Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V013182 | pET28a-PPK | 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:
- pET28a-PPK
- Antibiotic Resistance:
- Kanamycin
- Length:
- 7297 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- E. coli
- Promoter:
- T7
- Growth Strain(s):
- DH5a
- Growth Temperature:
- 37℃
pET28a-PPK 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.
pET28a-PPK vector Sequence
LOCUS V013182 7297 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V013182
VERSION V013182
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 7297)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..7297
/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..2227)
/gene="ppk"
/label="Polyphosphate kinase"
/note="Polyphosphate kinase from Escherichia coli O157:H7.
Accession#: P0A7B2"
RBS complement(2234..2256)
/label="RBS"
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
protein_bind complement(2271..2295)
/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(2296..2314)
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 2623..2700
/label="lacI promoter"
CDS 2701..3780
/label="lacI"
/note="lac repressor"
protein_bind 3796..3817
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
CDS 4592..4780
/label="rop"
/note="Rop protein, which maintains plasmids at low copy
number"
misc_feature 4885..5027
/label="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(5213..5801)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 5923..6735
/label="KanR"
/note="aminoglycoside phosphotransferase"
rep_origin complement(6831..7286)
/direction=LEFT
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"