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.
Check the wikipage of IISER_Berhampur 2021 to know more about the working of this part.
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).
We have used parts BBa_K3982001, BBa_K3982002, BBa_K3982003 to build this composite part.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1411
Illegal PstI site found at 201
Illegal PstI site found at 367 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1411
Illegal PstI site found at 201
Illegal PstI site found at 367 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1411
Illegal BamHI site found at 1005 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1411
Illegal PstI site found at 201
Illegal PstI site found at 367 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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