Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000994 | NL4-3 mCherry Luciferase | 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
The NL4 - 3 mCherry Luciferase vector is an important tool for studying HIV - 1 latency.
It combines mCherry and Luciferase genes connected by a T2A ribosomal skip sequence. Under the same LTR - driven mRNA, they are expressed as separate proteins with Nef under an IRES element.
This dual reporter gene system allows simultaneous monitoring of activated cell number (mCherry) and reactivation strength (Luciferase). It accurately reflects HIV - 1 states, providing a comprehensive perspective for latency mechanism study. Its advantages include comprehensive information acquisition, rapid latency establishment and reactivation detection in primary CD4 T cells and different subsets, and broad application prospects for evaluating activator effects and studying subset differences, supporting HIV latency research and treatment strategy development.
- Vector Name:
- NL4-3 mCherry Luciferase
- Antibiotic Resistance:
- Ampicillin
- Length:
- 17833 bp
- Type:
- Lentiviral
- Replication origin:
- ori
- Selection Marker:
- mCherry, Luciferase
- Promoter:
- EF-1 alpha– driven mCherry:Puromycin
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- from pNLENG1 5'
- 3' Primer:
- from pNLENG1 3'
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
NL4-3 mCherry Luciferase 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
- Lassen KG, Hebbeler AM, Bhattacharyya D, Lobritz MA, Greene WC. A flexible model of HIV-1 latency permitting evaluation of many primary CD4 T-cell reservoirs. PLoS One. 2012;7(1):e30176. doi: 10.1371/journal.pone.0030176. Epub 2012 Jan 24. PMID: 22291913; PMCID: PMC3265466.
NL4-3 mCherry Luciferase vector Sequence
LOCUS V000994 17833 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V000994
VERSION V000994
KEYWORDS NL4-3 mCherry Luciferase
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 17833)
AUTHORS Lassen KG, Hebbeler AM, Bhattacharyya D, Lobritz MA, Greene WC
TITLE A flexible model of HIV-1 latency permitting evaluation of many
primary CD4 T-cell reservoirs.
JOURNAL PLoS One. 2012;7(1):e30176. doi: 10.1371/journal.pone.0030176. Epub
2012 Jan 24.
PUBMED 22291913
REFERENCE 2 (bases 1 to 17833)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 17833)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
date: "2012"; pages: "
10.1371/journal.pone.0030176. Epub 2012 Jan 24"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..17833
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 377..634
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 681..806
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
CDS 790..2289
/label="HIV-1 gag"
/note="gag protein from human immunodeficiency virus 1"
CDS 2085..5093
/label="HIV-1 pol"
/note="pol protein from human immunodeficiency virus 1"
CDS 5041..5616
/gene="vif"
/label="Virion infectivity factor"
/note="Virion infectivity factor from Human
immunodeficiency virus type 1 group M subtype B (isolate
NY5). Accession#: P12504"
CDS 5830..6003
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate BH5). Accession#: P04612"
/label="Protein Tat"
CDS 6221..8779
/gene="env"
/label="Envelope glycoprotein gp160"
/note="Envelope glycoprotein gp160 from Human
immunodeficiency virus type 1 group M subtype B (isolate
MFA). Accession#: P19551"
CDS 8787..9494
/label="mCherry"
/note="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
CDS 9501..9554
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 9558..11204
/label="luciferase"
/note="firefly luciferase"
misc_feature 11212..11794
/label="IRES2"
/note="internal ribosome entry site (IRES) of the
encephalomyocarditis virus (EMCV)"
CDS 11795..11821
/gene="nef"
/label="Protein Nef"
/note="Protein Nef from Human immunodeficiency virus type 1
group M subtype D (isolate Z84). Accession#: P12481"
LTR 12084..12717
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
primer_bind complement(14357..14374)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(14528..15116)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(15290..16147)
/label="AmpR"
/note="beta-lactamase"
promoter complement(16148..16252)
/label="AmpR promoter"
primer_bind 16320..16338
/label="pBRforEco"
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(16376..16398)
/label="pGEX 3'"
/note="pGEX vectors, reverse primer"
primer_bind 16498..16517
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"