PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-NH2-His-EKT-NcoI
ProductCode:OG211
Size(bp):4256bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
ThisvectoraddsaHisepitopetagtotheN-terminusofaproteinthatisencodedwithinthemultiplecloningsite.Thistagallowsthepurificationofataggedproteinbybindingtometalmatricessuchasnickelorcobalt.Thereisanenterokinasecleavagesite(DDDDK)immediatelydownstreamoftheHistagthatcanbeusedtoremovetheHistagfromapurifiedprotein.Itcleavesafterthelysineresidue.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.ForthisreasonwestockarangeofotherpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
Multiplecloningsitenotes:ThehistagisinframewiththeATGstartcodonthatiswithintheNcoIrestrictionsiteallowingfusionwithanygenesthatwesellintheMCSthatcontainanNcoIatthe5prime.ThistagispositionedimmediatelyupstreamoftheNcoIsitetominimisetheamountofextraaminoacidsthatareaddedtotheN-terminusofyourgene.
ThestartcodonthatiswithintheNcoIrestrictionsiteisinframewithandadjacenttothecodingsequencefortheHis/EKTpeptidetag.Shine-DalgarnosequencesandKOZAKsequencesarepositionedcorrectlyatthestartoftheHissignalpeptide.TheClaItoNheIsiteshaveotherfunctionssuchasaddingpeptidetagsorIRESelements.TheBsgIandBseRIrestrictionsitescleavewithinthestopcodonintheXbaIsiteandallowfusionwithourC-terminalpeptidetagsprovidedthatyourgenesstopcodonisalsopositionedhere.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
