Coding

Part:BBa_K2361000:Design

Designed by: Mart Bartelds   Group: iGEM17_Groningen   (2017-10-09)


spdCas9


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1100
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3379
    Illegal XhoI site found at 4115
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part originates from Streptococcus pyogenes Cas9 and it contains two amino acid substitutions (D10A & H840A) that make it catalytically dead. Also it contains one base substitution (T->A) that remove the illegal EcoRI site. The two amino acid substitutions were already present in the part we started with, so we made it biobrick compatible by removing the EcoRI site and adding the prefix and suffix.

Source

This part originates from the Addgene plasmid pJWV102-PL-dCas9. The dCas9 on this plasmid is a non-codon optimized variant of the S. pyogenes Cas9 gene.

References

High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Liu X, Gallay C, Kjos M, Domenech A, Slager J, van Kessel SP, Knoops K, Sorg RA, Zhang JR, Veening JW. Mol Syst Biol. 2017 May 10;13(5):931. doi: 10.15252/msb.20167449.