各类质粒载体图谱.pdf

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1、pGADT7VectorInformationPT3249-5GenBankAccession#:Submissioninprogress.Catalog#K1612-12moriPADH1HindIII(1480)rSV40NLSAmppGADT7GAL4AD8.0kbPT7TpUCADH1MCSoriLEU2HindIII(2280)HAepitopetagT7SequencingPrimer1858MATCHMAKER5'ADLD-InsertScreeningAmplimerGAL4•ActivationDomainT7Pr

2、omoterCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGATCTTTAATACGACTa.a.BglII8811915STARTinvitroHAEpitopeTag•CACTATAGGGCGAGCGCCGCCATGGAGTACCCATACGACGTACCAGATTACGCTNcoI1969•CATATGGCCATGGAGGCCAGTGAATTCCACCCGGGTGGGCATCGATACGGGATCCATNdeINcoISfiIEcoRISmaI/ClaIBamHIXmaI2026STOP•C

3、GAGCTCGAGCTGCAGATGAATCGTAGATACTGAAAAACCCCGCAAGTTCACTTCSacIXhoIPstIMATCHMAKER3'ADLD-InsertScreeningAmplimer2081•AACTGTGCATCGTGCACCATCT3'ADSequencingPrimerRestrictionMapandMultipleCloningSite(MCS)ofpGADT7.Uniquerestrictionsitesareinbold.Description:ThepGADT7vectorexpress

4、esproteinsfusedtoaminoacids768–881oftheGAL4activationdomain(AD).Inyeast,fusionproteinsareexpressedathighlevelsfromtheconstitutiveADH1promoter(P);transcriptionisterminatedattheADH1transcriptionterminationsignal(T).TheADH1ADH1fusionproteinistargetedtotheyeastnucleusbythe

5、SV40nuclearlocalizationsequencesthathavebeenaddedtotheactivationdomainsequence(1).pGADT7alsocontainstheT7promoter,anHAepitopetag,andaMCS.pGADT7replicatesautonomouslyinbothE.coliandS.cerevisiaefromthepUCand2mori,respectively.ThevectorcarriesAmprforselectioninE.coliandth

6、eLEU2nutritionalmarkerforselectioninyeast.Use:pGADT7istheADVectorincludedwithMATCHMAKERTwo-HybridSystem3.TheMCSofpGADT7hasuniquerestrictionsitesinframewiththe3'-endoftheGAL4ADforconstructingafusionproteinwitheitheraproteinofinterestorafusionproteinlibrary.Thebaitprotei

7、nisalsoexpressed(PR8Z151)pGADT7VectorInformationasafusiontoahemagglutinin(HA)epitopetag.HA-taggedproteinscanbeidentifiedwithantibodiesraisedtothiscommonepitope,eliminatingtheneedtogeneratespecificantibodiestonewproteins.TheT7promoterisusedforinvitrotranscriptionandtran

8、slationoftheepitopetaggedfusionproteinandalsoprovidesabindingsiteforsequencingusingtheT7SequencingPrimer.NotethattheA