Part:BBa_K4727008

dCas9 standardized

dead Cas9 (dCas9), in which the two nuclease domains, HNH and RuvC, are catalytically inactive. Therefore, the protein is unable to cleave the DNA but can only bind to the target DNA sequence that is complementary to the guide RNA [1]. The dCas9:sgRNA complex performs the transcription repression or the modulation of the expression of the target.

The coding sequence for dCas9, though, is not suitable as a standard part due to the presence of an EcoRI site in position 1339 5'-end of the dCas9 coding sequence. This new registered part solves this problem, as the EcoRI restriction site present in the sequence, has been removed and mutagenised altering the sequence with a silent mutation that maintains the codon usage in E. coli unvaried.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1099
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3378
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Mahas, Ahmed, C. Neal Stewart, e Magdy M. Mahfouz. «Harnessing CRISPR/Cas Systems for Programmable Transcriptional and Post-Transcriptional Regulation». Biotechnology Advances 36, fasc. 1 (gennaio 2018): 295–310. https://doi.org/10.1016/j.biotechadv.2017.11.008.