PlasmidInfo:
PlasmidInformation
ProductName:pSF-AOX1-A-factor-SnapFusion-Zeo
ProductCode:OG3586
Size(bp):5905bp
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.ThisvectorcontainstheSnapFusionMCSfortheeasyinsertionofpeptidetags.
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:
Multiplecloningsitenotes:1:SnapFusionCloning:
IfyouwouldliketofuseyourcodingsequencetothetagwithminimaladditionalbasesyoucanuseourSnapFusiontechnology.ThisprocessinvolvesamplifyingyourgenebyPCRtoaddspecificrestrictionsitesontotheends.WhenthesesitesarecuttheyproduceanoverhangthatiscompatiblewiththisplasmidcutwithBseRIorBsgI.
Toinsertyourgene:
1:Amplifyyourgenewithprimersdesignedusingthisspreadsheet
2:CuttheplasmidwitheitherBseRIorBsgI.*
3:Cutyourgenewiththeenzymeyouaddedusingthespreadsheet(anyofAcuIBpmIBpuEIBseRIBsgIEciI).
4:ClonethegeneintotheplasmidusingDNAligase.
UsingthismethodwithanN-terminaltagplasmidwillresultinthetagcodingsequenceimmediatelyfollowedbyyourgenesATGstartcodonatthejoin.Thisresultsinaseamlessfusionofthetwosequenceswithnoextrabasesbeingadded.UsingthismethodonC-terminaltagplasmidswillconvertyourgenesstopcodonintoaTAC(TyrY)codonfollowedbytheplasmidtagcodingsequence.Thisresultsinnoextrabasesbetweenyourgeneandthetag.Seethediagrambelowformoreinformation.
*PleasenotethatinsectexpressionplasmidscannotbecutwithBsgIonlyBseRIbecauseofunavoidableconflictingsitesinthebackbone.AlsoYeastplasmidscannotbecutwithBseRIbecauseofunavoidablerestrictionsitesinthebackbone.
Usingthistechniquewillcreateagenefragmentthatcanbeligatedintoanyorour>1500peptideandreportertagplasmids.Ifyouuseoneoftheothertechniquesbelow(GibsonInFusionSeamlessorLIC)youwillneednewprimersforeveryvectoryoucloneintobecausethearmsofhomologywillchangeaccordingtothetagplasmidyouarecloninginto.
Ifyoufindthatyourgenesequencehassitesinitthatmakeusingthiscloningstrategydifficultyoucanstilluseoneofthealternativemethodsbelow(e.g.standardcloningorGibsoncloning).
OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneinourSnapFusiontechnique.
2:StandardEnzymes:Ifyouarenotconcernedaboutleavingafewextrabasesbetweenthetagcodingsequenceandyourgeneyoucancloneyourgeneintothevectorusingstandardcloningrestrictionenzymes.Thisstrategywillrequireyoutochoosewhichenzymesyouwanttousetocloneyourgene.
OpenthePrimerDesignToolwhichprovidesprimerswithdifferentenzymechoicespositioningyourgeneasclosetothetagaspossibleineachcase.Pleasenotethatstandardenzymeswillalwaysleaveadditionalnucleotidesbetweenyourgeneandthetagbutusingthespreadsheetwillensurethetagandgeneareinframe.
3:Gibsoncloning/InfusionHD/GeneArtSeamless/LigaseIndependentCloning(LIC)Methods:ThesecloningtechniquesusereagentssoldbyothercompaniesandallowyoutofusesequencestogetherusingenzymesthatchewbacktheDNAtoleaveoverlappingends/overhangs.ThesubsequentmethodofjoiningtheDNAdependsonthekitused.Touseoneofthesetechniquesyoucaneitherdesignyourownprimersoryoucanusethespreadsheetbelowtohelpwiththedesign.
OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneusingGibsonassemblyInfusionHDGeneArtSeamlesscloningorLigaseIndependentCloning(LIC)techniques.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
