Part:BBa_K3602022

Cas12a w. tags, optimized for human cells

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_K3602015). The sequence was codon-optimized for human cells. 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
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 430
  Illegal PstI site found at 1492
  Illegal PstI site found at 3901
  Illegal PstI site found at 4099
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 430
  Illegal PstI site found at 1492
  Illegal PstI site found at 3901
  Illegal PstI site found at 4099
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 614
  Illegal BglII site found at 2156
  Illegal BglII site found at 2465
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 430
  Illegal PstI site found at 1492
  Illegal PstI site found at 3901
  Illegal PstI site found at 4099
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 430
  Illegal PstI site found at 1492
  Illegal PstI site found at 3901
  Illegal PstI site found at 4099
  Illegal NgoMIV site found at 3700
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 212