Coding

Part:BBa_K2429183:Design

Designed by: Nia Myrie   Group: iGEM17_MIT   (2017-10-26)


L. shahii Cas13a BioBrick


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 572
    Illegal BglII site found at 1196
    Illegal BglII site found at 1592
    Illegal BglII site found at 1883
    Illegal BglII site found at 1973
    Illegal BglII site found at 3134
    Illegal BglII site found at 3221
    Illegal BamHI site found at 4216
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4173
    Illegal NgoMIV site found at 4192
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 558
    Illegal SapI.rc site found at 664
    Illegal SapI.rc site found at 1534
    Illegal SapI.rc site found at 2368


Design Notes

Due to the gene's large size, dCas13a had many restriction sites that would not make it BioBrick Compatible. To account for this, our team decided to order g-blocks of dCas13a with slightly altered sequences to rid if prohibited restriction sites rather than apply multiple point mutations to the existing construct. After the g-blocks arrived, a teammate applied Gibson Assembly to put them together in the pSB1C3 backbone.

Source

Sequence for L. shahii Cas13a was found from Addgene(https://www.addgene.org/79150/), which is also where our team received the original plasmid from.

References