>
产品中心 >
Sandwich_method_ELISA >
Oxford Genetics/pSF-OXB20-COOH-Thr-Strep-6His (OG2863) C-terminal 6 His and Strep dual tag bacterial plasmid/OG2863/1 E
PlasmidInfo:
PlasmidInformation
ProductName:pSF-OXB20-COOH-Thr-Strep-6His
ProductCode:OG2863
Size(bp):3914bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:OXB20strongconstitutivebacterialpromoter
PlasmidPurpose:
ThisplasmidisdesignedtoexpresstaggedproteinsinE.coli.Theplasmidcontainsaconstitutivepromoter(OXB20)derivedfromtheregionupstreamoftheE.coliRecAgene.Itdoesnotrequireinductionoranyadditionalcomponentsforactivity.Itisthestrongestofthebacterialpromotersthatweprovideandthishighlevelofexpressioncancauseexpressionproblemswithsomeproteinswithpoorsolubility.Forthisreasonwesellarangeofbacterialpromoterswithdifferentexpressionlevels(OXB1(low)>OXB20(high))thatcanbeprovidedwiththepeptidetagsinthisplasmidonrequest.
AbouttheCleavageTag:Thisplasmidalsoencodesaproteasecleavagesitethatisdesignedtobepositionedbetweenyourgeneofinterestandthetagtoallowtheremovalofthetagfollowingproteinpurificationorisolation.ThisplasmidcontainsaThrombincleavagetag.Theproteinsequenceofthecleavagetagis:LVPR?GS.ItcleavespreferentiallybetweentheArgandGlyresidues.Offtargetcleavagecanoftenoccuratnon-specificsitesnormallyfromothercontaminatingproteases.Toensuremaximalproteinintegritytheenzymereagentmustbehighlypure.
Formoreinformationonwhichcleavagetagtouseseeourcleavagetagguide.
PromoterExpressionLevel:Thisplasmidcontainsaconstitutivebacterialpromoterthatdoesnotrequireinduction.Itisthestrongestbacterialpromoterwesellandthiscancausesolubilityandexpressionproblemswithsomeproteins.WealsoofferarangeofotherbacterialpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.
Thisplasmidcontainsac-terminalStrepepitopetagthatcanbefusedtoageneofinteresttoallowproteindetectionand/orpurification.Thesequenceofthetagis:WSHPQFEK
Formoreinformationonthemethodsthatcanbeusedtopurifyproteinspleaseseeourproteintagguide.
ThisplasmidalsocontainsasecondaryHexa-Histidine(6His)tagproteintag.Thesequenceofthistagis:HHHHHH
Weprovidearangeofdualpeptidetagplasmids.ThisisbecausesomepeptidetagsprovidespecificBIOLOGicalproperties(e.gsmallmoleculeaffinitynewepitopessolubilityorproteinsecretion)thatarenotprovidedbyothers.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
MakingProteinFusions:ThisplasmidhasbeendesignedtoallowthreetypesofcloningintothemainMCStojoinacodingsequencewiththetag.
SnapFusionCloning:IfyouwouldliketofuseyourcodingsequencetothetagwithminimaladditionalbasesyoucanuseourSnapFusiontechnology.ThisprocessinvolvesamplifyingyourgenebyPCRtoaddspecificrestrictionsitesontotheends.WhenthesesitesarecuttheyproduceanoverhangthatiscompatiblewiththisplasmidcutwithBseRIorBsgI.
Toinsertyourgene:
1:Amplifyyourgenewithprimersdesignedusingthisspreadsheet
2:CuttheplasmidwitheitherBseRIorBsgI.*
3:Cutyourgenewiththeenzymeyouaddedusingthespreadsheet(anyofAcuIBpmIBpuEIBseRIBsgIEciI).
4:ClonethegeneintotheplasmidusingDNAligase.
UsingthismethodwithanN-terminaltagplasmidwillresultinthetagcodingsequenceimmediatelyfollowedbyyourgenesATGstartcodonatthejoin.Thisresultsinaseamlessfusionofthetwosequenceswithnoextrabasesbeingadded.UsingthismethodonC-terminaltagplasmidswillconvertyourgenesstopcodonintoaTAC(TyrY)codonfollowedbytheplasmidtagcodingsequence.Thisresultsinnoextrabasesbetweenyourgeneandthetag.Seethediagrambelowformoreinformation.
*PleasenotethatinsectexpressionplasmidscannotbecutwithBsgIonlyBseRIbecauseofunavoidableconflictingsitesinthebackbone.AlsoYeastplasmidscanonlybecutwithBsgInotBseRIbecauseofconflictingsitesinthebackbone.
Usingthistechniquewillcreateagenefragmentthatcanbeligatedintoanyorour>1500peptideandreportertagplasmids.Ifyouuseoneoftheothertechniquesbelow(GibsonInFusionSeamlessorLIC)youwillneednewprimersforeveryvectoryoucloneintobecausethearmsofhomologywillchangeaccordingtothetagplasmidyouarecloninginto.
Ifyoufindthatyourgenesequencehassitesinitthatmakeusingthiscloningstrategydifficultyoucanstilluseoneofthealternativemethodsbelow(e.g.standardcloningorGibsoncloning).
OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneinourSnapFusiontechnique.
StandardEnzymes:Ifyouarenotconcernedaboutleavingafewextrabasesbetweenthetagcodingsequenceandyourgeneyoucancloneyourgeneintothevectorusingstandardcloningrestrictionenzymes.Thisstrategywillrequireyoutochoosewhichenzymesyouwanttousetocloneyourgene.
OpenthePrimerDesignToolwhichprovidesprimerswithdifferentenzymechoicespositioningyourgeneasclosetothetagaspossibleineachcase.Pleasenotethatstandardenzymeswillalwaysleaveadditionalnucleotidesbetweenyourgeneandthetagbutusingthespreadsheetwillensurethetagandgeneareinframe.
Gibsoncloning/InfusionHD/GeneArtSeamless/LigaseIndependentCloning(LIC)Methods:ThesecloningtechniquesusereagentssoldbyothercompaniesandallowyoutofusesequencestogetherusingenzymesthatchewbacktheDNAtoleaveoverlappingends/overhangs.ThesubsequentmethodofjoiningtheDNAdependsonthekitused.Touseoneofthesetechniquesyoucaneitherdesignyourownprimersoryoucanusethespreadsheetbelowtohelpwiththedesign.
OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneusingGibsonassemblyInfusionHDGeneArtSeamlesscloningorLigaseIndependentCloning(LIC)techniques.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
