Difference between revisions of "Part:BBa K3120018"
 
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<partinfo>BBa_K3120018 parameters</partinfo>
 
<partinfo>BBa_K3120018 parameters</partinfo>
 
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    This part is the homologous recombination plasmid of E-Cadherin. Using CRISPR/cas9 technology, homologous recombination can occur between the left arm and right arm of E-Cadherin and the corresponding fragments on the genome, so as to realize the insertion of tdTomato before the termination codon of E-Cadherin gene of homo sapiens WA09(human embryonic stem cell line).(Fig.1A ,Fig.1B)
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This part is the homologous recombination plasmid of E-Cadherin. Using CRISPR/cas9 technology, homologous recombination can occur between the left arm and right arm of E-Cadherin and the corresponding fragments on the genome, so as to realize the insertion of tdTomato before the termination codon of E-Cadherin gene of homo sapiens WA09(human embryonic stem cell line).<b>(Fig.1A ,Fig.1B)</b>
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    After co-transfection with plasmid pBluescriptll SK-Ecadherin-tdTomato into WA09 cells, monoclonal cloning, we identified the correct insertion of tdTomato at the genome level and mRNA level(cDNA).(Fig.1C)The correct subcellular localiza-tion of ECAD and tdTomato （Fusion expression）was observed by confocal micro-scope.（Fig.1D）
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[[Image:T--BM-AMU--part7.jpg|center|thumb|500px|'''Fig1 .  
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After co-transfection with plasmid pBluescriptll SK-Ecadherin-tdTomato into WA09 cells, monoclonal cloning, we identified the correct insertion of tdTomato at the genome level and mRNA level(cDNA).<b>(Fig1C)</b>The correct subcellular localiza-tion of ECAD and tdTomato （Fusion expression）was observed by confocal micro-scope.<b>（Fig.1D）</b>
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<b>( A)</b>Schematic of plasmid pBluescriptll SK-Ecadherin-tdTomato.  <b>(B)</b>Schematic of plasmid PX330. <b>(C)</b>The correct insertion of tdTomato at the genome level and mRNA level(cDNA) by PCR.<b>(D)</b>The correct subcellular localization of ECAD and tdTomato （Fusion expression）was observed by confocal microscope.
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[[Image:T--BM-AMU--part7.jpg|center|thumb|500px|'''Fig1 . <b>( A)</b>Schematic of plasmid pBluescriptll SK-Ecadherin-tdTomato.  <b>(B)</b>Schematic of plasmid PX330. <b>(C)</b>The correct insertion of tdTomato at the genome level and mRNA level(cDNA) by PCR.<b>(D)</b>The correct subcellular localization of ECAD and tdTomato （Fusion expression）was observed by confocal microscope.
 
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===Reference===
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1. Julia J,Feng Z, et al.Genome-scale CRISPR-Cas9 knockout and transcrip-tional activation screening. Nat Prot,12,828-863(2017).

Latest revision as of 01:24, 20 October 2019


E-cad LA+Glycine linker+tdTomato+E-cad RA

This part is the homologous recombination plasmid of E-Cadherin. Using crisper CAS9 technology, homologous recombination can occur between the left arm and right arm of E-Cadherin and the corresponding fragments on the genome, so as to realize the insertion of tdTomato before the termination codon of E-Cadherin gene of homo sapiens H9(human embryonic stem cell line).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 3994
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 333
    Illegal BsaI site found at 782
    Illegal BsaI site found at 4472
    Illegal BsaI site found at 4635
    Illegal BsaI site found at 4658
    Illegal BsaI.rc site found at 1647


This part is the homologous recombination plasmid of E-Cadherin. Using CRISPR/cas9 technology, homologous recombination can occur between the left arm and right arm of E-Cadherin and the corresponding fragments on the genome, so as to realize the insertion of tdTomato before the termination codon of E-Cadherin gene of homo sapiens WA09(human embryonic stem cell line).(Fig.1A ,Fig.1B)

After co-transfection with plasmid pBluescriptll SK-Ecadherin-tdTomato into WA09 cells, monoclonal cloning, we identified the correct insertion of tdTomato at the genome level and mRNA level(cDNA).(Fig1C)The correct subcellular localiza-tion of ECAD and tdTomato （Fusion expression）was observed by confocal micro-scope.（Fig.1D）

Fig1 . ( A)Schematic of plasmid pBluescriptll SK-Ecadherin-tdTomato. (B)Schematic of plasmid PX330. (C)The correct insertion of tdTomato at the genome level and mRNA level(cDNA) by PCR.(D)The correct subcellular localization of ECAD and tdTomato （Fusion expression）was observed by confocal microscope.

Reference

1. Julia J,Feng Z, et al.Genome-scale CRISPR-Cas9 knockout and transcrip-tional activation screening. Nat Prot,12,828-863(2017).