Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V011613 | pIZT/V5-His | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
An insect cell expression vector featuring a constitutive promoter driving a GFP-Zeocin resistance fusion gene cassette. This system enables both selection (via Zeocin) and visual monitoring (via GFP) of stably transfected cells. It is designed for constitutive protein expression and optionally incorporates a C-terminal V5-6xHis tag for detection and purification.
- Vector Name:
- pIZT/V5-His
- Antibiotic Resistance:
- Zeocin
- Length:
- 3336 bp
- Type:
- Insect Cell Vectors
- Replication origin:
- ori
- Source/Author:
- Invitrogen (Life Technologies)
- Copy Number:
- High copy number
- Promoter:
- OpIE-2
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pIZT/V5-His 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
- Liang Z, Lu Y, Qian Y, Zhu L, Kuang S, Chen F, Feng Y, Hu X, Cao G, Xue R, Gong C. Cultured cells and wing disc size of silkworm can be controlled by the Hippo pathway. Open Biol. 2018 Jul;8(7):180029. doi: 10.1098/rsob.180029. PMID: 29973396; PMCID: PMC6070717.
pIZT/V5-His vector Sequence
LOCUS pIZT_V5-His. 3336 bp DNA circular SYN 01-JAN-1980
DEFINITION Insect cell vector with a GFP-Zeocin(TM) resistance fusion gene, for
constitutive expression of proteins with an optional C-terminal
V5-6xHis tag.
ACCESSION .
VERSION .
KEYWORDS pIZT V5-His
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3336)
AUTHORS Invitrogen (Life Technologies)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 3336)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..3336
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 5..552
/label=OpIE-2 promoter
/note="strong constitutive baculovirus promoter for insect
cell expression"
misc_feature 567..656
/label=MCS
/note="MCS"
/note="multiple cloning site"
CDS 663..704
/label=V5 tag
/note="epitope tag from simian virus 5"
CDS 714..731
/label=6xHis
/note="6xHis affinity tag"
polyA_signal 749..878
/label=OpIE-2 poly(A) signal
/note="baculovirus polyadenylation signal"
rep_origin complement(1006..1594)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 1669..1960
/label=OpIE-1 promoter
/note="moderate constitutive baculovirus promoter for
insect cell expression"
promoter 1986..2033
/label=EM7 promoter
/note="synthetic bacterial promoter"
CDS 2052..2054
/codon_start=1
/product="start codon"
/label=start codon
/note="ATG"
/translation="M"
CDS 2067..2771
/label=Cycle 3 GFP
/note="Cycle 3 GFP (Crameri et al., 1996)"
CDS 2766..3143
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=Sh ble from Streptoalloteichus hindustanus
/note="BleoR"
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MDAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRD
DVTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWG
REFALRDPAGNCVHFVAEEQD"
polyA_signal 3207..3336
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"