PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-TOPO
ProductCode:OG108
Size(bp):4343bp
BacterialAntibioticSelection:AmpR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
Aversatilecloningplasmidfortheexpressionofgenesinmammaliancells.Thisplasmidcontainsthemultiplecloningsite(MCS)fromLifeTechnologiesTOPOvectorhoweverithasbeenmodifiedslightlytoaccommodatesomerestrictionsitesintheSnapFastTMsystem.ThesechangesaredescribedinthefullplasmiddetailsPDF.TheuseofthisMCSinsteadofthenormalSnapFastTMvectorMCSwilllimittheABIlitytousesomeoftheinsertsthatwesellthatimmediatelyflankorareinsertedwithinthestandardMCS.InthisvectortheselimitationsmainlyextendtoNcoIalignedpeptidetagsandsignalpeptideshoweverthesesameinsertscanstillbeligatedintotheNotIandHindIIIsitesinthisMCS.MostotherinsertsshouldstillbecompatIBLewiththisplasmid.TOPOisaregisteredtrademarkofLifeTechnologies.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.Forthisreasonwestockarangeofotherpromotersthatarecompatiblewiththisplasmidandareavailableonrequest.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
Multiplecloningsitenotes:Thisvectorcontainsadifferentmultiplecloningsite(MCS)fromthestandardSnapFastTMvector.TheMCSisderivedfromTOPO.TheorderoftherestrictionsitesinthisvectorarealmostthesameasinTOPOhoweverthesequenceisnot.TheTOPOMCShasalsobeenmodifiedasfollows:
- TheAflIIsitethesecondEcoRIsitethePstIsiteshavebeenremovedtoallowcompatibilitywithasmanyofourproductsaspossible.
- TheNotIsitethatisnormallyinthisMCShasbeenreplacedwithaPspOMIsitewhichhascompatiblecohesiveendswithNotIdigestedfragmentswhencleaved.TheNotIhasbeenmovedtothe5primeofthemultiplecloningsiteasthisallowstheuseofmanyofour5primecodingsequencesandtags.
- TheXbaIsitehasbeenmoveddownstreambyonerestrictionsite.
- TheBamHIsitethatisnormallyfoundinallSnapFastTMvectors(inthedownstreamfusionMCS)hasbeenreplacedwithaBclIsitebecausetheTOPOMCScontainsthissite.BamHIandBclIproducecompatiblecohesiveendswhencleavedalthoughBclIismethylatedinthisvectorandwillrequiregrowthinaDammethylasenegativebacterialstrain(suchasJM110cells)beforeitcanbeused.
- InthenormalversionofthismultiplecloningsiteanNsiIsitecanbefoundatthe5prime.Howeverthissitecontainsastartcodonandsoithasbeenablatedtopreventprematureribosomaltranslationinitiation.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
