Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012696 | pLVX-mCherry-N1 | In stock, instant shipping |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pLVX-mCherry-N1 is an HIV-1-based, lentiviral expression vector that allows you to express your gene of interest fused to mCherry, a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (1). The excitation and emission maxima of the native mCherry protein are 587 nm and 610 nm, respectively. Genes cloned into the multiple cloning site (MCS), located upstream of the mCherry coding sequence, are expressed as N-terminal mCherry fusion proteins. Expression of the fusion protein is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE), located just upstream of the MCS. Lentiviral particles derived from the vector allow the expression of mCherry fusion proteins in virtually any cell type, including primary cells. The unmodified vector expresses mCherry, and may be used to produce marker virus to optimize infection protocols. pLVX-mCherry-N1 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 and transgene RNA (2), leading to increased viral titers from packaging cells, and enhanced expression of your gene of interest in target 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-mCherry-N1 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). In addition to lentiviral elements, pLVX-mCherry-N1 contains a puromycin resistance gene (Puror) under the control of the murine phosphoglycerate kinase (PGK) promoter (PPGK) for the selection of stable transductants. 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-mCherry-N1
- Antibiotic Resistance:
- Ampicillin
- Length:
- 8778 bp
- Type:
- Viral Expression & Packaging Vectors
- Replication origin:
- ori
- Source/Author:
- Clontech
- Selection Marker:
- Puromycin
- Copy Number:
- High copy number
- Promoter:
- mPGK
- Cloning Method:
- Enzyme Cut
- Fusion Tag:
- mCherry
- Growth Strain(s):
- stbl3
- Growth Temperature:
- 37℃
- Expression Method:
- Constiutive, Stable
pLVX-mCherry-N1 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.
pLVX-mCherry-N1 vector Sequence
LOCUS V012696 8778 bp DNA circular SYN 08-APR-2021
DEFINITION Exported.
ACCESSION V012696
VERSION V012696
KEYWORDS pLVX-mCherry-N1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 8778)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 8778)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..8778
/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"
enhancer 2201..2504
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 2505..2708
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
misc_feature 2793..2873
/label="MCS"
/note="multiple cloning site"
CDS 2875..3582
/label="mCherry"
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
promoter 3613..4112
/label="PGK promoter"
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 4133..4729
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 4746..5334
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 5541..6174
/label="5' LTR"
/note="5' long terminal repeat (LTR) from HIV-1"
primer_bind complement(6303..6319)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(6327..6343)
/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(6351..6381)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(6396..6417)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(6705..7293)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(7467..8324)
/label="AmpR"
/note="beta-lactamase"
promoter complement(8325..8429)
/label="AmpR promoter"
polyA_signal 8477..8611
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"