pETDuet-1

Novagen•ORDERING800-526-7319•TECHNICALSUPPORT800-207-0144pETUpstreamPrimer#69214-3T7promoter-1AGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAAGCCAGGATCCGAATTCGAGCTCGGCGCGCCTGCAGGTCGACAAGCTTGCGGCCGCATAATGCTTAAGTCGAACAGAAAGTAATCGTATTGTACACGGCCSerGlnAspProAsnSerSerSerAlaArgLeuGlnValAspLysLeuAlaAlaAlaEndBamHIEcoRISacISse8387IAscIPstIEcl136ISalIBspMINotIHindIIIAflIIDuetUP2Primer#71180-3DuetDOWN1Primer#71179-3BsrGIGCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATNdeIDuetUP2Primer#71180-3DuetDOWN1Primer#71179-3T7transcriptionstart-2lacoperatorrbsATGGCAGATCTCAATTGGATATCGGCCGGCCACGCGATCGCTGACGTCGGTACCCTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGMetAlaAspLeuAsnTrpIleSerAlaGlyHisAlaIleAlaAspValGlyThrLeuGluSerGlyLysGluThrAlaAlaAlaLysPheGluArgGlnNdeIMunIFseISgfIKpnIXhoIBglIIEcoRVNgoAIVAatIIAvaIS•TagCACATGGACTCGTCTACTAGCGCAGCTTAATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGHisMetAspSerSerThrSerAlaAlaEndBpu1102IPacIAvrIIT7TerminatorPrimer#69337-3T7terminatorS•TagT7promoter-2SgrAIClaIGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCACCATCATCACCACMetGlySerSerHisHisHisHisHisHisNcoIHis•TagT7transcriptionstart-1lacoperatorrbsXbaITB337RevA0903pETDuet-1VectorpETDuet™-1isdesignedforthecoexpressionoftwotargetgenes.Thevectorcon-tainstwomultiplecloningsites(MCS),eachofwhichisprecededbyaT7promoter/lacoperatorandaribosomebindingsite(rbs).ThevectoralsocarriesthepBR322-derivedColE1replicon,lacIgeneandampicillinresistancegene.ThisvectorcanbeusedincombinationwithpACYCDuet™-1(Cat.No.71147-3)inanappropriatehoststrainforthecoexpressionofupto4targetgenes.GenesinsertedintoMCS1canbesequencedusingthepETUpstreamPrimer(Cat.No.69214-3)andDuetDOWN1Primer(Cat.No.71179-3).GenesinsertedintoMCS2canbesequencedusingtheDuetUP2Primer(Cat.No.71180-3)andT7TerminatorPrimer(Cat.No.69337-3).pETDuet-1sequencelandmarksT7promoter-15404–5420T7transcriptionstart-11His•Tag®codingsequence83–100Multiplecloningsites-1(NcoI–AflII)69–168T7promoter-2214–230T7transcriptionstart-2231Multiplecloningsites-2(NdeI–AvrII)297–438S•Tag™codingsequence366–410T7terminator462–509lacIcodingsequence3931–5013pBR322origin2737bla(Ap)codingsequence1119–1976f1origin547–994BsrGI(190)ClaI(5383)SgrAI(5339)MluI(4658)BclI(4644)BstEII(4476)ApaI(4455)HpaI(4156)Psp5II(3789)Bpu10I(3689)Tth111I(3052)SapI(2912)BspLU11I(2794)AlwNI(2385)ScaI(1669)AhdI(1189)SspI(984)DraIII(779)Bst1107I(3027)BsaI(1250)BglI(1309)FspI(1411)EcoNI(5126)SphI(5191)T7lacNdeI(298)BglII(305)MunI(311)EcoRV(319)NgoAIVI(324)FseI(328)SgfI(337)AatII(346)KpnI(352)XhoI(354)S•TagPacI(429)AvrII(433)T7terminatorlacI(3931–5013)Ap(1119–1976)f1ori(547–994)oriMCS2T7lacNcoI(69)His•TagBamHI(106)EcoRI(112)SacI(122)BspMI(124)AscI(125)Sse8387I(135)PstI(135)SalI(137)HindIII(143)NotI(150)AflII(163)MCS1pETDuet-1(5420bp)pETDuet-1cloning/expressionregionsNovagenCat.No.pETDuet-1DNA71146-3Enzyme#SitesLocationsAatII1346AccI31384113026AciI78AflII1163AflIII227944658AhdI11189AluI25Alw26I71250202731534043443045564961AlwI15AlwNI12385ApaI14455ApaLI41856248029804678ApoI51123849599704383AscI1125AvaI1354AvaII513271549351037894107AvrII1433BamHI1106BanI93487351137388940194738520153155336BanII5122705445552685282BbsI3367641734512BbvI28BcgI4162172832234338BclI14644BfaI103141543446262310441379230137823817BglI11309BglII1305BpmI41259327343374826Bpu10I13689Bpu1102I1451BsaAI27763046BsaBI3360153795389BsaHI7343172640204703520253165337BsaI11250BsaJI7694334732634402352155221BsaWI7528148024412588360538364339BsgI3364046134813BsiEI915319932533715591708246028843879BsiHKAI81221775186024842984380846825166BslI22BsmBI231534043BsmFI355735235197Bsp1286I12BspEI25283605BspLU11I12794BspMI1124BsrBI51324363220312865BsrDI41250142442514617BsrFI63246711269497253395348BsrGI1190BsrI24BssHII21254247BssSI218532621Bst1107I13027BstEII14476BstXI3461247354864BstYI11Cac8I36ClaI15383CviJI84DdeI1026245111461686211125202987352736894087DpnI28Enzyme#SitesLocationsDraI3105510741766DraIII1779DrdI382326923107DsaI2695221EaeI81501963223261577398452185350EagI3150196322EarI3198429115041Ecl136II1120Eco47III235445257Eco57I218562252EcoNI15126EcoO109I347837895226EcoRI1112EcoRII81022633264627674022407946194934EcoRV1319EheI44021520353175338FauI16Fnu4HI48FokI101155133616233108324934353513357546034612FseI1328FspI11411HaeII13HaeIII24HgaI12HhaI44HincII21394156HindIII1143HinfI16HpaI14156HphI18KpnI1352MaeIII17MboII13MluI14658MnlI25MseI33MslI9144116001959322536163811429243224610MspA1I1037545018242211245630883207396940624632MspI29MunI1311MwoI34NarI44020520253165337NciI12NcoI169NdeI1298NgoAIV33246715348NlaIII26NlaIV22NotI1150NspI42798316534575191PacI1429PflMI24015083PleI12Psp1406I59891415178834714998Psp5II13789PstI1135PvuI23371559PvuII3320739694062RcaI41993202520745260RsaI5192350166929924515SacI1122SalI1137SapI12911Sau3AI28Sau96I15ScaI11669ScrFI20Enzyme#SitesLocationsSfaNI20SfcI71312265531430233825295416SgfI1337SgrAI15339SphI15191Sse8387I1135SspI1984StyI369433473TaiI15TaqI16TfiI4