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Oxford Genetics/pSF-PGK-Cas9WT-EMCV-Puro (OG3575) CRISPR/Cas9 Expression Plasmid/OG3575/1 Ea
PlasmidInfo:
PlasmidInformation
ProductName:pSF-PGK-Cas9WT-EMCV-Puro
ProductCode:OG3575
Size(bp):9440bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:MousePhosphoglycerateKinase(PGK)Promoter
PlasmidPurpose:
Plasmidvectorforexpressingwild-typeStreptococcuspyogenesCas9proteininmammalincellsfromaPGKpromoterexpressioncassetteinthemainpSFMCS.Alsocontainsadown-streamEncephalomyocarditisvirus(EMCV)internalribosomeentrysite(IRES)fortheco-expressionofthepuromycinresistancegene.
PromoterExpressionLevel:Themurinephosphoglyceratekinase(PGK)promoterdrivingexpressionallowsforlongtermstableexpressioninvitroandinvivoinmosttypes.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
Cloninginagene:Thisplasmidcontainsagenewithinthemainmultiplecloningsite(NotI-ClaI).Anyplasmidthatwesellwherethegeneisthisconfigurationwillbelocatedintheexactsamepositioninrelationtothestartandstopcodonofthegeneintheplasmid.TheonlyexceptionstothisrulearefusionsproteinswherethefusiongenemaybepositionedatthefrontorendoftheMCStoallowgenefusion.
Bypositioningallofourgenesinthesamelocationitallowsthemtobetransferredbetweenplasmidsusingthesamecloningmethodandrestrictionsitesregardlessoftheplasmidbeingusedfromourproductrange.InsertinganewgeneintothisplasmidshouldbeeasilypossIBLeusingarangeofstandardrestrictionenzymesitesthatflankthegenecurrentlyinthevector.
Thisplasmidalsocontainsaninternalribosomeentrysite(IRES)followedbyapuromycinselectiongene(Puro).TheIRESallowstheco-expressionoftwogenesfromthesamemRNA,inthiscasebothCas9andPuro.Whendesigningallourplasmidsweaimtoremoveallconflictingresitrictionsitestomakecloningsimpler,however,theIREScontainsanumberofrestrictionsitesthatarewithincoformationallyimportanthairpins,wehavethereforebeenunabletoremovethem.therefore,pleasecheckthisplasmidmapbeforecloningtoensurenoneoftheserestrictionsiteswillcausecloningproblems.
Multiplecloningsitenotes:InthemultiplecloningsitetherearetwoimportantrestrictionsitescalledBsgIandBseRIsites.ThesesitesbothcuttheDNAatthesamepositionandcleavethestopcodonofthegeneinthemultiplecloningsiteinthisplasmidtherebyproducingaTAoverhang.Thisoverhangiscompatiblewithanyofourpeptideorreporterfusiontagplasmidsalsocutwitheitheroftheseenzymes.ThisallowsseamlessC-terminalfusionstobemadewiththegeneinthismultiplecloningsiteusingasinglecloningstepfromourC-terminalpeptideandreportertagproductrange.NormallytheeasiestmethodistoclonetheC-terminaltagfromourotherplasmidproductsintothisplasmidusingBsgI or BseRIandthedownstreamClaIrestrictionsite.
BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsites.Thisallowsthemtocuttheupstreamstopcodoninthegeneinthisplasmidregardlessofthegenesequence.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastProplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
