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pET16B.Pfu vector (V013296) Gene synthesis in pET16B.Pfu backbone

Price Information

Cat No. Plasmid Name Availability Buy one, get one free! (?)
V013296 pET16B.Pfu In stock, instant shipping

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

Basic Vector Information

pET16B.Pfu is an expression vector carrying the N-(His)₆-DNA polymerase gene from Pyrococcus furiosus. It is used to express the high-fidelity Pfu polymerase in Escherichia coli, providing DNA synthesis activity for PCR systems lacking dNTPs.

Vector Name:
pET16B.Pfu
Antibiotic Resistance:
Ampicillin
Length:
8033 bp
Type:
Protein expression
Replication origin:
ori
Host:
E. coli
Promoter:
tet
Growth Strain(s):
DH10B
Growth Temperature:
37℃

pET16B.Pfu vector Map

Copy Sequence View Map
pET16B.Pfu8033 bp400800120016002000240028003200360040004400480052005600600064006800720076008000bomropCAP binding sitelacIlacI promoterT7 promoterlac operatorRBS9xHisFactor Xa siteDNA polymeraseT7 terminatortet promoterAmpR promoterAmpRori

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

  • Loan TD, Easton CJ, Alissandratos A. DNA amplification with in situ nucleoside to dNTP synthesis, using a single recombinant cell lysate of E. coli. Sci Rep. 2019 Oct 30;9(1):15621. doi: 10.1038/s41598-019-51917-z. PMID: 31666578; PMCID: PMC6821818.

pET16B.Pfu vector Sequence

LOCUS       V013296                 8033 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V013296
VERSION     V013296
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 8033)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8033
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    complement(5..144)
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(249..437)
                     /label="rop"
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     protein_bind    complement(1715..1736)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(1752..2831)
                     /label="lacI"
                     /note="lac repressor"
     promoter        complement(2832..2909)
                     /label="lacI promoter"
     promoter        3218..3236
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3237..3261
                     /label="lac operator"
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be
                     relieved by adding lactose or
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             3276..3298
                     /label="RBS"
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3311..3337
                     /label="9xHis"
                     /note="9xHis affinity tag"
     CDS             3353..3364
                     /label="Factor Xa site"
                     /note="Factor Xa recognition and cleavage site"
     CDS             3368..5692
                     /gene="pol"
                     /label="DNA polymerase"
                     /note="DNA polymerase from Pyrococcus furiosus (strain ATCC
                     43587 / DSM 3638 / JCM 8422 / Vc1). Accession#: P61875"
     terminator      5765..5812
                     /label="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA
                     polymerase"
     promoter        complement(5987..6015)
                     /label="tet promoter"
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     promoter        6128..6232
                     /label="AmpR promoter"
     CDS             6233..7090
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      7264..7852
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"