Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V012715 | pLVX-shRNA2 | In stock, 1 week for quality controls |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pLVX-shRNA2 is an HIV-1-based, lentiviral expression vector designed to express a small hairpin RNA (shRNA) for RNA interference (RNAi) studies. Expression of your shRNA is driven by the RNA Pol III-dependent, human U6 promoter (PU6), located just upstream of the MCS. pLVX-shRNA2 can be used as a plasmid expression vector and transfected into cells, or it can be packaged into viral particles and transduced into cells. Lentiviral particles derived from the vector allow the expression of shRNAs in virtually any cell type, including primary cells. In addition to expressing shRNAs, pLVX-shRNA2 also expresses the fluorescent protein ZsGreen1, a human codon-optimized variant of the reef coral Zoanthus sp. green fluorescent protein, ZsGreen (1). Expression of ZsGreen1 is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE), allowing it to be used as an indicator of transfection or transduction efficiency, as well as a marker for cell sorting. pLVX-shRNA2 contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral RNA (2), leading to increased viral titers from packaging cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (3). Finally, pLVX-shRNA2 also contains a central polypurine tract/central termination sequence element (cPPT/CTS). During target cell infection, this element creates a central DNA flap that increases nuclear import of the viral genome, resulting in improved vector integration and more efficient transduction (4). The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.
- Vector Name:
- pLVX-shRNA2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7880 bp
- Type:
- Viral Expression & Packaging Vectors
- Replication origin:
- ori
- Source/Author:
- Clontech
- Selection Marker:
- ZsGreen1
- Copy Number:
- High copy number
- Promoter:
- U6
- Cloning Method:
- Enzyme Cut
pLVX-shRNA2 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
- He, X., He, Q., Yu, W., Huang, J., Yang, M., Chen, W., & Han, W. (2021). Optimized protocol for high-titer lentivirus production and transduction of primary fibroblasts. Journal of basic microbiology, 61(5), 430–442. https://doi.org/10.1002/jobm.202100008
pLVX-shRNA2 vector Sequence
LOCUS V012715 7880 bp DNA circular SYN 08-APR-2021
DEFINITION Exported.
ACCESSION V012715
VERSION V012715
KEYWORDS pLVX-shRNA2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 7880)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7880)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7880
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 1..634
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1303..1536
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1721..1765
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 1914..1955
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2027..2144
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2196..2436
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
enhancer 2522..2825
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 2826..3029
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 3074..3766
/label="ZsGreen1"
/note="Zoanthus green fluorescent protein"
misc_feature 3802..4390
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 4597..5230
/label="5' LTR"
/note="5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(5358..5374)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(5382..5398)
/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(5406..5436)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(5451..5472)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(5760..6348)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6522..7379)
/label="AmpR"
/note="beta-lactamase"
promoter complement(7380..7484)
/label="AmpR promoter"
polyA_signal 7532..7666
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"