Composite

Part:BBa_K3406006:Design

Designed by: Xiaojie Zhou   Group: iGEM20_ZJUT_China_B   (2020-09-02)


6xHis/Twin strep -SUMO-PsmCas13b


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1266
    Illegal BglII site found at 3600
    Illegal BamHI site found at 144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 644
    Illegal NgoMIV site found at 1622
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 880
    Illegal SapI.rc site found at 2611


Design Notes

The sequence of PsmCas13 protein contains rare codons,so in order to be successfully expressed,a host which is enriched in tRNAs for rare codons is required(such as E. coli Rosetta2(DE3)). 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,so we attached a solubility tag to the protein coding sequence.

Source

Prevotella sp.MA2016

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.