pRetroX-Tet3G vector (Cat. No.: V010381)

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Basic Information

Note: Retroviral vector to be used in conjunction with a Tet-responsive vector for doxycycline-inducible expression of a gene.

Name:: pRetroX-Tet3G

Antibiotic Resistance:: Ampicillin

Length:: 8136 bp

Type:: Viral Expression & Packaging Vectors

Replication origin:: ori

Source/Author:: Clontech

Selection Marker:: Neomycin/G418(Geneticin)

Copy Number:: High copy number

Promoter:: CMV

$ 249.6

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

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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.

pRetroX-Tet3G vector (Cat. No.: V010381) Sequence

LOCUS       pRetroX-Tet3G.        8136 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Retroviral vector to be used in conjunction with a Tet-responsive 
            vector for doxycycline-inducible expression of a gene.
ACCESSION   .
VERSION     .
KEYWORDS    pRetroX-Tet3G
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8136)
  AUTHORS   Clontech
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8136)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8136
                     /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"
     enhancer        1604..1907
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1908..2111
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             2290..3033
                     /label=Tet-On(R) 3G
                     /note="modified rtTA protein that binds tightly to
                     promoters containing the tet operator in the presence of 
                     doxycycline"
     misc_feature    3045..3630
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             3631..4422
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase from Tn5"
     LTR             4842..5267
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     promoter        5533..5862
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      complement(6155..6743)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6917..7774)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(7775..7879)
                     /label=AmpR promoter
     enhancer        8065..8136
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"