Difference between revisions of "Part:BBa K3219000"
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<partinfo>BBa_K3219000 short</partinfo>
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<partinfo>BBa_K3219000 short</partinfo> <br>
 
This is a part improvement of BBa_K1689013 (https://parts.igem.org/Part:BBa_K1689013). <br>
 
This is a part improvement of BBa_K1689013 (https://parts.igem.org/Part:BBa_K1689013). <br>
  
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dCas9 enzyme is also known as a catalytically dead Cas9 enzyme<ref> Larson, M. H. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, 2180–2196.</ref>. Different from traditional CRISPR Cas9 enzymes, dCas9 lacks endonuclease activity. It does not cleave DNA. Instead, with the help of a guide RNA, it specifically binds to the target, usually 20 -30 bp, and blocks transcript elongation by RNA polymerase.
 
dCas9 enzyme is also known as a catalytically dead Cas9 enzyme<ref> Larson, M. H. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, 2180–2196.</ref>. Different from traditional CRISPR Cas9 enzymes, dCas9 lacks endonuclease activity. It does not cleave DNA. Instead, with the help of a guide RNA, it specifically binds to the target, usually 20 -30 bp, and blocks transcript elongation by RNA polymerase.
  
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In this part, a GFP is added to the C-terminus of the dCas9, connected using an SGAAAAGGS linker. The GFP is added so that the expression of both proteins could be checked easier.  
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In this part, a GFP is added to the C-terminus of the dCas9, connected using an SGAAAAGGS linker. The GFP is added so that the expression of both proteins could be checked easier. <br>
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We did not add ribosome binding sites or promoters to this sequence to allow larger flexibility for users to choose the promoter and RBS that is suitable for their chassis.  
  
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This sequence can be used by cloning it into a desired vector with a promoter and ribosome binding site.
 
  
 
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===Usage and Biology===
 
===Usage and Biology===
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This sequence can be used by cloning it into a desired vector with a promoter and ribosome binding site.
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 08:03, 13 October 2019


dCas9-GFP
This is a part improvement of BBa_K1689013 (https://parts.igem.org/Part:BBa_K1689013).

Original Part BBa_K1689013

Part BBa_K1689013 is an N-terminal fragment of β-lactamase fused with dCas9. Team iGEM15_Peking designed it to be resistance against several antibiotics. However, β-lactamase may not be applicable to each and every project. For example, in our project, the plasmid already confers Kanamycin resistance gene. β-lactamase may not be applicable in this situation. Other than using β-lactamase as a selection method, we hope to provide more options for CRISPR imaging. The GFP will allow visual confirmation of successful transformation and indicates that the dCas9 enzyme has been successfully expressed.

BBa_K3219000

dCas9 enzyme is also known as a catalytically dead Cas9 enzyme[1]. Different from traditional CRISPR Cas9 enzymes, dCas9 lacks endonuclease activity. It does not cleave DNA. Instead, with the help of a guide RNA, it specifically binds to the target, usually 20 -30 bp, and blocks transcript elongation by RNA polymerase.

In this part, a GFP is added to the C-terminus of the dCas9, connected using an SGAAAAGGS linker. The GFP is added so that the expression of both proteins could be checked easier.

We did not add ribosome binding sites or promoters to this sequence to allow larger flexibility for users to choose the promoter and RBS that is suitable for their chassis.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1837
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 4116
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 644


  1. ↑ Larson, M. H. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols, 2180–2196.