Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V012723 | pLVX-IRES-mCherry | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pLVX-IRES-mCherry is an HIV-1-based, lentiviral expression vector that allows the simultaneous expression of your protein of interest and mCherry in virtually any mammalian cell type, including primary cells. mCherry is a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (1). The vector expresses the two proteins from a bicistronic mRNA transcript, allowing mCherry to be used as an indicator of transduction efficiency and a marker for selection by flow cytometry. Expression of the bicistronic transcript is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE) located just upstream of the MCS. An encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), positioned between the MCS and mCherry, facilitates cap-independent translation of mCherry from an internal start site at the IRES/mCherry junction (1). pLVX-IRES-mCherry contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, 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-IRES-mCherry 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-IRES-mCherry
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8172 bp
- Type:
- Viral Expression & Packaging Vectors
- Replication origin:
- ori
- Source/Author:
- Clontech
- Selection Marker:
- mCherry
- Copy Number:
- High copy number
- Promoter:
- CMV
- Cloning Method:
- Enzyme Cut
- Expression Method:
- Constiutive, Stable
pLVX-IRES-mCherry 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
- Mereby SA, Maehigashi T, Holler JM, Kim DH, Schinazi RF, Kim B. Interplay of ancestral non-primate lentiviruses with the virus-restricting SAMHD1 proteins of their hosts. J Biol Chem. 2018 Oct 19;293(42):16402-16412.
pLVX-IRES-mCherry vector Sequence
LOCUS Exported 8172 bp DNA circular SYN 10-SEP-2025
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8172)
AUTHORS hfytfiu
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..8172
/mol_type="other DNA"
/organism="synthetic DNA construct"
source join(6340..8172,1..6339)
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 195..311
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1 (lacking the first T)"
enhancer 368..671
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 672..875
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 1010..1582
/label=IRES
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 1584..2294
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
misc_feature 2308..2896
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(2779..2790)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 3103..3736
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
primer_bind complement(3864..3880)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind 3888..3904
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(3912..3942)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 3957..3978
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(4266..4854)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5025..5885)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5886..5990)
/gene="bla"
/label=AmpR promoter
polyA_signal 6038..6172
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
LTR 6340..6973
/label=3' LTR
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 7020..7145
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7642..7875
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."