Part:BBa_K3738026:Design

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1134
  Illegal PstI site found at 1989
  Illegal PstI site found at 2928
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1134
  Illegal PstI site found at 1989
  Illegal PstI site found at 2928
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 415
  Illegal BglII site found at 1351
  Illegal BglII site found at 1561
  Illegal BglII site found at 1615
  Illegal BglII site found at 1846
  Illegal BglII site found at 2119
  Illegal BglII site found at 2605
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1134
  Illegal PstI site found at 1989
  Illegal PstI site found at 2928
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1134
  Illegal PstI site found at 1989
  Illegal PstI site found at 2928
  Illegal NgoMIV site found at 2067
  Illegal NgoMIV site found at 2703
  
• 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