Basic Vector Information
Bacterial expression of WT L. buccalis C2c2 CRISPR effector. Codon optimized for E. Coli expression. His-MBP fusion tag can be removed with TEV protease.
p2CT-His-MBP-Lbu_C2c2_WT vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
p2CT-His-MBP-Lbu_C2c2_WT vector Sequence
LOCUS p2CT-His-MBP-Lbu 9437 bp DNA circular SYN 03-JUL-2018 DEFINITION Bacterial expression of WT L. buccalis C2c2 CRISPR effector.. ACCESSION . VERSION . KEYWORDS p2CT-His-MBP-Lbu-C2c2-WT. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9437) AUTHORS East-Seletsky A, O'Connell MR, Knight SC, Burstein D, Cate JH, Tjian R, Doudna JA TITLE Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. JOURNAL Nature. 2016 Sep 26. doi: 10.1038/nature19802. PUBMED 27669025 REFERENCE 2 (bases 1 to 9437) TITLE Direct Submission REFERENCE 3 (bases 1 to 9437) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 2016 Sep 26. doi: 10.1038/nature19802." COMMENT SGRef: number: 2; type: "Journal Article" FEATURES Location/Qualifiers source 1..9437 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 104..208 /label=AmpR promoter CDS 209..1066 /label=AmpR /note="beta-lactamase" rep_origin 1240..1828 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" misc_feature complement(2014..2156) /label=bom /note="basis of mobility region from pBR322" CDS complement(2261..2449) /label=rop /note="Rop protein, which maintains plasmids at low copy number" promoter 4010..4028 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" RBS 4117..4139 /label=RBS /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 4159..4176 /label=6xHis /note="6xHis affinity tag" CDS 4186..5286 /label=MBP /note="maltose binding protein from E. coli" CDS 5341..5361 /label=TEV site /note="tobacco etch virus (TEV) protease recognition and cleavage site" CDS 5368..8844 /gene="cas13a" /label=cas13a /note="CRISPR-associated endoribonuclease Cas13a from Leptotrichia buccalis (strain ATCC 14201 / DSM 1135 / JCM 12969 / NCTC 10249 / C-1013-b). Accession#: C7NBY4" terminator 8987..9034 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(9400..9428) /label=tet promoter /note="E. coli promoter for tetracycline efflux protein gene"
This page is informational only.