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pRetroX-Tight-Pur vector (V010377)

Price Information

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V010377 pRetroX-Tight-Pur In stock, instant shipping

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

Basic Vector Information

Mammalian cells, retrovirus expression vector. It is inducible and self-inactivating.

Vector Name:
pRetroX-Tight-Pur
Antibiotic Resistance:
Ampicillin
Length:
6567 bp
Type:
Viral Expression & Packaging Vectors
Replication origin:
ori
Source/Author:
Clontech
Copy Number:
High copy number
Promoter:
mPGK
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

pRetroX-Tight-Pur vector Map

Copy Sequence View Map
pRetroX-Tight-Pur6567 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300CMV enhancerCMV promoter5' LTR (truncated)MMLV Psigag (truncated)tight TRE promoterMCSPGK promoterPuroR3' LTR (Delta-U3)SV40 promoteroriAmpRAmpR promoterCMV enhancer

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

  • Sekine Y, Houston R, Eckl EM, Fessler E, Narendra DP, Jae LT, Sekine S. A mitochondrial iron-responsive pathway regulated by DELE1. Mol Cell. 2023 Jun 15;83(12):2059-2076.e6. doi: 10.1016/j.molcel.2023.05.031. PMID: 37327776; PMCID: PMC10329284.

pRetroX-Tight-Pur vector Sequence

LOCUS       pRetroX-Tight-Pu        6567 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Retroviral vector for expressing a gene with the Tet-On(R) Advanced 
            or Tet-Off(R) Advanced system.
ACCESSION   .
VERSION     .
KEYWORDS    pRetroX-Tight-Pur
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6567)
  AUTHORS   Clontech
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 6567)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..308
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        309..512
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     LTR             513..688
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    751..950
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus
                     (MMLV)"
     CDS             1151..1567
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag
                     gene lacking the start codon"
     promoter        1603..1917
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     misc_feature    1925..1968
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     promoter        1973..2472
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     CDS             2493..3089
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
     LTR             3273..3698
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     promoter        3964..4293
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      complement(4586..5174)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5348..6205)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6206..6310)
                     /label=AmpR promoter
     enhancer        6496..6567
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"