PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-pGem
ProductCode:OG109
Size(bp):4354bp
BacterialAntibioticSelection:AmpR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
Aversatilecloningvectorfortheexpressionofgenesinmammaliancells.Thisvectorcontainsthemultiplecloningsite(MCS)fromthePromegapGEMvectorhoweverithasbeenmodifiedslightlytoaccommodatesomerestrictionsitesintheSnapFastsystem.Thesechangesaredescribedinthecloningtab.TheuseofthisMCSinsteadofthenormalSnapFastvectorMCSwilllimittheABIlitytousesomeoftheinsertsthatwesellinotherplasmidthatimmediatelyflankorareinsertedwithinthestandardMCS.Inthisvectortheselimitationsmainlyextendto5primetagsandsignalpeptides.MostotherinsertsshouldstillbecompatIBLewiththisvector.pGEMisaregisteredtrademarkofthePromegaCorporation.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.Forthisreasonwestockarangeofotherpromotersthatarecompatiblewiththisplasmidandareavailableonrequest.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
Multiplecloningsitenotes:Thisvectorcontainsadifferentmultiplecloningsite(MCS)fromthestandardSnapFastvector.TheMCSisderivedfrompGEM.TheorderoftherestrictionsitesinthisvectorarealmostthesameasinpGEMhoweverthesequenceisnot.ThepGEMMCShasalsobeenmodifiedasfollows:
- ThenormalNotISbfIandPstIsiteshavebeenablatedinthisMCS.TheNotIsitehasbeenmovedtothe5primeoftheMCSthisallowstheuseofmanyofour5primecodingsequencesandtags.
- AnXbaIsitehasbeenaddedtotheendoftheMCS.
- TheBamHIsitethatisnormallyfoundinallSnapFastvectors(inthedownstreamfusionMCS)hasbeenreplacedwithaBclIsite.BamHIandBclIproducecompatiblecohesiveendswhencleavedalthoughBclIismethylatedinthisvectorandwillrequiregrowthinaDammethylasenegativebacterialstrain(suchasJM110cells)beforeitcanbeused.
TheClaItoNheIsiteshaveotherfunctionssuchasaddingpeptidetagsorIRESelements.TheBsgIandBseRIrestrictionsitescleavewithinthestopcodonintheXbaIsiteandallowtheretrospectivefusionofcodingsequences.Thesesitesarenormallyonlyusedongenesthatwesellinthemainmultiplecloningsite.Thisconceptisexplainedinmoredetailonourwebsiteusingthedropdownboxonthehomepageandselecting:Fuseacodingsequencetothe3primeendofagenealreadyintheSnapFastplasmid.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
