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Part:BBa_K1903023:Design

Designed by: Rafael Montenegro Marín   Group: iGEM16_TEC-Costa_Rica   (2016-10-18)


Insertion 2 for dCas9 version B


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part is a protein insertion design to be assembled in dCas9 version B's (BBa_K1903002) N888 amino acid hotspot by a Golden Gate reaction. The part is flanked by two BbsI recognition sites and it has the fusion sites AATG and TACT at the start and end of the sequence, as its shown below (Insertion B).

Although this part is a 2xGGGS linker-SSp DnaB-NSplit Tev-Npu DnaE-SSp DnaB-2xGGGS linker insertion, its sequence can be adapted to insert any protein into dCas9 version B's first insertion frame. For adapting this part, you should translate the sequence right after the first BbsI fusion site (AATG) and replace the amino acids from 9 to 294 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.

Source

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.