Coding

Part:BBa_K3454038:Design

Designed by: Ruijie Xiang   Group: iGEM20_ShanghaiTech_China   (2020-10-26)


AsCas12a_R


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 196
    Illegal PstI site found at 3667
    Illegal PstI site found at 3865
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 886
    Illegal PstI site found at 196
    Illegal PstI site found at 3667
    Illegal PstI site found at 3865
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3855
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 196
    Illegal PstI site found at 3667
    Illegal PstI site found at 3865
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 196
    Illegal PstI site found at 3667
    Illegal PstI site found at 3865
    Illegal NgoMIV site found at 3399
    Illegal AgeI site found at 2296
    Illegal AgeI site found at 2767
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1039
    Illegal BsaI.rc site found at 2701
    Illegal SapI.rc site found at 289


Design Notes

This is designed with MBP and his tag that can help AsCas12a mutant expression, folding and purification.

According to "Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing", we introduced mutation S542R

Source

It comes from expression of plasmid DNA(contains designed mutation ) of E.coli

References

Kleinstiver BP, Sousa AA, Walton RT, et al. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing [published correction appears in Nat Biotechnol. 2020 Jul;38(7):901]. Nat Biotechnol. 2019;37(3):276-282. doi:10.1038/s41587-018-0011-0