pRevTRE vector (V007373)

Price Information

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

Basic Vector Information

pRevTRE is a retroviral Tet response vector that expresses a gene of interest from the Tetresponse element (TRE). This vector is derived from pLNCX, a retroviral vector created using elements of Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma virus (MoMuSV) as described (1). The TRE contains seven direct repeats of the tetO operator sequence, upstream of a minimal CMV promoter, which can be bound by the tTA and rtTA transactivators. The 5' viral LTR controls expression of the transcript that contains Ψ+ (the extended viral packaging signal) and the hygromycin resistance (Hygr) gene for antibiotic selection in mammalian cells. The TRE is derived from vectors described previously (2, 3). pRevTRE also includes the E. coli Ampr gene for antibiotic selection in bacteria.The complete RevTet-Off and RevTet-On Systems also include the control vector pRevTRELuc, which was constructed by cloning the firefly luciferase gene into the Hind III/Cla I sites in the MCS of pRevTRE.pRevTRE can be used to establish inducible Tet Systems via retrovirus-mediated gene transfer (4). Retroviral gene transfer allows the highly efficient transduction of virtually all dividing cell types. The RevTetTM Systems are also suitable for establishing transgenic animals. In combination with the pRevTet-On or pRevTet- Off regulatory vector, a gene of interest can be inducibly expressed at high levels in response to varying concentrations of tetracycline (Tc) or Tc derivatives such as doxycycline (Dox). tTA and rtTA bind to the Tetresponse element (TRE) and activate transcription from the minimal promoter in the absence or presence of Dox, respectively. pRevTRE lacks the viral genes gag, pol, and env, which are supplied by the packaging cell line. It can be transfected into a high titer packaging cell line and thereby mediate production of infectious, replication-incompetent retroviral particles (1, 6–7). The transcript produced by the pRevTRE construct is recognized by the viral structural proteins expressed in a packaging cell line and packaged into infectious retroviral particles. Because the RNA transcript packaged in these particles does not contain the viral genes, it cannot replicate in the target cells that it infects.The level of induction in cell populations infected with this vector depends on the efficiency of infection, the site of integration, and the titer of the virus. Viral supernatants with titers >105 cfu/ml should be produced to achieve high-level induction.

pRevTRE 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.

pRevTRE vector Sequence

LOCUS       40924_36898        6487 bp DNA     circular SYN 13-JAN-2022
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6487)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 6487)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6487
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             2..589
                     /label=5' LTR
                     /note="long terminal repeat from Moloney murine sarcoma
                     virus"
     misc_feature    652..851
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus
                     (MMLV)"
     CDS             1052..1468
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag
                     gene lacking the start codon"
     CDS             1524..2540
                     /label=HygR
                     /note="aminoglycoside phosphotransferase from E. coli"
     protein_bind    2847..3117
                     /label=tetracycline response element
                     /note="contains seven copies of the tetracycline operator
                     tetO"
     promoter        3150..3187
                     /note="minimal CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     misc_feature    3281..3337
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     LTR             3389..3982
                     /label=LTR
                     /note="long terminal repeat from Moloney murine leukemia
                     virus"
     misc_feature    4193..4333
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(4519..5107)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5281..6138)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6139..6243)
                     /label=AmpR promoter