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pYM44 vector (V010197) Gene synthesis in pYM44 backbone

Price Information

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V010197 pYM44 In stock, instant shipping

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

Basic Vector Information

Vector Name:
pYM44
Antibiotic Resistance:
Ampicillin
Length:
4749 bp
Type:
Yeast Plasmids
Replication origin:
ori
Host:
Yeast
Source/Author:
Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, Maekawa H,
Copy Number:
High copy number
Promoter:
TEF
Growth Strain(s):
Top10

pYM44 vector Map

Copy Sequence View Map
pYM444749 bp600120018002400300036004200yeGFPADH1 terminatorHIS3MX6T7 promoteroriAmpRAmpR promoterSP6 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.

pYM44 vector Sequence

LOCUS       40924_48208        4749 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid with a HIS3MX6 marker that provides a template for PCR to 
            fuse yeast enhanced GFP (yeGFP) to the C-terminus of a protein of 
            interest.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4749)
  AUTHORS   Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, Maekawa H, 
            Moreno-Borchart A, Doenges G, Schwob E, Schiebel E, Knop M.
  TITLE     A versatile toolbox for PCR-based tagging of yeast genes: new 
            fluorescent proteins, more markers and promoter substitution 
            cassettes.
  JOURNAL   Yeast 2004;21:947-62.
  PUBMED    15334558
REFERENCE   2  (bases 1 to 4749)
  AUTHORS   EUROSCARF
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 4749)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Yeast"; 
            date: "2004"; volume: "21"; pages: "947-62"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4749
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             82..795
                     /codon_start=1
                     /label=yeGFP
                     /note="yeast-enhanced green fluorescent protein"
                     /translation="LSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKTDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      820..1007
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae
                     alcohol dehydrogenase 1 (ADH1) gene"
     gene            1082..2282
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        complement(2387..2405)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(2663..3251)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(3425..4282)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4283..4387)
                     /label=AmpR promoter
     promoter        4733..4749
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"