Oxford Genetics/pSF-TEF1-NH2-6His-STag-TEV (OG1692) N-terminal 6 His and S-Tag dual tag yeast plasmid/OG1692/1 Ea

PlasmidInfo:

PlasmidPurpose:

Thisplasmidisdesignedtoexpresstaggedproteinsinyeastcells(Saccharomycescerevisiae).TheplasmidcontainsanauxotrophicUracilselectionexpressioncassette(URA3)thatallowsforthepositiveselectionofyeastthataredeficientintheURA3gene(YEL021W).ThisistypicallyachievedbygrowingtheyeastinminimalmediathatisreconstitutedwiththeessentialaminoacidsandnucleotidesbutexcludingUracil.

Thisplasmidalsoencodesaproteasecleavagesitethatisdesignedtobepositionedbetweenyourgeneofinterestandthetagtoallowtheremovalofthetagfollowingproteinpurificationorisolation.ThisplasmidcontainsaTEVcleavagetag.Theproteinsequenceofthecleavagetagis:ENLYFQG.CleavageoccursbetweentheGluandGlyresidues.TEVisoftenreportedtohavebetterspecificityforitsrecognitionsitecomparedtoEKTThrombinorFaxtorXa.

Formoreinformationonwhichcleavagetagtouseseeourcleavagetagguide.

ThisplasmidcontainstheYeastElongationFactorAlphapromoter(TEF1).Thisisthestrongestoftheyeastpromotersthatwesell.Itisaconstitutivepromoterandrequiresnoinduction.IfyouareinterestedinweakerpromoterslevelsthanwealsostockplasmidsthatcontainthefollowingpromotersinorderofdecreasingstrengthTPI(strong)ADHI(medium)STE5(weak).WealsostockGalactoseinducIBLepromoterplasmidsifinducibleexpressionisrequired.Pleasecontactusforfurtherinformation.

Thisplasmidcontainsann-terminalSTagepitopetagthatcanbefusedtoageneofinteresttoallowproteindetectionand/orpurification.Thesequenceofthetagis:KETAAAKFERQHMDS

Formoreinformationonthemethodsthatcanbeusedtopurifyproteinspleaseseeourproteintagguide.

ThisplasmidalsocontainsasecondaryHexa-Histidine(6His)proteintag.Thesequenceofthistagis:HHHHHH

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