pLP2 vector (V010443)

Price Information

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

Basic Vector Information

pLP2 is a lentiviral packaging plasmid containing the HIV-1 rev gene.

pLP2 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

• Sun L, Zhang Y, Huang CX, Qu XL, Zhang Y, Zhang JC, Wei X, Zhuang Y, Zhai S, Peng MJ, Jiang H, Hao CQ, Li YH, Lu J, Lian JQ, Sun YT, Bai XF. Therapeutic effect of RANTES-KDEL on inhibition of HIV-1 in CD34(+) human hematopoietic stem cells (hHSC). J Virol Methods. 2008 Dec;154(1-2):194-9. doi: 10.1016/j.jviromet.2008.07.003. Epub 2008 Oct 28. PMID: 18706447.
• Zhang H, Zhu WQ, Zhao SJ, Li YL, Hou J, Bian CR, Bao CM, Wei Z, Sun J, Li B, Li BA. [Construction and application of lentivirus overexpression vector with two labeling genes fused with CopGFP and PuroR]. Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2022 Mar;38(2):108-112. Chinese.
• Yang GH, Wu W, Li YC, Ni L, Wang ZQ, Song XT, Liu CW. [Establishment and functional identification of a stable cell line overexpression heme oxygenase-1]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2014 Aug;36(4):420-5. Chinese. doi: 10.3881/j.issn.1000-503X.2014.04.013. PMID: 25176212.

pLP2 vector Sequence

LOCUS       pLP2.        4180 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Lentiviral packaging plasmid containing the HIV-1 rev gene.
ACCESSION   .
VERSION     .
KEYWORDS    pLP2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4180)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4180)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4180
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..262
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     CDS             391..738
                     /label=Rev
                     /note="human immunodeficiency virus 1 (HIV-1) Rev protein"
     primer_bind     complement(825..841)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    850..971
                     /label=HIV-1 poly(A)
                     /note="HIV-1 poly(A)"
                     /note="polyadenylation signal from the LTR of HIV-1"
     rep_origin      1173..1628
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1910..2014
                     /label=AmpR promoter
     CDS             2015..2872
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      3046..3634
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    3922..3943
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        3958..3988
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3996..4012
                     /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)."
     primer_bind     4020..4036
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"