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pHRW32 vector (V014703)

Price Information

Cat No. Plasmid Name Availability Add to cart
V014703 pHRW32 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:
pHRW32
Antibiotic Resistance:
Ampicillin
Length:
10858 bp
Type:
Protein expression
Replication origin:
ori
Host:
Yeast
Selection Marker:
Zeo
Promoter:
TEF
Growth Temperature:
37℃

pHRW32 vector Vector Map

pHRW3210858 bp5001000150020002500300035004000450050005500600065007000750080008500900095001000010500Homothallic switching endonuclease13xMycCYC1 terminatorlac promoterCAP binding siteoriAmpRAmpR promoterTEF promoterBleoRTEF terminatorARS1CEN4tTACMV promoterCMV enhancerADH1 terminatortet operator

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.

pHRW32 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       V014703                10858 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V014703
VERSION     V014703
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 10858)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..10858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             116..1873
                     /gene="HO"
                     /label="Homothallic switching endonuclease"
                     /note="Homothallic switching endonuclease from
                     Saccharomyces cerevisiae (strain ATCC 204508 / S288c).
                     Accession#: P09932"
     CDS             1922..2419
                     /label="13xMyc"
                     /note="13 tandem Myc epitope tags"
     terminator      2612..2859
                     /label="CYC1 terminator"
                     /note="transcription terminator for CYC1"
     promoter        complement(2930..2960)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(2975..2996)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(3284..3872)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(4046..4903)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(4904..5008)
                     /label="AmpR promoter"
     promoter        5216..5559
                     /label="TEF promoter"
                     /note="Ashbya gossypii TEF promoter"
     CDS             5560..5931
                     /label="BleoR"
                     /note="antibiotic-binding protein"
     terminator      5940..6137
                     /label="TEF terminator"
                     /note="Ashbya gossypii TEF terminator"
     rep_origin      6480..7317
                     /label="ARS1"
                     /note="S. cerevisiae autonomously replicating sequence
                     ARS1/ARS416"
     misc_feature    7453..7745
                     /label="CEN4"
                     /note="S. cerevisiae CEN4 centromere"
     CDS             complement(8549..9553)
                     /label="tTA"
                     /note="tetracycline-controlled transactivator, comprising a
                     fusion of the tetracycline repressor TetR with the
                     C-terminal activation domain of herpes simplex virus VP16"
     promoter        complement(9659..9862)
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     enhancer        complement(9863..10242)
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     terminator      10355..10539
                     /label="ADH1 terminator"
                     /note="transcription terminator for the S. cerevisiae
                     alcohol dehydrogenase 1 (ADH1) gene"
     protein_bind    10597..10615
                     /label="tet operator"
                     /note="bacterial operator O2 for the tetR and tetA genes"