pYC12 vector (Cat. No.: V001924)
Note: pYC12, as a yeast expression vector, mediates the efficient expression of target genes, thereby facilitating research into microbial functions and metabolic mechanisms.
- Name:
- pYC12
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5682 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Host:
- Yeast
- Source/Author:
- Yanez Carrillo P, Castano Navarro I.
- Promoter:
- TEF1
- Growth Strain(s):
- Top10
Resources
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
- Maldonado G, García A, Herrero S, Castaño I, Altmann M, Fischer R, Hernández G. The gene YEF3 function encoding translation elongation factor eEF3 is partially conserved across fungi. Front Microbiol. 2024 Aug 23;15:1438900. doi: 10.3389/fmicb.2024.1438900. PMID: 39247690; PMCID: PMC11378755.
pYC12 vector (Cat. No.: V001924) Sequence
LOCUS Exported 5682 bp DNA circular SYN 16-DEC-2025
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5682)
AUTHORS 11111111
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..5682
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 2811..3280
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 666..737
/gene="bla"
/label=AmpR promoter
CDS 738..1598
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1769..2357
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 2645..2666
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 2681..2711
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2719..2735
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
primer_bind 2743..2759
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 2780..2798
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 2815..3214
/label=TEF1 promoter promoter for EF-1-alpha
/label=TEF1 promoter
/note="promoter for EF-1-alpha"
primer_bind 3218..3234
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(3268..3280)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(3699..3717)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(3727..3743)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin 3884..4339
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
CDS complement(4470..5273)
/codon_start=1
/gene="S. cerevisiae URA3"
/product="orotidine-5'-phosphate decarboxylase, required
for uracil biosynthesis"
/label=URA3
/note="yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
promoter complement(5274..5489)
/gene="S. cerevisiae URA3"
/label=URA3 promoter