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pCT302 vector (V014486)

Price Information

Cat No. Plasmid Name Availability Add to cart
V014486 pCT302 In stock, 1 week for quality controls

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

Basic Vector Information

Vector Name:
pCT302
Antibiotic Resistance:
Ampicillin
Length:
7032 bp
Type:
Protein expression
Replication origin:
ori
Host:
Yeast
Selection Marker:
TRP1
Promoter:
GAL1,10
Growth Temperature:
37℃

pCT302 vector Map

Copy Sequence View Map
pCT3027032 bp30060090012001500180021002400270030003300360039004200450048005100540057006000630066006900TRP1TRP1 promoterCEN/ARSAmpR promoterAmpRoriCAP binding sitelac promoterlac operatorM13 revT3 promoterGAL1,10 promoterA-agglutinin-binding subunitFactor Xa siteHAT7 promoterM13 fwdf1 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.

pCT302 vector Sequence

LOCUS       V014486                 7032 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V014486
VERSION     V014486
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 7032)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7032
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(162..833)
                     /label="TRP1"
                     /note="phosphoribosylanthranilate isomerase, required for
                     tryptophan biosynthesis"
     promoter        complement(834..1114)
                     /label="TRP1 promoter"
     misc_feature    complement(1370..1873)
                     /label="CEN/ARS"
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     promoter        1910..2014
                     /label="AmpR promoter"
     CDS             2015..2872
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      3046..3634
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     protein_bind    3922..3943
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        3958..3988
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    3996..4012
                     /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     4020..4036
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     promoter        4057..4075
                     /label="T3 promoter"
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        4240..4904
                     /label="GAL1,10 promoter"
                     /note="divergent inducible promoter, regulated by Gal4"
     CDS             4944..5204
                     /gene="AGA2"
                     /label="A-agglutinin-binding subunit"
                     /note="A-agglutinin-binding subunit from Saccharomyces
                     cerevisiae (strain ATCC 204508 / S288c). Accession#:
                     P32781"
     CDS             5223..5234
                     /label="Factor Xa site"
                     /note="Factor Xa recognition and cleavage site"
     CDS             5235..5261
                     /label="HA"
                     /note="HA (human influenza hemagglutinin) epitope tag"
     promoter        complement(6453..6471)
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6478..6494)
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     rep_origin      6639..7032
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"