Coding

Part:BBa_K3406003:Design

Designed by: Xiaojie Zhou   Group: iGEM20_ZJUT_China_B   (2020-08-27)


LbaCas13a coding sequence


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2168
    Illegal BglII site found at 3683
    Illegal BamHI site found at 95
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1738
    Illegal AgeI site found at 2815
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 409
    Illegal BsaI.rc site found at 1492


Design Notes

The sequence of LbaCas13 protein contains rare codens,so in order to be successfully expressed,host which is enriched in tRNAs for rare codons is required(such as E.coli Rosetta2).In addition,Cas13 protein might be toxic to the host,so pLysS is also needed to reduce it's basic expression.Meanwhile,this protein is not very soluble and should be tagged with a solubility tag, such as SUMO or MBP.


Source

Lachnospirace ae bacterium NK4A179

References

Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018 Apr 27;360(6387):439-444. doi: 10.1126/science.aaq0179. Epub 2018 Feb 15. PMID: 29449508; PMCID: PMC5961727.