Part:BBa_K3219000

dCas9-GFP

This part consists of the sequence of dead Cas9-GFP complex connected using linker 2A, a 7x His-tag and a ribosome binding site. A dead Cas9 enzyme (dCas9) is a mutation of the CRISPR Cas9 enzyme that lacks endonuclease activity. With the help of a guide RNA, the dCas9 specifically binds to the target DNA and blocks transcript elongation by RNA polymerase, and thus repress the expression of the target gene. The GFP is added for easier selection of positive colonies and to indicate that the dCas9-enzyme has been expressed. Between the dCas9 and GF, we added a linker 2A, which is a self-cleaving linker. This allows the GFP and the dCas9 to fold independently. The 7x His-Tag is for protein purification.

This sequence can be used by cloning it into a desired vector with a promoter.

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