Coding

Part:BBa_K1179009:Design

Designed by: Nelson Hall   Group: iGEM13_MIT   (2013-09-15)


(Cas9-VP16) A non-nuclease Cas9 with a C-terminal VP16 fusion for activation


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3343
    Illegal PstI site found at 1732
    Illegal PstI site found at 1966
    Illegal PstI site found at 3178
    Illegal PstI site found at 3482
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3343
    Illegal PstI site found at 1732
    Illegal PstI site found at 1966
    Illegal PstI site found at 3178
    Illegal PstI site found at 3482
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3343
    Illegal BglII site found at 708
    Illegal BamHI site found at 1526
    Illegal XhoI site found at 3064
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3343
    Illegal PstI site found at 1732
    Illegal PstI site found at 1966
    Illegal PstI site found at 3178
    Illegal PstI site found at 3482
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3343
    Illegal PstI site found at 1732
    Illegal PstI site found at 1966
    Illegal PstI site found at 3178
    Illegal PstI site found at 3482
    Illegal NgoMIV site found at 2260
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The BsaI digestion and ligation was a tricky process to nail down, so expect some failed attempts when trying to construct Cas9-VP16 if following the same construction method.

Source

It is a fusion between the mutant DNA-binding Cas9 protein from Streptococcus pyogenes and the viral VP16 transactivation domain from the herpes simplex virus. This entry vector was synthesized though the BsaI sites in part BBa_K1179008 and in the coding sequence of VP16 attached with a BsaI site at the front of the sequence, which was made in a gBlock by IDT.

References