Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V009891 | pET-12b | 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
Hosts: E.coli. Related vectors: pBR322.
- Vector Name:
- pET-12b
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4673 bp
- Type:
- E. coli Expression Vectors
- Replication origin:
- ori
- Copy Number:
- High copy number
- Promoter:
- tet
- Cloning Method:
- Enzyme digestion and ligation
- 5' Primer:
- 5'-TAATACGACTCACTATAGGG-3'
pET-12b 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.
pET-12b vector Sequence
LOCUS 40924_17656 4673 bp DNA circular SYN 13-JAN-2022
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4673)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 4673)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4673
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 10..38
/label=tet promoter
/note="E. coli promoter for tetracycline efflux protein
gene"
terminator complement(404..451)
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
RBS complement(588..610)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
promoter complement(642..660)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 2225..2413
/codon_start=1
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
/translation="VTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
misc_feature 2518..2660
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(2846..3434)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(3608..4465)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(4466..4570)
/label=AmpR promoter