PlasmidInfo:
PlasmidInformation
ProductName:pSF-EF1-Alpha-Cas9WT
ProductCode:OG3539
Size(bp):8977bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:HumanElongationFactorAlphaPromoter
PlasmidPurpose:
ThisplasmidisdesignedtoexpresswildtypeCas9inmammaliancells.ExpressionisdrivenfromaEF1-AlphapromoterinthemainMCS.
PromoterExpressionLevel:TheEF1Alphapromoterisarelativelystrongmammalianpromotershowinggoodactivityinmostmammaliancelllines.AlthoughitexhibitsweakerexpressionlevelscomparedtopromoterssuchasCMVorCAG,itisreportedtomaintainexpressionlevelsforlongerinextendedcultureorinvivo.Thisisinpartduetothelargeintronwithinthepromoter.
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
