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pGEX-5X-2 vector (V010914)

Price Information

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V010914 pGEX-5X-2 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:
pGEX-5X-2
Antibiotic Resistance:
Ampicillin
Length:
4973 bp
Type:
pGEX Vectors (GE Healthcare)
Replication origin:
ori
Source/Author:
GE Healthcare
Copy Number:
High copy number
Growth Strain(s):
Top10
Growth Temperature:
37℃

pGEX-5X-2 vector Map

Copy Sequence View Map
pGEX-5X-24973 bp6001200180024003000360042004800tac promoterlac operatorGSTFactor Xa siteMCSstop codonsAmpR promoterAmpRorilacIq promoterlacICAP binding sitelac promoterlacZ-alpha

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

  • Chauhan V, Chaudhary D, Rao MK, Dhaked RK. Expression, Purification and Immunological Characterization of Recombinant Shiga Toxin A Subunit. Protein Pept Lett. 2015;22(9):844-52. doi: 10.2174/0929866522666150707115007. PMID: 26282729.

pGEX-5X-2 vector Sequence

LOCUS       pGEX-5X-2.        4973 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for expressing GST fusion proteins with a Factor Xa
            site. For other reading frames, use pGEX-5X-1 or pGEX-5X-3.
ACCESSION   .
VERSION     .
KEYWORDS    pGEX-5X-2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4973)
  AUTHORS   GE Healthcare
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4973)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        183..211
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /label=GST
                     /note="glutathione S-transferase from Schistosoma
                     japonicum"
     CDS             921..932
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
     misc_feature    934..970
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     misc_feature    975..985
                     /label=stop codons
                     /note="stop codons"
                     /note="stop codons in all three reading frames"
     promoter        1276..1380
                     /label=AmpR promoter
     CDS             1381..2238
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      2412..3000
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        3244..3321
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             3322..4401
                     /label=lacI
                     /note="lac repressor"
     protein_bind    4417..4438
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        4453..4483
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     CDS             4527..4700
                     /label=lacZ-alpha
                     /note="LacZ-alpha fragment of beta-galactosidase"