Basic Vector Information
JDS246 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 ℃.
JDS246 vector Sequence
LOCUS JDS246. 7614 bp DNA circular SYN 01-JAN-1980 DEFINITION Plasmid for expressing FLAG(R)-tagged human codon-optimized Cas9 in mammalian cells. ACCESSION . VERSION . KEYWORDS JDS246. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7614) AUTHORS Joung Lab / Addgene #43861 TITLE Direct Submission REFERENCE 2 (bases 1 to 7614) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..7614 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 132..335 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" promoter 377..395 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" CDS 409..4512 /label=Cas9 /note="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" CDS 4519..4539 /codon_start=1 /product="nuclear localization signal of SV40 large T antigen" /note="SV40 NLS" /translation="PKKKRKV" CDS 4546..4611 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=three tandem FLAG /note="3xFLAG" /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 4629..4646 /label=6xHis /note="6xHis affinity tag" polyA_signal 4675..4899 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" primer_bind complement(4970..4986) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind complement(4994..5010) /label=lac operator /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(5018..5048) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(5063..5084) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(5372..5960) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(6134..6991) /label=AmpR /note="beta-lactamase" promoter complement(6992..7096) /label=AmpR promoter enhancer 7366..7614 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer"
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