pYC2/CT vector (Cat. No.: V010213)
Note: pYC2-CT is a low-copy shuttle plasmid for Saccharomyces cerevisiae. It has CEN6/ARSH4 for single-copy stability, a galactose-inducible (glucose-repressed) GAL1 promoter, a URA3 marker for screening, and C-terminal V5/6×His tags for protein detection/purification. It’s widely used in yeast gene expression control, protein localization and interaction studies.
- Name:
- pYC2/CT
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4627 bp
- Type:
- Yeast Plasmids
- Replication origin:
- ori
- Source/Author:
- Invitrogen (Life Technologies)
- Copy Number:
- High copy number
- Promoter:
- GAL1
- Growth Strain(s):
- DH10B
Resources
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.
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.
pYC2/CT vector (Cat. No.: V010213) Sequence
LOCUS pYC2_CT. 4627 bp DNA circular SYN 01-JAN-1980
DEFINITION Centromeric vector with a URA3 marker for galactose-inducible
expression of C-terminally tagged proteins in S. cerevisiae.
ACCESSION .
VERSION .
KEYWORDS pYC2 CT
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4627)
AUTHORS Invitrogen (Life Technologies)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 4627)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4627
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 2..443
/label=GAL1 promoter
/note="inducible promoter, regulated by Gal4"
promoter 475..493
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 501..594
/label=MCS
/note="MCS"
/note="multiple cloning site"
CDS 607..648
/label=V5 tag
/note="epitope tag from simian virus 5"
CDS 658..675
/label=6xHis
/note="6xHis affinity tag"
terminator 709..956
/label=CYC1 terminator
/note="transcription terminator for CYC1"
rep_origin complement(1204..1792)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(1966..2823)
/label=AmpR
/note="beta-lactamase"
CDS complement(2922..3722)
/label=URA3
/note="orotidine-5'-phosphate decarboxylase, required for
uracil biosynthesis"
promoter complement(3723..3943)
/label=URA3 promoter
misc_feature 3971..4474
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"