pNL4-3.Luc.R-E- vector (V012797)

Price Information

Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Firefly luciferase gene was inserted into the pNL4-3 nef gene. Two frameshifts (5'Env and Vpr aa 26) render this clone Env- and Vpr-. Competent for a single round of replication. Requires cotransfection with env expression vector to produce infectious virus. Virus can be produced by transfecting 2x10^6 293 or 293T cells with 20 ug of the NL4-3 DNA, or with 10 ug NL4-3 DNA and 10 ug env expression vector DNA. Transfections can be performed in a 10 cm^2 tissue culture dish using standard calcium phosphate protocols. Virus is typically harvested 48 hours post-transfection. Infections should be performed in a total volume of 0.5 ml. Amphotropic pseudotypes generally have much higher infectivity than those bearing HIV-1 env. Cultures infected with the luciferase viruses can be lysed 2-5 days post-infection and assayed using commercial lysis buffer and luciferase reagents

pNL4-3.Luc.R-E- 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

• Wang B, Wang B, Liu P, Li T, Si W, Xiu J, Liu H. Package of NDV-pseudotyped HIV-Luc virus and its application in the neutralization assay for NDV infection. PLoS One. 2014 Jun 17;9(6):e99905. doi: 10.1371/journal.pone.0099905. PMID: 24937158; PMCID: PMC4061091.

pNL4-3.Luc.R-E- vector Sequence

LOCUS       Exported               16389 bp DNA     circular SYN 29-AUG-2024
DEFINITION  Exported.
ACCESSION   V012797
VERSION     .
KEYWORDS    pnl4-3-luc-r-e-
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 16389)
  AUTHORS   Triple Threat
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 16389)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..16389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        669..773
                     /label=AmpR promoter
     CDS             774..1631
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1805..2393
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     LTR             complement(4203..4836)
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     CDS             complement(5026..6672)
                     /label=luciferase
                     /note="firefly luciferase"
     CDS             complement(6698..9256)
                     /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             complement(9474..9647)
                     /label=Protein Tat
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate BH5). Accession#: P04612"
     CDS             complement(9861..10436)
                     /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             complement(10384..13392)
                     /label=HIV-1 pol
                     /note="pol protein from human immunodeficiency virus 1"
     CDS             complement(13188..14687)
                     /label=HIV-1 gag
                     /note="gag protein from human immunodeficiency virus 1"
     misc_feature    complement(14671..14796)
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     LTR             complement(14843..15100)
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"