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牛津遗传学/pSF-OXB20-NH2-Strep-6His-EKT（OG2818）N端Strep和6-His双标签细菌质粒/OG2818/1 Ea
PlasmidInfo:
PlasmidInformation
ProductName:pSF-OXB20-NH2-Strep-6His-EKT
ProductCode:OG2818
Size(bp):3911bp
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.ThisplasmidcontainsaEKTcleavagetag.Theproteinsequenceofthecleavagetagis:DDDDK.Enterokinase(EKT)proteasecleavesaftertheLysineresidue.Itcancleaveatotherbasicresiduesbutthisisdependentonproteinconfirmation.Ifaprolinefollowsthesiteitwillnotcut.Noneofourproductscontainaprolineafterthesite.
Formoreinformationonwhichcleavagetagtouseseeourcleavagetagguide.
PromoterExpressionLevel:Thisplasmidcontainsaconstitutivebacterialpromoterthatdoesnotrequireinduction.Itisthestrongestbacterialpromoterwesellandthiscancausesolubilityandexpressionproblemswithsomeproteins.WealsoofferarangeofotherbacterialpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.
Thisplasmidcontainsann-terminalHexa-Histidine(6His)affinitytagthatcanbefusedtoageneofinteresttoallowproteindetectionand/orpurification.Thesequenceofthetagis:HHHHHH
Formoreinformationonthemethodsthatcanbeusedtopurifyproteinspleaseseeourproteintagguide.
ThisplasmidalsocontainsasecondaryStrepproteintag.Thesequenceofthistagis:WSHPQFEK
Weprovidearangeofdualpeptidetagplasmids.ThisisbecausesomepeptidetagsprovidespecificBIOLOGicalproperties(e.gsmallmoleculeaffinitynewepitopessolubilityorproteinsecretion)thatarenotprovidedbyothers.
SequenceandMap:
OtherInfo:
TranscriptionTermination:Thisplasmidcontainsthreealternativetranscriptionterminatorsformammalianbacterialandbacteriophage(T7)expression.Thismeansthatonlythepromoterneedstobechangedtoaltertheexpressionsystemyouareusing.Wesellmultiplepromotersthatcanbeusedineachofthesesystems.Thepresenceofeachterminatordoesnotreduceexpressioninthealternativesystems.
Cloning:
MakingProteinFusions:ThisplasmidhasbeendesignedtoallowthreetypesofcloningintothemainMCStojoinacodingsequencewiththetag.
1:SnapFusionCloning:IfyouwouldliketofuseyourcodingsequencetothetagwithminimaladditionalbasesyoucanuseourSnapFusiontechnology.ThisprocessinvolvesamplifyingyourgenebyPCRtoaddspecificrestrictionsitesontotheends.WhenthesesitesarecuttheyproduceanoverhangthatiscompatiblewiththisplasmidcutwithBseRIorBsgI.
Toinsertyourgene:
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.
2:StandardEnzymes:Ifyouarenotconcernedaboutleavingafewextrabasesbetweenthetagcodingsequenceandyourgeneyoucancloneyourgeneintothevectorusingstandardcloningrestrictionenzymes.Thisstrategywillrequireyoutochoosewhichenzymesyouwanttousetocloneyourgene.
OpenthePrimerDesignToolwhichprovidesprimerswithdifferentenzymechoicespositioningyourgeneasclosetothetagaspossibleineachcase.Pleasenotethatstandardenzymeswillalwaysleaveadditionalnucleotidesbetweenyourgeneandthetagbutusingthespreadsheetwillensurethetagandgeneareinframe.
3:Gibsoncloning/InfusionHD/GeneArtSeamless/LigaseIndependentCloning(LIC)Methods:ThesecloningtechniquesusereagentssoldbyothercompaniesandallowyoutofusesequencestogetherusingenzymesthatchewbacktheDNAtoleaveoverlappingends/overhangs.ThesubsequentmethodofjoiningtheDNAdependsonthekitused.Touseoneofthesetechniquesyoucaneitherdesignyourownprimersoryoucanusethespreadsheetbelowtohelpwiththedesign.
OpenthePrimerDesignTooltohelpyoudesignprimersforcloningyourgeneusingGibsonassemblyInfusionHDGeneArtSeamlesscloningorLigaseIndependentCloning(LIC)techniques.
IPStatus:
IntellectualPropertyStatusThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere
