PlasmidInfo:
PlasmidInformation
ProductName:pSF-CAG-Ub-Hygro
ProductCode:OG507
Size(bp):7694bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:CAGsyntheticmammalianpromoterandUbiquitinpromoter/ChickenBetaActin(CBA)promoter/HumanUbiquitinPromoter
PlasmidPurpose:
AversatilecloningvectorthatcontainsthestrongconstitutivemammalianpromoterCAGtodrivetransgeneexpression.ItalsocontainsahygromycinselectioncassettedrivenbythehighlyactiveUbiquitinpromotertoallowforstablecelllinestobegeneratedexpressingageneofinterest.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCAGpromoterwhichisasyntheticcompositeoftheCMVimmediateearlyenhancerfollowedbytheCBApromoterandtherabbitbetaglobinintron.TheChickenbetaactincontainsaCpGislandthatcanhelptokeepthepromoteractiveforlongerinstableculturewhencomparedtotheCMVpromoter.Theubiquitinpromoterthatisdrivingexpressionofthehygromycingenedemonstratesahighlevelofexpressioninmostcelltypes.ItdemonstratessimiliarlevelsofexpressiontothecommonlyusedEF1-Alphapromoter.
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
