PlasmidInfo:
PlasmidInformation
ProductName:pSF-AOX1-Zeo
ProductCode:OG3584
Size(bp):5646bp
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.
PromoterExpressionLevel:ThispromoterisinducIBLebymethanolandexhibitshighexpressionundertheseconditions.Intheabsenceofmethanolthepromoteristightlyregulatedandshowsonlybasallevelsofexpression.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthepoly-adenylationsignalfromtheAlcoholOxidase1Promoter(AOX1)genefromPichiapastoristoterminatetranscription.
Cloning:
Cloninginagene:Thisplasmidhasbeendesignedtobecompatiblewitharangeofcloningtechniques.Themultiplecloningsitecontainsarangeofstandardcommonlyusedrestrictionsitesforcloning.UsingthesesitesgenescanbeinsertedusingstandardcloningmethodswithDNAligase.Othermethodssuchasligaseindependentcloning(LIC)GibsonAssemblyInFusionHDorSeamlessGeneArtcanalsobeusedandbecauseallofourplasmidsarebasedonthesamebackbonethesamemethodcanbeusedforcloningintoallofourcataloguevectors.
Multiplecloningsitenotes:ThereareafewimportantsiteswithintheMCS.TheseincludetheNcoIsitetheXbaIsiteandtheBsgIandBseRIsites.TheNcoIsitecontainsastartcodonthatisimmediatelydownstreamofbothaKozakandShine-Dalgarnoribosomalbindingsite.Theseallowforoptimalpositioningofgeneswhenthestartcodonisplacedinthislocation.IfthisisnotrequiredandyouwishtouseadownstreamsiteforgenecloningyoucanremovetheNcoIsitebycleavingtheplasmidwithKpnI.
BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsites.Thisallowsthemtocuttheupstreamstopcodoninthegeneinthisplasmidregardlessofthegenesequence.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
