Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V003233 | pSH62 | 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
Cre-expressing (pGAL1-cre) CEN/ARS plasmid, marker gene: pScHIS3-ScHIS3-tScHIS3, selectable phenotype: histidine prototrophy
- Vector Name:
- pSH62
- Antibiotic Resistance:
- Ampicillin
- Length:
- 7051 bp
- Type:
- Recombinase expression vector
- Replication origin:
- ori
- Source/Author:
- Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH.
- Promoter:
- GAL1
pSH62 vector 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
- Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH. A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. Nucleic Acids Res. 2002 Mar 15;30(6):e23. doi: 10.1093/nar/30.6.e23.
pSH62 vector Sequence
LOCUS 40924_40077 7051 bp DNA circular SYN 18-DEC-2018
DEFINITION Recombinase expression vector pSH62, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7051)
AUTHORS Gueldener U, Heinisch J, Koehler GJ, Voss D, Hegemann JH.
TITLE A second set of loxP marker cassettes for Cre-mediated multiple gene
knockouts in budding yeast
JOURNAL Nucleic Acids Res. 30 (6), E23 (2002)
PUBMED 11884642
REFERENCE 2 (bases 1 to 7051)
AUTHORS Gueldener U, Heinisch JH, Voss D, Koehler G, Hegemann JH.
TITLE Direct Submission
JOURNAL Submitted (23-AUG-2000) Institut fuer Mikrobiologie,
Heinrich-Heine-Universitaet, Universtitaetsstr. 1, Duesseldorf
40225, Germany
REFERENCE 3 (bases 1 to 7051)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 7051)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic
Acids Res."; date: "2002"; volume: "30"; issue: "6"; pages: "E23"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(23-AUG-2000) Institut fuer Mikrobiologie,
Heinrich-Heine-Universitaet, Universtitaetsstr. 1, Duesseldorf
40225, Germany"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..7051
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 317..503
/label=HIS3 promoter
CDS 504..1160
/codon_start=1
/label=HIS3
/note="imidazoleglycerol-phosphate dehydratase, required
for histidine biosynthesis"
/translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
rep_origin complement(1423..1878)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 2023..2039
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 2049..2067
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
terminator complement(2086..2333)
/label=CYC1 terminator
/note="transcription terminator for CYC1"
CDS complement(2655..3683)
/codon_start=1
/label=Cre
/note="site-specific recombinase"
/translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRTLDSETGAMVRLLE
DGD"
primer_bind complement(3781..3797)
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
promoter complement(3812..4253)
/label=GAL1 promoter
/note="inducible promoter, regulated by Gal4"
promoter complement(4277..4295)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(4316..4332)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(4340..4356)
/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(4364..4394)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(4409..4430)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(4718..5306)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(5480..6337)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6338..6442)
/label=AmpR promoter
misc_feature 6479..6982
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"