pIRES2-EGFP

UnitedStates/Canada800.662.2566AsiaPacific+1.650.919.7300Europe+33.(0)1.3904.6880Japan+81.(0)77.543.6116ClontechLaboratories,Inc.ATakaraBioCompany1290TerraBellaAve.MountainView,CA94043TechnicalSupport(US)E-mail:[e-mail](PR29951;published03October2002)RestrictionMapandMultipleCloningSite(MCS)ofpIRES2-EGFPVector.Uniquerestrictionsitesareinbold.NotethattheEco47IIIsitehasnotbeenconfirmedinthefinalconstruct.Description:pIRES2-EGFPcontainstheinternalribosomeentrysite(IRES;1,2)oftheencephalomyocarditisvirus(ECMV)betweentheMCSandtheenhancedgreenfluorescentprotein(EGFP)codingregion.Thispermitsboththegeneofinterest(clonedintotheMCS)andtheEGFPgenetobetranslatedfromasinglebicistronicmRNA.pIRES2-EGFPisdesignedfortheefficientselection(byflowcytometryorothermethods)oftransientlytransfectedmammaliancellsexpressingEGFPandtheproteinofinterest.ThisvectorcanalsobeusedtoexpressEGFPaloneortoobtainstablytransfectedcelllineswithouttime-consumingdrugandclonalselection.EGFPisared-shiftedvariantofwild-typeGFP(3–5)whichhasbeenoptimizedforbrighterfluorescenceandhigherexpressioninmammaliancells.(Excitationmaximum=488nm;emissionmaximum=507nm.)EGFPencodestheGFPmut1variant(6)whichcontainsthedouble-amino-acidsubstitutionofPhe-64toLeuandSer-65toThr.ThecodingsequenceoftheEGFPgenecontainsmorethan190silentbasechangeswhichcorrespondtohumancodon-usagepreferences(7).SequencesflankingEGFPhavebeenconvertedtoaKozakconsensustranslationinitiationsite(8)tofurtherincreasethetranslationefficiencyineukaryoticcells.TheMCSinpIRES2-EGFPisbetweentheimmediateearlypromoterofcytomegalovirus(PCMVIE)andtheIRESsequence.SV40polyadenylationsignalsdownstreamoftheEGFPgenedirectproperprocessingofthe3'endofthebicistronicmRNA.ThevectorbackbonealsocontainsanSV40originforreplicationinmammaliancellsexpressingtheSV40Tantigen.Aneomycin-resistancecassette(Neor),consistingoftheSV40earlypromoter,theneomycin/kanamycinresistancegeneofTn5,andpolyadenylationsignalsfromtheherpessimplexvirusthymidinekinase(HSVTK)gene,allowsstablytransfectedeukaryoticcellstobeselectedusingG418.AbacterialpromoterupstreamofthiscassetteexpresseskanamycinresistanceinE.coli.ThepIRES2-EGFPbackbonealsoprovidesapUCoriginofreplicationforpropagationinE.coliandanf1originforsingle-strandedDNAproduction.pIRES2-EGFPreplaces(butisnotderivedfrom)thepIRES-EGFPVectorpreviouslysoldbyBDBiosciencesClontech.pIRES2-EGFPisfunctionallysimilarlytopIRES-EGFP;however,pIRES2-EGFPgivesbrighterEGFPfluorescencethantheoldervector.NotethattheXbaIsiteatpositionGCTAGCGCTACCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCC591•611•601•621•631•641•661•651•HindIIIXhoIApaIBsp120IKpnIAsp718IBamHIXmaISmaISacIISalIAccISacIEcl136IIEcoRIPstIEco47IIIBglIINheI...IRESpIRES2-EGFP5.3kbpUCoriHSVTKpolyAf1oriSV40polyAEGFPPCMVIEPSV40eSV40oriPKanr/NeorMCS(591–666)IRES1987ismethylatedintheDNAprovidedbyBDBiosciencesClontech.Ifyouwishtodigestthevectorwiththisenzyme,youwillneedtotransformthevectorintoadam–hostandmakefreshDNA.Use:GenesinsertedintotheMCSshouldincludetheinitiatingATGcodon.pIRES2-EGFPanditsderivativescanbeintroducedintomammaliancellsusinganystandardtransfectionmethod.Ifrequired,stabletransformantscanbeselectedusingG418(9).Locationoffeatures:•Humancytomegalovirus(CMV)immediateearlypromoter:1–589Enhancerregion:59–465;TATAbox:554–560;Transcriptionstartpoint:583C→GmutationtoremoveSacIsite:569•MCS:591–665•IRESsequence:666–1250•Enhancedgreenfluorescentprotein(EGFP)geneKozakconsensustranslationinitiationsite:1247–1257Startcodon(ATG):1254–1256;Stopcodon:1971–1973InsertionofValatposition2:1257–1259GFPmut1chromophoremutations(Phe-64toLeu;Ser-65toThr):1446–1451His-231toLeumutation(A→T):1948•SV40earlymRNApolyadenylationsignalPolyadenylationsignals:2127–2132&2156–2161;mRNA3'ends:2165&2177•f1single-strandDNAorigin:2224–2679(PackagesthenoncodingstrandofEGFP.)•BacterialpromoterforexpressionofKanrgene:–35region:2741–2746;–10region:2764–2769Transcriptionstartpoint:2776•SV40originofreplication:3020–3155•SV40earlypromoter/enhancer72-bptandemrepeats:2853–2996;21-bprepeats(3):3000–3063Earlypromoterelement:3076–3082•Kanamycin/neomycinresistancegene:3204–3998G→AmutationtoremovePstIsite:3386;C→A(ArgtoSer)mutationtoremoveBssHIIsite:3732•Herpessimplexvirus(HSV)thymidinekinase(TK)polyadenylationsignals:4234–4252•pUCplasmidreplicationorigin:4583–5226PropagationinE.coli•Suitablehoststrains:DH5α,HB101,andothergeneralpurposestrains.Single-strandedDNAproductionrequiresahostcontaininganFplasmidsuchasJM101orXL1-Blue.•Selectablemarker:plasmidconfersresistancetokanamycin(30µg/ml)toE.colihosts.•E.colireplicationorigin:pUC•Copynumber:~500•Plasmidincompatibilitygroup:pMB1/ColE1References:1.Jackson,R.J.,etal.(1990)TrendsBiochem.Sci.15:477–483.2.Jang,S.K.,etal.(1990)J.Virol.62:2636–2643.3.Cormack,B.,etal.(1996)Gene173:33–38.4.Yang,T.T.,etal.(1996)NucleicAcidsRes.24:4592–4593.5.Haas,J.,etal.(1996)Curr.Biol.6:315–324.6.Jackson,R.J.,etal.(1990)TrendsBiochem.15:477–483.7.Jang,S.K.,etal.(1988)