PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-FMDV-iLumena
ProductCode:OG523
Size(bp):5347bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
ThisexpressionvectorcontainstheCMVpromoterfortheexpressionofgenesinmammaliancells.Theplasmidalsoincludestheinternalribosomeentrysite(IRES)fromFootandMouthDiseaseVirus(FMDV)thatenablestwoproteinstobeproducedfromthesamemRNA.InthisvectorthereisasecretedluciferasethatwehavedevelopedimmediatelydownstreamfromtheIRESthatallowsplasmidactivitytobemeasuredinthesupernatantofmammaliancultures.UsingIRESexpressionsystemscanoftenproducelowlevelsofexpressionparticularlyinsomecelltypesforthisreasonwecanprovideotherplasmidswithhigherlevelsofreportergeneexpressionsuchaspSF-CMV-PGK-iLumenaorpSF-CMV-Ub-iLumena.
PromoterandIRESExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.Forthisreasonwestockarangeofotherpromotersthatarecompatiblewiththisplasmidandareavailableonrequest.TheIRESthatisusedtodriveiLumenasecretedluciferaseexpressionfromtheCMVpromoterisconsiderablyweakerthantheCMVpromoterandtypicallyyields>20foldlowerproteinlevels.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
CloninginaGene:ThisplasmidhasbeendesignedtobecompatIBLewitharangeofcloningtechniques.Themultiplecloningsitecontainsarangeofstandardcommonlyusedrestrictionsitesforcloning.UsingthesesitesgenescanbeinsertedusingstandardcloningmethodswithDNAligase.Othermethodssuchasligaseindependentcloning(LIC)GibsonAssemblyInFusionHDorSeamlessGeneArtcanalsobeusedandbecauseallofourplasmidsarebasedonthesamebackbonethesamemethodcanbeusedforcloningintoallofourcataloguevectors.
Multiplecloningsitenotes:ThereareafewimportantsiteswithintheMCS.TheseincludetheNcoIsitetheXbaIsiteandtheBsgIandBseRIsites.TheNcoIsitecontainsastartcodonthatisimmediatelydownstreamofbothaKozakandShine-Dalgarnoribosomalbindingsite.Theseallowforoptimalpositioningofgeneswhenthestartcodonisplacedinthislocation.
TheXbaIsitecontainsastopcodon.ThisstopcodonispositionedinaspecificpositioninrelationtotheBsgIandBseRIsitesthatareimmediatelydownstream.WheneitherBseRIorBsgIcleavetheplasmidtheyproduceaTAoverhangfromthestopcodonintheXbaIsitethatiscompatiblewithallofourpeptidetagplasmidscutwiththesamesites.BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsite.
WheneverwecloneageneintoourmultiplecloningsitewealwayspositionthestartandstopcodoninthesamepositionsintheMCS.IfthestartandendsofthegenesarenotcompatiblewithNcoIandXbaIweextendthesequencetothenearestexternalsitesbutkeepthestartandstopcodonslocationsconsistent.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
