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科学研究使用,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。

Cre-expressing (pGAL1-cre) CEN/ARS plasmid, marker gene: pScURA3-ScURA3-tScURA3, selectable phenotype: uracil prototrophy

载体名称:
pSH47
载体抗性:
Ampicillin
载体长度:
6979 bp
载体类型:
Recombinase expression vector
复制子:
ori
载体来源:
Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH.
启动子:
GAL1
感受态:
stbl3
培养温度:
37℃

pSH47 载体图谱

pSH476979 bp30060090012001500180021002400270030003300360039004200450048005100540057006000630066006900URA3 promoterURA3f1 oriM13 fwdT7 promoterCYC1 terminatorCreSK primerGAL1 promoterT3 promoterM13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR promoterCEN/ARS

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。

pSH47 载体序列

LOCUS       40924_40067        6979 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Recombinase expression vector pSH47, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6979)
  AUTHORS   Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH.
  TITLE     A new efficient gene disruption cassette for repeated use in budding
            yeast
  JOURNAL   Nucleic Acids Res. 24 (13), 2519-2524 (1996)
  PUBMED    8692690
REFERENCE   2  (bases 1 to 6979)
  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   3  (bases 1 to 6979)
  AUTHORS   Gueldener U, Hegemann JH, Heck S, Fiedler T, Beinhauer JD.
  TITLE     Direct Submission
  JOURNAL   Submitted (23-AUG-2000) Institut fuer Mikrobiologie, 
            Heinrich-Heine-Universitaet, Universtitaetsstr. 1, Duesseldorf 
            40225, Germany
REFERENCE   4  (bases 1 to 6979)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 6979)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "1996"; volume: "24"; issue: "13"; pages: 
            "2519-2524"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "2002"; volume: "30"; issue: "6"; pages: "E23"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (23-AUG-2000) Institut fuer Mikrobiologie, 
            Heinrich-Heine-Universitaet, Universtitaetsstr. 1, Duesseldorf 
            40225, Germany"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6979
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        258..478
                     /label=URA3 promoter
     CDS             479..1279
                     /codon_start=1
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1413..1868)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     2013..2029
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2039..2057
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(2076..2323)
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     CDS             complement(2645..3673)
                     /codon_start=1
                     /label=Cre
                     /note="site-specific recombinase"
                     /translation="MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRTLDSETGAMVRLLE
                     DGD"
     primer_bind     complement(3771..3787)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(3802..4243)
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     promoter        complement(4267..4285)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4306..4322)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4330..4346)
                     /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(4354..4384)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4399..4420)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4708..5296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5470..6327)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6328..6432)
                     /label=AmpR promoter
     misc_feature    6469..6972
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"