PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-NH2-FLAG-EKT-NcoI
ProductCode:OG96
Size(bp):4247bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
ThisplasmidaddsaFLAGepitopetagtotheN-terminusofaproteinthatisencodedwithinthemultiplecloningsite.ThistagallowsthedetectionandpurificationofataggedproteinusingantibodiesraisedagainsttheFLAGepitope.TheFLAGtagcodingsequenceisDYKDDDDK.Thereisanenterokinasecleavagesite(DDDDK)withintheFLAGtagsequencethatcanbeusedtoremoveitfromapurifiedproteinifrequired.Itcleavesafterthelysineresidue.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.ForthisreasonwestockarangeofotherpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.
FLAGisaregisteredtrademarkoftheSigmaAldrichCorporation.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
CloninginaGene:Thisvectorhasbeendesignedtoallowtheadditionofapeptidetagtotheendofaproteinofinterestusingstandardcloningtechniques.
MultipleCloningSiteNotes:ThereisastartcodonintheNcoIsitecanberemovedbydigestionwithKpnIifrequired.TheMCSforgeneinsertionsextendsfromNotItoXbaIhoweverthetagresidesbetweentheNotIandHindIIIsites.ThereareShine-DalgarnosequencesandKOZAKsequencesalignedwiththestartcodonofthepeptidetag.
TheClaItoNheIsiteshaveotherfunctionssuchasaddingC-terminalpeptidetagssecondpromotersorIRESexpressioncomponents.TheBsgIandBseRIrestrictionsitescleavewithinthestopcodonintheXbaIsiteandallowtheretrospectivefusionofC-terminalpeptidetagssequencesifthestopcodonisplacedinthisposition.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
