Plasmid

Part:BBa_K3602021

Designed by: Flora Fuglsang   Group: iGEM20_SDU-Denmark   (2020-10-23)


Cas12a composite part, optimized for E. coli K12

The full Cas12a sequence was combined with the positively regulated lac promoter (BBa_R0011). To help translate the protein we chose an effective ribosomal binding site (RBS) which was BBa_B0034. After the promoter and the RBS, the Cas12a protein sequence was placed (BBa_k3602014). The sequence was codon-optimized for E. coli K12 to yield a greater harvest of protein. For a terminator sequence, the part (BBa_B1006) which forms an 8 bp stem, 6 nt loop is used.

Cas12a is a protein with endonuclease activity. In complex with a sgRNA, Cas12a cleaves nearby ssDNA upon complementary binding of a target sequence matching the sgRNA. This can be utilized for detection of different targets, as a DNA reporter can be used for the cleavage. It is noteworthy that IDT was not allowed to produce a part of the Cas12a sequence as it comes from a pathogenic bacterium and therefore requires a special license to ship outside of the United States.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2312
    Illegal BamHI site found at 1557
    Illegal XhoI site found at 1192
    Illegal XhoI site found at 1594
    Illegal XhoI site found at 3055
    Illegal XhoI site found at 3373
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 886
    Illegal AgeI site found at 2377
    Illegal AgeI site found at 3775
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 949


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Categories
Parameters
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