Coding

Part:BBa_K1723000:Design

Designed by: Gregoire Thouvenin   Group: iGEM15_EPF_Lausanne   (2015-08-10)
Revision as of 11:57, 25 August 2015 by Gregthouvenin (Talk | contribs) (→‎Source)

dCas9-ω


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
    Illegal BamHI site found at 4212
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 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. Cas9-ω contains an EcoRI restriction site which was removed via site-directed mutagenesis. Fusion of the omega subunit (rpoZ) to dCas9 was achieved by Gibson assembly.

Source

A plasmid containing the omega subunit (pWJ66) was given to the iGEM15_EPFL team by David Bikard. The dCas9 protein was obtained from Addgene pdCas9-bacteria (Plasmid #44249)