pCI vector (V011274) Gene synthesis in pCI backbone

Price Information

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

Basic Vector Information

The pCI Mammalian Expression Vector enables constitutive expression of cloned DNA inserts in mammalian cells. Its key difference from the pSI Vector lies in the enhancer/promoter region regulating inserted gene expression: pCI contains the human cytomegalovirus (CMV) major immediate-early enhancer/promoter, plus a β-globin/IgG chimeric intron downstream to boost expression. Its late SV40 polyadenylation signal increases mRNA steady-state levels ~5-fold more than the early SV40 signal.
Suitable for both transient and stable expression, pCI requires co-transfection with a selectable gene-containing vector for stable expression.

pCI 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.

References

• Brinster, R.L. et al. (1988) Proc. Natl. Acad. Sci. USA 85, 836–40.
• Choi, T. et al. (1991) Mol. Cell. Biol. 11, 3070–4.
• Palmiter, R.D. et al. (1991) Proc. Natl. Acad. Sci. USA 88, 478–82.
• Carswell, S. and Alwine, J.C. (1989) Mol. Cell. Biol. 9, 4248–58.

pCI vector Sequence

LOCUS       pCI.        4006 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Mammalian cell expression vector with the CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    pCI
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4006)
  AUTHORS   Promega
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4006)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4006
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        139..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..721
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     intron          857..989
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        1034..1052
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    1052..1104
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     polyA_signal    complement(1120..1241)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      1422..1877
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2209..2313
                     /label=AmpR promoter
     CDS             2314..3171
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      3345..3933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"