Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V010718 | pJH001 | 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:
- pJH001
- Antibiotic Resistance:
- Ampicillin
- Length:
- 11928 bp
- Type:
- Yeast Expression, CRISPR
- Replication origin:
- ori
- Host:
- Yeast
- Selection Marker:
- LEU2
- Copy Number:
- High Copy
- Promoter:
- GAP
pJH001 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.
pJH001 vector Sequence
LOCUS 40924_26101 11928 bp DNA circular SYN 13-MAY-2021
DEFINITION High copy Cas9 expression vector with LEU2 marker for yeast
transformation.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11928)
AUTHORS Laughery MF, Hunter T, Brown A, Hoopes J, Ostbye T, Shumaker T,
Wyrick JJ
TITLE New vectors for simple and streamlined CRISPR-Cas9 genome editing in
Saccharomyces cerevisiae.
JOURNAL Yeast. 2015 Dec;32(12):711-20. doi: 10.1002/yea.3098. Epub 2015 Sep
21.
PUBMED 26305040
REFERENCE 2 (bases 1 to 11928)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 11928)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi: "10.1002/yea.3098";
journalName: "Yeast"; date: "2015-12"; volume: "32"; issue: "12";
pages: "711-20"
COMMENT SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..11928
/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"
CDS complement(667..1758)
/codon_start=1
/label=LEU2
/note="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
/translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
DLGGSNSTTEVGDAVAEEVKKILA"
promoter complement(1759..2166)
/gene="S. cerevisiae LEU2"
/label=LEU2 promoter
rep_origin complement(2467..2922)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 3048..3070
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 3062..3079
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 3063..3079
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 3089..3107
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind 3132..3148
/label=pBluescriptKS
/note="For pBluescript vector"
primer_bind 3133..3149
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
promoter 3194..3860
/label=GAP promoter
/note="promoter for glyceraldehyde-3-phosphate
dehydrogenase; also known as the TDH3 promoter"
CDS 3895..7998
/codon_start=1
/label=Cas9
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 8008..8028
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
terminator 8056..8243
/label=ADH1 terminator
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
promoter complement(8323..8341)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(8362..8378)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8362..8378)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(8375..8397)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 8386..8402
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/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(8410..8440)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(8455..8476)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(8593..8610)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(8764..9352)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(9526..10383)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(10384..10488)
/label=AmpR promoter
rep_origin complement(10515..11861)
/direction=LEFT
/label=2u ori
/note="yeast 2u plasmid origin of replication"
primer_bind 11901..11919
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"