YEplac195 vector (V010167)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V010167	YEplac195	In stock, instant shipping	Buy one, get one free!

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

Basic Vector Information

The YEplac195 is a yeast episomal plasmid that carries a URA3 marker. Similar vectors include YEplac112 and YEplac181.

Vector Name:: YEplac195

Antibiotic Resistance:: Ampicillin

Length:: 5241 bp

Type:: Yeast Plasmids

Replication origin:: ori

Host:: Yeast

Source/Author:: Gietz RD, Sugino A.

Copy Number:: High copy number

Promoter:: URA3

5' Primer:: M13 fwd

3' Primer:: M13 rev

Growth Strain(s):: DH10B

Growth Temperature:: 37℃

YEplac195 vector Map

Copy Sequence View Map

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

Mioka T, Fujimura-Kamada K, Mizugaki N, Kishimoto T, Sano T, Nunome H, Williams DE, Andersen RJ, Tanaka K. Phospholipid flippases and Sfk1p, a novel regulator of phospholipid asymmetry, contribute to low permeability of the plasma membrane. Mol Biol Cell. 2018 May 15;29(10):1203-1218. doi: 10.1091/mbc.E17-04-0217. Epub 2018 Mar 22. PMID: 29540528; PMCID: PMC5935070.

YEplac195 vector Sequence

LOCUS       40924_49662        5241 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Yeast episomal plasmid with a URA3 marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5241)
  AUTHORS   Gietz RD, Sugino A.
  TITLE     New yeast-Escherichia coli shuttle vectors constructed with in vitro
            mutagenized yeast genes lacking six-base pair restriction sites.
  JOURNAL   Gene 1988;74:527-34.
  PUBMED    3073106
REFERENCE   2  (bases 1 to 5241)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 5241)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Gene"; 
            date: "1988"; volume: "74"; pages: "527-34"
COMMENT     SGRef: number: 2; type: "Journal Article"
COMMENT     The sequence of this plasmid was reconstructed using the description
            from Gietz and Sugino.
FEATURES             Location/Qualifiers
     source          1..5241
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    106..127
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        142..172
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    180..196
                     /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     204..220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(233..289)
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(290..306)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(743..2089)
                     /direction=LEFT
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     CDS             complement(2203..3003)
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(3004..3219)
                     /label=URA3 promoter
     promoter        3335..3439
                     /label=AmpR promoter
     CDS             3440..4297
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4471..5059
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"