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Oxford Genetics/pSF-CAG-Ub-Puro (OG600) CAG Promoter Puromycin Resistant Vector/OG600/1 Ea
PlasmidInfo:
PlasmidInformation
ProductName:pSF-CAG-Ub-Puro
ProductCode:OG600
Size(bp):7265bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:CAGsyntheticmammalianpromoterandUbiquitinpromoter/ChickenBetaActin(CBA)promoter/HumanUbiquitinPromoter
PlasmidPurpose:
InthisvectortheCAGpromoterisupstreamoftheMCS(whereyourgeneofinterestcanbeeasilyinserted)andpuromycinresistancegeneisundercontroloftheubiquitinpromoter.CAGisasyntheticcompositeoftheCMVimmediateearlyenhancerfollowedbytheCBApromoterandtherabbitbetaglobinintronandprovidehighlevelanddurableexpressioninmammaliancells.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCAGpromoterwhichisasyntheticcompositeoftheCMVimmediateearlyenhancerfollowedbytheCBApromoterandtherabbitbetaglobinintron.TheChickenbetaactincontainsaCpGislandthatcanhelptokeepthepromoteractiveforlongerinstableculturewhencomparedtotheCMVpromoter.Theubiquitinpromoterthatisdrivingexpressionofthepuromycingenedemonstratesahighlevelofexpressioninmostcelltypes.ItdemonstratessimiliarlevelsofexpressiontothecommonlyusedEF1-Alphapromoter.
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
