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pYC6/CT vector (V010208)

Price Information

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V010208 pYC6/CT In stock, instant shipping

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

Basic Vector Information

The pYC6/CT is a centromeric vector designed for S. cerevisiae, featuring a blasticidin resistance marker. It facilitates galactose-inducible expression of C-terminally tagged proteins.

Vector Name:
pYC6/CT
Antibiotic Resistance:
Ampicillin
Length:
4504 bp
Type:
Yeast Plasmids
Replication origin:
ori
Source/Author:
Invitrogen (Life Technologies)
Copy Number:
High copy number
Promoter:
GAL1
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

pYC6/CT vector Map

Copy Sequence View Map
pYC6/CT4504 bp600120018002400300036004200GAL1 promoterT7 promoterMCSV5 tag6xHisCYC1 terminatororiAmpRBSDEM7 promoterTEF1 promoterCEN/ARS

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

  • Miura T, Yamana Y, Usui T, Ogawa HI, Yamamoto MT, Kusano K. Homologous recombination via synthesis-dependent strand annealing in yeast requires the Irc20 and Srs2 DNA helicases. Genetics. 2012 May;191(1):65-78. doi: 10.1534/genetics.112.139105. Epub 2012 Feb 23. PMID: 22367032; PMCID: PMC3338270.

pYC6/CT vector Sequence

LOCUS       pYC6_CT.        4504 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Centromeric vector with a blasticidin resistance marker for 
            galactose-inducible expression of C-terminally tagged proteins in S.
            cerevisiae.
ACCESSION   .
VERSION     .
KEYWORDS    pYC6 CT
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4504)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 4504)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..443
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     promoter        475..493
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    495..606
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             607..648
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
     CDS             658..675
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      709..956
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(1204..1792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1966..2823)
                     /label=AmpR
                     /note="beta-lactamase"
     CDS             complement(2946..3341)
                     /label=BSD
                     /note="blasticidin S deaminase"
     promoter        complement(3360..3407)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter"
     promoter        complement(3414..3821)
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     misc_feature    3848..4351
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"