PlasmidInfo:

PlasmidPurpose:

ThisvectorencodestheSEAPpolypeptideasapositivecontrolforsecretedproteinexpression.Thisvectorisdesignedfortheexpressionofageneofinterest(GOI)inthemethylotrophicyeast,Pichiapastoris.OncetheGOIhasbeenclonedintotheMCS,theplasmidisthenlinearisedandtransformedintoanappropriateP.pastorisstrainbyelectroporation.Theexpressionconstructbecomesintegratedintothegenome.ExpressionoftheGOIisinducedbyadditionofmethanoltotheculturemedium.ThevectorcontainsaHIS4Markerfornutritionalselectionofintegrantsinahis4P.pastorisstrain.ThisvectorenablessecretionofyourGOIusingtheupstreamalpha-factorsecretionsignal.

ThispromoterisinducIBLebymethanolandexhibitshighexpressionundertheseconditions.Intheabsenceofmethanolthepromoteristightlyregulatedandshowsonlybasallevelsofexpression.

ThisplasmidcontainsanfulllengthAlphaFactor(A-FactorFL)secretorypeptidetoallowproteinstobeexportedfromthecytosol.Alphafactorisanendogenousyeastproteinthatisnaturallysecretedintothesupernatant.Thewild-typeAlphaFactorproteincontainsaseriesofrepeatingproteinsequencesthatarecleavedoutbycellularproteasesthatrecogniseaspecificsite.Thisproducesaseriesofsmallpeptidesthatcontainthematurealphapeptidesequence.Thisplasmidcontainthewild-typesignalpeptidefromthefulllengthAlphafactorgenebutalsothecodingsequencethatleadsuptothefirstendogenousproteasecleavagesite.Theadditionofthesesequenceshasbeendemonstratedtoprovidemoreconsistentsecretionofproteinsincomparisontousingjustthesignalpeptidealone.Allofthewild-typeproteinsequenceswillberemovedduringproteinmaturationandexportbyendogenousproteases.

SequenceandMap:

OtherInfo:

Thisplasmidcontainsthepoly-adenylationsignalfromtheAlcoholOxidase1Promoter(AOX1)genefromPichiapastoristoterminatetranscription.

Cloning:

Thisplasmidisnotdesignedtocloneinagene,insteaditisdesignedfortestingsecretioninPichiapastoris.

IPStatus:

ThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere