Oxford Genetics/pSFCMVUbZeo_AscI (OG23) Zeocin Selection Plasmid/OG23/1 Ea

PlasmidInfo:

PlasmidPurpose:

ThisvectorcontainsaZeocin(Zeo)resistanceexpressioncassetteundercontroloftheUbiquitin(Ub)promoterthatisdesignedasanadd-ontoanyofourvectors.TheentireUbZeocassetteisflankedbyAscIsite.Thisvectorcanbeusedtoforcerecombinationintothegenomeofmammaliancellsfollowingtransfectionandselectionalthoughthismethodcanbeinefficient.ItcanalsobeusedconjunctionwiththeSV40originofreplicationincellsexpressingtheSV40largeTantigen(suchas293Tcells)tomakestablecellslinesfollowingtransfectionandselection.

ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.Forthisreasonwestockarangeofotherpromotersthatarecompatiblewiththisplasmidandareavailableonrequest.

SequenceandMap:

OtherInfo:

Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(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