Basic Vector Information
- Vector Name:
- Cas9m4
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9553 bp
- Type:
- CRISPR Plasmids
- Replication origin:
- ori
- Source/Author:
- Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang
- Selection Marker:
- Neomycin/G418(Geneticin)
- Copy Number:
- High copy number
- Promoter:
- CMV
Cas9m4 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 ℃.
Cas9m4 vector Sequence
LOCUS Cas9m4. 9553 bp DNA circular SYN 01-JAN-1980
DEFINITION Plasmid for expression in mammalian cells of catalytically dead
Cas9m4.
ACCESSION .
VERSION .
KEYWORDS Cas9m4.
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9553)
AUTHORS Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang
L, Church GM.
TITLE CAS9 transcriptional activators for target specificity screening and
paired nickases for cooperative genome engineering.
JOURNAL Nat. Biotechnol. 2013;31:833-8.
PUBMED 23907171
REFERENCE 2 (bases 1 to 9553)
AUTHORS Church Lab / Addgene #47316
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9553)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat.
Biotechnol."; date: "2013"; volume: "31"; pages: "833-8"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9553
/mol_type="other DNA"
/organism="synthetic DNA construct"
protein_bind 107..128
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 143..173
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 181..197
/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)."
primer_bind 205..221
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
polyA_signal complement(258..391)
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
CDS complement(570..1361)
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
promoter complement(1428..1757)
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin complement(1771..2199)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
polyA_signal complement(2401..2449)
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
CDS complement(2548..2568)
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
CDS complement(2581..6684)
/label=Cas9m4
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
promoter complement(6799..7002)
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
enhancer complement(7003..7382)
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 7648..7752
/label=AmpR promoter
CDS 7753..8610
/label=AmpR
/note="beta-lactamase"
rep_origin 8784..9372
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
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