>
产品中心 >
Specialty_ELISA_kits >
Oxford Genetics/pSF-CMV-EMCV-KrYFP (OG518) EMCV IRES YFP Mammalian Plasmid/OG518/1 Ea
PlasmidInfo:
PlasmidInformation
ProductName:pSF-CMV-EMCV-KrYFP
ProductCode:OG518
Size(bp):5514bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter
PlasmidPurpose:
ThisplasmidallowsfortheexpressionoftransgenesinmammaliancellsunderthecontroloftheCMVpromoter.ThevectoralsocontainstheEncephalomyocarditisvirusInternalRibosomeEntrySite(IRES)sequencewhichallowsfortheexpressionofasecondinsertedaftertheIRESsequence.BoththeupstreamgeneandthegenecontrolledbytheIRESaretranslatedfromthesamemRNAmolecule.Inthisplasmidthereisayellowfluorescentprotein(YFP)underthecontroloftheIRES.ThereportergeneiscalledKringleYFPandwasdevelopedbyDNA2.0.
IRESexpressionistypicallysignificantlylower(10-30foldlesstotalproteinbyweightdependingoncelltype)than5primecap-dependenttranslation(theupstreamgene).Forthisreasondetectioncanbedifficultincelllinesthatarehardtotransfect.AlternativeplasmidsinourproductrangeincludepSF-CMV-PGK-KrYFPwhichtypicallyexhibitshigherexpressionlevels.
PromoterandIRESExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.ForthisreasonwestockarangeofotherpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.TheIRESthatisusedtodriveYFPexpressionfromtheCMVpromoterisconsiderablyweakerthantheCMVpromoterandtypicallyyields15-20foldlowerproteinlevels.
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
