Part:BBa_K3738027:Design

Lbu-Cas13a with an N-terminal Anionic Tag and C-Terminal 6xHistidine Tag and crRNA

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1162
  Illegal PstI site found at 2017
  Illegal PstI site found at 2956
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1162
  Illegal PstI site found at 2017
  Illegal PstI site found at 2956
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 443
  Illegal BglII site found at 1379
  Illegal BglII site found at 1589
  Illegal BglII site found at 1643
  Illegal BglII site found at 1874
  Illegal BglII site found at 2147
  Illegal BglII site found at 2633
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1162
  Illegal PstI site found at 2017
  Illegal PstI site found at 2956
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1162
  Illegal PstI site found at 2017
  Illegal PstI site found at 2956
  Illegal NgoMIV site found at 2095
  Illegal NgoMIV site found at 2731
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This part has been codon optimized for E.coli. The part must be in vitro transcribed to obtain the direct-repeat stem loop RNA structure and complex with Cas13a.

Source

The Mcy gene cluster comes from Microcystic Aeruginosa, however the crRNA originates from Leptrotrichia buccalis. The Cas13a protein comes from the Gram-negative bacteria Leptotrichia buccalis (Lbu).

References

Glasgow, J., Capehart, S., Francis, M., and Tullman, D (2012) Osmolyte-Mediated Encapsulation of Proteins inside MS2 Viral Capsids. ACS Nano. 10, 8658-8664.

Huynh, N., Depner, N., Larson, R. et al. A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila. Genome Biol 21, 279 (2020). https://doi.org/10.1186/s13059-020-02193-y

McDade Joel. CRISPR 101: Targeting RNA with Cas13a (C2c2). Retrieved from https://blog.addgene.org/crispr-101-targeting-rna-with-cas1