pet-32a图谱

Novagen•ORDERING800-526-7319•TECHNICALSUPPORT800-207-0144TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAGC...315bp...CTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCTMetSer105aa...LeuAlaGlySerGlySerGlyHisMetHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAGGlyMetLysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerProAspLeuGlyThrAspAspAspAspLysGCCATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAAAlaMetAlaAspIleGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEndGCCATGGCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAApET-32b(+)AlaMetAlaIleSerAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEndGCCATGGGATATCTGTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAApET-32c(+)AlaMetGlyTyrLeuTrpIleArgIleArgAlaProSerThrSerLeuArgProHisSerSerThrThrThrThrThrThrGluIleArgLeuLeuThrCAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGLysProGluArgLysLeuSerTrpLeuLeuProProLeuSerAsnAsnEndBamHIHindIIISacINcoINotISalIXhoIEcoRIEcoRVAvaIEagITrx•TagT7terminatorBpu1102IT7terminatorprimer#69337-3T7promoterrbsXbaIS•Tagprimer#69945-3His•TagthrombinS•TagenterokinaseNspVlacoperatorHis•TagMscIKpnIBglIIpET-32a(+)ThepET-32seriesisdesignedforcloningandhigh-levelexpressionofpeptidesequencesfusedwiththe109aaTrx•Tag™thioredoxinprotein(1).CloningsitesareavailableforproducingfusionproteinsalsocontainingcleavableHis•Tag®andS•Tag™sequencesfordetectionandpurification.Uniquesitesareshownonthecirclemap.NotethatthesequenceisnumberedbythepBR322con-vention,sotheT7expressionregionisreversedonthecirclemap.Thecloning/expressionregionofthecodingstrandtranscribedbyT7RNApolymeraseisshownbelow.Thef1originisorientedsothatinfectionwithhelperphagewillproducevirionscontainingsingle-strandedDNAthatcorre-spondstothecodingstrand.Therefore,single-strandedsequencingshouldbeperformedusingtheT7terminatorprimer(Cat.No.69337-3).1.LaVallie,E.R.,DiBlasio,E.A.,Kovacic,S.,Grant,K.L.,Schendel,P.F.andMcCoy,J.M.(1993)Bio/Technology11,187–193.trxA(366-692)lacI(1171-2250)ori(3684)Ap(4445-5302)f1origin(5434-5889)Bpu1102I(80)AvaI(158)XhoI(158)EagI(166)NotI(166)HindIII(173)SalI(179)SacI(190)EcoRI(192)BamHI(198)EcoRV(206)NcoI(212)KpnI(238)BglII(241)NspV(268)MscI(351)RsrII(589)XbaI(729)SgrAI(840)SphI(996)EcoNI(1056)ApaBI(1205)MluI(1521)BclI(1535)BstEII(1702)BmgI(1730)ApaI(1732)BssHII(1932)HpaI(2027)PshAI(2366)Psp5II(2628)BspGI(3148)Tth111I(3367)Bst1107I(3393)SapI(3506)BspLU11I(3622)AlwNI(4038)BsaI(4576)PstI(4760)PvuI(4885)ScaI(4995)DraIII(5658)pET-32a-c(+)cloning/expressionregionTB12212/98pET-32a(+)sequencelandmarksT7promoter764-780T7transcriptionstart763Trx•Tagcodingsequence366-692His•Tagcodingsequence327-344S•Tagcodingsequence249-293Multiplecloningsites(NcoI-XhoI)158-217His•Tagcodingsequence140-157T7terminator26-72lacIcodingsequence1171-2250pBR322origin3684blacodingsequence4445-5302f1origin5434-5889ThemapsforpET-32b(+)andpET-32c(+)arethesameaspET-32a(+)(shown)withthefollowingexceptions:pET-32b(+)isa5899bpplasmid;subtract1bpfromeachsitebeyondBamHIat198.pET-32c(+)isa5901bpplasmid;add1bptoeachsitebeyondBamHIat198exceptforEcoRV,whichcutsat209.pET-32a-c(+)VectorspET-32a(+)(5900bp)Eam1105I(4357)–Cat.No.pET-32aDNA69015-3pET-32bDNA69016-3pET-32cDNA69017-3TB12212/98pET-32a(+)RestrictionSitesEnzyme#SitesLocationsAccI21803392AceIII812882016234731313272357448145498AciI85AflIII215213622AluI25AlwI17Alw21I101591904141021150526163440394051015186Alw44I54101501343639365182AlwNI14038ApaI11732ApaBI11205ApoI5192270179654605471AvaI1158AvaII85892073244925372628290746534875BamHI1198BanI11BanII519090591917325733BbsI41667200623802740BbvI29BccI14Bce83I721233525053713401142525120BcefI510401381200841245684BcgI14BclI11535BfaI77073026364117437047055809BglI225854635BglII1241BmgI11730BpmI513591848248231494585Bpu10I12728Bpu1102I180BsaI14576BsaAI233745658BsaBI37948042819BsaHI6844865979147821615052BsaJI957212469496958964215625943782BsaWI72184023432811382839754806BsaXI221805606BsbI233385565BscGI14BsgI3137215722782BsiI237955179BsiEI616923063538396248855034BslI25BsmAI71218162317492136326345765352BsmBI221363263BsmFI4982252328935873BsoFI50Bsp24I1081184313621394166416964115414742934325Bsp1286I14BspEI222811BspGI13148BspLU11I13622BsrI25BsrBI4750355553565802BsrDI41568193445764750BsrFI836383184012072419257945955759BssHII11932Bst1107I13393BstEII11702Enzyme#SitesLocationsBstXI3132314521575BstYI12Cac8I39CjeI26CjePI18ClaI2508798CviJI90CviRI28DdeI11DpnI32DraI3438144005092DraIII15658DrdI3331537305613DrdII335712445663DsaI42124699582594EaeI716634936582996121954903EagI1166Eam1105I14515EarI3113935065310EciI41298369638424670Eco47III392624272876Eco57I241705182EcoNI11056EcoO109I3539542628EcoRI1192EcoRII93674951244155920992156364837693782EcoRV1206FauI17FokI13FspI226034737GdiII616636582996121954903HaeI7217351124925703637