Part:BBa_K1903021:Design

Insertion 2 for dCas9 version A

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 153
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 474
  

Design Notes

This part is a protein insertion design to be assembled in dCas9 version A's (BBa_K1903001) E802 amino acid hotspot by a Golden Gate reaction. The part is flanked by two BbsI recognition sites and it has the fusion sites GAGC and TGCC at the start and end of the sequence, as its shown below (Insertion B).

Although this part is a 2xGGGS linker-SSp DnaB-Split TEV-Npu DnaE-SSp DnaB-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 at the first BbsI fusion site's (GAGC) cytosine and replace the amino acids from 23 to 308 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.