Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V017705 | pCMV-PE6c | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pCMV-PE6c is a plasmid used for expression in mammalian cells. It was constructed by the David Liu laboratory and has an Addgene plasmid number of 207853. Its function is to express the PE6c prime editor in mammalian cells.
- Vector Name:
- pCMV-PE6c
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9279 bp
- Type:
- Gene-editing vector
- Replication origin:
- ori
- Host:
- Mammalian cells
- Promoter:
- CMV
- 5' Primer:
- BGH
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pCMV-PE6c 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.
pCMV-PE6c vector Sequence
LOCUS . 9279 bp DNA circular UNK 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION
VERSION
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
enhancer 54..433
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 434..637
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
promoter 679..697
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 747..767
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 765..4868
/label="Cas9(H840A)"
/note="nickase mutant of the Cas9 endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 4926..4946
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 6531..6551
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 6561..6587
/label="c-myc NLS"
/note="nuclear localization signal of human c-Myc
proto-oncogene (Dang and Lee, 1988)"
CDS 6596..6613
/label="6xHis"
/note="6xHis affinity tag"
polyA_signal 6642..6866
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
primer_bind complement(6937..6953)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6961..6977)
/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-β-D-thiogalactopyranoside (IPTG)."
promoter complement(6985..7015)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(7030..7051)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(7339..7927)
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8101..8958)
/label="AmpR"
/note="β-lactamase"
promoter complement(8959..9063)
/label="AmpR promoter"