Basic Vector Information
- Vector Name:
- pML104
- Antibiotic Resistance:
- Ampicillin
- Length:
- 11240 bp
- Type:
- Yeast Expression, CRISPR
- Replication origin:
- ori
- Host:
- Yeast
- Selection Marker:
- URA3
- Copy Number:
- High Copy
- Promoter:
- GAP
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- T3
pML104 vector Vector Map
Plasmid Resuspension 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 ℃.
pML104 vector Sequence
LOCUS 40924_31075 11240 bp DNA circular SYN 13-MAY-2021
DEFINITION Expresses Cas9 and contains guide RNA expression cassette with
BclI-SwaI cloning sites for guide sequence cloning; Contains URA3
marker for yeast transformation.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 11240)
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 11240)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 11240)
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..11240
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 1..4104
/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
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEKEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 4114..4134
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
terminator 4162..4349
/label=ADH1 terminator
/note="transcription terminator for the S. cerevisiae
alcohol dehydrogenase 1 (ADH1) gene"
terminator complement(4467..4486)
/label=SUP4 terminator
/note="transcription terminator for the S. cerevisiae SUP4
tRNA gene"
promoter complement(4568..4836)
/label=SNR52 promoter
/note="promoter for the S. cerevisiae small nucleolar RNA
gene SNR52"
promoter complement(4867..4885)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind complement(4906..4922)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4906..4922)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(4919..4941)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 4930..4946
/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(4954..4984)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(4999..5020)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(5137..5154)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(5308..5896)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(6070..6927)
/codon_start=1
/label=AmpR
/note="beta-lactamase"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(6928..7032)
/label=AmpR promoter
rep_origin complement(7059..8401)
/direction=LEFT
/label=2u ori
/note="yeast 2u plasmid origin of replication"
primer_bind 8442..8460
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(8498..8520)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 8620..8639
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
promoter 8663..8878
/label=URA3 promoter
CDS 8879..9679
/codon_start=1
/label=URA3
/note="orotidine-5'-phosphate decarboxylase, required for
uracil biosynthesis"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
rep_origin complement(9813..10268)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind 10409..10425
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 10435..10453
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind 10479..10495
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
promoter 10540..11206
/label=GAP promoter
/note="promoter for glyceraldehyde-3-phosphate
dehydrogenase; also known as the TDH3 promoter"
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