Coding

Part:BBa_K2477000

Designed by: Jacob Premo   Group: iGEM17_UMaryland   (2017-10-25)


iGEM GFP (E0040) mutant with added PAM site

The GFP gene was originally obtained from the iGEM 2017 DNA kit. Site directed mutagenesis was used to create a synonymous mutation at base pair 186 where a cytosine was incorporated into the top strand to replace a thymine. This acted to create a PAM site for Cas9 from S. pyogenes (5' NGG 3') on the bottom strand. This mutation was made in order to allow for the design of an sgRNA capable of interacting with the chromophore encoding region of GFP. The biobrick number of the sgRNA designed by our team for this purpose is BBa_K2477001.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 644


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Parameters
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