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Oxford Genetics/pSF-CMV-Puro-NH2-MBP-6His-EKT (OG1173) N-terminal MBP and 6 His dual tag mammalian plasmid/OG1173/1 Ea188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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Oxford Genetics/pSF-CMV-Puro-NH2-MBP-6His-EKT (OG1173) N-terminal MBP and 6 His dual tag mammalian plasmid/OG1173/1 Ea

PlasmidInfo:

PlasmidInformation

ProductName:pSF-CMV-Puro-NH2-MBP-6His-EKT

ProductCode:OG1173

Size(bp):7286bp

BacterialAntibioticSelection:KanR

OriginandCompatibility:pUChighcopyderivedfrompBR322

BacterialCopyNumber:500-700percell

Promoter:Cytomegalovirus(CMV)immediateearlypromoter/HumanUbiquitinPromoter

PlasmidPurpose:

Thisplasmidisdesignedtoexpresstaggedproteinsinmammaliancellseitherbytransienttransfectionorbycreatingstablecelllines.ItcontainsapuromycinresistanceexpressioncassetteusingthehumanUbiquitinpromotertodriveexpressionandallowfortheselectionofcellscontainingtheplasmid.

AbouttheCleavageTag:

Thisplasmidalsoencodesaproteasecleavagesitethatisdesignedtobepositionedbetweenyourgeneofinterestandthetagtoallowtheremovalofthetagfollowingproteinpurificationorisolation.ThisplasmidcontainsaEKTcleavagetag.Theproteinsequenceofthecleavagetagis:DDDDK.Enterokinase(EKT)proteasecleavesaftertheLysineresidue.Itcancleaveatotherbasicresiduesbutthisisdependentonproteinconfirmation.Ifaprolinefollowsthesiteitwillnotcut.Noneofourproductscontainaprolineafterthesite.

Formoreinformationonwhichcleavagetagtouseseeourcleavagetagguide.

PromoterExpressionLevel:

ThisplasmidcontainsthemammalianCMVpromotertodrivegeneexpression.WehavetestedallofourmammalianpromotersinarangeofcelltypesandCMVisconsistentlythestrongestinthosewehavestudied.HowevertherearemanyreportsoftheCMVpromoterdemonstratingsilencingbymethylationinlong-termculture.ForthisreasonwestockarangeofotherpromotersthatarecompatIBLewiththisplasmidandareavailableonrequest.

AboutthePeptideTag:

Thisplasmidcontainsann-terminalHexa-Histidine(6His)affinitytagthatcanbefusedtoageneofinteresttoallowproteindetectionand/orpurification.Thesequenceofthetagis:HHHHHH

Formoreinformationonthemethodsthatcanbeusedtopurifyproteinspleaseseeourproteintagguide.

ThisplasmidalsocontainsasecondaryMaltoseBindingProtein(MBP)proteintag.Thesequenceofthistagis:

EEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSS

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:

IntellectualPropertyStatus

ThisproductispartofourSnapFastplasmidrange,formoreinformationontheIntellectualpropertystatusofthisplasmidpleaseclickhere

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