ProductName:pSF-CMV-Ub-Neo_G418-SV40_Ori_SbfI
ProductCode:OG138
Size(bp):6532bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:Cytomegalovirus(CMV)immediateearlypromoter/HumanUbiquitinPromoter
ThisvectorcontainsaNeomycin(Neo)resistanceexpressioncassetteundertheUbpromoter.ItalsocontainstheSimianVirus40(SV40originofreplication.Thiscanbeusedtoincreasetransgenesignalfollowingtransienttransfection.ItisalsoreportedthatitispossIBLetomakecelllinescontainingtheplasmidasamulti-copyepisomehoweverwehavenotvalidateditforthispurpose.TheUbNeocassetteisflankedbyAscIsitesinthisplasmidandisnotdesignedtobesplitup.IfyouwouldliketheNeomycingenealoneortheUbpromoteralonepleaseseethemammalianselectiongenesectionorthepromotersectiononourwebsite.ThisvectorcanalsobeusedtoforcerecombinationintothegenomeofmammaliancellsnotexpressingSV40largeTantigenfollowingtransfectionandNeomycinselectionalthoughthismethodcanbeinefficient.IfcellsareexpressingtheSV40largeTantigenthosethatintegratetheplasmidwouldlikelyexperiencechromosomalinstABIlitybecausethelargeTantigenwouldinitiateDNAreplicationfromwithinthehostchromosome.
PromoterExpressionLevel:ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.Forthisreasonwestockarangeofotherpromotersthatarecompatiblewiththisplasmidandareavailableonrequest.
Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Thisplasmidhasbeendesignedtobecompatiblewitharangeofcloningtechniques.Themultiplecloningsitecontainsarangeofstandardcommonlyusedrestrictionsitesforcloning.UsingthesesitesgenescanbeinsertedusingstandardcloningmethodswithDNAligase.Othermethodssuchasligaseindependentcloning(LIC)GibsonAssemblyInFusionHDorSeamlessGeneArtcanalsobeusedandbecauseallofourplasmidsarebasedonthesamebackbonethesamemethodcanbeusedforcloningintoallofourcataloguevectors.
Multiplecloningsitenotes:ThereareafewimportantsiteswithintheMCS.TheseincludetheNcoIsitetheXbaIsiteandtheBsgIandBseRIsites.TheNcoIsitecontainsastartcodonthatisimmediatelydownstreamofbothaKozakandShine-Dalgarnoribosomalbindingsite.Theseallowforoptimalpositioningofgeneswhenthestartcodonisplacedinthislocation.IfthisisnotrequiredandyouwishtouseadownstreamsiteforgenecloningyoucanremovetheNcoIsitebycleavingtheplasmidwithKpnI.
TheXbaIsitecontainsastopcodon.ThisstopcodonispositionedinaspecificpositioninrelationtotheBsgIandBseRIsitesthatareimmediatelydownstream.WheneitherBseRIorBsgIcleavetheplasmidtheyproduceaTAoverhangfromthestopcodonintheXbaIsitethatiscompatiblewithallofourpeptidetagplasmidscutwiththesamesites.BseRIandBsgIsitesarenon-palindromicandcleaveadefinednumberofbasesawayfromtheirbindingsite.
WheneverwecloneageneintoourmultiplecloningsitewealwayspositionthestartandstopcodoninthesamepositionsintheMCS.IfthestartandendsofthegenesarenotcompatiblewithNcoIandXbaIweextendthesequencetothenearestexternalsitesbutkeepthestartandstopcodonslocationsconsistent.
ThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
SouthernBiotech/Goat Anti-Human Lambda-Alexa Fluor® 555/2070-32/1.0 mg
vectorlabs/Biotinylated Aleuria Aurantia Lectin (AAL)/B-1395/1 mg
vectorlabs/VECTASTAIN® Elite® ABC-HRP Kit (Peroxidase, Mouse IgG)/PK-6102/1 kit
vectorlabs/VECTASTAIN® Elite® ABC-HRP Kit (Peroxidase, Universal)/PK-6200/1 kit
vectorlabs/Unconjugated Musa Paradisiaca (Banana) Lectin (BanLec)/L-1410/5 mg
GiottoBiotech/Calmodulin N60D/5 mg/G02CLM60cn