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Oxford Genetics/pSF-TEF1-NH2-GAL4-DBD-Cmyc-2Micron-TRP1 (OG3588)/OG3588/1 Ea
PlasmidInfo:
PlasmidInformation
ProductName:pSF-TEF1-NH2-GAL4-DBD-Cmyc-2Micron-TRP1
ProductCode:OG3588
Size(bp):7091bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:YeastElongationFactorAlpha-1(TEF-1)promoter
PlasmidPurpose:
Yeasttwo-hybridbaitvector.ThisvectorexpressesgenesfusedtotheDNAbindingdomain(aminoacids1-147)ofGAL4.AC-Mycepitopetagisalsopresenttoaiddetection.ProteinexpressionisdrivenfromtheconstitutiveyeastTEF1promoter.ThevectorreplicatesautonomouslyinS.cerevisiaeandE.colifrom2micrometerandpUCoriginsrespectively.ThevectorhastheKanRandTRP1MarkersforselectioninE.coliandS.cerevisiaerespectively.
PromoterExpressionLevel:ThisplasmidcontainstheYeastElongationFactorAlphapromoter(TEF1).Thisisthestrongestoftheyeastpromotersthatwesell.Itisaconstitutivepromoterandrequiresnoinduction.IfyouareinterestedinweakerpromoterslevelsthanwealsostockplasmidsthatcontainthefollowingpromotersinorderofdecreasingstrengthTPI(strong)ADHI(medium)STE5(weak).WealsostockGalactoseinducIBLepromoterplasmidsifinducibleexpressionisrequired.Pleasecontactusforfurtherinformation.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthepoly-adenylationsignalfromtheAlcoholOxidase1Promoter(AOX1)genefromPichiapastoristoterminatetranscription.
Cloning:
Cloninginagene:ThisplasmidhasbeendesignedtoallowthreetypesofcloningintothemainMCStojoinacodingsequencewiththetag.
Multiplecloningsitenotes:1:SnapFusionCloning:
IfyouwouldliketofuseyourcodingsequencetothetagwithminimaladditionalbasesyoucanuseourSnapFusiontechnology.ThisprocessinvolvesamplifyingyourgenebyPCRtoaddspecificrestrictionsitesontotheends.WhenthesesitesarecuttheyproduceanoverhangthatiscompatiblewiththisplasmidcutwithBseRIorBsgI.
Toinsertyourgene:
1:Amplifyyourgenewithprimersdesignedusingthisspreadsheet
2:CuttheplasmidwitheitherBseRIorBsgI.*
3:Cutyourgenewiththeenzymeyouaddedusingthespreadsheet(anyofAcuIBpmIBpuEIBseRIBsgIEciI).
4:ClonethegeneintotheplasmidusingDNAligase.
UsingthismethodwithanN-terminaltagplasmidwillresultinthetagcodingsequenceimmediatelyfollowedbyyourgenesATGstartcodonatthejoin.Thisresultsinaseamlessfusionofthetwosequenceswithnoextrabasesbeingadded.UsingthismethodonC-terminaltagplasmidswillconvertyourgenesstopcodonintoaTAC(TyrY)codonfollowedbytheplasmidtagcodingsequence.Thisresultsinnoextrabasesbetweenyourgeneandthetag.Seethediagrambelowformoreinformation.
*PleasenotethatinsectexpressionplasmidscannotbecutwithBsgIonlyBseRIbecauseofunavoidableconflictingsitesinthebackbone.AlsoYeastplasmidscannotbecutwithBseRIbecauseofunavoidablerestrictionsitesinthebackbone.
Usingthistechniquewillcreateagenefragmentthatcanbeligatedintoanyorour>1500peptideandreportertagplasmids.Ifyouuseoneoftheothertechniquesbelow(GibsonInFusionSeamlessorLIC)youwillneednewprimersforeveryvectoryoucloneintobecausethearmsofhomologywillchangeaccordingtothetagplasmidyouarecloninginto.
Ifyoufindthatyourgenesequencehassitesinitthatmakeusingthiscloningstrategydifficultyoucanstilluseoneofthealternativemethodsbelow(e.g.standardcloningorGibsoncloning).
OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneinourSnapFusiontechnique.
2:StandardEnzymes:
Ifyouarenotconcernedaboutleavingafewextrabasesbetweenthetagcodingsequenceandyourgeneyoucancloneyourgeneintothevectorusingstandardcloningrestrictionenzymes.Thisstrategywillrequireyoutochoosewhichenzymesyouwanttousetocloneyourgene.
OpenthePrimerDesignToolwhichprovidesprimerswithdifferentenzymechoicespositioningyourgeneasclosetothetagaspossibleineachcase.Pleasenotethatstandardenzymeswillalwaysleaveadditionalnucleotidesbetweenyourgeneandthetagbutusingthespreadsheetwillensurethetagandgeneareinframe.
3:Gibsoncloning/InfusionHD/GeneArtSeamless/LigaseIndependentCloning(LIC)Methods:ThesecloningtechniquesusereagentssoldbyothercompaniesandallowyoutofusesequencestogetherusingenzymesthatchewbacktheDNAtoleaveoverlappingends/overhangs.ThesubsequentmethodofjoiningtheDNAdependsonthekitused.Touseoneofthesetechniquesyoucaneitherdesignyourownprimersoryoucanusethespreadsheetbelowtohelpwiththedesign.
OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneusingGibsonassemblyInfusionHDGeneArtSeamlesscloningorLigaseIndependentCloning(LIC)techniques.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
