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Oxford Genetics/pSF-TEFI-TPI1-Fluc-URA3 (OG545) Firefly Luciferase Yeast Vector/OG545/1 Ea
PlasmidInfo:
PlasmidInformation
ProductName:pSF-TEFI-TPI1-Fluc-URA3
ProductCode:OG545
Size(bp):9227bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:YeastElongationFactorAlpha-1(TEF-1)promoter/Yeast(strong)constitutivetriosephosphateisomerase(TPI1)gene
PlasmidPurpose:
AdualtransgeneSaccharomycescerevisiaeyeastexpressionplasmidthatallowstheco-expressionofluciferasealongsideageneofinterest.Thevectoralsocontainsagenethatisessentialforuracilsynthesisallowingtheplasmidstobemaintainedincellsthataredefectiveforthisgenewhentheyaregrowninmediathatismissinguracil.
PromoterExpressionLevel:Thisplasmidcontainstheyeasttranslationelongationfactor1promoter.ItisthestrongestpromoterthatweprovideforexpressioninSaccharomycescerevisiae.Italsocontainsthestrongyeastconstitutivetriosephosphateisomerase(TPI1)genepromotertodrivetheexpressionofthereportergene.TheTPIpromoterdemonstratessimilarlevelsofexpressiontothetranslationelongationfactor1promoter(TEF-1).
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsforyeastbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
CloninginaGene:ThisplasmidhasbeendesignedtobecompatIBLewitharangeofcloningtechniques.Themultiplecloningsitecontainsarangeofstandardcommonlyusedrestrictionsitesforcloning.UsingthesesitesgenescanbeinsertedusingstandardcloningmethodswithDNAligase.Othermethodssuchasligaseindependentcloning(LIC)GibsonAssemblyInFusionHDorSeamlessGeneArtcanalsobeusedandbecauseallofourplasmidsarebasedonthesamebackbonethesamemethodcanbeusedforcloningintoallofourcataloguevectors.
Multiplecloningsitenotes:ThereareafewimportantsiteswithintheMCS.TheseincludetheNcoIsitetheXbaIsiteandtheBsgIandBseRIsites.TheNcoIsitecontainsastartcodonthatisimmediatelydownstreamofbothaKozakandShine-Dalgarnoribosomalbindingsite.Theseallowforoptimalpositioningofgeneswhenthestartcodonisplacedinthislocation.IfthisisnotrequiredandyouwishtouseadownstreamsiteforgenecloningyoucanremovetheNcoIsitebycleavingtheplasmidwithKpnI.
TheXbaIsitecontainsastopcodon.ThisstopcodonispositionedinaspecificpositioninrelationtotheBsgIandBseRIsitesthatareimmediatelydownstream.WheneitherBseRIorBsgIcleavetheplasmidtheyproduceaTAoverhangfromthestopcodonintheXbaIsitethatiscompatiblewithallofourpeptidetagplasmidscutwiththesamesites.BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsite.
WheneverwecloneageneintoourmultiplecloningsitewealwayspositionthestartandstopcodoninthesamepositionsintheMCS.IfthestartandendsofthegenesarenotcompatiblewithNcoIandXbaIweextendthesequencetothenearestexternalsitesbutkeepthestartandstopcodonslocationsconsistent.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
