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pET-15b vector (V011029)

Price Information

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V011029 pET-15b 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:
pET-15b
Antibiotic Resistance:
Ampicillin
Length:
5708 bp
Type:
pET & Duet Vectors (Novagen)
Replication origin:
ori
Source/Author:
Novagen (EMD Millipore)
Copy Number:
High copy number
Growth Strain(s):
DH10B
Growth Temperature:
37℃

pET-15b vector Map

Copy Sequence View Map
pET-15b5708 bp60012001800240030003600420048005400tet promoterT7 terminatorMCSthrombin site6xHisATGRBSlac operatorT7 promoterlacI promoterlacICAP binding siteropbomoriAmpRAmpR 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.

pET-15b vector Sequence

LOCUS       pET-15b.        5708 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for inducible expression of N-terminally 
            6xHis-tagged proteins.
ACCESSION   .
VERSION     .
KEYWORDS    pET-15b
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5708)
  AUTHORS   Novagen (EMD Millipore)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5708)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        10..38
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_feature    319..335
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             complement(336..353)
                     /label=thrombin site
                     /note="thrombin recognition and cleavage site"
     CDS             complement(363..380)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(390..392)
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     RBS             complement(399..421)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    complement(436..460)
                     /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)."
     promoter        complement(461..479)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        788..865
                     /label=lacI promoter
     CDS             866..1945
                     /label=lacI
                     /note="lac repressor"
     protein_bind    1961..1982
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             3260..3448
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    3553..3695
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(3881..4469)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4643..5500)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(5501..5605)
                     /label=AmpR promoter