Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000020 | pBIS-GALkFLP(URA) | In stock, 1 week for quality controls |
Buy one, get one free! |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pBIS-GALkFLP(URA)
- Antibiotic Resistance:
- Ampicillin
- Length:
- 6861 bp
- Type:
- Yeast Expression
- Replication origin:
- ori
- Selection Marker:
- URA3
- Promoter:
- GAL10
- 5' Primer:
- M13-F20
- 3' Primer:
- M13R
pBIS-GALkFLP(URA) vector 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.
pBIS-GALkFLP(URA) vector Sequence
LOCUS 40924_6551 6861 bp DNA circular SYN 13-MAY-2021
DEFINITION overespression of Flp step-arrest mutant in yeast, URA selection.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6861)
AUTHORS Tsalik EL, Gartenberg MR
TITLE Curing Saccharomyces cerevisiae of the 2 micron plasmid by targeted
DNA damage.
JOURNAL Yeast. 1998 Jun 30;14(9):847-52.
PUBMED 9818722
REFERENCE 2 (bases 1 to 6861)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 6861)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Yeast.";
date: "1998-06-30"; volume: "14(9)"; pages: "847-52"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6861
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(29..51)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 151..170
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
promoter 196..416
/label=URA3 promoter
CDS 417..1217
/codon_start=1
/label=URA3
/note="orotidine-5'-phosphate decarboxylase, required for
uracil biosynthesis"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
rep_origin complement(1351..1806)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 1951..1967
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 1977..1995
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind 2021..2037
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(2250..2580)
/label=GAL10 promoter
/note="inducible promoter, regulated by Gal4"
CDS 2588..3856
/codon_start=1
/label=FLP
/note="site-specific recombinase"
/translation="MPQFGILCKTPPKVLVRQFVERFERPSGEKIALCAAELTYLCWMI
THNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKKLIPAWEFTI
IPYYGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESIWEITEKILN
SFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGVIIQCLVTET
KTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEYQLLKDNLVR
SYNKALKKNAPYSIFAIKNGPKSLIGRHLMTSFLSMKGLTELTNVVGNWSDKRASAVAR
TTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQLKGSAEGSIR
YPAWNGIISQEVLDYLSSYINRRI"
promoter complement(4087..4105)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(4126..4142)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(4150..4166)
/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)."
promoter complement(4174..4204)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(4219..4240)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(4357..4374)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4528..5116)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5290..6147)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6148..6252)
/label=AmpR promoter
misc_feature 6289..6792
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
primer_bind 6834..6852
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"