pLV2-TRE3GS-PtpB(H37Rv)-TetOne-Hyg vector (V014837)

Price Information

Cat No. Plasmid Name Availability Add to cart
V014837 pLV2-TRE3GS-PtpB(H37Rv)-TetOne-Hyg In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
pLV2-TRE3GS-PtpB(H37Rv)-TetOne-Hyg
Antibiotic Resistance:
Ampicillin
Length:
10550 bp
Type:
Protein expression, Tetracycline inducible
Replication origin:
ori
Host:
Mammalian cells, Lentivirus
Selection Marker:
Hyg
Promoter:
hPGK
Growth Temperature:
37℃

pLV2-TRE3GS-PtpB(H37Rv)-TetOne-Hyg vector Map

pLV2-TRE3GS-PtpB(H37Rv)-TetOne-Hyg10550 bp5001000150020002500300035004000450050005500600065007000750080008500900095001000010500SV40 poly(A) signalAmpR promoterAmpRoriCAP binding sitelac promoter5' LTRWPREHygRSV40 promoterTet-On(R) 3GhPGK promoterTRE3GS promoterFLAGTriple specificity protein phosphatase PtpBSV40 poly(A) signalcPPT/CTSProtein Tatgp41 peptideRREHIV-1 Psi3' LTR

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.

pLV2-TRE3GS-PtpB(H37Rv)-TetOne-Hyg vector Sequence

LOCUS       V014837                10550 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V014837
VERSION     V014837
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 10550)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10550
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    complement(216..350)
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     promoter        398..502
                     /label="AmpR promoter"
     CDS             503..1360
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      1534..2119
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     protein_bind    2407..2428
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2443..2473
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     LTR             complement(2650..3283)
                     /label="5' LTR"
                     /note="5' long terminal repeat (LTR) from HIV-1"
     misc_feature    complement(3490..4078)
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     CDS             complement(4095..5117)
                     /label="HygR"
                     /note="aminoglycoside phosphotransferase from E. coli"
     promoter        complement(5166..5495)
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     CDS             complement(5509..6252)
                     /label="Tet-On(R) 3G"
                     /note="modified rtTA protein that binds tightly to
                     promoters containing the tet operator in the presence of
                     doxycycline"
     promoter        complement(6271..6781)
                     /label="hPGK promoter"
                     /note="human phosphoglycerate kinase 1 promoter"
     promoter        6798..7165
                     /label="TRE3GS promoter"
                     /note="3rd-generation Tet-responsive promoter that can be
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription
                     factors"
     CDS             7195..7218
                     /label="FLAG"
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             7219..8046
                     /gene="ptpB"
                     /label="Triple specificity protein phosphatase PtpB"
                     /note="Triple specificity protein phosphatase PtpB from
                     Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv).
                     Accession#: I6WXK4"
     polyA_signal    complement(8230..8364)
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     misc_feature    complement(8407..8524)
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     CDS             complement(8596..8637)
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     CDS             complement(8786..8830)
                     /label="gp41 peptide"
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
                     al., 2013)"
     misc_feature    complement(9015..9248)
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     misc_feature    complement(9745..9870)
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     LTR             complement(9917..10550)
                     /label="3' LTR"
                     /note="3' long terminal repeat (LTR) from HIV-1"