Oxford Genetics/pSFCMVNH2HisEKT3 (OG332) NTerminal His Tag Plasmid/OG332/1 Ea

PlasmidInfo:

PlasmidPurpose:

ThisvectoraddsaHistagtotheN-terminusofaproteinthatisencodedwithinthemultiplecloningsite.Thistagallowsthepurificationofataggedproteinbybindingtometalmatricessuchasnickelorcobalt.TheHisisfollowedbyanenterokinasecleavagesite(DDDDK)withinitthatallowstheremovaloftheN-terminaltagifrequired.Itcleavesafterthelysineresidue.

ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.ForthisreasonwestockarangeofotherpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.

SequenceandMap:

OtherInfo:

Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.

Cloning:

Thisvectorhasbeendesignedtoallowtheadditionofapeptidetagtotheendofaproteinofinterestusingstandardcloningtechniques.

ThereisastartcodonintheNcoIsitecanberemovedbydigestionwithKpnIifrequired.TheMCSforgeneinsertionsextendsfromNotItoXbaIhoweverthetagresidesbetweentheNotIandHindIIIsites.ThereareShine-DalgarnosequencesandKOZAKsequencesalignedwiththestartcodonofthepeptidetag.

TheClaItoNheIsiteshaveotherfunctionssuchasaddingC-terminalpeptidetagssecondpromotersorIRESexpressioncomponents.TheBsgIandBseRIrestrictionsitescleavewithinthestopcodonintheXbaIsiteandallowtheretrospectivefusionofC-terminalpeptidetagssequencesifthestopcodonisplacedinthisposition.

IPStatus:

ThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere