PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-NH2-InsulinSP1
ProductCode:OG85
Size(bp):4328bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
Thisplasmidaddsthesecretorysignalpeptidethatnormallyinducesthesecretionofinsulininhumans.ItwilltrafficanyproteintowhichitisattachedattheN-terminusintotheendoplasmicreticulum.Itispositionedupstreamofthemultiplecloningsite.Ifyourproteinhasnoorganelleretentionsignalsorspecifictraffickingsequencesitwillmostlikelybesecretedintothemediumbybulkflowexocytosismechanisms.
Thistagwilltranslocatetheproteintowhichitisattachedintotheendoplasmicreticulum(ER)ofaeukaryoticcellafterwhichthesignalpeptidewillbecleavedoff.ThesignalsthattraffictheproteintotheERarewithinthetagasarethesignalsthatmediateitscleavagefromthedownstreamprotein.Itsactivityisbasedmoreonthehydropathyofthesignalpeptidesequenceratherthantheactualorderoftheaminoacidswithinit.Theaminoacidsequenceofthesignalpeptideis:MALWMRLLPLLALLALWGPDPAAA.Cleavageoccursimmediatelyafterthefinalalanineresidue.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.ForthisreasonwestockarangeofotherpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
CloninginaGene:Thisvectorhasbeendesignedtoallowtheadditionofapeptidetagtotheendofaproteinofinterestusingstandardcloningtechniques.
MultipleCloningSiteNotes:ThereisastartcodonintheNcoIsitecanberemovedbydigestionwithKpnIifrequired.TheMCSforgeneinsertionsextendsfromNotItoXbaIhoweverthetagresidesbetweentheNotIandHindIIIsites.ThereareShine-DalgarnosequencesandKOZAKsequencesalignedwiththestartcodonofthepeptidetag.
TheClaItoNheIsiteshaveotherfunctionssuchasaddingC-terminalpeptidetagssecondpromotersorIRESexpressioncomponents.TheBsgIandBseRIrestrictionsitescleavewithinthestopcodonintheXbaIsiteandallowtheretrospectivefusionofC-terminalpeptidetagssequencesifthestopcodonisplacedinthisposition.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
