pET-26b(+) TB071 10/98 - synthesisgene.com

1 Novagen ORDERING800-526-7319 TECHNICAL SUPPORT800-207-0144pET-26b(+) cloning/expression regionpET-26b(+) VectorTB071 12/98 The pET-26b(+) vector (Cat. No. 69862-3) carries an N-terminal pelBsignal sequence for potential periplasmic localization, plus optional C-terminal His Tag sequence. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression region is reversed on the circular map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore, single-stranded sequencing should be performedusing the T7 terminator primer (Cat.)

2 No. 69337-3).pET-26b(+) sequence landmarksT7 promoter361-377T7 transcription start360pelB coding sequence224-289 Multiple cloning sites(NcoI - XhoI)158-225 His Tag coding sequence140-157T7 terminator26-72lacI coding sequence764-1843pBR322 origin3277 Kan coding sequence3986-4798f1 origin4894-5349lac operatorMsc IAva I*AGATCTCGATCCCGCGAAATTAATACGACTCACTATAG GGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAAT TTTGTTTAACTTTAAGAAGGAGATATACATATGAAATACC TGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGC CCAGCCGGCGATGGCCATGGATATCGGAATTAATTCGGAT CCGAATTCGAGCTCC MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuL euLeuAlaAlaGlnProAlaMetAlaMetAspIleGlyIl eAsnSerAspProAsnSerSerSerGTCGACAAGCTTGCG GCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGG CTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGC CACCGCTGAGCAATAACValAspLysLeuAlaAlaAlaLe uGluHisHisHisHisHisHisEndTAGCATAACCCCTTG GGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGNde IEcoR IXho IpelB

3 LeaderT7 terminatorrbsNco IBamH ISal IHind IIINot IBgl IIXba ISac IEag IBpu1102 IBspM Isignal peptidaseT7 promoterT7 promoter primer #69348-3T7 terminator primer #69337-3 His TagpET-26b(+)(5360bp)lacI (764-1843)ori (3277)kan (3986-4798)f1 origin (4894-5349)Bpu1102 I(80)Xho I(158)Eag I(166)Not I(166)Hind III(173)Sal I(179)Sac I(190)EcoR I(192)BamH I(198)Nco I(220)Msc I(225)BseR I(260)BspM I(268)Nde I(288)Xba I(326)Bgl II(392)SgrA I(433)Sph I(589)ApaB I(798)Mlu I(1114)Bcl I(1128)BstE II(1295)Bmg I(1323)Apa I(1325)BssH II(1525)Hpa I(1620)PshA I(1959)Pfl1108 I(2001)Fsp I(2196)Psp5 II(2221)BspG I(2741)Tth111 I(2960)Bst1107 I(2986)Sap I(3099)BspLU11 I(3215)Bsi I(3388)AlwN I(3631)Eco57 I(3763)Nru I(4074)Cla I(4108)Sma I(4291)Pvu I(4417)Sgf I(4417)Dra III(5118)

4 Enzyme# Sites LocationsAccI21802985 AceIII7881160919402724286531674958 AciI74 AflIII211143215 AluI22 AlwI13 Alw21I71591906141098220930333533 Alw44I3109430293529 AlwNI13631 ApaI11325 ApaBI1798 ApoI619213894030421449204931 AvaI21584289 AvaII516662042213022212500 BamHI1198 BanI843645757110341753188320095155 BanII6190498512132540725193 BbsI41260159919732333 BbvI28 BccI14 Bce83I62119282098330636043845 BcefI66339741601371747365144 BcgI8160194140614401940197427922826 BclI11128 BfaI6703272229371040175269 BglI22372178 BglII1392 BmgI11323 BpmI4952144120752742 Bpu10I223214434 Bpu1102I180 BsaAI229675118 BsaBI33913972412 BsaHI543745857210711754 BsaJI10572205515571749218733754288428946 90 BsaWI72143319362404342135684552 BsaXI217735066 BsbI229315025 BscGI11 BseRI1260 BsgI396511652375 BsiI13388 BsiEI61692711899313135554417 BslI23 BsmI243014378 BsmAI681112161342172928564433 BsmBI3172928564433 BsmFI4575211624865333 BsoFI46 Bsp24I1040443695598712571289370837403886 3918 Bsp1286I12 BspEI222404 BspGI12741 BspLU11I 13215 BspMI1268 BsrI21 BsrBI4347314848165262 BsrDI211611527 Enzyme# Sites LocationsBsrFI82314244338002012217243715 219 BssHII11525 Bst1107I12986 BstEII11295 BstXI391610451168 BstYI913219839267818902407385638674666 Cac8I40 CjeI24 CjePI18 ClaI14108 CviJI84 CviRI22 DdeI11 DpnI21 DraIII15118 DrdI3290833235073 DrdII28375123 DsaI32205512187 EaeI51662234225541788 EagI1166 EarI373230994230 EciI389132893435 Eco47 III351920202469 Eco57I13763 EcoNI26494329

5 EcoO109I3535472221 EcoRI1192 EcoRII9837115216921749324133623375430546 62 EcoRV22171564 FauI17 FokI911601169243424962574276029014055466 1 FspI12196 GdiII41664225541788 HaeI722584221633230324136934504 HaeII14 HaeIII24 HgaI11 HgiEII27123801 HhaI46 Hin4I3101341034645 HincII21811620 HindIII1173 HinfI18 HpaI11620 HphI16 MaeII14 MaeIII16 MboII12 MluI11114 MmeI73430361440594253461546245095 MnlI26 MscI1225 MseI26 MslI6116614541484220223972788 MspI30 MspA1I9842671144171418072806292535573802 MwoI40 NarI44374585721754 NciI12 NcoI1220 NdeI1288 NgoAIV5231424201221725219 NlaIII25 NlaIV21 Enzyme# Sites LocationsNotI1166 NruI14074 NsiI242674533 NspI4589256028523219 Pfl1108I12001 PflMI26964680 PleI9375663750154631093594464950535061 PshAI11959 Psp5II12221 Psp1406I4776214425404903 PvuI14417 PvuII3171418072806 RcaI351239354810 RsaI3126130214252 SacI1190 SalI1179 SapI13099 Sau96I14 Sau3AI21 ScrFI21 SfaNI23 SfcI4360348036715337 SgfI14417 SgrAI1433 SmaI14291 SphI1589 SspI243424910 StyI257220 TaqI15 TaqII6102212401913311746715022 TfiI917932095226527693190432843844556464 7 ThaI35 TseI28 Tsp45I71295212326542867296245645291 Tsp509I22 Tth111I12960 Tth111II89531646267638053812384442534380 UbaJI18 VspI62073751799185846164805 XbaI1326 XcmI397014861504 XhoI1158 XmnI320827734806 Enzymes that do not cut pET-26b(+).

6 AatIIAflIIAgeIAscIAvrIIBaeIBsaIBsrGIBsu3 6 IDraIEam1105I FseIKpnIMunINheINspVPacIPmeIPmlIPstIRleA IRsrIISacIIScaISexAISfiISnaBISpeISrfISse 8387 IStuISunISwaIpET-26b(+) Restriction MapTB071 12/98 Novagen FAX608-238-1388