质粒编号
质粒名称
规格
价格
V001375
pML104
5 μg (冻干粉)
我们所展示的质粒图谱主要是从文献和开放数据库中收集而来，主要是为了方便研究工作，其中一小部分质粒进行了质量控制，可供科学家使用。
所有的产品都严格仅供学术研究使用，不能应用于临床试验，包括人体摄入、注射或外用的药理学用途。
确保质粒的关键元件正确，但是我们并不能保证实验结果。页面展示的图谱序列为理论序列，可能与测序结果不一致，请自行比对后确定是否满足要求。(如果测过序，本页面一般会提供下载)
开放数据库中的大多数载体序列都没有被完全测序。如果实际序列与参考序列的相似度超过99%，则将其视为正确。
由于科学研究是在探索未知，具有很大的不确定性，在任何情况下，我们都不承担超出质粒本身的额外经济损失或责任。
Note:
- 载体名称:
- pML104
- 载体抗性:
- Ampicillin
- 载体长度:
- 11240 bp
- 载体类型:
- Yeast Expression, CRISPR
- 复制子:
- ori
- 宿主:
- Yeast
- 筛选标记:
- URA3
- 拷贝数:
- High Copy
- 启动子:
- GAP
- 克隆方法:
- Restriction Enzyme
- 5'引物:
- T3
- 感受态:
- DH10B
- 培养温度:
- 37℃
pML104 载体图谱
质粒操作方法
1. 发货形式：质粒干粉(常温运输，存于-20度，请务必先转化提质粒后使用)
2. 收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O溶解质粒;（质粒复测的浓度有时候与标称值差距较大，这可能是因为冻干质粒在管中的位置、复溶效率、测量偏差以及管壁的吸附导致，因此建议先转化提质粒后再使用）
3. 取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；
4. 加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min (30℃培养1-1.5小时);
5. 6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上;
6. 将平板倒置37℃培养14h，如果要求是30℃则培养20h; (菌落过多则将质粒稀释后再转化；没有菌落则加入10μl质粒转化；建议不要直接转表达感受态， 要先转克隆感受态，重提质粒后再导入表达感受态);
7. 挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
pML104 载体序列
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"