Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010233 | pRS305 | 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
Useful for chromosomal integration of a device into yeast strains. This will insert the vector at the specific locus of the LEU2 gene. To be used in conjunction with leucine drop-out media for positive selection of transformed cells. Transformation using this vector requires linearization of the plasmid by cutting with BstEII.
- Vector Name:
- pRS305
- Antibiotic Resistance:
- Ampicillin
- Length:
- 5504 bp
- Type:
- Yeast Plasmids
- Replication origin:
- ori
- Host:
- Yeast
- Source/Author:
- Sikorski RS, Hieter P.
- Copy Number:
- High copy number
- Promoter:
- LEU2
- 5' Primer:
- M13 fwd
- 3' Primer:
- M13 rev
pRS305 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.
References
- Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19-27. doi: 10.1093/genetics/122.1.19. PMID: 2659436; PMCID: PMC1203683.
pRS305 vector Sequence
LOCUS 40924_37793 5504 bp DNA circular SYN 01-JAN-1980
DEFINITION Yeast integrative vector with a LEU2 marker and an MCS derived from
pBLUESCRIPT.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5504)
AUTHORS Sikorski RS, Hieter P.
TITLE A system of shuttle vectors and yeast host strains designed for
efficient manipulation of DNA in Saccharomyces cerevisiae.
JOURNAL Genetics 1989;122:19-27.
PUBMED 2659436
REFERENCE 2 (bases 1 to 5504)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 5504)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Genetics";
date: "1989"; volume: "122"; pages: "19-27"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..5504
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(666..1757)
/codon_start=1
/label=LEU2
/note="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
/translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
DLGGSNSTTEVGDAVAEEVKKILA"
promoter complement(1770..2174)
/label=LEU2 promoter
rep_origin complement(2474..2929)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 3074..3090
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 3097..3115
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
misc_feature 3124..3231
/label=MCS
/note="pBluescript multiple cloning site"
promoter complement(3244..3262)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(3283..3299)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(3307..3323)
/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(3331..3361)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(3376..3397)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(3685..4273)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(4447..5304)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(5305..5409)
/label=AmpR promoter