PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-NH2-InsulinSP-NcoI
ProductCode:OG92
Size(bp):4292bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
Thisplasmidaddsthesecretorysignalpeptidethatnormallyinducesthesecretionofinsulininhumans.ItwilltrafficanyproteintowhichitisattachedattheN-terminusintotheendoplasmicreticulum.ItispositionedupstreamofthemultiplecloningsitebutadjacenttotheNcoIrestrictionsite.Ifyourproteinhasnoorganelleretentionsignalsorspecifictraffickingsequencesitwillmostlikelybesecretedintothemediumbybulkflowexocytosismechanisms.
Thistagwilltranslocatetheproteintowhichitisattachedintotheendoplasmicreticulum(ER)ofaeukaryoticcellafterwhichthesignalpeptidewillbecleavedoff.ThesignalsthattraffictheproteintotheERarewithinthetagasarethesignalsthatmediateitscleavagefromthedownstreamprotein.Itsactivityisbasedmoreonthehydropathyofthesignalpeptidesequenceratherthantheactualorderoftheaminoacidswithinit.TheaminoacidsequenceofthesignalpeptideisMALWMRLLPLLALLALWGPDPAAA.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
