PlasmidInfo:
PlasmidInformation
ProductName:pSF-AOX1-A-factor-Zeo
ProductCode:OG3585
Size(bp):5898bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Yeast(strong)constitutivetriosephosphateisomerase(TPI1)gene
PlasmidPurpose:
Thisvectorisdesignedfortheexpressionofageneofinterest(GOI)inthemethylotrophicyeast,Pichiapastoris.OncetheGOIhasbeenclonedintotheMCS,theplasmidisthenlinearisedandtransformedintoanappropriateP.pastorisstrainbyelectroporation.Theexpressionconstructbecomesintegratedintothegenome.ExpressionoftheGOIisinducedbyadditionofmethanoltotheculturemedium.ThevectorcontainsaZeocinresistanceMarkerforselectionofintegrantsinanyPichiastrain.IncreasingtheZeocinconcentrationalsoenablesselectionofmulticopyintegration.ThisvectorenablessecretionofyourGOIusingtheupstreamalpha-factorsecretionsignal.
PromoterExpressionLevel:ThispromoterisinducIBLebymethanolandexhibitshighexpressionundertheseconditions.Intheabsenceofmethanolthepromoteristightlyregulatedandshowsonlybasallevelsofexpression.
AboutthePeptideTag:ThisplasmidcontainsanfulllengthAlphaFactor(A-FactorFL)secretorypeptidetoallowproteinstobeexportedfromthecytosol.Alphafactorisanendogenousyeastproteinthatisnaturallysecretedintothesupernatant.Thewild-typeAlphaFactorproteincontainsaseriesofrepeatingproteinsequencesthatarecleavedoutbycellularproteasesthatrecogniseaspecificsite.Thisproducesaseriesofsmallpeptidesthatcontainthematurealphapeptidesequence.Thisplasmidcontainthewild-typesignalpeptidefromthefulllengthAlphafactorgenebutalsothecodingsequencethatleadsuptothefirstendogenousproteasecleavagesite.Theadditionofthesesequenceshasbeendemonstratedtoprovidemoreconsistentsecretionofproteinsincomparisontousingjustthesignalpeptidealone.Allofthewild-typeproteinsequenceswillberemovedduringproteinmaturationandexportbyendogenousproteases.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthepoly-adenylationsignalfromtheAlcoholOxidase1Promoter(AOX1)genefromPichiapastoristoterminatetranscription.
Cloning:
Cloninginagene:ThisplasmidisdesignedformakingfusionproteinswiththeAlphafactorsecretionsignal.Itcontainsourstandardmultiplecloningsitewhichallowstheinsertionofgenesusingrestrictionenzymebasedapproaches.TomakeamoreseamlessfusiontryourSnapFusionversionofthisplasmid,OG3586.
Multiplecloningsitenotes:
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
