Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010997 | pT3-EF1a-c-Met | 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:
- pT3-EF1a-c-Met
- Antibiotic Resistance:
- Ampicillin
- Length:
- 9743 bp
- Type:
- Mammalian Expression
- Replication origin:
- ori
- Copy Number:
- Low Copy
- Promoter:
- EF-1α
- Cloning Method:
- Gateway Cloning
- 5' Primer:
- EF-1a-Fwd
pT3-EF1a-c-Met 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.
pT3-EF1a-c-Met vector Sequence
LOCUS V010997 9743 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V010997
VERSION V010997
KEYWORDS pT3-EF1a-c-Met
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 9743)
AUTHORS Tward AD, Jones KD, Yant S, Cheung ST, Fan ST, Chen X, Kay MA, Wang
R, Bishop JM
TITLE Distinct pathways of genomic progression to benign and malignant
tumors of the liver.
JOURNAL Proc Natl Acad Sci U S A. 2007 Sep 11;104(37):14771-6. Epub 2007 Sep
4.
PUBMED 17785413
REFERENCE 2 (bases 1 to 9743)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 9743)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl
Acad Sci U S A."; date: "2007-09-11"; volume: "104(37)"; pages:
"14771-6. Epub 2007 Sep 4"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..9743
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind 174..193
/label="pBR322ori-F"
/note="pBR322 origin, forward primer"
primer_bind 427..444
/label="L4440"
/note="L4440 vector, forward primer"
protein_bind 561..582
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 597..627
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 635..651
/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)."
primer_bind 659..675
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(1101..1120)
/label="EBV-rev"
/note="SV40 polyA terminator, reverse primer"
protein_bind 1173..1206
/label="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
promoter 1347..2525
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
promoter 2542..2560
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 2639..2663
/label="attB1"
/note="recombination site for the Gateway(R) BP reaction"
CDS 2684..6853
/gene="MET"
/label="Hepatocyte growth factor receptor"
/note="Hepatocyte growth factor receptor from Homo sapiens.
Accession#: P08581"
protein_bind complement(6855..6879)
/label="attB2"
/note="recombination site for the Gateway(R) BP reaction"
CDS 6932..6973
/codon_start=1
/product="epitope tag from simian virus 5"
/label="V5 tag"
/translation="GKPIPNPLLGLDST"
polyA_signal 7017..7241
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
protein_bind complement(7311..7344)
/label="loxP"
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
primer_bind 7400..7420
/label="pBABE 3'"
/note="SV40 enhancer, reverse primer for pBABE vectors"
primer_bind complement(7802..7818)
/label="M13 fwd"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8027..8046)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 8146..8168
/label="pGEX 3'"
/note="pGEX vectors, reverse primer"
primer_bind complement(8206..8224)
/label="pBRforEco"
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 8292..8396
/label="AmpR promoter"
CDS 8397..9254
/label="AmpR"
/note="beta-lactamase"
rep_origin 9428..9743
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"