Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V014486 | pCT302 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pCT302 is the yeast display platform's core vector, driving linear peptide epitopes to fuse with yeast surface anchors like Aga2p, facilitating their display, with a selection marker for screening, supporting high-throughput peptide-MHC-II binding assessment.
- Vector Name:
- pCT302
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7036 bp
- Type:
- Protein expression
- Replication origin:
- ori
- Host:
- Yeast
- Selection Marker:
- TRP1
- Promoter:
- GAL1,10
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 37℃
pCT302 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
- Huisman BD, Balivada PA, Birnbaum ME. Yeast display platform with expression of linear peptide epitopes for high-throughput assessment of peptide-MHC-II binding. J Biol Chem. 2023 Mar;299(3):102913. doi: 10.1016/j.jbc.2023.102913. Epub 2023 Jan 14. PMID: 36649909; PMCID: PMC9971316.
pCT302 vector Sequence
LOCUS Exported 7036 bp DNA circular SYN 25-SEP-2025
DEFINITION Exported.
ACCESSION V014486
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7036)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 7036)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7036
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(159..833)
/codon_start=1
/gene="S. cerevisiae TRP1"
/product="phosphoribosylanthranilate isomerase, required
for tryptophan biosynthesis"
/label=TRP1
/note="yeast auxotrophic marker"
/translation="MSVINFTGSSGPLVKVCGLQSTEAAECALDSDADLLGIICVPNRK
RTIDPVIARKISSLVKAYKNSSGTPKYLVGVFRNQPKEDVLALVNDYGIDIVQLHGDES
WQEYQEFLGLPVIKRLVFPKDCNILLSAASQKPHSFIPLFDSEAGGTGELLDWNSISDW
VGRQESPESLHFMLAGGLTPENVGDALRLNGVIGVDVSGGVETNGVKDSNKIANFVKNA
KK"
CDS complement(162..833)
/label=TRP1
/note="phosphoribosylanthranilate isomerase, required for
tryptophan biosynthesis"
promoter complement(834..1114)
/gene="S. cerevisiae TRP1"
/label=TRP1 promoter
misc_feature complement(1374..1877)
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
promoter 1914..2018
/label=AmpR promoter
CDS 2019..2879
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
CDS 2019..2876
/label=AmpR
/note="beta-lactamase"
rep_origin 3050..3638
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 3926..3947
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 3962..3992
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 4000..4016
/label=lac operator
/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 4024..4040
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
promoter 4061..4079
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 4244..4908
/label=GAL1,10 promoter
/note="divergent inducible promoter, regulated by Gal4"
protein_bind 4460..4577
/label=UAS
/bound_moiety="Gal4"
/note="upstream activating sequence mediating
Gal4-dependent induction"
CDS 4948..5208
/gene="AGA2"
/label=A-agglutinin-binding subunit
/note="A-agglutinin-binding subunit from Saccharomyces
cerevisiae (strain ATCC 204508 / S288c). Accession#:
P32781"
CDS 5227..5238
/label=Factor Xa site
/note="Factor Xa recognition and cleavage site"
CDS 5239..5265
/label=HA
/note="HA (human influenza hemagglutinin) epitope tag"
CDS 6094..6123
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
promoter complement(6457..6475)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(6482..6498)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
rep_origin join(6643..7036,1..62)
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
rep_origin 6643..7036
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"