PlasmidInfo:

PlasmidPurpose:

ThisplasmidisdesignedtoexpresstaggedproteinsinE.coli.Theplasmidcontainsaconstitutivepromoter(OXB20)derivedfromtheregionupstreamoftheE.coliRecAgene.Itdoesnotrequireinductionoranyadditionalcomponentsforactivity.Itisthestrongestofthebacterialpromotersthatweprovideandthishighlevelofexpressioncancauseexpressionproblemswithsomeproteinswithpoorsolubility.Forthisreasonwesellarangeofbacterialpromoterswithdifferentexpressionlevels(OXB1(low)>OXB20(high))thatcanbeprovidedwiththepeptidetagsinthisplasmidonrequest.

Thisplasmidalsoencodesaproteasecleavagesitethatisdesignedtobepositionedbetweenyourgeneofinterestandthetagtoallowtheremovalofthetagfollowingproteinpurificationorisolation.ThisplasmidcontainsaThrombincleavagetag.Theproteinsequenceofthecleavagetagis:LVPR?GS.ItcleavespreferentiallybetweentheArgandGlyresidues.Offtargetcleavagecanoftenoccuratnon-specificsitesnormallyfromothercontaminatingproteases.Toensuremaximalproteinintegritytheenzymereagentmustbehighlypure.

Formoreinformationonwhichcleavagetagtouseseeourcleavagetagguide.

Thisplasmidcontainsaconstitutivebacterialpromoterthatdoesnotrequireinduction.Itisthestrongestbacterialpromoterwesellandthiscancausesolubilityandexpressionproblemswithsomeproteins.WealsoofferarangeofotherbacterialpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.

FLAGisaregisteredtrademarkoftheSigmaAldrichCorporation.

ThisplasmidcontainsanPectateLyaseB(PelB)secretorysignalpeptide(SP)toallowproteinstobeexportedfromthecytosol.Duringtranslocationfromthecytosolthesignalpeptideisremovedfromtheproteinbyendogenousproteases.

ThisplasmidalsocontainsasecondaryFLAGproteintag.Thesequenceofthistagis:DYKDDDDK

Weprovidearangeofdualpeptidetagplasmids.ThisisbecausesomepeptidetagsprovidespecificBIOLOGicalproperties(e.gsmallmoleculeaffinitynewepitopessolubilityorproteinsecretion)thatarenotprovidedbyothers.

SequenceandMap:

OtherInfo:

Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.

Cloning:

ThisplasmidhasbeendesignedtoallowthreetypesofcloningintothemainMCStojoinacodingsequencewiththetag.

IfyouwouldliketofuseyourcodingsequencetothetagwithminimaladditionalbasesyoucanuseourSnapFusiontechnology.ThisprocessinvolvesamplifyingyourgenebyPCRtoaddspecificrestrictionsitesontotheends.WhenthesesitesarecuttheyproduceanoverhangthatiscompatiblewiththisplasmidcutwithBseRIorBsgI.

1:Amplifyyourgenewithprimersdesignedusingthisspreadsheet
2:CuttheplasmidwitheitherBseRIorBsgI.*
3:Cutyourgenewiththeenzymeyouaddedusingthespreadsheet(anyofAcuIBpmIBpuEIBseRIBsgIEciI).
4:ClonethegeneintotheplasmidusingDNAligase.

UsingthismethodwithanN-terminaltagplasmidwillresultinthetagcodingsequenceimmediatelyfollowedbyyourgenesATGstartcodonatthejoin.Thisresultsinaseamlessfusionofthetwosequenceswithnoextrabasesbeingadded.UsingthismethodonC-terminaltagplasmidswillconvertyourgenesstopcodonintoaTAC(TyrY)codonfollowedbytheplasmidtagcodingsequence.Thisresultsinnoextrabasesbetweenyourgeneandthetag.Seethediagrambelowformoreinformation.

*PleasenotethatinsectexpressionplasmidscannotbecutwithBsgIonlyBseRIbecauseofunavoidableconflictingsitesinthebackbone.AlsoYeastplasmidscannotbecutwithBseRIbecauseofunavoidablerestrictionsitesinthebackbone.

Usingthistechniquewillcreateagenefragmentthatcanbeligatedintoanyorour>1500peptideandreportertagplasmids.Ifyouuseoneoftheothertechniquesbelow(GibsonInFusionSeamlessorLIC)youwillneednewprimersforeveryvectoryoucloneintobecausethearmsofhomologywillchangeaccordingtothetagplasmidyouarecloninginto.

Ifyoufindthatyourgenesequencehassitesinitthatmakeusingthiscloningstrategydifficultyoucanstilluseoneofthealternativemethodsbelow(e.g.standardcloningorGibsoncloning).

OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneinourSnapFusiontechnique.

Ifyouarenotconcernedaboutleavingafewextrabasesbetweenthetagcodingsequenceandyourgeneyoucancloneyourgeneintothevectorusingstandardcloningrestrictionenzymes.Thisstrategywillrequireyoutochoosewhichenzymesyouwanttousetocloneyourgene.

OpenthePrimerDesignToolwhichprovidesprimerswithdifferentenzymechoicespositioningyourgeneasclosetothetagaspossibleineachcase.Pleasenotethatstandardenzymeswillalwaysleaveadditionalnucleotidesbetweenyourgeneandthetagbutusingthespreadsheetwillensurethetagandgeneareinframe.

ThesecloningtechniquesusereagentssoldbyothercompaniesandallowyoutofusesequencestogetherusingenzymesthatchewbacktheDNAtoleaveoverlappingends/overhangs.ThesubsequentmethodofjoiningtheDNAdependsonthekitused.Touseoneofthesetechniquesyoucaneitherdesignyourownprimersoryoucanusethespreadsheetbelowtohelpwiththedesign.

OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneusingGibsonassemblyInfusionHDGeneArtSeamlesscloningorLigaseIndependentCloning(LIC)techniques.

IPStatus:

ThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere

Oxford Genetics公司位于英国牛津，由来自生物各领域中具有多年基因工程经验的专家组建而成，专注于为全球生物不同领域的科研用户和生物技术工业用户提供多种DNA表达载体。 其所有产品基于一个中心质粒骨架(SnapFast )SnapFast 载体经过精心设计，不同的构建中配以不同的DNA元件，在特异酶切位点更换需要的元件，可进行上千种改造，满足各种生物学研究的需要。该公司还为科学家提 供万能克隆平台和DNA插入匹配库，有针对哺乳动物，细菌和噬菌体等多个系统的各种元件，包括启动子、报告基因、抗性标记、标签蛋白、核糖体位点、复制起 点、多克隆位点、融合蛋白、信号肽、终止子及其他DNA元件(SV40原点、P2A肽段、CMV增强子、小启动子等)。所有的元件都可以在不同载体间互换 使用，并且和很多市场上应用的克隆载体和穿梭载体都兼容，利于基因片段的转移。该系统非常灵活、高效、经济、知识产权友好，可解决目前基因工程研究中普遍 存在的克隆问题。