pCMS-EGFP vector (V006577)

Price Information

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

Basic Vector Information

pCMS-EGFP is a mammalian expression vector that allows you to express your gene of interest at high levels by cloning it into a multiple cloning site (MCS) downstream of the immediate early promoter of cytomegalovirus (PCMV IE). The intervening sequence (IVS) between PCMV IE and the MCS is an intron that is efficiently spliced out following transcription. SV40 polyadenylation signals downstream of the MCS direct proper processing of the 3' end of the mRNA from your gene of interest. Bacteriophage T7 and T3 promoters are located upstream and downstream of the MCS, respectively.pCMS-EGFP uses the enhanced green flourescent protein (EGFP) as a transformation marker. EGFP is a red-shifted variant of wild-type GFP from Aquorea victoria (1–3). EGFP has been optimized for brighter fluorescence and higher expression in mammalian cells. (Excitation maximum = 488 nm; emission maximum = 507 nm.) EGFP encodes the GFPmut1 variant (4) which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 to Thr. The coding sequence of the EGFP gene contains more than 190 silent base changes which correspond to human codon-usage preferences (5). Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (6) to further increase the translation efficiency in eukaryotic cells. EGFP is expressed from the SV40 enhancer/promoter, and a polyadenylation signal from the bovine growth hormone (BGH) gene directs proper processing of the 3' end of the EGFP mRNA. The SV40 origin also allows for replication in mammalian cells expressing the SV40 T antigen.The vector backbone also contains the β-lactamase gene for ampicillin resistance and a ColE1 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.The name of the vector comes from the order of four critical elements: the CMV IE promoter; the MCS; the SV40 promoter; and EGFP.Genes cloned into the MCS must contain an ATG start codon. pCMS-EGFP and derivatives can be introduced into mammalian cells by any standard method. Transfected cells can be monitored and/or selected by flow cytometry based on the fluorescence of EGFP. Sense or antisense RNA can be transcribed from the T7 and T3 promoters, respectively.

pCMS-EGFP vector 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.

pCMS-EGFP vector Sequence

LOCUS       40924_11461        5541 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 5541)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 5541)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..5541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        139..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1141..1158)
                     /note="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    complement(1174..1295)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      1481..1936
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        2055..2412
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             2449..3165
                     /label=EGFP
                     /note="enhanced GFP"
     polyA_signal    3200..3424
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        3744..3848
                     /label=AmpR promoter
     CDS             3849..4706
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      4880..5468
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"