Part:BBa_K3982006

6xHis - Tagged Cas14a1 with TEV site

Cas14a1 is a type V-F exceptionally compact RNA-guided nuclease. Cas14 forms a complex with RNA-guided nuclease and it can cleave ssDNA without PAM specificity. It is tagged with 6xHis and has the Tobacco Etch Virus (TEV) protease and cleavage site for easy purification. In our project CODE M, Cas14a1 and sgRNA complex is used to detect SNPs in the Mycobacterium tuberculosis genome in a sputum sample. Upon detection, it triggers cleavage of a Fluorescence-Quencher pair ssDNA reporter and thus fluorescence is emitted which confirms the presence of SNPs in the genome

Usage and Biology

Cas14a1 (also called as Un1Cas12f1) is a miniature cas protein used in CRISPR/Cas technology identified from the immune system against viral genome in archaebacteria and bacteria. It is a RNA guided nuclease which cleaves ssDNA without any Protospacer Adjacent Motif (PAM) specificity i.e. PAM independent ssDNA cleavage. This enables high-fidelity Single-nucleotide polymorphism (SNP) genotyping. It is guided by a single guide RNA (sgRNA) which has two components - CRISPR RNA (crRNA) and Trans-activating crispr RNA (tracrRNA).

6xHis tagged Cas14a1 gene with TEV site

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1411
  Illegal PstI site found at 201
  Illegal PstI site found at 367
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1411
  Illegal PstI site found at 201
  Illegal PstI site found at 367
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1411
  Illegal BamHI site found at 1005
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1411
  Illegal PstI site found at 201
  Illegal PstI site found at 367
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1411
  Illegal PstI site found at 201
  Illegal PstI site found at 367
  Illegal AgeI site found at 661
  
• 1000
  COMPATIBLE WITH RFC[1000]