Coding

Part:BBa_K1723021:Design

Designed by: Cyril Pulver   Group: iGEM15_EPF_Lausanne   (2015-09-16)

dCas9_VP64


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 4
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4153
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 4
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 4
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

dCas9 is a Cas9 double mutant, with mutations at amino acid positions D10A and H840A. These mutations inactivate Cas9 nuclease and nickase activities. SV40 nuclear localization sequence recruits dCas9_VP64 to the nucleus, thus allowing it to interact with the genome. VP64 eukaryotic transcription factor recruits RNA polymerase II, thus enabling the transcription factor activity of dCas9_VP64. This construct is yeast codon optimized[1].


Source

dCas9_VP64 was extracted from plasmid pTPGI_dCas9_VP64 which was a gift from Timothy Lu (Addgene plasmid # 49013).

References

[1] Farzadfard, F., Perli, S.D., Lu, T.K., 2013. Tunable and Multifunctional Eukaryotic Transcription Factors Based on CRISPR/Cas, ACS Synthetic Biology (ACS publications), http://pubs.acs.org/doi/pdf/10.1021/sb400081r (16.09.2015)