各类质粒载体图谱

pGADT7VectorInformationPT3249-5GenBankAccession#:Submissioninprogress.Catalog#K1612-1(PR8Z151)RestrictionMapandMultipleCloningSite(MCS)ofpGADT7.Uniquerestrictionsitesareinbold.Description:ThepGADT7vectorexpressesproteinsfusedtoaminoacids768–881oftheGAL4activationdomain(AD).Inyeast,fusionproteinsareexpressedathighlevelsfromtheconstitutiveADH1promoter(PADH1);transcriptionisterminatedattheADH1transcriptionterminationsignal(TADH1).ThefusionproteinistargetedtotheyeastnucleusbytheSV40nuclearlocalizationsequencesthathavebeenaddedtotheactivationdomainsequence(1).pGADT7alsocontainstheT7promoter,anHAepitopetag,andaMCS.pGADT7replicatesautonomouslyinbothE.coliandS.cerevisiaefromthepUCand2μori,respectively.ThevectorcarriesAmprforselectioninE.coliandtheLEU2nutritionalmarkerforselectioninyeast.Use:pGADT7istheADVectorincludedwithMATCHMAKERTwo-HybridSystem3.TheMCSofpGADT7hasuniquerestrictionsitesinframewiththe3'-endoftheGAL4ADforconstructingafusionproteinwitheitheraproteinofinterestorafusionproteinlibrary.ThebaitproteinisalsoexpressedCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGATCTTTAATACGACTCACTATAGGGCGAGCGCCGCCATGGAGTACCCATACGACGTACCAGATTACGCTCATATGGCCATGGAGGCCAGTGAATTCCACCCGGGTGGGCATCGATACGGGATCCATCGAGCTCGAGCTGCAGATGAATCGTAGATACTGAAAAACCCCGCAAGTTCACTTCAACTGTGCATCGTGCACCATCTT7PromoterHAEpitopeTagSfiIEcoRISmaI/XmaINdeINcoIBamHIClaIPstISTOPXhoI1915•1969•2026•2081•3'ADSequencingPrimerNcoISTARTinvitroMATCHMAKER5'ADLD-InsertScreeningAmplimerMATCHMAKER3'ADLD-InsertScreeningAmplimera.a.881GAL4ActivationDomain1858•SacIT7SequencingPrimerBglIIHAepitopetagpGADT78.0kbLEU2Ampr2μoriGAL4ADpUCoriPADH1PT7TADH1MCSSV40NLSHindIII(1480)HindIII(2280)pGADT7VectorInformationCLONTECHLaboratories,Inc.INTERNET::•Full-lengthS.cerevisiaeADH1promoter(PADH1):7–1479•GAL4ADpolypeptidewithSV40NuclearLocalizationSignal(NLS)NLS:1501–1557GAL4aminoacids768–881:1561–1899•T7RNApolymerasepromoter:1905–1927•HAepitopetag:1942–1968•MultipleCloningSites:1969–2041•TranscriptionterminationsignalFragmentcarryingtheS.cerevisiaeADH1terminator(TADH1):2280–2605•LEU2codingsequences:3814–2723•pUCplasmidreplicationorigin:4581–5418•Ampicillinresistancegene:6432–5575•Yeast2μreplicationorigin:6998–7988Locationofprimers:•T7SequencingPrimer:1905–1925•3'ADSequencingPrimer:2102–2083•MATCHMAKER5'ADLD-InsertScreeningAmplimer(#9103-1):1858–1889•MATCHMAKER3'ADLD-InsertScreeningAmplimer(#9103-1):2078–2046PropagationinE.coli:•Suitablehoststrains:DH5α,DH10&othergeneralpurposestrains•Selectablemarker:plasmidconfersresistancetoampicillin(100μg/ml)toE.colihosts•E.colireplicationorigin:pUC•Copynumber:~500•Plasmidincompatibilitygroup:pMB1/ColE1PropagationinS.cerevisiae:•Suitablehoststrains:Y187(α),Y190(a),SFY526(a),CG1945(a),HF7c(a),orAH109(a)•Selectablemarker:LEU2•S.cerevisiaeorigin:2μReference:1.Chien,C.T.,Bartel,P.L.,Sternglanz,R.&Fields,S.(1991)Proc.Natl.Acad.Sci.USA88:9578–9582.Note:Theattachedsequencefilehasbeencompiledfrominformationinthesequencedatabases,publishedliterature,andothersources,togetherwithpartialsequencesobtainedbyCLONTECH.Thisvectorhasnotbeencompletelysequenced.©1999,CLONTECHLaboratories,Inc.NoticetoPurchaserThisproductisintendedforresearchpurposesonly.Itisnottobeusedfordrugordiagnosticpurposesnorisitintendedforhumanuse.CLONTECHproductsmaynotberesold,modifiedforresale,orusedtomanufacturecommercialproductswithoutwrittenapprovalofCLONTECH.asafusiontoahemagglutinin(HA)epitopetag.HA-taggedproteinscanbeidentifiedwithantibodiesraisedtothiscommonepitope,eliminatingtheneedtogeneratespecificantibodiestonewproteins.TheT7promoterisusedforinvitrotranscriptionandtranslationoftheepitopetaggedfusionproteinandalsoprovidesabindingsiteforsequencingusingtheT7SequencingPrimer.NotethattheADisnotexpressedduringtheinvitrotranscriptionandtranslationreactions.TheNcoIandPstIsitesmaybeusedtoshuttleinsertsfrompGADT7intopGBKT7,theMATCHMAKERTwo-HybridSystem3DNA-BDVector.TheMCSinpGADT7iscompatiblewiththoseinpMyc-CMVandpHA-CMV,CLONTECH'sepitopetaggedmammalianexpressionvectorset(#K6003-1).Asaresult,thetargetgenecanbeshuttledintothesevectorsinordertoconfirmproteininteractionsinvivo.pGBKT7VectorInformation PT3248-5 Cat. No. 630303 (PR631431; published 30 March 2006) RestrictionMapandMultipleCloningSite(MCS)ofpGBKT7.Unique restriction sites are in bold.Description: The pGBKT7 vector expresses proteins fused to amino acids 1–147 of the GAL4 DNA binding do-main (DNA-BD). In yeast, fusion proteins are expressed at high levels from the constitutive ADH1 promoter (PADH1); transcription is terminated by the T7 and ADH1 transcription termination signals (TT7 & ADH1). pGBKT7 also contains the T7 promoter, a c-Myc epitope tag, and a MCS. pGBKT7 rep-licates autonomously in both E.coli and S.cerevisiae from the pUC and 2 μ ori, respectively. The vector carries the Kanr for selection in E.coli and the TRP1nutritional marker for selection in yeast. Yeast strains containing pGBKT7 exhibit a higher transformation efficiency than strains carrying other DNA-