pGEMEX-1 vector (V011630)

Price Information

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

Basic Vector Information

The pGEMEXVectors(a,b) are T7 expression vectors that can be used for cloning, as templates for in vitro transcription and for production of single-stranded DNA (ssDNA). Expression from the pGEMEXVectors is based on the T7 expression system (a) developed by Studier (1), which uses a convenient vector/host combination for high-level expression of cloned genes in vivo. Sequences cloned into the pGEMEX Vectors are expressed as T7 gene 10 fusion proteins in JM109(DE3)(a) or BL21(DE3)pLysS(c) host strains containing an inducible gene for T7 RNA polymerase. JM109(DE3) is a specially constructed host strain that contains an IPTGinducible gene for T7 RNA polymerase; JM109(DE3) and JM109 are supplied with the pGEMEXVectors. The pGEMEX-1 and pGEMEX-2 Vectors differ by only two base pairs resulting in a shift in the reading frame (Figure 1). The bacteriophage T7 gene 10 leader peptide (260 amino acids) is expressed very efficiently in this host and can accumulate to greater than 50% of total cell protein in three hours or less. Typical levels of fusion protein production are on the order of 10mg per liter of induced culture.

pGEMEX-1 vector Map

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.

pGEMEX-1 vector Sequence

LOCUS       40924_21389        3995 bp DNA     circular SYN 13-JAN-2022
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3995)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 3995)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3995
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(88..105)
                     /note="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(873..905)
                     /label=T7 tag (gene 10 leader)
                     /note="leader peptide from bacteriophage T7 gene 10"
     RBS             complement(913..935)
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     promoter        complement(967..985)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(1248..1836)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2010..2867)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(2868..2972)
                     /label=AmpR promoter
     rep_origin      3306..3761
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     terminator      complement(3860..3907)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3979..3995
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"