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Oxford Genetics/pSF-PGK-EMCV-Puro (OG612) PGK Promoter EMCV IRES Puromycin Vector/OG612/1 Ea
PlasmidInfo:
PlasmidInformation
ProductName:pSF-PGK-EMCV-Puro
ProductCode:OG612
Size(bp):5335bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:MousePhosphoglycerateKinase(PGK)Promoter
PlasmidPurpose:
Thisplasmidvectorcontainsthephosphoglyceratekinase(PGK)promoterupstreamoftheMCSandEMCVIRESregulatingexpressionofpuromycinresistance
PromoterExpressionLevel:Themurinephosphoglyceratekinase(PGK)promoterdrivingexpressionallowsforlongtermstableexpressioninvitroandinvivoinmosttypes.TheIRESthatisusedtodrivepuromycinresistancedownstreamofthePGKpromoterproducessignificantlyweakerexpressioncomparedtotheupstreamgene(typically15-20foldlowerthantheupstreamgene).
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
