Oxford Genetics/pSFTEFIURA3 (OG534) Uracil Yeast Selection Plasmid/OG534/1 Ea

PlasmidInfo:

PlasmidPurpose:

ThisyeastexpressionplasmidisdesignedfortheproductionproteinsinSaccharomycescerevisiaewithselectiononmediathatisdeficientinthemetaboliteuracil.ThevectorcontainstheconstitutiveTEF1yeastpromotertodrivetheexpressionofageneofinterest.Italsocontainsagenethatisanessentialcomponentoftheuracilsynthesispathway.Thisallowstheplasmidtobemaintainedinyeastcellsthathavethisgenedeletedonmediathatdoesnotcontainuracil.ThisisthemostcommonlyusedselectionmethodforSaccharomycescerevisiae.Wealsoprovideothermetaboliteselectionyeastplasmidsthatusehistidineleucineortryptophanastheselectionmethod.Wealsoprovideplasmidsusingsmallmoleculeselectionsuchaspuromycinandblasticidin.

Thisplasmidcontainstheyeasttranslationelongationfactor1promoter.ItisthestrongestpromoterthatweprovideforexpressioninSaccharomycescerevisiae.

SequenceandMap:

OtherInfo:

Thisplasmidcontainsthreealternativetranscriptionterminatorsforyeastbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.

Cloning:

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

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

TheXbaIsitecontainsastopcodon.ThisstopcodonispositionedinaspecificpositioninrelationtotheBsgIandBseRIsitesthatareimmediatelydownstream.WheneitherBseRIorBsgIcleavetheplasmidtheyproduceaTAoverhangfromthestopcodonintheXbaIsitethatiscompatiblewithallofourpeptidetagplasmidscutwiththesamesites.BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsite.

WheneverwecloneageneintoourmultiplecloningsitewealwayspositionthestartandstopcodoninthesamepositionsintheMCS.IfthestartandendsofthegenesarenotcompatiblewithNcoIandXbaIweextendthesequencetothenearestexternalsitesbutkeepthestartandstopcodonslocationsconsistent.

IPStatus:

ThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere