PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-pUC19
ProductCode:OG106
Size(bp):4309bp
BacterialAntibioticSelection:AmpR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
Aversatilecloningplasmidfortheexpressionofgenesinmammaliancells.Thisplasmidcontainsthemultiplecloningsite(MCS)frompUC19howeverithasbeenmodifiedslightlytoaccommodatesomerestrictionsitesintheSnapFastTMsystem.ThesechangesaredescribedinthefullplasmiddetailsPDF.TheuseofthisMCSinsteadofthenormalSnapFastTMvectorMCSwilllimittheABIlitytousesomeoftheinsertsthatwesellthatimmediatelyflankorareinsertedwithinthestandardMCS.ThisprimarilyincludesN-terminaltagsandsignalpeptides.ThisisbecausetheseinsertsareflankedbyrestrictionsitesthatarenotcompatIBLewiththepUC19MCS.Mostotherinsertsshouldstillbecompatiblewiththisplasmid.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.Forthisreasonwestockarangeofotherpromotersthatarecompatiblewiththisplasmidandareavailableonrequest.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
Multiplecloningsitenotes:Thisvectorcontainsadifferentmultiplecloningsite(MCS)fromourstandardvectors.TheMCSisderivedfrompUC19.TheorderoftherestrictionsitesinthisvectorarealmostthesameasinpUC19howeverthesequenceisnot.ThepUC19MCShasalsobeenmodifiedasfollows.
- TheXbaIsiteinthepUC19MCShasbeenreplacedwithaSpeIsite(thesesitesproducecompatiblecohesiveendswhencleaved).
- TheXbaIsiteinthepUC19MCShasbeenreplacedwithaSpeIsite(thesesitesproducecompatiblecohesiveendswhencleaved).
- TheSbfIandPstIsiteshavebeenreplacedwithanNsiIsite(whichproducescompatiblecohesiveendswithSbfIandPstIwhencleaved).
- TheBamHIsitethatisnormallyfoundinallSnapFastvectors(inthedownstreamfusionMCS)hasbeenreplacedwithaBclIsitebecausethepUC19MCScontainsthissite.BamHIandBclIproducecompatiblecohesiveendswhencleavedalthoughBclIismethylatedinthisvectorandwillrequiregrowthinaDammethylasenegativebacterialstrain(suchasJM110cells)beforeitcanbeused.
- TheClaItoNheIsiteshaveotherfunctionssuchasaddingpeptidetagsorIRESelements.TheBsgIandBseRIrestrictionsitescleavewithinthestopcodonintheXbaIsiteandallowtheretrospectivefusionofcodingsequences.Thesesitesarenormallyonlyusedongenesthatwesellinthemainmultiplecloningsite.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
