PlasmidInfo:
PlasmidInformation
ProductName:pSF-OXB11
ProductCode:OG554
Size(bp):3858bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:OXB11bacterialpromoter
PlasmidPurpose:
AnintermediatestrengthconstitutivebacterialpromoterthatallowsforproteinexpressioninE.coli.ThispromoterwasderivedbymodificationoftheRecAE.colipromotertoremovetheLexArepressorsitefollowedbyrandommutagenesistocreateapaelofpromoter.ThispromoterwasfoundtodemonstrateintermediateexpressionlevelswhengeneexpressionwasanalysedduringE.colilogphasegrowthinshakingculture.ThepromoterinthisplasmidiscalledOXB11becauseitdemonstratesroughlyintermediatelevelsofexpressionbetweenOXB1andOXB20whicharethepromotersshowingthelowestandhighestlevelsofpromoteractivityinthisproductrangerespectively.
PromoterExpressionLevel:ThisplasmidcontainsamediumstrengthconstitutiveE.colipromoterthatwasderivedfromtheRecApromoterbyrandommutagenesis.Itispartofourconstitutivebacterialpromoterrange.Thispromoter(OXB11)showstheintermediatelevelsofexpressionintherangewithOXB1showingthelowestlevelandOXB20showingthehighestlevelofexpression.Theyrequirenoinducingagentforexpression.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(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
