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pYES2/NT A vector (V010206)

Price Information

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V010206 pYES2/NT A In stock, instant shipping

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

Basic Vector Information

Vector Name:
pYES2/NT A
Antibiotic Resistance:
Ampicillin
Length:
6038 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℃

pYES2/NT A vector Map

Copy Sequence View Map
pYES2/NT A6038 bp30060090012001500180021002400270030003300360039004200450048005100540057006000GAL1 promoterT7 promoterATG6xHisT7 tag (gene 10 leader)MCSV5 tag6xHisCYC1 terminatororiAmpRURA3URA3 promoter2u oriM13 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.

pYES2/NT A vector Sequence

LOCUS       pYES2_NT_A.        6038 bp DNA     circular SYN 01-JAN-1980
DEFINITION  High-copy episomal vector for galactose-inducible expression of 
            N-terminally tagged proteins in S. cerevisiae. For other reading 
            frames, use pYES2/NT B or pYES2/NT C.
ACCESSION   .
VERSION     .
KEYWORDS    pYES2 NT A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6038)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 6038)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6038
                     /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"
     CDS             510..512
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             522..539
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             543..575
                     /label=T7 tag (gene 10 leader)
                     /note="leader peptide from bacteriophage T7 gene 10"
     CDS             579..602
                     /label=Xpress(TM) tag
                     /note="Xpress(TM) epitope tag, including an enterokinase 
                     recognition and cleavage site"
     misc_feature    602..669
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             682..723
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
     CDS             733..750
                     /label=6xHis
                     /note="6xHis affinity tag"
     terminator      784..1031
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(1279..1867)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(2041..2898)
                     /label=AmpR
                     /note="beta-lactamase"
     CDS             complement(2997..3797)
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
     promoter        complement(3798..4018)
                     /label=URA3 promoter
     rep_origin      complement(4617..5497)
                     /direction=LEFT
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     rep_origin      complement(5508..6021)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"