Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V006636 | pET-28a-hPKM1 | 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:
- pET-28a-hPKM1
- Antibiotic Resistance:
- Kanamycin
- Length:
- 6967 bp
- Type:
- Bacterial Expression
- Replication origin:
- ori
- Promoter:
- T7
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- T7 Promoter Primer
- 3' Primer:
- T7 Terminator Primer
pET-28a-hPKM1 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-28a-hPKM1 vector Sequence
LOCUS V006636 6967 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V006636
VERSION V006636
KEYWORDS pET-28a-hPKM1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 6967)
AUTHORS Christofk HR, Vander Heiden MG, Wu N, Asara JM, Cantley LC
TITLE Pyruvate kinase M2 is a phosphotyrosine-binding protein.
JOURNAL Nature. 2008 Mar 13;452(7184):181-6. doi: 10.1038/nature06667.
PUBMED 18337815
REFERENCE 2 (bases 1 to 6967)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6967)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName:
"Nature"; date: "2008-03-13- 13"; volume: "452"; issue: "7184";
pages: "181-6"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6967
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 55..73
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 74..98
/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)."
RBS 113..135
/label="RBS"
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 154..171
/label="6xHis"
/note="6xHis affinity tag"
CDS 181..198
/label="thrombin site"
/note="thrombin recognition and cleavage site"
CDS 202..234
/label="T7 tag (gene 10 leader)"
/note="leader peptide from bacteriophage T7 gene 10"
CDS 250..1842
/gene="PKM"
/label="Pyruvate kinase PKM"
/note="Pyruvate kinase PKM from Pongo abelii. Accession#:
Q5NVN0"
CDS 1886..1903
/label="6xHis"
/note="6xHis affinity tag"
terminator 1970..2017
/label="T7 terminator"
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 2054..2509
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(2604..3416)
/label="KanR"
/note="aminoglycoside phosphotransferase"
rep_origin 3538..4126
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 4280..4297
/label="L4440"
/note="L4440 vector, forward primer"
misc_feature complement(4312..4451)
/label="bom"
/note="basis of mobility region from pBR322"
primer_bind 4537..4559
/label="pGEX 3'"
/note="pGEX vectors, reverse primer"
CDS complement(4556..4744)
/label="rop"
/note="Rop protein, which maintains plasmids at low copy
number"
protein_bind complement(5519..5540)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(5556..6635)
/label="lacI"
/note="lac repressor"
promoter complement(6636..6713)
/label="lacI promoter"
primer_bind 6919..6938
/label="pBRrevBam"
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"