PlasmidInfo:

PlasmidPurpose:

Thisvectorisdesignedfortheexpressionofageneofinterest(GOI)inthemethylotrophicyeast,Pichiapastoris.OncetheGOIhasbeenclonedintotheMCS,theplasmidisthenlinearisedandtransformedintoanappropriateP.pastorisstrainbyelectroporation.Theexpressionconstructbecomesintegratedintothegenome.ExpressionoftheGOIisinducedbyadditionofmethanoltotheculturemedium.ThevectorcontainsaHIS4Markerfornutritionalselectionofintegrantsinahis4P.pastorisstrain.

ThispromoterisinducIBLebymethanolandexhibitshighexpressionundertheseconditions.Intheabsenceofmethanolthepromoteristightlyregulatedandshowsonlybasallevelsofexpression.

SequenceandMap:

OtherInfo:

Thisplasmidcontainsthepoly-adenylationsignalfromtheAlcoholOxidase1Promoter(AOX1)genefromPichiapastoristoterminatetranscription.

Cloning:

Thisplasmidhasbeendesignedtobecompatiblewitharangeofcloningtechniques.Themultiplecloningsitecontainsarangeofstandardcommonlyusedrestrictionsitesforcloning.UsingthesesitesgenescanbeinsertedusingstandardcloningmethodswithDNAligase.Othermethodssuchasligaseindependentcloning(LIC)GibsonAssemblyInFusionHDorSeamlessGeneArtcanalsobeusedandbecauseallofourplasmidsarebasedonthesamebackbonethesamemethodcanbeusedforcloningintoallofourcataloguevectors.

ThereareafewimportantsiteswithintheMCS.TheseincludetheNcoIsitetheXbaIsiteandtheBsgIandBseRIsites.TheNcoIsitecontainsastartcodonthatisimmediatelydownstreamofbothaKozakandShine-Dalgarnoribosomalbindingsite.Theseallowforoptimalpositioningofgeneswhenthestartcodonisplacedinthislocation.IfthisisnotrequiredandyouwishtouseadownstreamsiteforgenecloningyoucanremovetheNcoIsitebycleavingtheplasmidwithKpnI.

BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsites.Thisallowsthemtocuttheupstreamstopcodoninthegeneinthisplasmidregardlessofthegenesequence.

IPStatus:

ThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere