCGAAGACGAAACCGCTCAGATCCCAGCTGAAGCCGTCATCGGTTACCTTGACCTTGAGGGTGACTTCGATGTCGCCGTCCTTCCATTCTCTAACAGTACCAACAACGGTCTTTTGTTCATTAACACCACCATTGCTTCTATCGCCGCTAAGGAGGAGGGTGTCTCTCTTGACAAGAGACATCACCACCACCATCACAGTGCTAAGTCTAGTTTCATCTCTACCACCACTACCGACCTTACCTCTATCAACACCAGTGCCTACAGTACCGGTTCTATCTCTACCGTCGAAACCGGAAACAGAACCACCTCTGAGGTCATCTCTCATGTCGTCACCACCTCTACCAAGCTTAGTCCTACCGCCACCACCAGTCTTACCATCGCTCAAACTTCTATTTATAGTACCGACTCTAATATTACCGTCGGTACCGACATCCACACCACCAGTGAAGTCATCTCTGACGTCGAAACCATCTCTAGGGAAACCGCCTCTACCGTTGTTGCCGCCCCAACTTCTACCACTGGTTGGACCGGTGCCATGAACACTTACATCTCTCAATTCACTTCTTCTAGTTTTGCTACTATTAACTCTACCCCTATCATCTCTTCTAGTGCCGTCTTCGAAACCAGTGACGCCTCTATCGTCAACGTCCATACCGAGAACATCACCAACACTGCTGCCGTTCCATCTGAGGAGCCTACCTTCGTCAACGCCACTAGAAACTCTCTTAATTCTTTTTGTTCTTCTAAACAACCATCTTCTCCTAGTTCTTATACTTCTTCTCCTTTGGTTTCTTCTTTGTCTGTTTCTAAGACCTTGTTGTCTACCTCTTTCACCCCTTCTGTCCCAACTTCTAATACTTATATTAAGACCAAGAACACTGGTTACTTCGAGCACACTGCCTTGACCACCTCTAGTGTTGGATTGAATAGTTTCTCTGAAACCGCCGTCTCTAGTCAAGGTACCAAGATTGACACCTTTTTGGTTTCTAGTTTGATCGCCTACCCATCTTCTGCCTCTGGTTCTCAGCTTAGTGGTATCCAGCAAAATTTTACCTCTACTTCTTTGATGATCAGTACCTACGAGGGTAAGGCCAGTATCTTCTTCTCTGCCGAGTTGGGTTCTATTATTTTTTTGTTGCTTAGTTATTTGTTGTTTTAAAACGGGTACTGTACAGTTAGTACATTGAGTCGAAATATACGAAATTATTGTTCATAATTTTCATCCTGGCTCTTTTTTTCTTCAACCATAGTTAAATGGACAGTTCATATCTTAACTCTAATAATACTTTTCTAGTTCTTATCCTTTTCCGTCTCACCGCAGATTTTATCATAGTATTAAATTTATATTTTGTTCGTAAAAAGAAAAATTTGTGAGCGTTACCGCTCGTTTCATTACCCGAAGGCTGTTTCAGTAGACCACTGATTAAGTAAGTAGATGAAAAAATTTCATCACCATGAAAGAGTTCGATGAGAGCTACTTTTTCAAATGCTTAACAGCTAACCGCCATTCAATAATGTTACGCTCTCTTCATTCTGCGGCTACGTTATCTAACAAGAGGTTTTACTCTCTCATATCTCATTCAAATAGAAAGAACATAATCAAAAGGATCCTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGGCCTTACTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

#### pSB1C3-BBa\_K3126019 (pSB1C3-nixA/ kanR)

The sequence of pSB1C3-BBa\_K3126019

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTATCCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCCTTACTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

#### pSB1C3-BBa\_K3126020 (pSB1C3-TgMTP1t2/ kanR)

The sequence of pSB1C3 BBa\_K3126020

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\_K3126014 | 25 μL |
| *Spe*I | 3 μL |
| *Pst*I | 3 μL |
| Cutsmart Buffer | 5 μL |
| ddH2O | 14 μL |

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126017 | 25 μL |
| *Xba*I | 3 μL |
| *Pst*I | 3 μL |
| Cutsmart Buffer | 5 μL |
| ddH2O | 14 μL |

The enzyme system we use:

|  |  |
| --- | --- |
| BBa\_K3126014 linear plasmid | 2 μL |
| kanR part | 6 μL |
| T4 DNA Ligase | 0.5 μL |
| T4Buffer | 1 μL |
| ddH2O | 0.5 μL |

pSB1C3-BBa\_K3126019 (pSB1C3-nixA/ kanR)

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126015 | 25 μL |
| *Spe*I | 3 μL |
| *Pst*I | 3 μL |
| Cutsmart Buffer | 5 μL |
| ddH2O | 14 μL |

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126017 | 25 μL |
| XbaI | 3 μL |
| *Pst*I | 3 μL |
| Cutsmart Buffer | 5 μL |
| ddH2O | 14 μL |

The enzyme system we use:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126015 linear plasmid | 2 μL |
| kanR part | 4 μL |
| T4 DNA Ligase | 0.5 μL |
| T4Buffer | 1 μL |
| ddH2O | 0.5 μL |

pSB1C3-BBa\_K3126020 (pSB1C3-TgMTP1t2/ kanR)

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126016 | 25 μL |
| *Spe*I | 3 μL |
| *Pst*I | 3 μL |
| Cutsmart Buffer | 5 μL |
| ddH2O | 14 μL |

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126017 | 25 μL |
| XbaI | 3 μL |
| *Pst*I | 3 μL |
| Cutsmart Buffer | 5 μL |
| ddH2O | 14 μL |

The enzyme system we use:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126016 linear plasmid | 2 μL |
| kanR part | 6 μL |
| T4 DNA Ligase | 0.5 μL |
| T4Buffer | 1 μL |
| ddH2O | 0.5 μL |

![](https://static.igem.org/mediawiki/2019/3/31/T--HBUT-China--photoPlasmid--figure--BBa-k3126009.png)
![](https://static.igem.org/mediawiki/2019/b/ba/T--HBUT-China--photoPlasmid--figure--BBa-k3126010.png)
![](https://static.igem.org/mediawiki/2019/6/6e/T--HBUT-China--photoPlasmid--figure--BBa-k3126011.png)

# 2019.7.1 ~ 7.10

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

#### pSB1C3-BBa\_K3126021 (pSB1C3- hexa-His/ nixA/ kanR)

The sequence of pSB1C3-BBa\_K3126021

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCCCTTGCCAACAGGGAGTTCTTCAGAGACATGGAGGCTCAAAACGAAATTATTGACAGCCTAGACATCAATAGTCATACAACAGAAAGCGACCACCCAACTTTGGCTGATAATAGCGTATAAACAATGCATACTTTGTACGTTCAAAATACAATGCAGTAGATATATTTATGCATATTACATATAATACATATCACATAGGAAGCAACAGGCGCGTTGGACTTTTAATTTTCGAGGACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGACACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCATCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAAGGATCCATGAGATTCCCTAGTATCTTCACCGCCGTCTTGTTCGCTGCTAGTTCTGCTTTGGCTGCTCCAGTCAACACTACCACCGAAGACGAAACCGCTCAGATCCCAGCTGAAGCCGTCATCGGTTACCTTGACCTTGAGGGTGACTTCGATGTCGCCGTCCTTCCATTCTCTAACAGTACCAACAACGGTCTTTTGTTCATTAACACCACCATTGCTTCTATCGCCGCTAAGGAGGAGGGTGTCTCTCTTGACAAGAGACATCACCACCACCATCACAGTGCTAAGTCTAGTTTCATCTCTACCACCACTACCGACCTTACCTCTATCAACACCAGTGCCTACAGTACCGGTTCTATCTCTACCGTCGAAACCGGAAACAGAACCACCTCTGAGGTCATCTCTCATGTCGTCACCACCTCTACCAAGCTTAGTCCTACCGCCACCACCAGTCTTACCATCGCTCAAACTTCTATTTATAGTACCGACTCTAATATTACCGTCGGTACCGACATCCACACCACCAGTGAAGTCATCTCTGACGTCGAAACCATCTCTAGGGAAACCGCCTCTACCGTTGTTGCCGCCCCAACTTCTACCACTGGTTGGACCGGTGCCATGAACACTTACATCTCTCAATTCACTTCTTCTAGTTTTGCTACTATTAACTCTACCCCTATCATCTCTTCTAGTGCCGTCTTCGAAACCAGTGACGCCTCTATCGTCAACGTCCATACCGAGAACATCACCAACACTGCTGCCGTTCCATCTGAGGAGCCTACCTTCGTCAACGCCACTAGAAACTCTCTTAATTCTTTTTGTTCTTCTAAACAACCATCTTCTCCTAGTTCTTATACTTCTTCTCCTTTGGTTTCTTCTTTGTCTGTTTCTAAGACCTTGTTGTCTACCTCTTTCACCCCTTCTGTCCCAACTTCTAATACTTATATTAAGACCAAGAACACTGGTTACTTCGAGCACACTGCCTTGACCACCTCTAGTGTTGGATTGAATAGTTTCTCTGAAACCGCCGTCTCTAGTCAAGGTACCAAGATTGACACCTTTTTGGTTTCTAGTTTGATCGCCTACCCATCTTCTGCCTCTGGTTCTCAGCTTAGTGGTATCCAGCAAAATTTTACCTCTACTTCTTTGATGATCAGTACCTACGAGGGTAAGGCCAGTATCTTCTTCTCTGCCGAGTTGGGTTCTATTATTTTTTTGTTGCTTAGTTATTTGTTGTTTTAAAACGGGTACTGTACAGTTAGTACATTGAGTCGAAATATACGAAATTATTGTTCATAATTTTCATCCTGGCTCTTTTTTTCTTCAACCATAGTTAAATGGACAGTTCATATCTTAACTCTAATAATACTTTTCTAGTTCTTATCCTTTTCCGTCTCACCGCAGATTTTATCATAGTATTAAATTTATATTTTGTTCGTAAAAAGAAAAATTTGTGAGCGTTACCGCTCGTTTCATTACCCGAAGGCTGTTTCAGTAGACCACTGATTAAGTAAGTAGATGAAAAAATTTCATCACCATGAAAGAGTTCGATGAGAGCTACTTTTTCAAATGCTTAACAGCTAACCGCCATTCAATAATGTTACGCTCTCTTCATTCTGCGGCTACGTTATCTAACAAGAGGTTTTACTCTCTCATATCTCATTCAAATAGAAAGAACATAATCAAAAGGATCCTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAGG**GCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTATCCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACG**AGGCCTTACTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

#### pSB1C3-BBa\_K3126022 (pSB1C3- nixA/ TgMTP1t2/ kanR)

The sequence of pSB1C3-BBa\_K3126022

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGGGTCTCATGTCTATCCCAGAAACTCAAAAAGGTGTTATCTTCTACGAATCCCGCCAAACTTTTCGGTTAACACATGCAGTGATGCACGCGCGATGGTGCTAAGTTACATATATATATATATATATATATATATATATATATAGCCATAGTGATGTCTAAGTAACCTTTATGGTATATTTCTTAATGTGGAAAGATACTAGCGCGCGCACCCACACACAAGCTTCGTCTTTTCTTGAAGAAAAGAGGAAGCTCGCTAAATGGGATTCCACTTTCCGTTCCCTGCCAGCTGATGGAAAAAGGTTAGTGGAACGATGAAGAATAAAAAGAGAGATCCACTGAGGTGAAATTTCAGCTGACAGCGAGTTTCATGATCGTGATGAACAATGGTAACGAGTTGTGGCTGTTGCCAGGGAGGGTGGTTCTCAACTTTTAATGTATGGCCAAATCGCTACTTGGGTTTGTTATATAACAAAGAAGAAATAATGAACTGATTCTCTTCCTCCTTCTTGTCCTTTCTTAATTCTGTTGTAATTACCTTCCTTTGTAATTTTTTTTGTAATTATTCTTCTTAATAATCCAAACAAACACACATATTACAATAGGATCCATGATTAAGTACCATGTCATGAAAGAAGGTTACATCTTGACTGTTTTTAAAAATGAAAGGTTGAGTTGGTTGCCATATATCGCCATCGTCATTTTGTTGCACGTTATTGGTTTCTCTTTCTTGTGGATCGCCGGTAAGGACCATCACATTTTGTTCGGTATGGGTATCCTTGCCTACACTCTTGGTTTGAGGCATGCCTTTGATGCCGACCATATCGCCGCTATCGACAACACCGTTAGAAAGTTGTTGCAGCAGAGAAAGGACCCATCTGGTGTCGGTTTCTACTTCTCTATTGGACATTCTTCTGTCGTCTTCTTGATGGCCGTTTTCCTTGGTGTCTCTGTCAAGTGGGCCAAGGACGAACTTCCTCACTTCCAAGATATCGGAGGTACCATTGGAACCCTTGTTTCTGGTTTTTTCTTGGTTCTTATTGGAGTTCTTAATCTTATTATTCTTATTTCTTTGATTAACTTGTTCGCCAAGTTGAGGAGGGAACACATCGAAGAAGCTGAGGTCGATGCCTTGTTGGAGTCTAGGGGTCTTGTCTCTAGGTTCGTCGGACCATACTTTAAGTTGATCACCAGAAGTTGGCACGTCTTGCCTCTTGGATTCCTTTTCGGATTGGGATTCGACACCGCTTCTGAGATCGCCTTGCTTGCCCTTAGTAGTGGAGCCTCTCAGCAAGCTATCTCTTTCATTGGTATTTTGTCTTTGCCTATCTTGTTCGCCAGTGGTATGAGTCTTCTTGACACCCTTGACGGTGTCGTCATGAAGTACGCCTACAACTGGGCCTTCTTCAACCCTATCAGAAAAATCTATTACAACATTACCATTACCGCCATCTCTGTTATGGCCGCCCTTGTTATCGGTATGATCGAATTGCTTCAAATTCTTGCCGACAAACTTGACTTGCATGGAGCCTTCTGGGCCTTCATTGGAAGTATCGAGTTCGATTATTTGGGTTATATCTTGGTTGCCTTGTTTCTTATTACTTGGTTGATCAGTTCTTTGATTTGGAAATTTGGAAGAATTGAACACAAATGGAGTAGATAAGGATCCATAAAGCAATCTTGATGAGGATAATGATTTTTTTTTGAATATACATAAATACTACCGTTTTTCTGCTAGATTTTGTGATGACGTAAATAAGTACATATTACTTTTTAAGCCAAGACAAGATTAAGCATTAACTTTACCCTTTTCTTTCTAAGTTTCAATATTAGTTATCACTGTTTAAAAGTTATGGCGAGAACGTCGGCGGTTAAAATATATTACCCTGAACGAGGCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAAGGATCCATGGAGAGTTCTTCTCATATTATTGAAGTCAACGGAGGTAGATCTGACGAGGAAAGAAGGGCTGTCGCTAGTAAGGTCTGCGGAGAAGCTCCTTGCGGTTTCAGTGACGCTAAGAACGCTTCTGGTGACGCCCAAGAAAGGAACGCCTCTATGAGGAAATTGTGCATCGCCGTCGTCTTGTGCCTTGTCTTCATGTCTGTCGAGGTCGTTGGTGGTGTCAAGGCCAACAGTTTGGCCATCATGACCGATGCCGCTCATCTTTTGAGTGACGTTGCCGCTTTCGCCATCTCTTTGTTCAGTCTTTGGGCTGCCGGTTGGGAGGCTACCCCAAGGCACACCTACGGATTCTTCAGAATCGAAATTCTTGGAGCTCTTGTTTCTATCCAATTGATTTGGTTGCTTACTGGTATCCTTGTTTACGAGGCCATCATTAGACTTTTGACCGAAACCTCTGAGGTCAACGGTTTCTTGATGTTCGCTGTCGCCACTTTCGGTTTGCTTGTCAACATTATCATGGCTGTCATGCTTGGTCACGACCATGACAATGGTCACGGTCATAGTCACGGTGAGGACAAACACGATGAAGCCCATGGTGACGTCACTGAACAGCTTCTTGAAAAGCCAAAACAGCAGAAGGAAAAAAAGAAGAGGAACATCAACGCCCAAGGTGCCTATTTGCACGTCTTGGGTGACTCTATCCAGTCTGTCGGAGTCATGATCGGTGGTGCTGCTATCTGGTACAATCCAAAGTGGAAGATCATTGACTTGATCTGCACCTTGGTCTTCTCTGTCATCGTCTTGGGAACCACCATCAATATGATCAGAAGTATTCTTGAGGTCTTGATGGAGAGTACCCCAAGAGAAATCGACGCCACCAAGCTTGAGAAGGGTCTTCTTGAGATGGAGGAGGTCGTCGCTGTTCACGAATTGCACATCTGGGCCTCTACCGTTGGTAAGGTCTTGCTTGCTTGCCATGTCAACGCCAGACCAGATGCCGATGCCGATATGGTCCTTAACAAAGTCGTTGACTATATTAGGAGAGAATATAACATTTCTCATGTTACTATCCAAATTGAAAGATAAGGATCCATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAAATTCGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTTCCCATTCGATATTTCTATGTAGGCCTTACTAGAGTTGATAGGTCAAGATCAATGTAAACAATTACTTTGTTATGTAGAGTTTTTTTAGCTACCTATATTCCACCATAACATCAATCATGCGGTTGCTGGTGTATTTACCAATAATGTTTAATGTATATATATATATATATATATGGGGCCGTATACTTACATATAGTAGATGTCAAGCGTAGGCGCTTCCCCTGCCGGCTGTGAGGGCGCCATAACCAAGGTATCTATAGACCGCCAATCAGCAAACTACCTCCGTACATTCATGTTGCACCCACACATTTATACACCCAGACCGCGACAAATTACCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACCAAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCTTCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGGTTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTTTTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTTTTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGTTTTTAGAATATACGGTCAACGAACTATAATTAACTAAACGGATCCATAACTTCGTATAATGTATGCTATACGAAGTTATATGGGAAAGGAGAAGACCCACGTCAGTAGACCAAGGCTTAACAGTAACATGGACGCCGACTTGTACGGATACAAGTGGGCCAGAGACAACGTCGGTCAGTCTGGAGCTACCATCTATAGACTTTACGGAAAGCCAGACGCCCCAGAGTTGTTCCTTAAGCACGGTAAGGGATCTGTTGCCAACGACGTCACCGATGAGATGGTTAGACTTAACTGGCTTACCGAGTTCATGCCACTTCCAACCATCAAGCACTTCATTAGAACCCCAGATGACGCTTGGTTGTTGACCACTGCCATCCCCGGTAAGACCGCCTTTCAAGTTTTGGAAGAGTACCCAGACTCTGGAGAGAACATCGTTGACGCCTTGGCCGTCTTCCTTAGGAGGTTGCACTCTATCCCAGTTTGCAACTGCCCATTCAATAGTGATAGAGTCTTCAGACTTGCCCAAGCCCAGTCTAGGATGAACAACGGATTGGTCGATGCCTCTGACTTCGACGACGAGAGAAATGGATGGCCAGTCGAGCAAGTTTGGAAGGAGATGCATAAGCTTTTGCCTTTTTCTCCAGACAGTGTTGTCACCCACGGTGACTTTAGTTTGGACAACCTTATTTTCGACGAGGGTAAATTGATCGGTTGCATCGACGTCGGTAGAGTTGGTATCGCCGATAGATACCAAGACTTGGCCATCTTGTGGAACTGCTTGGGTGAGTTCTCTCCAAGTCTTCAAAAAAGATTGTTCCAAAAATATGGTATTGACAACCCAGATATGAATAAATTGCAATTTCATTTGATGTTGGATGAGTTTTTCTAAATAACTTCGTATAATGTATGCTATACGAAGTTATGGATCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCTTATTTTTTCATCATAGTTTAGAACACTTTATATTAACGAATAGTTTATGAATCTATTTAGGTTTAAAAATTGATACAGTTTTATAAGTTACTTTTTCAAAGACTCGTGCTGTCTATTGCATAATGCACTGGAAGGGGAAAAAAAAGGTGCACACGCGTGGCTTTTTCTTGAATTTGCAGTTTGAAAAATAAAGGGTCAAATCGTTGGTAGATACGTTGTTGACACTTCTAAATAAGAGACCTACTAGTAGCGGCCGCTGCA

#### pSB1C3-BBa\_K3126023 (pSB1C3- hexa-His/ nixA/ TgMTP1t2/ kanR)

The sequence of pSB1C3-BBa\_K3126023

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

ACCTATCAACTCTAGTA**AGGCCT**CGTTCAGGGTAATATATTTT)

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126015/SpeⅠ *Pst*Ⅰ | 1 μL |
| P-nixA-T\_F | 2 μL |
| P-nixA-T\_R | 2 μL |
| 2×PCR mix | 20 μL |
| ddH2O | 15 μL |

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126018 | 15 μL |
| *Stu*I | 3 μL |
| buffer | 5 μL |
| ddH2O | 27 μL |

Seamless cloning system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126018/*Stu*Ⅰ | 2 μL |
| pGPM1-nixA-tTDH1 | 4 μL |
| 5×CEⅡ Buffer | 2 μL |
| Exnase Ⅱ | 1 μL |
| ddH2O | 1 μL |

pSB1C3-BBa\_K3126022 (pSB1C3- nixA/ TgMTP1t2/ kanR)

The PCR system of pTDH3-TgMTP1t2-tPGK1

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126016/SpeⅠ *Pst*Ⅰ | 1 μL |
| P-TgMTP1t2-T\_F | 2 μL |
| P-TgMTP1t2-T\_R | 2 μL |
| 2×PCR mix | 20 μL |
| ddH2O | 15 μL |

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126021 | 15 μL |
| *Stu*I | 3 μL |
| buffer | 5 μL |
| ddH2O | 27 μL |

Seamless cloning system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126021/*Stu*Ⅰ | 2 μL |
| pTDH3-TgMTP1t2-tPGK1 | 4 μL |
| 5×CEⅡ Buffer | 2 μL |
| Exnase Ⅱ | 1 μL |
| ddH2O | 1 μL |

pSB1C3-BBa\_K3126023 (pSB1C3- hexa-His/ nixA/ TgMTP1t2/ kanR)

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126021 | 15 μL |
| *Stu*I | 3 μL |
| buffer | 5 μL |
| ddH2O | 27 μL |

Seamless cloning system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126021/*Stu*Ⅰ | 2 μL |
| pTDH3-TgMTP1t2-tPGK1 | 4 μL |
| 5×CEⅡ Buffer | 2 μL |
| Exnase Ⅱ | 1 μL |
| ddH2O | 1 μL |

![](https://static.igem.org/mediawiki/2019/4/4e/T--HBUT-China--photoPlasmid--figure--BBa-k3126014.png)
![](https://static.igem.org/mediawiki/2019/3/3b/T--HBUT-China--photoPlasmid--figure--BBa-k3126013.png)
![](https://static.igem.org/mediawiki/2019/6/65/T--HBUT-China--photoPlasmid--figure--BBa-k3126012.png)

# 2019.7.20 ~ 7.25

#### The plasmid is digested with *Bsa*1 to get the fragment to be integrated.

pSB1C3- hexa-His- kanR/ *Bsa*Ⅰ

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126018 | 25μL |
| *Bsa*I | 3 μL |
| buffer | 5 μL |
| ddH2O | 17 μL |

![](https://static.igem.org/mediawiki/2019/c/ce/T--HBUT-China--photoBBa-k3126009.png)

pSB1C3-nixA- kanR/ *Bsa*Ⅰ

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126019 | 25μL |
| *Bsa*I | 3 μL |
| buffer | 5 μL |
| ddH2O | 17 μL |

![](https://static.igem.org/mediawiki/2019/a/a8/T--HBUT-China--photoBBa-k3126010.png)

pSB1C3-TgMTP1t2- kanR/ *Bsa*Ⅰ

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126020 | 25μL |
| *Bsa*I | 3 μL |
| buffer | 5 μL |
| ddH2O | 17 μL |

![](https://static.igem.org/mediawiki/2019/0/0d/T--HBUT-China--photoBBa-k3126011.png)

pSB1C3-hexa-His-nixA- kanR/ *Bsa*Ⅰ

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126021 | 25μL |
| *Bsa*I | 3 μL |
| buffer | 5 μL |
| ddH2O | 17 μL |

![](https://static.igem.org/mediawiki/2019/9/9e/T--HBUT-China--photoBBa-k3126012.png)

pSB1C3-nixA-TgMTP1t2- kanR/ *Bsa*Ⅰ

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126022 | 25μL |
| *Bsa*I | 3 μL |
| buffer | 5 μL |
| ddH2O | 17 μL |

![](https://static.igem.org/mediawiki/2019/d/dd/T--HBUT-China--photoBBa-k3126013.png)

pSB1C3-hexa-His-nixA-TgMTP1t2- kanR/ *Bsa*Ⅰ

Enzyme digestion system:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K3126023 | 25μL |
| *Bsa*I | 3 μL |
| buffer | 5 μL |
| ddH2O | 17 μL |

![](https://static.igem.org/mediawiki/2019/e/ec/T--HBUT-China--photoBBa-k3126014.png)

# 2019.7.25 ~ 8.5

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

![](https://static.igem.org/mediawiki/2019/d/d3/T--HBUT-China--photoIntegration.png)

# 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.

![](https://static.igem.org/mediawiki/2019/e/e1/T--HBUT-China--notebook-new1.png)

![](https://static.igem.org/mediawiki/2019/1/1c/T--HBUT-China--notebook-new2.png)

# Improvement

# 2019.9.20 ~ 2019.9.30

**[Protocols]**

1.Digested the pSB1C3-BBa\_K2652001 circular plasmid with *Bam*HI.

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 |
| *Bam*HI | 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/ *Bam*HⅠ | 3 μL |
| *His-Tag-nikABCDE- His-Tag* | 4 μL |
| Buffer | 2 μL |
| ExnaseⅡ | 1 μL |

**Enzyme Digestion picture:**

![](https://static.igem.org/mediawiki/2019/0/06/T--HBUT-China--_BBa-k3126001.png)

**[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.

#### The sequence of BBa\_K2652001

GTTTCTTC**GAATTCGCGGCCGCTTCTAGAG**TTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATC**GGATCC**tactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcg**CTCGAGTACTAGTAGCGGCCGCTGCAGGAAGAAAC**

#### The sequence of BBa\_K3126026

![](https://static.igem.org/mediawiki/2019/1/12/T--HBUT-China--photoBBak3126026.png)

CATCATCATCATCATCACATGCTCTCCACACTCCGCCGCACTCTATTTGCGCTGCTGGCTTGTGCGTCTTTTATCGTCCATGCCGCTGCACCAGATGAAATCACCACCGCCTGGCCGGTGAATGTCGGGCCACTAAACCCGCACCTTTACACGCCTAACCAGATGTTCGCCCAGAGCATGGTTTATGAACCATTGGTGAAATATCAGGCAGACGGTTCGGTGATCCCGTGGCTGGCAAAAAGCTGGACTCATTCAGAAGATGGTAAAACCTGGACCTTCACCCTGCGTGATGACGTGAAATTCTCCAACGGTGAACCGTTCGATGCCGAGGCGGCGGCAGAAAACTTCCGCGCAGTGCTCGATAACCGTCAACGTCACGCCTGGCTGGAGCTGGCAAACCAGATTGTTGATGTTAAAGCACTCAGTAAAACAGAGCTGCAAATTACCCTGAAAAGCGCCTACTATCCTTTCCTGCAAGAACTGGCCCTGCCCCGTCCTTTCCGTTTTATCGCTCCCTCGCAGTTTAAAAACCATGAAACCATGAACGGAATTAAAGCGCCGATTGGCACCGGACCGTGGATTTTGCAGGAATCGAAACTGAATCAGTACGATGTCTTCGTCCGTAACGAAAACTACTGGGGCGAAAAGCCAGCGATTAAAAAGATCACCTTTAACGTCATCCCGGACCCGACTACCCGCGCGGTGGCGTTTGAAACTGGCGATATCGACCTGCTGTACGGAAACGAAGGGTTATTACCGCTCGATACCTTCGCCCGCTTTAGCCAGAATCCGGCTTACCACACCCAACTGTCACAGCCGATCGAAACCGTGATGCTGGCGCTCAATACCGCCAAAGCCCCCACCAACGAGCTGGCAGTACGTGAAGCTCTTAATTACGCGGTAAACAAAAAATCGCTGATTGATAACGCGTTGTATGGCACCCAGCAGGTCGCCGACACCCTGTTTGCCCCTTCTGTGCCCTACGCCAACCTCGGCCTGAAACCGAGCCAGTACGATCCGCAAAAAGCGAAAGCGTTGCTGGAAAAAGCCGGTTGGACGCTGCCTGCGGGCAAAGACATCCGCGAGAAAAATGGTCAGCCGCTGCGCATTGAACTTTCGTTCATCGGCACCGATGCGTTAAGCAAATCGATGGCGGAAATCATTCAGGCTGATATGCGCCAGATTGGCGCAGATGTCTCGCTGATTGGCGAAGAAGAGAGCAGTATCTATGCTCGTCAGCGCGACGGTCGTTTTGGCATGATTTTCCACCGCACCTGGGGCGCGCCATATGATCCACACGCCTTCCTCAGTTCAATGCGCGTACCGTCACACGCTGACTTCCAGGCACAGCAAGGATTAGCCGACAAACCGCTGATTGATAAAGAGATCGGCGAAGTGCTGGCGACCCATGACGAAACGCAACGTCAGGCGCTGTATCGCGACATTCTGACCCGTCTGCATGACGAGGCGGTTTATCTGCCTATCAGTTACATCTCAATGATGGTGGTATCAAAACCGGAGCTGGGTAACATCCCCTACGCGCCGATCGCCACCGAAATTCCGTTCGAACAGATTAAACCGGTGAAACCTTAAATGTTGCGTTACGTATTACGCCGCTTTCTGCTGCTGATCCCGATGGTGCTCGCCGCCTCGGTGATCATTTTTCTGATGCTGCGCCTCGGTACCGGCGACCCGGCGCTCGATTATTTGCGTCTGTCTAACCTGCCGCCGACGCCGGAGATGCTGGCCTCTACCCGCACCATGCTGGGACTGGATCAGCCGCTGTACGTCCAGTACGGCACCTGGTTGTGGAAGGCGCTGCATCTTGACTTTGGTATCTCATTCGCCAGCCAACGCCCGGTACTGGACGATATGCTGAACTTCCTGCCCGCCACGCTGGAACTTGCAGGTGCGGCGCTGGTATTAATTCTGCTCACTTCCGTACCGCTCGGTATCTGGGCGGCGCGCCATCGCGACCGTCTGCCGGATTTCGCCGTACGTTTCATCGCGTTTCTTGGCGTGTCGATGCCTAACTTCTGGCTGGCGTTTTTACTGGTGATGGCGTTTTCGGTGTATCTGCAATGGCTACCCGCGATGGGTTACGGCGGCTGGCAGCACATCATTTTGCCTGCGGTTTCCATTGCCTTTATGTCGCTGGCGATTAACGCGCGTTTACTGCGCGCCAGTATGCTGGACGTCGCCGGTCAGCGTCACGTCACCTGGGCGCGTCTGCGCGGCCTGAACGACAAACAGACCGAACGTCGCCACATCCTGCGCAATGCCTCGCTGCCGATGATCACCGCCGTGGGGATGCATATCGGCGAACTGATTGGCGGGACGATGATTATCGAAAACATCTTTGCCTGGCCGGGCGTCGGGCGCTATGCGGTGTCGGCGATTTTTAACCGTGACTATCCGGTGATCCAGTGCTTTACGCTGATGATGGTGGTGGTTTTTGTGGTCTGTAATTTGATTGTCGATTTGCTCAACGCCGCGCTGGACCCGCGCATTCGTCGTCATGAAGGAGCGCACGCGTGAGTGAACTTTTTCCTCTCTTCCCGCTGGTCGGTACGCCTGGCGCTGATCATTATCGCCCTGCTGGCGCTGATTGCGCTCACCAGCCAGTGGTGGCTGCCGTATGACCCACAGGCGATTGATTTGCCGTCGCGCCTGCTTTCGCCGGATGCGCAGCACTGGCTGGGCACCGATCACTTAGGTCGCGATATTTTCTCGCGGCTGATGGCAGCGACCCGCGTGTCGCTCGGTTCGGTAATGGCCTGCCTGCTGCTGGTGCTGACATTAGGGCTGGTTATTGGCGGCAGCGCCGGGTTGATTGGCGGGCGCGTTGATCAGGCCACCATGCGCGTCGCCGATATGTTTATGACCTTCCCGACCTCGATTCTGTCGTTCTTTATGGTTGGCGTGCTCGGCACCGGGCTGACCAACGTAATTATCGCCATCGCCCTGTCGCACTGGGCGTGGTATGCACGCATGGTGCGCAGCCTGGTGATTTCACTACGCCAACGCGAGTTTGTGCTGGCGTCACGGCTTTCCGGTGCGGGCCATGTGCGGGTGTTTGTCGATCATCTGGCAGGCGCGGTGATCCCTTCGCTGCTGGTGCTGGCAACGCTGGATATCGGCCATATGATGCTGCACGTCGCGGGGATGTCTTTCCTTGGCCTCGGTGTGACCGCGCCGACCGCCGAATGGGGCGTGATGATTAACGACGCGCGCCAGTATATCTGGACCCAGCCGCTGCAAATGTTCTGGCCGGGGCTGGCGCTGTTTATCAGCGTGATGGCCTTTAACCTGGTGGGTGACGCACTGCGCGATCATCTGGACCCTCATCTGGTGACGGAGCACGCACACTAAATGCCGCAACAGATTGAACTACGTAATATCGCGCTACAGGCCGCGCAGCCGCTGGTACACGGTGTATCGTTAACCCTGCAACGCGGGCGCGTGCTGGCGTTAGTCGGCGGTAGCGGCAGCGGGAAATCATTAACCTGCGCCGCAACGCTGGGCATTTTGCCCGCTGGCGTTCGCCAGACGGCGGGGGAAATTTTAGCCGATGGCAAACCGGTTTCGCCTTGCGCCCTGCGCGGCATCAAAATTGCCACCATCATGCAGAACCCGCGCAGCGCCTTTAATCCACTGCACACCATGCACACCCACGCGCGGGAAACCTGCCTGGCGTTAGGGAAACCCGCCGATGACGCTACGCTTACCGCTGCCATAGAAGCGGTGGGGCTGGAAAACGCCGCGCGCGTGCTGAAGCTGTACCCGTTCGAGATGAGCGGCGGCATGTTGCAGCGCATGATGATTGCGATGGCGGTGCTGTGTGAATCACCGTTTATCATCGCCGATGAACCGACCACCGACCTTGACGTGGTAGCACAGGCGCGCATCCTCGATCTGCTGGAAAGCATTATGCAAAAACAAGCGCCGGGAATGCTGCTGGTGACCCATGATATGGGCGTTGTGGCGCGTCTGGCGGATGACGTGGCGGTGATGTCTGACGGTAAGATTGTCGAACAGGGCGATGTAGAAACGCTGTTTAACGCCCCCAAACATACAGTGACGCGCAGCCTGGTTTCCGCTCATCTCGCCCTCTACGGTATGGAGCTGGCATCATGAATGACTTTACTTAACATCTCCGGCCTTTCCCATCACTATGCGCACGGTGGATTTAACGGAAAACATCAACATCAGGCGGTGCTGAATAACGTTTCCCTGACCCTGAAAAGCGGCGAAACCGTCGCCCTGCTGGGGCGCAGCGGCTGCGGGAAAAGTACCCTCGCGCGGTTGCTGGTGGGTTTAGAATCGCCCGCGCAGGGGAATATTAGCTGGCGTGGCGAACCGCTGGCGAAACTCAATCGCGCCCAGCGTAAAGCGTTCCGCCGCGATATTCAGATGGTATTTCAGGACTCCATCAGCGCCGTGAATCCGCGCAAAACCGTGCGCGAGATCCTGCGTGAACCGATGCGCCACCTGCTATCACTGAAAAAATCCGAACAACTGGCGCGCGCCAGCGAAATGCTGAAGGCGGTCGATCTCGATGACAGCGTTCTCGACAAACGCCCACCGCAGTTAAGCGGCGGCCAGCTCCAGCGCGTCTGCCTGGCTCGCGCGCTGGCGGTCGAACCGAAACTACTGATTCTGGATGAAGCCGTTTCTAACCTTGATCTCGTGTTACAGGCGGGTGTCATTCGCCTGCTGAAAAAGCTACAACAACAGTTTGGCACCGCCTGCCTGTTCATCACCCACGACTTACGCCTGGTAGAACGCTTTTGCCAGCGGGTAATGGTTATGGACAACGGACAAATCGTCGAAACCCAGGTGGTGGGAGAGAAATTAACCTTTTCCTCTGACGCCGGACGTGTGCTACAAAACGCGGTATTACCCGCATTCCCCGTGCGCCGTCGCACCACAGAAAAGGTTCACCACCACCACCACCACTAA

#### The sequence of pSB1C3-BBa\_K3126026

![](https://static.igem.org/mediawiki/2019/f/f3/T--HBUT-China--photoBBa--k3126026.png)

GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCCATCATCATCATCATCACATGCTCTCCACACTCCGCCGCACTCTATTTGCGCTGCTGGCTTGTGCGTCTTTTATCGTCCATGCCGCTGCACCAGATGAAATCACCACCGCCTGGCCGGTGAATGTCGGGCCACTAAACCCGCACCTTTACACGCCTAACCAGATGTTCGCCCAGAGCATGGTTTATGAACCATTGGTGAAATATCAGGCAGACGGTTCGGTGATCCCGTGGCTGGCAAAAAGCTGGACTCATTCAGAAGATGGTAAAACCTGGACCTTCACCCTGCGTGATGACGTGAAATTCTCCAACGGTGAACCGTTCGATGCCGAGGCGGCGGCAGAAAACTTCCGCGCAGTGCTCGATAACCGTCAACGTCACGCCTGGCTGGAGCTGGCAAACCAGATTGTTGATGTTAAAGCACTCAGTAAAACAGAGCTGCAAATTACCCTGAAAAGCGCCTACTATCCTTTCCTGCAAGAACTGGCCCTGCCCCGTCCTTTCCGTTTTATCGCTCCCTCGCAGTTTAAAAACCATGAAACCATGAACGGAATTAAAGCGCCGATTGGCACCGGACCGTGGATTTTGCAGGAATCGAAACTGAATCAGTACGATGTCTTCGTCCGTAACGAAAACTACTGGGGCGAAAAGCCAGCGATTAAAAAGATCACCTTTAACGTCATCCCGGACCCGACTACCCGCGCGGTGGCGTTTGAAACTGGCGATATCGACCTGCTGTACGGAAACGAAGGGTTATTACCGCTCGATACCTTCGCCCGCTTTAGCCAGAATCCGGCTTACCACACCCAACTGTCACAGCCGATCGAAACCGTGATGCTGGCGCTCAATACCGCCAAAGCCCCCACCAACGAGCTGGCAGTACGTGAAGCTCTTAATTACGCGGTAAACAAAAAATCGCTGATTGATAACGCGTTGTATGGCACCCAGCAGGTCGCCGACACCCTGTTTGCCCCTTCTGTGCCCTACGCCAACCTCGGCCTGAAACCGAGCCAGTACGATCCGCAAAAAGCGAAAGCGTTGCTGGAAAAAGCCGGTTGGACGCTGCCTGCGGGCAAAGACATCCGCGAGAAAAATGGTCAGCCGCTGCGCATTGAACTTTCGTTCATCGGCACCGATGCGTTAAGCAAATCGATGGCGGAAATCATTCAGGCTGATATGCGCCAGATTGGCGCAGATGTCTCGCTGATTGGCGAAGAAGAGAGCAGTATCTATGCTCGTCAGCGCGACGGTCGTTTTGGCATGATTTTCCACCGCACCTGGGGCGCGCCATATGATCCACACGCCTTCCTCAGTTCAATGCGCGTACCGTCACACGCTGACTTCCAGGCACAGCAAGGATTAGCCGACAAACCGCTGATTGATAAAGAGATCGGCGAAGTGCTGGCGACCCATGACGAAACGCAACGTCAGGCGCTGTATCGCGACATTCTGACCCGTCTGCATGACGAGGCGGTTTATCTGCCTATCAGTTACATCTCAATGATGGTGGTATCAAAACCGGAGCTGGGTAACATCCCCTACGCGCCGATCGCCACCGAAATTCCGTTCGAACAGATTAAACCGGTGAAACCTTAAATGTTGCGTTACGTATTACGCCGCTTTCTGCTGCTGATCCCGATGGTGCTCGCCGCCTCGGTGATCATTTTTCTGATGCTGCGCCTCGGTACCGGCGACCCGGCGCTCGATTATTTGCGTCTGTCTAACCTGCCGCCGACGCCGGAGATGCTGGCCTCTACCCGCACCATGCTGGGACTGGATCAGCCGCTGTACGTCCAGTACGGCACCTGGTTGTGGAAGGCGCTGCATCTTGACTTTGGTATCTCATTCGCCAGCCAACGCCCGGTACTGGACGATATGCTGAACTTCCTGCCCGCCACGCTGGAACTTGCAGGTGCGGCGCTGGTATTAATTCTGCTCACTTCCGTACCGCTCGGTATCTGGGCGGCGCGCCATCGCGACCGTCTGCCGGATTTCGCCGTACGTTTCATCGCGTTTCTTGGCGTGTCGATGCCTAACTTCTGGCTGGCGTTTTTACTGGTGATGGCGTTTTCGGTGTATCTGCAATGGCTACCCGCGATGGGTTACGGCGGCTGGCAGCACATCATTTTGCCTGCGGTTTCCATTGCCTTTATGTCGCTGGCGATTAACGCGCGTTTACTGCGCGCCAGTATGCTGGACGTCGCCGGTCAGCGTCACGTCACCTGGGCGCGTCTGCGCGGCCTGAACGACAAACAGACCGAACGTCGCCACATCCTGCGCAATGCCTCGCTGCCGATGATCACCGCCGTGGGGATGCATATCGGCGAACTGATTGGCGGGACGATGATTATCGAAAACATCTTTGCCTGGCCGGGCGTCGGGCGCTATGCGGTGTCGGCGATTTTTAACCGTGACTATCCGGTGATCCAGTGCTTTACGCTGATGATGGTGGTGGTTTTTGTGGTCTGTAATTTGATTGTCGATTTGCTCAACGCCGCGCTGGACCCGCGCATTCGTCGTCATGAAGGAGCGCACGCGTGAGTGAACTTTTTCCTCTCTTCCCGCTGGTCGGTACGCCTGGCGCTGATCATTATCGCCCTGCTGGCGCTGATTGCGCTCACCAGCCAGTGGTGGCTGCCGTATGACCCACAGGCGATTGATTTGCCGTCGCGCCTGCTTTCGCCGGATGCGCAGCACTGGCTGGGCACCGATCACTTAGGTCGCGATATTTTCTCGCGGCTGATGGCAGCGACCCGCGTGTCGCTCGGTTCGGTAATGGCCTGCCTGCTGCTGGTGCTGACATTAGGGCTGGTTATTGGCGGCAGCGCCGGGTTGATTGGCGGGCGCGTTGATCAGGCCACCATGCGCGTCGCCGATATGTTTATGACCTTCCCGACCTCGATTCTGTCGTTCTTTATGGTTGGCGTGCTCGGCACCGGGCTGACCAACGTAATTATCGCCATCGCCCTGTCGCACTGGGCGTGGTATGCACGCATGGTGCGCAGCCTGGTGATTTCACTACGCCAACGCGAGTTTGTGCTGGCGTCACGGCTTTCCGGTGCGGGCCATGTGCGGGTGTTTGTCGATCATCTGGCAGGCGCGGTGATCCCTTCGCTGCTGGTGCTGGCAACGCTGGATATCGGCCATATGATGCTGCACGTCGCGGGGATGTCTTTCCTTGGCCTCGGTGTGACCGCGCCGACCGCCGAATGGGGCGTGATGATTAACGACGCGCGCCAGTATATCTGGACCCAGCCGCTGCAAATGTTCTGGCCGGGGCTGGCGCTGTTTATCAGCGTGATGGCCTTTAACCTGGTGGGTGACGCACTGCGCGATCATCTGGACCCTCATCTGGTGACGGAGCACGCACACTAAATGCCGCAACAGATTGAACTACGTAATATCGCGCTACAGGCCGCGCAGCCGCTGGTACACGGTGTATCGTTAACCCTGCAACGCGGGCGCGTGCTGGCGTTAGTCGGCGGTAGCGGCAGCGGGAAATCATTAACCTGCGCCGCAACGCTGGGCATTTTGCCCGCTGGCGTTCGCCAGACGGCGGGGGAAATTTTAGCCGATGGCAAACCGGTTTCGCCTTGCGCCCTGCGCGGCATCAAAATTGCCACCATCATGCAGAACCCGCGCAGCGCCTTTAATCCACTGCACACCATGCACACCCACGCGCGGGAAACCTGCCTGGCGTTAGGGAAACCCGCCGATGACGCTACGCTTACCGCTGCCATAGAAGCGGTGGGGCTGGAAAACGCCGCGCGCGTGCTGAAGCTGTACCCGTTCGAGATGAGCGGCGGCATGTTGCAGCGCATGATGATTGCGATGGCGGTGCTGTGTGAATCACCGTTTATCATCGCCGATGAACCGACCACCGACCTTGACGTGGTAGCACAGGCGCGCATCCTCGATCTGCTGGAAAGCATTATGCAAAAACAAGCGCCGGGAATGCTGCTGGTGACCCATGATATGGGCGTTGTGGCGCGTCTGGCGGATGACGTGGCGGTGATGTCTGACGGTAAGATTGTCGAACAGGGCGATGTAGAAACGCTGTTTAACGCCCCCAAACATACAGTGACGCGCAGCCTGGTTTCCGCTCATCTCGCCCTCTACGGTATGGAGCTGGCATCATGAATGACTTTACTTAACATCTCCGGCCTTTCCCATCACTATGCGCACGGTGGATTTAACGGAAAACATCAACATCAGGCGGTGCTGAATAACGTTTCCCTGACCCTGAAAAGCGGCGAAACCGTCGCCCTGCTGGGGCGCAGCGGCTGCGGGAAAAGTACCCTCGCGCGGTTGCTGGTGGGTTTAGAATCGCCCGCGCAGGGGAATATTAGCTGGCGTGGCGAACCGCTGGCGAAACTCAATCGCGCCCAGCGTAAAGCGTTCCGCCGCGATATTCAGATGGTATTTCAGGACTCCATCAGCGCCGTGAATCCGCGCAAAACCGTGCGCGAGATCCTGCGTGAACCGATGCGCCACCTGCTATCACTGAAAAAATCCGAACAACTGGCGCGCGCCAGCGAAATGCTGAAGGCGGTCGATCTCGATGACAGCGTTCTCGACAAACGCCCACCGCAGTTAAGCGGCGGCCAGCTCCAGCGCGTCTGCCTGGCTCGCGCGCTGGCGGTCGAACCGAAACTACTGATTCTGGATGAAGCCGTTTCTAACCTTGATCTCGTGTTACAGGCGGGTGTCATTCGCCTGCTGAAAAAGCTACAACAACAGTTTGGCACCGCCTGCCTGTTCATCACCCACGACTTACGCCTGGTAGAACGCTTTTGCCAGCGGGTAATGGTTATGGACAACGGACAAATCGTCGAAACCCAGGTGGTGGGAGAGAAATTAACCTTTTCCTCTGACGCCGGACGTGTGCTACAAAACGCGGTATTACCCGCATTCCCCGTGCGCCGTCGCACCACAGAAAAGGTTCACCACCACCACCACCACTAAGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCA

# Characterization

# 2019.9.10 ~ 2019.9.20

We used the pSB1C3-BBa\_K2652001 from last year and digested with *Bam*H I enzyme.

The pSB1C3 digestion system we used:

|  |  |
| --- | --- |
| pSB1C3-BBa\_K2652001 | 15μL |
| *Bam*HI | 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/ *Bam*HI | 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/ *Bam*HI | 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/*Bam*HI | 2μL |
| BBa\_E0032 | 4 μL |
| 5×CEⅡ Buffer | 2 μL |
| Exnase Ⅱ | 1 μL |
| ddH2O | 1 μL |

|  |  |
| --- | --- |
| pSB1C3-BBa\_K2652001/*Bam*HI | 2μL |
| BBa\_E0034 | 4 μL |
| 5×CEⅡ Buffer | 2 μL |
| Exnase Ⅱ | 1 μL |
| ddH2O | 1 μL |

|  |  |
| --- | --- |
| pSB1C3-BBa\_K2652001/*Bam*HI | 2μL |
| GFP | 4 μL |
| 5×CEⅡ Buffer | 2 μL |
| Exnase Ⅱ | 1 μL |
| ddH2O | 1 μL |

#### The sequence of BBa\_K2652001

GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCAGGAAGAAAC

#### The sequence of BBa\_E0032

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctttagtagcttaataa

#### The sequence of BBa\_K3126028

TTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctttagtagcttaataaGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcg

![](https://static.igem.org/mediawiki/2019/2/21/T--HBUT-China--fragmentBBa_K3126028.png)

#### The sequence of pSB1C3-BBa\_K3126028

**GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCC**atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctttagtagcttaataa**GGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCA**

![](https://static.igem.org/mediawiki/2019/5/55/T--HBUT-China--plasmidBBa_K3126028.png)

#### The sequence of BBa\_E0034

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctgctgctgtttaataa

#### The sequence of BBa\_K3126029

**TTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctgctgctgtttaataaGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcg**

![](https://static.igem.org/mediawiki/2019/e/e1/T--HBUT-China--fragmentBBa_K3126029.png)

#### The sequence of pSB1C3-BBa\_K3126029

**GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCC**atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgcaatgcttcgcccgctaccccgaccacatgaagctgcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaggcctgctgcaaacgacgaaaactacgctgctgctgtttaataa**GGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCA**

![](https://static.igem.org/mediawiki/2019/4/4c/T--HBUT-China--plasmidBBa_K3126029.png)

#### The sequence of GFP

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

#### The sequence of BBa\_K3126030

**TTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcg**

![](https://static.igem.org/mediawiki/2019/b/b3/T--HBUT-China--fragmentBBa_K3126030.png)

#### The sequence of pSB1C3-BBa\_K3126030

**GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACGGCTAGCTCAGCCCTAGGTATTATGCTAGCACATTTCCAACACTAACCCAAGGGAGCTTTAAATCGGATCC**ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
**GGATCCtactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgCTCGAGTACTAGTAGCGGCCGCTGCA**

![](https://static.igem.org/mediawiki/2019/3/3d/T--HBUT-China--plasmidBBa_K3126030.png)

### option date

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


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:HBUT-China/Experiments

[![](https://static.igem.org/mediawiki/2019/0/07/T--HBUT-China--weblogo.png)](https://2019.igem.org/Team%3AHBUT-China)

* [Home](https://2019.igem.org/Team%3AHBUT-China)
* Project
  + [Inspiration & Description](https://2019.igem.org/Team%3AHBUT-China/Description)
  + [Design](https://2019.igem.org/Team%3AHBUT-China/Design)
  + [Improvement](https://2019.igem.org/Team%3AHBUT-China/Improvement)
  + [Characterization](https://2019.igem.org/Team%3AHBUT-China/Characterization)
  + [Future plan](https://2019.igem.org/Team%3AHBUT-China/Future_Plan)
* Lab
  + [Notebook](https://2019.igem.org/Team%3AHBUT-China/Notebook)
  + [Results](https://2019.igem.org/Team%3AHBUT-China/Result)
  + [Parts](https://2019.igem.org/Team%3AHBUT-China/Parts)
  + [Safety](https://2019.igem.org/Team%3AHBUT-China/Safety)
* [Model](https://2019.igem.org/Team%3AHBUT-China/Model)
* Human Practices
  + [Overview](https://2019.igem.org/Team%3AHBUT-China/Human_Practices#Overview)
  + [Integrated Hunan Practices](https://2019.igem.org/Team%3AHBUT-China/Human_Practices#Integrated_Hunan_Practices)
  + [Education & Public Engagement](https://2019.igem.org/Team%3AHBUT-China/Human_Practices#Education_&_Public_Engagement)
* Team
  + [Team Member](https://2019.igem.org/Team%3AHBUT-China/TeamMember)
  + [Attributions](https://2019.igem.org/Team%3AHBUT-China/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3AHBUT-China/Collaborations)
* Device
  + [Mini Factory](https://2019.igem.org/Team%3AHBUT-China/Mini_Factory)
  + [Demonstrate](https://2019.igem.org/Team%3AHBUT-China/Demonstrate)
  + [3D Printing Technology](https://2019.igem.org/Team%3AHBUT-China/3DPrintingTechnology)
  + [Automatic Control System](https://2019.igem.org/Team%3AHBUT-China/AutomaticControlSystem)
* Awards
  + [Medals](https://2019.igem.org/Team%3AHBUT-China/Medals)
  + [Judging Form](https://igem.org/2019_Judging_Form?id=3126)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:HBUT-China/Results

[![](https://static.igem.org/mediawiki/2019/0/07/T--HBUT-China--weblogo.png)](https://2019.igem.org/Team%3AHBUT-China)

* [Home](https://2019.igem.org/Team%3AHBUT-China)
* Project
  + [Inspiration & Description](https://2019.igem.org/Team%3AHBUT-China/Description)
  + [Design](https://2019.igem.org/Team%3AHBUT-China/Design)
  + [Improvement](https://2019.igem.org/Team%3AHBUT-China/Improvement)
  + [Characterization](https://2019.igem.org/Team%3AHBUT-China/Characterization)
  + [Future plan](https://2019.igem.org/Team%3AHBUT-China/Future_Plan)
* Lab
  + [Notebook](https://2019.igem.org/Team%3AHBUT-China/Notebook)
  + [Results](https://2019.igem.org/Team%3AHBUT-China/Result)
  + [Parts](https://2019.igem.org/Team%3AHBUT-China/Parts)
  + [Safety](https://2019.igem.org/Team%3AHBUT-China/Safety)
* [Model](https://2019.igem.org/Team%3AHBUT-China/Model)
* Human Practices
  + [Overview](https://2019.igem.org/Team%3AHBUT-China/Human_Practices#Overview)
  + [Integrated Hunan Practices](https://2019.igem.org/Team%3AHBUT-China/Human_Practices#Integrated_Hunan_Practices)
  + [Education & Public Engagement](https://2019.igem.org/Team%3AHBUT-China/Human_Practices#Education_&_Public_Engagement)
* Team
  + [Team Member](https://2019.igem.org/Team%3AHBUT-China/TeamMember)
  + [Attributions](https://2019.igem.org/Team%3AHBUT-China/Attributions)
  + [Collaborations](https://2019.igem.org/Team%3AHBUT-China/Collaborations)
* Device
  + [Mini Factory](https://2019.igem.org/Team%3AHBUT-China/Mini_Factory)
  + [Demonstrate](https://2019.igem.org/Team%3AHBUT-China/Demonstrate)
  + [3D Printing Technology](https://2019.igem.org/Team%3AHBUT-China/3DPrintingTechnology)
  + [Automatic Control System](https://2019.igem.org/Team%3AHBUT-China/AutomaticControlSystem)
* Awards
  + [Medals](https://2019.igem.org/Team%3AHBUT-China/Medals)
  + [Judging Form](https://igem.org/2019_Judging_Form?id=3126)

# Result

## Overview of the result

### 1. Construction of six engineering *S. cerevisiae* and test function

### 2. Improvement of nikABCDE

### 3. Characterization of two EYFP proteins

## Test the function of engineering *S. cerevisiae*

### Adsorption effect on simulated Ni2+ concentration (15 mg/L)

In our Human Practical activities, we went to the automobile factory and obtained the nickel ions polluted
water samples from the real life. We measured that the nickel ion concentration of the direct effluent
discharge was about 15 mg/L. In order to be closer to the real-life situation, we also designed the nickel
ion solution with a concentration of 15 mg/L and a suitable yeast dosage of 2 g/L for simulation adsorption
experiments.

We carried out adsorption experiments with the original yeast and the six kinds of engineering yeast we
constructed at the same time and made sure that the other conditions were exactly the same. We also
constructed the original yeast and six engineered yeasts and ensured that the other conditions were
identical.

The experimental results are as follows：

![](https://static.igem.org/mediawiki/2019/e/ea/T--HBUT-China--result1.png)

Fig 1. Adsorption capacity of simulated
Ni2+ (15 mg/L) by engineering yeast after 45 minutes treatment.

![](https://static.igem.org/mediawiki/2019/a/a4/T--HBUT-China--result2.png)

Fig 2. Adsorption curve of simulated
Ni2+ (15 mg/L) by engineering yeast with time.

We can see that the adsorption capacity of nickel ion in our engineering yeast is higher than that in the
original yeast. And the adsorption capacity of *S.cerevisiae*/BBa\_k3126022 (nixA+TgMTP1t2) and *S.
cerevisiae*/BBa\_k3126023 (hexa-His+nixA+TgMTP1t2) for nickel ion is stronger, and the removal rate
is 80%.

And the adsorption equilibrium qe and adsorption equilibrium constant K were obtained by fitting the
enrichment rate model： t/qt=1/(k\*qe2)+t/qe

|  |  |  |
| --- | --- | --- |
|  | qe(mg/g) | Adsorption equilibrium constant K |
| Original *S.cerevisiae* | 2.44 | 0.3897 |
| *S.cerevisiae*/BBa\_k3126018(hexa-His) | 3.663 | 0.1696 |
| *S.cerevisiae*/BBa\_k3126019(nixA) | 3.3245 | 0.2817 |
| *S.cerevisiae*/BBa\_k3126020(TgMTP1t2) | 3.201 | 0.4694 |
| *S.cerevisiae*/BBa\_k3126021(hexa-His+nixA) | 3.701 | 0.298 |
| *S.cerevisiae*/BBa\_k3126022(nixA+TgMTP1t2） | 5.136 | 0.135 |
| *S.cerevisiae*/BBa\_k3126023(hexa-His+nixA+TgMTP1t2) | 6.34 | 0.17 |

From the chart above, we can see that the equilibrium enrichment of engineering yeast has been greatly
improved, and the original yeast is easy to reach adsorption saturation.

### Adsorption effect on high concentration of Ni2+(200 mg/L)

Through the tolerance experiment of S. cerevisiae to nickel ion, we know that our chassis organism has a
strong tolerance to nickel ions, so we increase the concentration of nickel ion solution in the experiment,
and further explore the adsorption capacity of our engineering yeast for nickel ion. We increased the
concentration of nickel ion solution to 200 mg / L, and the designed yeast dosage was changed to 7g/L.

The experimental results are as follows：

![](https://static.igem.org/mediawiki/2019/9/9b/T--HBUT-China--result3.png)

Fig 3. Adsorption capacity of
Ni2+ (200 mg/L) by engineering yeast after 45 minutes treatment.

![](https://static.igem.org/mediawiki/2019/a/a0/T--HBUT-China--result4.png)

Fig 4. Adsorption curve of simulated
Ni2+ (200 mg/L) by engineering yeast with time.

Based on our results, all types of engineering yeasts showed their advantages, and adsorbed more nickel ions
compared with original control yeast under the same conditions. Among all the types of engineering yeast,
*S.cerevisiae*/BBa\_k3126023 (hexa-His+nixA+TgMTP1t2) have highest absorption effect on nickel ions.

### Adsorption effect on actual nickel-containing effluent

The engineering yeast is embedded to absorbed 200 ml, nickel-containing effluent with a concentration of
about 15mg/L in the device.

The engineering yeast used is *S. cerevisiae*/BBa\_k3126023 (hexa-His+nixA+TgMTP1t2)

Yeast dosaget: 0.5 g/L×2

Operating process: Place yeast in the treatment pool, and the nickel-containing solution reacted from the
injection pool to the treatment pool. After adsorption for 20 min, measure the water samples in the
detection pool and take out the immobilization spheres which reached the adsorption equilibrium. Release the
new immobilization spheres, After 15 minutes of adsorption, measure the water samples in the detection pool,
and finally the waste water is discharged into a water outlet detection pool.

Results: It has been found that yeast can quickly absorb a lot of nickel ions. The nickel concentration in
the first batch of water samples after adsorption for 20 ~ 30 minutes was 3.7561 mg/L, and the nickel
concentration in the second batch was 0.4659mg/L after 15 minutes of adsorption.

![](https://static.igem.org/mediawiki/2019/1/17/T--HBUT-China--result5.jpg)

Adsorption of nickel-containing effluent by immobilization pellets in the device.

Our engineering yeast can adsorb the nickel-containing effluent of 15mg/L to only about 0.5 mg/L in 45
minutes, which basically meets the national discharge standard of nickel ion, and our method has the
advantages of high efficiency and low cost.

## Improvement of nikABCDE

### Two kinds of engineering bacteria were cultured in 30 mg/L nickel ion solution. Within 30 minutes, the original bacteria *(nikABCDE)* reduced the concentration of nickel ions from 30 mg/L to 15 mg/L, but the improved engineered bacteria *(His-Tag-nikABCDE-His-Tag)* reduced the concentration of nickel ions from 30 mg/L to 10 mg/L. It can be seen from the figure that the improved engineered bacteria has a stronger nickel adsorption ability than the original bacteria.

![](https://static.igem.org/mediawiki/2019/b/b2/T--HBUT-China--result6.png)

Fig 6. Adsorption curve of Ni2+ (30 mg/L) by two kinds of engineering bacteriat with time.

## Characterization of two EYFP proteins

1. We successfully connected the gene fragment between the promoter and terminator in pSB1C3 and send it for
sequencing. The result showed that we correctly connected the gene fragment.

2. The protein expression of E0032, E0034 and blank control in E.coli cells were detected by laser scanning
confocal microscope analysis.

![](https://static.igem.org/mediawiki/2019/4/47/T--HBUT-China--Characterization--1.png)

![](https://static.igem.org/mediawiki/2019/c/cf/T--HBUT-China--Characterization--2.png)

![](https://static.igem.org/mediawiki/2019/a/a8/T--HBUT-China--Characterization--3.png)

The results indicated that both E0032, E0034 and blank control had fluorescence, but the fluorescence is
very weak.

3. In order to confirm the expression of these two yellow fluorescent proteins. Another fluorescent protein,
green fluorescent protein was ligated in the same vector, with the same promoter and terminator. The
fluorescent microplate reader was used to measure the fluorescence of the three bacteria.

From the curve we can see that using these two promoter and terminator, the expression levels of these two
yellow fluorescent proteins are very low, lower than the green fluorescent protein, slightly higher than the
blank bacteria. We are not sure if other promoters and terminators can express higher values.

![](https://static.igem.org/mediawiki/2019/8/85/T--HBUT-China--Characterization--4.png)

![](https://static.igem.org/mediawiki/2019/f/fd/T--HBUT-China--Characterization--5.png)

## Conclusion

We can see fluorescence signals under the laser scanning confocal microscope, which tells that we have
correctly built the plasmid. However, comparing with GFP, using the same promoter and terminator, E0034 and
E0032 has a very low level of expression. Other promoters and terminators are not tried, this conclusion is
only based on the results of this experiment.

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