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Oxford Genetics/pSF-PGK-Cas9WT (OG3541) CRISPR/Cas9 Expression Plasmid/OG3541/1 Ea
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PlasmidInfo:PlasmidInformationProductName:pSF-PGK-Cas9WTProductCode:OG3541Size(bp):8277bpBacterialAntibioticSelection:KanROriginandCompatibility:pUChighcopyderivedfrompBR322BacterialCopyNumber:500-700percellPromoter:MousePhosphoglycerateKinase(PGK)PromoterPlasmidPurpose:ThisplasmidisdesignedtoexpresswildtypeCas9inmammaliancells.ExpressionisdrivenfromaPGKpromoterinthemainMCS.PromoterExpressionLevel:Themurinephosphoglyceratekinase(PGK)promoterdrivingexpressionallowsforlongtermstableexpressioninvitroandinvivoinmosttypes. SequenceandMap:OtherInfo:TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.Cloning:Cloninginagene:Thisplasmidcontainsagenewithinthemainmultiplecloningsite(NotI-ClaI).Anyplasmidthatwesellwherethegeneisthisconfigurationwillbelocatedintheexactsamepositioninrelationtothestartandstopcodonofthegeneintheplasmid.TheonlyexceptionstothisrulearefusionsproteinswherethefusiongenemaybepositionedatthefrontorendoftheMCStoallowgenefusion.Bypositioningallofourgenesinthesamelocationitallowsthemtobetransferredbetweenplasmidsusingthesamecloningmethodandrestrictionsitesregardlessoftheplasmidbeingusedfromourproductrange.InsertinganewgeneintothisplasmidshouldbeeasilypossIBLeusingarangeofstandardrestrictionenzymesitesthatflankthegenecurrentlyinthevector.Multiplecloningsitenotes:InthemultiplecloningsitetherearetwoimportantrestrictionsitescalledBsgIandBseRIsites.ThesesitesbothcuttheDNAatthesamepositionandcleavethestopcodonofthegeneinthemultiplecloningsiteinthisplasmidtherebyproducingaTAoverhang.Thisoverhangiscompatiblewithanyofourpeptideorreporterfusiontagplasmidsalsocutwitheitheroftheseenzymes.ThisallowsseamlessC-terminalfusionstobemadewiththegeneinthismultiplecloningsiteusingasinglecloningstepfromourC-terminalpeptideandreportertagproductrange.NormallytheeasiestmethodistoclonetheC-terminaltagfromourotherplasmidproductsintothisplasmidusingBsgI or BseRIandthedownstreamClaIrestrictionsite.BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsites.Thisallowsthemtocuttheupstreamstopcodoninthegeneinthisplasmidregardlessofthegenesequence.IPStatus:IntellectualPropertyStatusThisproductispartofourSnapFastProplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere

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