PlasmidInfo:
PlasmidInformation
ProductName:pSF-CAG-Kan
ProductCode:OG505
Size(bp):5358bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:CAGsyntheticmammalianpromoterandUbiquitinpromoter/ChickenBetaActin(CBA)promoter
PlasmidPurpose:
ThismammalianexpressionvectorcontainsthechimericCAGpromoterwhichisacompositeoftheCMVenhancerthechickenbetaactinpromoter(CBA)andtherabbitbetaglobinintron.ThispromoterisoftenusedinsteadoftheCMVpromoterbecauseinsomecelltypestheCMVpromotercangetsilenced.TopreventthistheCAGpromotercontainstheCpGislandfromtheCBApromotertohelppreventpromotermethylation.TheplasmidcanbeusedtodriveproteinexpressioninarangeofmammaliancelltypesandwehaveconsistentlyfoundthatexpressionlevelsareequaltoCMVinshorttermexperimentsinstandardcelltypes.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCAGpromoterwhichisasyntheticcompositeoftheCMVimmediateearlyenhancerfollowedbytheCBApromoterandtherabbitbetaglobinintron.TheChickenbetaactincontainsaCpGislandthatcanhelptokeepthepromoteractiveforlongerinstableculturewhencomparedtotheCMVpromoter.
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
