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Oxford Genetics/pSF-TEF1-COOH-TEV-GST (OG1918) C-terminal GST tag yeast plasmid/OG1918/1 Ea
PlasmidInfo:
PlasmidInformation
ProductName:pSF-TEF1-COOH-TEV-GST
ProductCode:OG1918
Size(bp):7392bp
BacterialAntibioticSelection:KanR
OriginandCompatibility:pUChighcopyderivedfrompBR322
BacterialCopyNumber:500-700percell
Promoter:YeastElongationFactorAlpha-1(TEF-1)promoter
PlasmidPurpose:
Thisplasmidisdesignedtoexpresstaggedproteinsinyeastcells(Saccharomycescerevisiae).TheplasmidcontainsanauxotrophicUracilselectionexpressioncassette(URA3)thatallowsforthepositiveselectionofyeastthataredeficientintheURA3gene(YEL021W).ThisistypicallyachievedbygrowingtheyeastinminimalmediathatisreconstitutedwiththeessentialaminoacidsandnucleotidesbutexcludingUracil.
AbouttheCleavageTag:Thisplasmidalsoencodesaproteasecleavagesitethatisdesignedtobepositionedbetweenyourgeneofinterestandthetagtoallowtheremovalofthetagfollowingproteinpurificationorisolation.ThisplasmidcontainsaTEVcleavagetag.Theproteinsequenceofthecleavagetagis:ENLYFQG.CleavageoccursbetweentheGluandGlyresidues.TEVisoftenreportedtohavebetterspecificityforitsrecognitionsitecomparedtoEKTThrombinorFaxtorXa.
Formoreinformationonwhichcleavagetagtouseseeourcleavagetagguide.
PromoterExpressionLevel:ThisplasmidcontainstheYeastElongationFactorAlphapromoter(TEF1).Thisisthestrongestoftheyeastpromotersthatwesell.Itisaconstitutivepromoterandrequiresnoinduction.IfyouareinterestedinweakerpromoterslevelsthanwealsostockplasmidsthatcontainthefollowingpromotersinorderofdecreasingstrengthTPI(strong)ADHI(medium)STE5(weak).WealsostockGalactoseinducIBLepromoterplasmidsifinducibleexpressionisrequired.Pleasecontactusforfurtherinformation.
AboutthePeptideTag:Thisplasmidcontainsac-terminalGlutathione-S-Transferase(GST)affinitytagthatcanbefusedtoageneofinteresttoallowproteindetectionand/orpurification.Thesequenceofthetagis:
SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKS
Formoreinformationonthemethodsthatcanbeusedtopurifyproteinspleaseseeourproteintagguide.
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
