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Oxford Genetics/pSF-TEFI-TPI1-iLumena (OG551) Secreted Luciferase Yeast Vector/OG551/1 Ea
PlasmidInfo:
PlasmidInformation
ProductName:pSF-TEFI-TPI1-iLumena
ProductCode:OG551
Size(bp):8210bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:YeastElongationFactorAlpha-1(TEF-1)promoter/Yeast(strong)constitutivetriosephosphateisomerase(TPI1)gene
PlasmidPurpose:
Thisplasmidencodesasecretedluciferasereportergene(iLumena)thatallowsforthemeasurementofplasmidactivityinthesupernatantofcellculturesfromSaccharomycescerevisiaeyeast.Thisreporterisuniquetoourproductrange.Thereporterusescoelenterazineasthesubstrateandsubstratesdesgiendforguassialuciferasewillworkwellwiththisreporter.ReportergeneexpressionisdrivenfromtheTPI1cassette.ThereisaTEF1promoterupstreamofthemultiplecloningsitetodrivetheexpressionofageneofintererst.Thesepromotersshowsimilarlevelsofexpression.TheplasmidismaintainedinyeastcellsusingtheURA3selectionmethodwherebytheyeaststrainusedisdefectiveforthisgene(whichisessentialforuracilsynthesis)andthecellsaregrowninuracildeficientmedia.
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
