Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V011006 | pET-27b(+) | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The pET-27b(+) is a bacterial expression vector designed to produce C-terminally HSV-tagged proteins in the periplasm. It includes a signal sequence for proper protein targeting and a kanamycin resistance marker for selection.
- Vector Name:
- pET-27b(+)
- Antibiotic Resistance:
- Kanamycin
- Length:
- 5410 bp
- Type:
- pET & Duet Vectors (Novagen)
- Replication origin:
- ori
- Source/Author:
- Novagen (EMD Millipore)
- Copy Number:
- High copy number
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pET-27b(+) 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.
References
- Li T, Xu L, Ren G, et al. A neutralization scFv antibody against IL-1β isolated from a NIPA-based bacterial display library. Curr Pharm Biotechnol. 2013;14(6):571-581. doi:10.2174/138920101131400223
pET-27b(+) vector Sequence
LOCUS Exported 5410 bp DNA circular SYN 05-SEP-2024
DEFINITION Bacterial vector with a signal sequence and a kanamycin resistance
marker for expression of C-terminally HSV-tagged proteins in the
periplasm.
ACCESSION .
VERSION .
KEYWORDS pET-27b(+)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5410)
AUTHORS Novagen (EMD Millipore)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 5410)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5410)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5410
/mol_type="other DNA"
/organism="synthetic DNA construct"
terminator complement(26..73)
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
CDS complement(140..157)
/label=6xHis
/note="6xHis affinity tag"
CDS complement(164..196)
/label=HSV tag
/note="HSV (herpes simplex virus) epitope tag"
misc_feature 212..279
/label=MCS
/note="MCS"
/note="multiple cloning site"
sig_peptide complement(278..343)
/label=pelB signal sequence
/note="leader peptide for secretion"
RBS complement(351..373)
/label=RBS
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(388..412)
/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(413..431)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 740..817
/label=lacI promoter
CDS 818..1897
/label=lacI
/note="lac repressor"
protein_bind 1913..1934
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 2709..2897
/label=rop
/note="Rop protein, which maintains plasmids at low copy
number"
misc_feature 3002..3141
/label=bom
/note="basis of mobility region from pBR322"
rep_origin complement(3327..3915)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 4037..4849
/label=KanR
/note="aminoglycoside phosphotransferase"
rep_origin complement(4944..5399)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"