pTRE2hyg-Stuffer vector (V012830)

Price Information

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V012830 pTRE2hyg-Stuffer In stock, instant shipping

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

Basic Vector Information

Vector Name:
pTRE2hyg-Stuffer
Antibiotic Resistance:
Ampicillin
Length:
6966 bp
Type:
Mammalian Expression Vectors
Replication origin:
ori
Selection Marker:
Hygromycin
Copy Number:
High copy number
Promoter:
SV40
Cloning Method:
Enzyme Cut
Fusion Tag:
Luc
Growth Strain(s):
DH10B
Growth Temperature:
37℃

pTRE2hyg-Stuffer vector Map

pTRE2hyg-Stuffer6966 bp30060090012001500180021002400270030003300360039004200450048005100540057006000630066006900tetracycline response elementminimal CMV promoterluciferasebeta-globin intronHemoglobin subunit betabeta-globin poly(A) signaloriAmpRAmpR promoterSV40 poly(A) signalHygRSV40 promoter

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.

pTRE2hyg-Stuffer vector Sequence

LOCUS       V012830                 6966 bp    DNA     circular SYN 13-DEC-2023
DEFINITION  Exported.
ACCESSION   V012830
VERSION     V012830
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 6966)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    15..285
                     /label="tetracycline response element"
                     /note="contains seven copies of the tetracycline operator
                     tetO"
     promoter        318..356
                     /label="minimal CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     CDS             480..2129
                     /label="luciferase"
                     /note="firefly luciferase"
     intron          2195..2767
                     /label="beta-globin intron"
                     /note="intron from rabbit beta-globin gene"
     CDS             2771..2893
                     /gene="HBB"
                     /label="Hemoglobin subunit beta"
                     /note="Hemoglobin subunit beta from Colobus guereza.
                     Accession#: Q9TT33"
     polyA_signal    2964..3019
                     /label="beta-globin poly(A) signal"
                     /note="rabbit beta-globin polyadenylation signal (Gil and
                     Proudfoot, 1987)"
     rep_origin      complement(3581..4169)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(4343..5200)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(5201..5305)
                     /label="AmpR promoter"
     polyA_signal    complement(5406..5487)
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     CDS             complement(5607..6629)
                     /label="HygR"
                     /note="aminoglycoside phosphotransferase from E. coli"
     promoter        complement(join(6638..6966,1))
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"