pPICZαA质粒图谱

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1、出品公司： 载体名称： 质粒类型： 表达水平： 启动子： 克隆方法： 载体大小： 5'测序引物及序列 3'测序引物及序列 载体标签： 载体抗性： 筛选标记： 备注： 产品目录号： 稳定性： 组成型： 病毒/非病毒： pPICZaa载体基本信息 Invitrogen pPICZalphaA,pPICZaA,pPICZalphaA,pPICZ“A,pPICZaA, pPICZa-A 毕赤酵母蛋白表达载体 高拷贝 AOX1 多克隆位点，限制性内切酶 3593bp 5AOX1:5-GACTGGTTCCAATTGACAAGC-3' 3AOX1:5

2、-GCAAATGGCATTCTGACATCC-3' C-Myc,C-His Zeocin博来霉素 HIS4 插入基因是必需包含起始密码子ATGo V195-20 稳定Stable 组成型Constitutive 非病毒 c-mycepitope6xHis 一eqx二ON一一。(tjs♦一OCX一3一8;dco<-cnESCQ 一汽二Ed-zo。山*薨<-eo a-factor MultipleCioningBelowisthemu

3、ltiplecluninmsitetorpllc'ZaA.RestrictionsitesarelabeledtoSiteofpPICZaAindicatethecleavagesite.Thebox叩niielectidesindicatethevanableregion. ThemultipLccloningsiteliasbeenconfirmedbvsequencingandfunctionaltestin4(hecompleteseqiienceofpPtC-ZirAh

4、kalSupport(seepageForamapandadescriptionofthefeaturesofpPICZarefertopages21-22. 5end01AOX1mRHA5\4QX7primigsite MlIAACCTTTTTTTTTATCATCATTATTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGA I CAA^CTU'I'TGATTTTAACGACTrTTAACGACAACTTGAGAA.GATCAAAAAACAACTAATT ATTCGAAACGATGA随TTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCAMet

5、ArgPheProSerHePheThrALaVaiLeuPheAlaAla W3TCCTCCGCATTAGCTGCTCCAGTCAACACTACAA2AGAAGATGAAACGGCA SerSerAlaLeuAlaAlaProVaiAsnThrTtirThrGluAspGluThrAla u-factorsignalsequence KHICAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTC GinlieProAlaGluAlaVaiILeGlyTyrSerAspLeuGluGlyAspPhe H1K5GATGTTGCTGTTTT

6、GCCATTTTCCAACAGCACAAATAACGGGTTATTGTTT AspVaiAl3Va1LeuProPheSerAsnSerThrAsnAsnGlyL仁uLeuPile Xhor 115ftATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTAICTCTClieAsnThrT^trlieAisSerHeAlaAlaL7sGluGinGlyVaiSerLeu Kex2由gn\cleavage产R|PnHSfi\酶严IAspT^I 118r ]2bl GAGj\AAAGATGAGGCTGAAGC^GAAITCACGTGGCCCAGCCG

7、GCCGTCTCGGATCGGTGluLygArgGluAla^GluAla^ S也signalcleavage KpnIXboISacIINotIXteIomyew闭ope ACCTCGAGCCGCGGCGGCCGCCAGCTTTCTAGAACAAAAACTCATCTCAGAAGAG CLuGinLysLeulieSerGluGlupdyhstidinetag1I1 I2,JL>GATCTGAATAGCGCCGTCGACCATCATCAT*CATCATCATTGAGTTTGTAGCCAspLeuAsnSerAlaVaiAgpHisHisHisHisHisHis*** 1351TT

8、AGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGA3"A0X1primingsits Ml'lTTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGAT3'tJOlyaoefi/latioriEite bFIACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTckTTTTGTTTCTTCTCGT 'Tovourproteinwithnati\eM-teriiunui,vouinu^tLisePLRandutilizethe X/

9、w]siteupstreaji'LcitheKex2cleavagesitetccloneynur^eneflushwitihtheKex2 sitelset?p*i^e4formnredet3i]s pPICZaA,B和C是3.6kb的毕赤酵母蛋白分泌表达载体。表达的重组蛋白是融合蛋白，含 有一个N-端多肽，编码酿酒酵母(Saccharomycescerevisiae)”-因子分泌信号。载体能够在毕赤酵母中利用甲醇诱导的高水平的表达目的蛋白，并且可以用在任何毕赤酵母中，包才X33, GS115菌株，SMD1168H,KM71H。 pPICZaA,B和C系列载体包含以下元素： ?5

10、'端含有AOX1启动子的严格调控，利用甲醇诱导表达任何感兴趣的基因(Ellis等，1985;Koutz 等人，1989;tschopp等人，1987A)。 ?0-因子分泌信号能够分泌性表达目的蛋白。 ?Zeocin抗性基因在大肠杆菌(Escherichiacoli)和毕赤酵母都能用于筛选(Baron等人，1992; Drocourt等人，1990)。 ?C-端含Myc和His标签，可以用于检测和纯化重组蛋白。 ?pPICZA,B,C三种读码框使得可以将基因克隆入载体而不发生任何移码突变。 pPICZaa载体序列 LOCUSpPICZAA3593bpDNAcircularSYN2

11、-SEP-2003 SOURCE ORGANISM COMMENTThisfileiscreatedbyVectorNTI COMMENTVNTAUTHORNAME|| FEATURESLocation/Qualifiers source1..3593 /invitrogen="25" /vntifkey="98" primercomplement(1423..1443) /invitrogen="1880000" /vntifkey="27" /label=3'\AOX1\primer primer855..875 /invitrogen="1890000"

12、 /vntifkey="27" /label=5'\AOX1\primer CDS1320..1337 /invitrogen="1010000" /vntifkey="4" /label=6xHis primer1144..1164 /invitrogen="1900000" /vntifkey="27" /label=alpha-factor\primer terminator1341..1682 /invitrogen="2550000" /vntifkey="43" /label=AOX1\transcription\terminator CDS1275..

13、1304 /invitrogen="1090000" /vntifkey="4" /label=c-myc\epitope terminator2538..2855 /invitrogen="2560000" /vntifkey="43" /label=CYC1\transcription\terminator promoter2095..2162 /invitrogen="2340000" /vntifkey="30" /label=EM7\promoter misc_feature1208..1274 /invitrogen="1460000" /vntifke

14、y="21" /label=MCS rep_origincomplement(2866..3539) /invitrogen="2540000" /vntifkey="33" /label=pUC\origin CDS941..1207 /invitrogen="1280000" /vntifkey="4" /label=alpha-factor\signal\peptide promoter1683..2094 /invitrogen="2300000" /vntifkey="29" /label=TEF1\promoter CDS2163..2537 /inv

15、itrogen="1370000" /vntifkey="4" /label=Zeo(R) promoter1..940 /invitrogen="2160000" /vntifkey="29" /label=AOX1\promoter BASECOUNT892a886c801g1014t ORIGIN 1agatctaacatccaaagacgaaaggttgaatgaaacctttttgccatccgacatccacag 61gtccattctcacacataagtgccaaacgcaacaggaggggatacactagcagcagaccgt 121tgcaaacg

16、caggacctccactcctcttctcctcaacacccacttttgccatcgaaaaacc 181agcccagttattgggcttgattggagctcgctcattccaattccttctattaggctacta 241acaccatgactttattagcctgtctatcctggcccccctggcgaggttcatgtttgttta 301tttccgaatgcaacaagctccgcattacacccgaacatcactccagatgagggctttctg 361agtgtggggtcaaatagtttcatgttccccaaatggcccaaaactgac

17、agtttaaacgct 421gtcttggaacctaatatgacaaaagcgtgatctcatccaagatgaactaagtttggttcg 481ttgaaatgctaacggccagttggtcaaaaagaaacttccaaaagtcggcataccgtttgt 541cttgtttggtattgattgacgaatgctcaaaaataatctcattaatgcttagcgcagtct 601ctctatcgcttctgaaccccggtgcacctgtgccgaaacgcaaatggggaaacacccgct 661ttttggatgattatgcattgtct

18、ccacattgtatgcttccaagattctggtgggaatact 721gctgatagcctaacgttcatgatcaaaatttaactgttctaacccctacttgacagcaat 781atataaacagaaggaagctgccctgtcttaaacctttttttttatcatcattattagctt 841actttcataattgcgactggttccaattgacaagcttttgattttaacgacttttaacga 901caacttgagaagatcaaaaaacaactaattattcgaaacgatgagatttccttcaatttt 9

19、61tactgctgttttattcgcagcatcctccgcattagctgctccagtcaacactacaacaga 1021agatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaagggga 1081tttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaa 1141tactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggc 1201tgaagctgaattcacgtggcccagccggccgtct

20、cggatcggtacctcgagccgcggcgg 1261ccgccagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgacc 1321atcatcatcatcatcattgagtttgtagccttagacatgactgttcctcagttcaagttg 1381ggcacttacgagaagaccggtcttgctagattctaatcaagaggatgtcagaatgccatt 1441tgcctgagagatgcaggcttcatttttgatacttttttatttgtaacctatatagtatag 1501gatt

21、ttttttgtcattttgtttcttctcgtacgagcttgctcctgatcagcctatctcgc 1561agctgatgaatatcttgtggtaggggtttgggaaaatcattcgagtttgatgtttttctt 1621ggtatttcccactcctcttcagagtacagaagattaagtgagaccttcgtttgtgcggat 1681cccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctc 1741ggactccgcgcatcgccgtaccacttcaaaacacccaagc

22、acagcatactaaattttccc 1801tctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaaga 1861gaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaag ttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaa

23、gaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggt

24、gcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaa

25、aggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttctt 3541 ttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatca

26、caaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacga

27、cttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgggg 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 tctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc