PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-COOH-EKT-HA3
ProductCode:OG71
Size(bp):4270bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
ThisvectoraddsaHAtagthatallowsproteindetectionorpurificationusingantibodiesagainsttheHApeptide.TheHAtagwillbepositionedatthe3primeend(Cterminus)ofagene(expressedprotein)thatiswithintheprimaryMCS(NotItoXbaI).TheHAtagcodingsequenceisYPYDVPDYA.Thereisanenterokinasecleavagesiteimmediatelyupstreamofthistag(DDDDK)thatcanbeusedtoremovethetagfromapurifiedproteinifrequired.Itcleavesafterthelysineresidue.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.ForthisreasonwestockarangeofotherpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
CloninginaGene:ThereisastartcodonintheNcoIsitecanberemovedbydigestionwithKpnIifrequired.TheMCSforgeneinsertionsextendsfromNotItoXbaI.TherearebuiltinShine-DalgarnoandKOZAKsequencesalignedwiththestartcodonwithintheNcoIrestrictionsite.Iftheseareremovedyouwillneedtoaddaribosomalentrysitetothe5primeendofyourgeneforoptimalexpression.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
