pGRFP vector (V005885)

Price Information

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

Basic Vector Information

The vector pGRFP was developed to report the integration of cloned inserts by shifting the ratio of green to red fluorescence. In the native vector, a fusion protein composed of GFP and rapidly maturing DsRed-T3 fluorescent protein is expressed.

pGRFP 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

• Juno Choe and others, A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting, Nucleic Acids Research, Volume 33, Issue 5, 1 March 2005, Page e49, https://doi.org/10.1093/nar/gni049

pGRFP vector Sequence

LOCUS       pGRFP.        3441 bp DNA     circular SYN 29-AUG-2023
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pGRFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3441)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..3441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    49..70
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        85..115
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             126..134
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             140..853
                     /label=yeGFP
                     /note="yeast-enhanced green fluorescent protein"
     primer_bind     873..889
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(918..934)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             953..1627
                     /label=DsRed-Express
                     /note="rapidly maturing tetrameric variant of DsRed
                     fluorescent protein (Bevis and Glick, 2002)"
     promoter        1673..1777
                     /label=AmpR promoter
     CDS             1778..2635
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      2809..3397
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"