Part:BBa_K3738021:Design

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1117
  Illegal PstI site found at 1972
  Illegal PstI site found at 2911
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1117
  Illegal PstI site found at 1972
  Illegal PstI site found at 2911
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 398
  Illegal BglII site found at 1334
  Illegal BglII site found at 1544
  Illegal BglII site found at 1598
  Illegal BglII site found at 1829
  Illegal BglII site found at 2102
  Illegal BglII site found at 2588
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1117
  Illegal PstI site found at 1972
  Illegal PstI site found at 2911
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1117
  Illegal PstI site found at 1972
  Illegal PstI site found at 2911
  Illegal NgoMIV site found at 2050
  Illegal NgoMIV site found at 2686
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This part is codon-optimized for E. coli.

The anionic tag is present to facilitate uptake into our delivery particle MS2 whereas the Histidine tag was added for the purpose of Nickel-Affinity chromatography in obtaining the purified protein.

Source

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-cas13a-c2c2.