Part:BBa_K3219002
Plasmid for in vivo expression of dCas9-sgRNA targeting mcyB
This plasmid consists of a cyanobacteria shuttle vector, ribosome binding sites, a dCas9-GFP complex and a sgRNA targeting mcyB of Microcystis Aeruginosa UTEX McyB.
The shuttle vector is from team 2016 iGEM team Nanjing_NFLS, part BBa_K1894001. It consists of two origin of replications, f1 ori and pUC ori, which allows it to replicate in both E.coli cells and cyanobacteria cells. It also consists of a t7 promoter, CaMV35S promoter, and Kanamycin resistance gene. The T7 promoter allows expression of the dCas9-GFP-sgRNA in E.coli and the CaMVS promoter allows expression in cyanobacteria.
The dCas9, also known as a catalytically dead Cas9 enzyme, is a mutated Cas9 enzyme without endonuclease activity. With the help of a single-guide RNA (sgRNA), it specifically binds to the target sequences and blocks transcript elongation by RNA polymerase. The GFP is added to the C-terminus of the dCas9, connected using a linker. There is also a 7x His-Tag, allowing for protein purification.
The sgRNA consists of a handle, a base-pairing region and a terminator. The base-pairing region is designed to target 25 base pairs of the McyB gene of Microcystis Aeruginosa UTEX2388.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3964
Illegal NheI site found at 12275
Illegal NotI site found at 777 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1685
Illegal XhoI site found at 716 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 7979
Illegal NgoMIV site found at 8139
Illegal NgoMIV site found at 11186 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 5721
Illegal SapI site found at 9059
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