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pCTcon2 vector (V011800)

Price Information

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V011800 pCTcon2 In stock, instant shipping

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

Basic Vector Information

It is the most frequently used plasmid for yeast display, which contains the insert CD20 and is published in Nat Protoc. 2006;1(2):755-68.

Vector Name:
pCTcon2
Antibiotic Resistance:
Ampicillin
Length:
6314 bp
Type:
Yeast Expression
Replication origin:
ori
Selection Marker:
TRP1
Copy Number:
Low Copy
Promoter:
GAL1
Growth Strain(s):
DH10B
Growth Temperature:
37℃

pCTcon2 vector Map

Copy Sequence View Map
pCTcon26314 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300GAL1 promoterA-agglutinin-binding subunitFactor Xa siteHAMycT7 promoterM13 fwdf1 oriTRP1TRP1 promoterpRS-markerpGEX 3'pBRforEcoCEN/ARSAmpR promoterAmpRoriL4440CAP binding sitelac promoterlac operatorM13 revT3 promoterGAL1,10 promoter

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

  • Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD. Isolating and engineering human antibodies using yeast surface display. Nat Protoc. 2006;1(2):755-68.

  • Zahradník J, Dey D, Marciano S, Kolářová L, Charendoff CI, Subtil A, Schreiber G. A Protein-Engineered, Enhanced Yeast Display Platform for Rapid Evolution of Challenging Targets. ACS Synth Biol. 2021 Dec 17;10(12):3445-3460. doi: 10.1021/acssynbio.1c00395. Epub 2021 Nov 22. PMID: 34809429; PMCID: PMC8689690.

  • Popovic M, Prodanovic R, Ostafe R, Schillberg S, Fischer R, Gavrovic-Jankulovic M. Yeast surface display is a novel tool for the rapid immunological characterization of plant-derived food allergens. Immunol Res. 2015 Mar;61(3):230-9. doi: 10.1007/s12026-014-8614-0. PMID: 25537533.

pCTcon2 vector Sequence

LOCUS       V011800                 6314 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V011800
VERSION     V011800
KEYWORDS    pCTcon2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 6314)
  AUTHORS   Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD
  TITLE     Isolating and engineering human antibodies using yeast surface
            display.
  JOURNAL   Nat Protoc. 2006;1(2):755-68.
   PUBMED   17406305
REFERENCE   2  (bases 1 to 6314)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6314)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat
            Protoc."; date: "2006"; volume: "1(2)"; pages: "755-68"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6314
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..442
                     /label="GAL1 promoter"
                     /note="inducible promoter, regulated by Gal4"
     CDS             489..749
                     /gene="AGA2"
                     /label="A-agglutinin-binding subunit"
                     /note="A-agglutinin-binding subunit from Saccharomyces
                     cerevisiae (strain ATCC 204508 / S288c). Accession#:
                     P32781"
     CDS             768..779
                     /label="Factor Xa site"
                     /note="Factor Xa recognition and cleavage site"
     CDS             780..806
                     /label="HA"
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             1059..1088
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label="Myc"
                     /translation="EQKLISEEDL"
     promoter        complement(1422..1440)
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1447..1463)
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     rep_origin      1604..2059
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     CDS             complement(2165..2836)
                     /label="TRP1"
                     /note="phosphoribosylanthranilate isomerase, required for
                     tryptophan biosynthesis"
     promoter        complement(2837..3118)
                     /label="TRP1 promoter"
     primer_bind     complement(3138..3157)
                     /label="pRS-marker"
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     3257..3279
                     /label="pGEX 3'"
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3317..3335)
                     /label="pBRforEco"
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     misc_feature    complement(3376..3879)
                     /label="CEN/ARS"
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     promoter        3916..4020
                     /label="AmpR promoter"
     CDS             4021..4878
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      5052..5640
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     primer_bind     5794..5811
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     protein_bind    5928..5949
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5964..5994
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    6002..6018
                     /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     6026..6042
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     promoter        6063..6081
                     /label="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6111..6314
                     /label="GAL1,10 promoter"
                     /note="divergent inducible promoter, regulated by Gal4"