PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-pUC18
ProductCode:OG107
Size(bp):4310bp
BacterialAntibioticSelection:AmpR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
Aversatilecloningplasmidfortheexpressionofgenesinmammaliancells.Thisplasmidcontainsthemultiplecloningsite(MCS)frompUC18howeverithasbeenmodifiedslightlytoaccommodatesomerestrictionsitesintheSnapFastTMsystem.ThesechangesaredescribedinthefullplasmiddetailsPDF.TheuseofthisMCSinsteadofthenormalSnapFastTMvectorMCSwilllimittheABIlitytousesomeoftheinsertsthatwesellthatimmediatelyflankorareinsertedwithinthestandardMCS.ThisprimarilyincludesN-terminaltagsandsignalpeptides.ThisisbecausetheseinsertsareflankedbyrestrictionsitesthatarenotcompatIBLewiththepUC18MCS.Mostotherinsertsshouldstillbecompatiblewiththisplasmid.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.Forthisreasonwestockarangeofotherpromotersthatarecompatiblewiththisplasmidandareavailableonrequest.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
Multiplecloningsitenotes:Thisvectorcontainsadifferentmultiplecloningsite(MCS)fromthestandardSnapFastvector.TheMCSisderivedfrompUC18.TheorderoftherestrictionsitesinthisvectorarealmostthesameasinpUC18howeverthesequenceisnot.ThepUC18MCShasalsobeenmodifiedasfollows:
- TheXbaIsiteinthepUC18MCShasbeenreplacedwithaSpeIsite(thesesitesproducecompatiblecohesiveendswhencleaved).
- TheSbfIandPstIsiteshavebeenreplacedwithanNsiIsite(whichproducescompatiblecohesiveendswithSbfIandPstIwhencleaved).
- TheBamHIsitethatisnormallyfoundinallSnapFastvectors(inthedownstreamfusionMCS)hasbeenreplacedwithaBclIsitebecausethepUC18MCScontainsthissite.BamHIandBclIproducecompatiblecohesiveendswhencleavedalthoughBclIismethylatedinthisvectorandwillrequiregrowthinaDammethylasenegativebacterialstrain(suchasJM110cells)beforeitcanbeused.
- TheClaItoNheIsiteshaveotherfunctionssuchasaddingpeptidetagsorIRESelements.TheBsgIandBseRIrestrictionsitescleavewithinthestopcodonintheXbaIsiteandallowtheretrospectivefusionofcodingsequences.Thesesitesarenormallyonlyusedongenesthatwesellinthemainmultiplecloningsite.Thisconceptisexplainedinmoredetailonourwebsiteusingthedropdownboxonthehomepageandselecting:Fuseacodingsequencetothe3primeendofagenealreadyintheSnapFastplasmid.
- TheClaItoNheIsiteshaveotherfunctionssuchasaddingpeptidetagsorIRESelements.TheBsgIandBseRIrestrictionsitescleavewithinthestopcodonintheXbaIsiteandallowtheretrospectivefusionofcodingsequences.Thesesitesarenormallyonlyusedongenesthatwesellinthemainmultiplecloningsite.Thisconceptisexplainedinmoredetailonourwebsiteusingthedropdownboxonthehomepageandselecting:Fuseacodingsequencetothe3primeendofagenealreadyintheSnapFastplasmid.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
