Part:BBa_K3738020:Design

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1089
  Illegal PstI site found at 1944
  Illegal PstI site found at 2883
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1089
  Illegal PstI site found at 1944
  Illegal PstI site found at 2883
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 370
  Illegal BglII site found at 1306
  Illegal BglII site found at 1516
  Illegal BglII site found at 1570
  Illegal BglII site found at 1801
  Illegal BglII site found at 2074
  Illegal BglII site found at 2560
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1089
  Illegal PstI site found at 1944
  Illegal PstI site found at 2883
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1089
  Illegal PstI site found at 1944
  Illegal PstI site found at 2883
  Illegal NgoMIV site found at 2022
  Illegal NgoMIV site found at 2658
  
• 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.