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pYES-mtGFP vector (V017264)

Price Information

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V017264 pYES-mtGFP In stock, instant shipping

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

Basic Vector Information

The pYES-mtGFP is a Yeast Expression vector fused with a mtGFP. The original backbone is pYES2.0, selection marker is URA3.

Vector Name:
pYES-mtGFP
Antibiotic Resistance:
Ampicillin
Length:
6759 bp
Type:
Gene expression
Replication origin:
ori
Host:
Yeast
Selection Marker:
URA3
Promoter:
GAL1
Growth Strain(s):
DH5a
Growth Temperature:
37℃

pYES-mtGFP vector Map

Copy Sequence View Map
pYES-mtGFP6759 bp3006009001200150018002100240027003000330036003900420045004800510054005700600063006600GAL1 promoterT7 promoterGFP (S65T)CYC1 terminatororiISSURA3URA3 promoter2u oriM13 ori

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

  • Westermann B, Neupert W. Mitochondria-targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae. Yeast. 2000;16(15):1421-1427. doi:10.1002/1097-0061(200011)16:15<1421::AID-YEA624>3.0.CO;2-U

pYES-mtGFP vector Sequence

LOCUS       62056_23080        6759 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6759)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..6759
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..444
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     promoter        476..494
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             736..1449
                     /codon_start=1
                     /label=GFP (S65T)
                     /note="S65T variant of Aequorea victoria green fluorescent 
                     protein (Heim et al., 1995)"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1512..1759
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(2000..2588)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    complement(3057..3435)
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene;
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     CDS             complement(3718..4518)
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(4519..4739)
                     /label=URA3 promoter
     rep_origin      complement(5338..6218)
                     /direction=LEFT
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     rep_origin      complement(6229..6742)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"