Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010067 | BE4-Gam | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- BE4-Gam
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9444 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- CMV
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- T7
- 3' Primer:
- M13R
BE4-Gam vector Vector Map
Plasmid 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 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
BE4-Gam vector Sequence
LOCUS V010067 9444 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V010067
VERSION V010067
KEYWORDS BE4-Gam
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 9444)
AUTHORS Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW,
Kim YB, Badran AH, Liu DR
TITLE Improved base excision repair inhibition and bacteriophage Mu Gam
protein yields C:G-to-T:A base editors with higher efficiency and
product purity.
JOURNAL Sci Adv. 2017 Aug 30;3(8):eaao4774. doi: 10.1126/sciadv.aao4774.
eCollection 2017 Aug.
PUBMED 28875174
REFERENCE 2 (bases 1 to 9444)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9444)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Sci Adv.";
date: "2017-08-30"; pages: "
10.1126/sciadv.aao4774. eCollection 2017 Aug"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9444
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 132..335
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 377..395
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 409..930
/gene="gam"
/label="Putative DNA ends protecting protein gam"
/note="Putative DNA ends protecting protein gam from
Escherichia phage Mu. Accession#: P06023"
CDS 979..1662
/label="APOBEC-1"
/note="cytidine deaminase (C to U editing enzyme) from rat"
CDS 1759..5859
/label="Cas9(D10A)"
/note="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 5890..6138
/label="UGI"
/note="uracil-DNA glycosylase inhibitor from a Bacillus
subtilis bacteriophage (Mol et al., 1995)"
CDS 6169..6417
/codon_start=1
/product="uracil-DNA glycosylase inhibitor from a Bacillus
subtilis bacteriophage (Mol et al., 1995)"
/label="UGI"
/translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
CDS 6430..6450
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 6459..6476
/label="6xHis"
/note="6xHis affinity tag"
polyA_signal 6505..6729
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(6800..6816)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6824..6840)
/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(6848..6878)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(6893..6914)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(7031..7048)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(7202..7790)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7964..8821)
/label="AmpR"
/note="beta-lactamase"
promoter complement(8822..8926)
/label="AmpR promoter"
primer_bind complement(9005..9024)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 9196..9444
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"