Part:BBa_K1903020:Design

Insertion 1 for dCas9 version A

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 29
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 142
  

Design Notes

This part is a protein insertion design to be assembled in dCas9 version A's (BBa_K1903001) L390 amino acid hotspot by a Golden Gate reaction. The part (insertion A) is flanked by two BbsI recognition sites and it has the fusion sites ACTA and TTAC at the start and end of the sequence, as it's represented below.

Although this part is a 2xGGGS linker-Intein SSp DnaE-2xGGGS linker insertion, its sequence can be adapted to insert any protein into dCas9 version A's first insertion frame. For adapting this part, you should translate the sequence right after the first BbsI fusion site (ACTA) and replace the amino acids from 47 to 206 for the sequence of the protein you are interested in inserting. It is important to add that the rest of the amino acid sequence of this BioBrick encodes for parts of the dCas9 that are lost when creating the insertion frame.

References

Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PloS one, 6(2), e16765.