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pGEM-HE vector (V012814)

Price Information

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V012814 pGEM-HE In stock, instant shipping

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

Basic Vector Information

Xenopus oocyte expression vector. It contains a 3'-UTR of the major beta-globin gene of Xenopus laevis. The pGEM-HE is a derivative of pGEM3Z vector.

Vector Name:
pGEM-HE
Antibiotic Resistance:
Ampicillin
Length:
3047 bp
Type:
Protein Expression plasmid
Replication origin:
ori
Selection Marker:
Ampicillin, 100 μg/mL
Copy Number:
High copy number
Promoter:
T7
Growth Strain(s):
DH5alpha
Growth Temperature:
37℃

pGEM-HE vector Map

Copy Sequence View Map
pGEM-HE3047 bp6001200180024003000M13 fwdT7 promoter5'UTR3'UTRSP6 promoterM13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR 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.

References

  • Loewen SK, Yao SY, Slugoski MD, Mohabir NN, Turner RJ, Mackey JR, Weiner JH, Gallagher MP, Henderson PJ, Baldwin SA, Cass CE, Young JD. Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes. Mol Membr Biol. 2004 Jan-Feb;21(1):1-10.

pGEM-HE vector Sequence

LOCUS       Exported                3047 bp DNA     circular SYN 23-OCT-2024
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pGEM-HE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3047)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 3047)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..62
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        69..87
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     5'UTR           119..161
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     3'UTR           220..345
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus
                     laevis"
     promoter        complement(457..475)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(493..509)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(517..533)
                     /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(541..571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(586..607)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(895..1483)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1657..2514)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(2515..2619)
                     /label=AmpR promoter