pET-20b(+) Vector TB037 12/98 The pET-20b(+) vector (Cat. No. 69739-3) carries an N-terminal pelB signal 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 performed using the T7 terminator primer (Cat. No. 69337-3). Xho I(158) Not I(166) Eag I(166) Hind III(173) Sal I(179) Hinc II(181) Sac I(190) EcoR I(192) BamH I(198) EcoR V(217) Nco I(220) BseR I(260) BspM I(268) Nde I(288) Xba I(326) Bpu1102 I(80) pET-20b(+) sequence landmarks T7 promoter T7 transcription start pelB coding sequence Multiple cloning sites (Nco I - Xho I) His•Tag coding sequence T7 terminator pBR322 origin bla coding sequence f1 origin 353-369 352 224-289 Drd II(3479) Dra III(3474) BsaX I(3422) 158-225 140-157 26-72 1500 2261-3118 3250-3705 f1 5) 0-370 325 ( n igi or Bgl II(384) Bpu10 I(544) Bbs I(556) Bsg I(598) Eco47 III(692) Sca I(2811) Pvu I(2701) Pst I(2576) -3118) (2261 Ap BsaH I(2868) pET-20b(+) BspG I(964) (3716bp) Pvu II(1029) BsmB I(1079) Tth111 I(1183) Bst1107 I(1209) Pfl1108 I(2349) Eam1105 I(2331) o HgiE II(2024) 15 ri ( 00 ) Sap I(1322) BspLU11 I(1438) Afl III(1438) AlwN I(1854) T7 promoter primer #69348-3 T7 promoter Bgl II rbs Xba I AGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA Nde I BspM I pelB leader Nco I EcoR V BamH I EcoR I Sac I TATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGATATCGGAATTAATTCGGATCCGAATTCGAGCTCC MetLysTyrLeuLeuProThrAlaAlaAlaGlyLeuLeuLeuLeuAlaAlaGlnProAlaMetAlaMetAspIleGlyIleAsnSerAspProAsnSerSerSer Hinc II Sal I Hind III Eag I Not I Xho I His•Tag signal peptidase Bpu1102 I GTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAAC ValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd T7 terminator primer #69337-3 T7 terminator TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG pET-20b(+) cloning/expression region Novagen • ORDERING 800-526-7319 • TECHNICAL SUPPORT 800-207-0144 pET-20b(+) Restriction Sites Enzyme AccI AceIII AciI AflIII AluI AlwI Alw21I # Sites 2 5 49 1 18 13 7 Alw44I AlwNI ApoI AvaI AvaII BamHI BanI BanII BbsI BbvI BccI 3 1 3 2 5 1 2 2 1 26 6 Bce83I BcefI BcgI 5 2 6 BfaI 7 BglI BglII BpmI Bpu10I Bpu1102I BsaI BsaAI BsaBI BsaHI BsaJI BsaWI BsaXI BsbI BscGI 2 1 2 1 1 2 2 2 1 4 5 1 2 10 BseRI BsgI BsiI BsiEI 1 1 2 6 BslI BsmAI BsmBI BsmFI BsoFI Bsp24I Bsp1286I 12 4 1 2 38 4 8 BspEI BspGI BspLU11I BspMI BsrI BsrBI BsrDI BsrFI Bst1107I BstYI 2 1 1 1 14 3 2 3 1 10 Cac8I CjeI 16 10 CjePI 10 CviJI CviRI 57 13 Locations 180 1208 947 1088 Enzyme DdeI 1390 2630 3314 1438 159 2917 1252 1854 192 158 402 198 2279 190 556 190 3002 1752 432 3276 388 444 3287 1256 1756 2998 723 2469 2691 3511 3549 227 3481 21 1940 160 2870 70 2521 237 384 965 544 80 351 1190 383 2868 57 2 3422 1154 44 2111 260 598 1611 169 2850 2368 2492 2779 3464 1529 3500 194 1827 2068 2936 1015 1049 2836 452 1933 2186 2995 271 1354 1778 351 1079 709 1079 2392 3168 1931 159 2917 2 964 1438 268 1963 190 3002 627 2109 432 3549 1371 2392 231 1209 132 2090 3172 2566 2411 3618 3575 198 2176 384 2188 630 2956 2079 2973 1451 2006 1925 2136 1484 2036 1957 2223 1925 2039 1958 2256 1958 2103 1990 2695 2003 2136 2103 2728 327 3625 2451 TB037 12/98 2401 2392 3474 635 220 627 410 1644 1598 1791 2622 3381 414 2332 799 2356 1132 2878 1765 3568 2701 3689 2141 1256 1756 # Sites Locations 10 80 101 1246 1713 DpnI 19 DraI 3 2197 2216 DraIII 1 3474 DrdI 3 1131 1546 DrdII 1 3479 DsaI 2 220 410 EaeI 4 166 223 EagI 1 166 Eam1105I 1 2331 EarI 2 1322 3126 EciI 3 1512 1658 Eco47III 1 692 Eco57I 2 1986 2998 EcoO109I 3 53 402 EcoRI 1 192 EcoRII 4 404 1464 EcoRV 1 217 FauI 8 368 453 1151 3611 FokI 8 657 719 2297 2478 FspI 2 419 2553 GdiII 2 166 2719 HaeI 5 225 409 HaeII 6 611 694 3633 HaeIII 14 HgaI 6 974 1131 3691 HgiEII 1 2024 HhaI 23 Hin4I 2 2330 2404 HincII 1 181 HindIII 1 173 HinfI 9 359 488 1809 2326 HphI 9 483 1058 2817 3023 MaeII 12 MaeIII 15 MboII 8 556 1309 3004 3113 MmeI 3 1653 1837 MnlI 20 MscI 2 225 409 MseI 23 MslI 6 425 620 3101 MspI 18 MspA1I 7 84 267 2025 2966 MwoI 19 NciI 7 448 776 2514 2865 NcoI 1 220 NdeI 1 288 NgoAIV 2 231 3575 NlaIII 15 NlaIV 15 NotI 1 166 NspI 3 783 1075 Pfl1108I 1 2349 PleI 6 367 1332 3417 Psp5II 2 402 444 Psp1406I 4 763 2557 PstI 1 2576 PvuI 1 2701 PvuII 1 1029 RcaI 2 2158 3166 136 2122 544 2288 706 2828 2908 Enzyme RsaI SacI SalI SapI Sau96I # Sites 2 1 1 1 10 Sau3AI ScaI ScrFI SfaNI SfcI SspI StyI 19 1 11 13 5 2 7 TaqII 6 TfiI ThaI TseI Tsp45I 3 16 26 6 Tsp509I Tth111I Tth111II UbaJI VspI XbaI XhoI XmnI 11 1 4 12 3 1 1 3 3429 407 2719 2486 444 1585 1598 734 3680 797 2765 920 1141 983 1124 1453 1316 1464 1686 1916 3625 1549 2127 2857 992 3401 1067 3058 1338 3423 2174 3475 1413 2100 3614 3451 2171 2926 1011 2583 2742 1029 1148 1780 1082 1117 1818 1817 2320 3409 2930 3259 2401 Locations 1244 2811 190 179 1322 53 402 2373 2452 444 2469 723 2691 910 3465 2811 352 3135 159 2982 1340 3378 488 1703 3266 180 3507 2679 1894 2572 3693 190 381 1538 2864 3017 3034 992 1413 877 3647 1090 1185 2587 2798 2028 2035 2067 367 2503 996 2930 1183 899 207 326 158 208 Enzymes that do not cut pET-20b(+): AatII AflII AgeI ApaI AscI AvrII BaeI BclI BsmI BsrGI BssHII BstEII Bsu36I ClaI EcoNI FseI KpnI MluI MunI NarI NruI NsiI NspV PacI PmeI PmlI PshAI RleAI SacII SexAI SfiI SgfI SmaI SnaBI SpeI SphI Sse8387I StuI SunI SwaI 1442 Novagen • FAX 608-238-1388 • E-MAIL [email protected] ApaBI BmgI BstXI HpaI NheI PflMI RsrII SgrAI SrfI XcmI
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