Sce48ri.com

www.
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com·info@neb.
comTECHNICALSUPPORTAPPENDIX10379RestrictionMapsPvuI555FspI702BsaI855SwaI1096PsiI1194PciI2310BstZ17I2480BstEII3916ApaI-PspOMI3891KasI-NarI-SfoI3459AfeI3194MluI4098EcoNI4562PmeI5606XbaI5683Acc65I-KpnI5782BaeI5782HindIII6224BmtI-NheI6589MscI6750StuI7150PmlI7175SacII7185BlpI7430SpeI7372AvaI-BsoBI1213DraIII1319BssHII3687BstBI7122MfeI6646BglII6255PstI7355SbfI7354EcoRI7348BamHI7342SalI7336EcoRV7330NotI7322NcoI7316NdeI7309Eco53KI-SacI7304BspQI-SapI7301BspEI7508ApRM13ori+-oriroplacIMCSStuI7150PmlI7175SacII7185SceinteinCBDpTYB21pTYB21isanE.
coliplasmidcloningvectordesignedforrecombinantproteinexpressionandpuricationusingtheIMPACTKit(NEB#E6901)(1,2).
ItcontainsthepMB1originofreplicationfrompBR322andismaintainedatasimilarcopynumbertopBR322;inaddition,pTYB21alsocontainsanM13originofreplication.
Themultiplecloningsite(MCS)ispositionedtoallowtranslationalfusionoftheSceVMAinteintagtotheN-terminusoftheclonedtargetprotein(2).
Thechitinbindingdomain(CBD)fromB.
circulans,facilitatespuricationoftheintein-targetproteinprecursor.
TranscriptionofthegenefusioniscontrolledbytheinducibleT7promoter,requiringE.
colistrainscontainingintegratedcopiesoftheT7RNApolymerasegene[e.
g.
,C2566,C2833orBL21(DE3)]forexpression.
BasalexpressionfromtheT7promoterisminimizedbythebindingoftheLacrepressor,encodedbythelacIgene,tothelacoperatorimmediatelydownstreamoftheT7promoter(3).
TranslationofthefusionReferences(1)Chongetal.
(1996)J.
Biol.
Chem.
,271,22159–22168(2)Chongetal.
(1998)NAR,26,5109–5115.
(3)Dubendorff,J.
W.
andStudier,F.
W.
(1991)J.
Mol.
Biol.
,219,45–59.
Sequenceleavailableatwww.
neb.
com.
Seepage205fororderinginformation.
FeatureCoordinatesSourcebla(ApR)140-1000Tn3M13origin1042-1555M13origin1666-2254pMB1rop2814-2623pMB1lacI4453-3371E.
coliT7promoter5637-5654T7expressionORF5725-7368–MCS7301-7361–SceVMAintein5770-7299S.
cerevisiaeCBD6595-6747B.
circulansori=originofreplicationAp=ampicillinutilizesthetranslationinitiationsignal(ShineDalgarnosequence)fromthestronglyexpressedT7gene10protein(φ10).
pTYB21containsaSapIsitewhichallowsforcloningofatargetgenewithoutanyextraaminoacids.
pTYB22isidenticaltopTYB21exceptfortheMCSregions(seebelow).
pTYB22containsanNdeIsiteoverlappingtheinitiatingmethioninecodonoftheinteinfusiongene.
pTYB21differsfrompTYB11inthatitcontainsauniversalMCSthatiscompatiblewithallNEBexpressionsystems.
Enzymeswithuniquerestrictionsitesareshowninboldtype.
LocationofsitesofallNEBrestrictionenzymescanbefoundontheNEBwebsite(chooseTechnicalReference>DNASequencesandMaps).
Restrictionsitecoordinatesrefertothepositionofthe5-mostbaseonthetopstrandineachrecognitionsequence.
Openreadingframe(ORF)coordinatesareintheform"translationalstart–translationalstop";numbersrefertopositionsonthetop(clockwise)strand,regardlessofthedirectionoftranscriptionandincludethestartandstopcodons.
ComponentgenesorregionsoffusionORFsareindentedbelowtheORFitself.
pMB1originofreplicationcoordinatesincludetheregionfromthe-35promotersequenceoftheRNAIItranscripttotheRNA/DNAswitchpoint.
FortheM13origin,thearrowshowsthedirectionofsynthesisofthe(+)strand,whichgetspackagedintophageparticles.
bla(ApR)genecoordinatesincludethesignalsequence.
Therearenorestrictionsitesforthefollowingenzymes:AarI(x),AatII,AII,AgeI,AscI,AsiSI,AvrII,BbvCI,BmgBI,BseRI,BsiWI,BsmI,BspDI,Bsu36I,ClaI,CspCI,FseI,FspAI(x),I-CeuI,I-SceI,NruI,NsiI,PI-PspI,PI-SceI,PacI,PaeR7I,PpuMI,PspXI,RsrII,SanDI(x),SexAI,SI,SgrAI,SmaI,SnaBI,SrfI(x),TliI,TspMI,XhoI,XmaI,ZraI(x)=enzymenotavailablefromNEBpTYB217,514bpSceVMAIntein.
.
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SDHQFLLGSQ.
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TAATACGACTCACTATAGGGGAATTGTG.
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GAAGACGATTATTATGGGATTACTTTATCTGATGATTCTGATCATCAGTTTTTGCTTGGATCTCAG5650722O724O726O7280T7UniversalPrimerSceVMAInteinSacISbfISapINdeINcoINotISalIBamHIEcoRIPstIGTTGTTGTACAGAACGGAAGAGCTCATATGTCCATGGGCGGCCGCGATATCGTCGACGGATCCGAATTCCCTGCAGGTAATTAAATAAC.
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VVVQNGRAHMSMGGRDIVDGSEFPAGN*SbfIBsmINdeINcoINotISalIBamHIEcoRIPstIGTTGTTGTACAGAATGCTGGTCATATGTCCATGGGCGGCCGCGATATCGTCGACGGATCCGAATTCCCTGCAGGTAATTAAATAAC.
.
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VVVQNAGHMSMGGRDIVDGSEFPAGN*pTYB21MCSpTYB22MCSSceVMAIntein