pET-32a-c(+) Vectors pET-32a DNA pET-32b DNA pET-32c DNA Cat. No. 69015- 3 69016- 3 69017- 3 TB122 12/98 The pET-32 series is designed for cloning and high-level expression of peptide sequences fused with the 109aa Trx'Tag™ thioredoxin protein (1). Cloning sites are available for producing fusion proteins also containing cleavable His'Tag® and S»Tag™ sequences for detection and purification. 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 circle map. The cloning/expression region of the coding strand transcribed by T7 RNA polymerase is shown below. The fl 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). 1. LaVallie, E.R, DiBlasio, E.A, Kovacic, S, Grant, KL, Schendel, P.F. and McCoy, J.M. (1993) Bio [Technology 11,187-193. pET-32a(+) sequence landmarks T7 promoter 764-780 T7 transc riptio n start 763 Trx'Tag coding sequence 366-692 His'Tag coding sequence 327-344 S»Tag coding sequence 249-293 Multiple cloning sites (Ncol-Xhol) 158-217 His'Tag coding sequence 140-157 T7 terminator 26-72 lad coding sequence 1171-2250 pBR322 origin 3684 bla coding sequence 4445-5302 fl origin 5434-5889 The maps for pET-32b(+) and pET-32c(+) are the same as pET-32a(+) (shown) with the following exceptions: pET-32b(+) is a 5899bp plasmid; subtract lbp from each site beyond BamH I at 198. pET-32c(+) is a 5901bp plasmid; add lbp to each site beyond .BamH I at 198 except for .EcoR V, which cuts at 209. Ava 1(158) Xho 1(158) Eag 1(166) Not 1(166) Hind 111(173) Sal 1(179) Sac 1(190) ECOR 1(192) BamH 1(198) ECOR V(206) NCO 1(212) Dra lll(5658) Sea 1(4995) Pvu 1(4885) Pst 1(4760) Bsa 1(4576) ■ Eam1105 1(4357) Xba 1(729) SgrA 1(840) AlwN 1(4038) Sph 1(996) EcoN 1(1056) ApaB 1(1205) U 1(1521) ^Bcl 1(1535) BstE 11(1702) Bmg 1(1730) Apa 1(1732) BssH 11(1932) Hpa 1(2027) BspLU1 1 1(3622) Sap 1(3506) Bst1107 1(3393) Tth1 1 1 1(3367) BspG 1(31 PshA 1(2366) Psp5 11(2628) T7 promoter lac operator Xdal TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA _Trx-Tag_ Afecl His-Tag_ TATACATATGAGC...315bp...CTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCT MetSer 105aa. . . LeuA I aG I ySerG I ySerG I yH i sMetH i sH i sH i sH i sH i sH i sSerSerG I yLeuVa I ProArgG I ySer ,,—-—-► S-Tag primer #69945-3 -1- _S-Tag WseV_ a5(7/|| Kpn\ thrombin GGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAG GIyMetLysGIuThrAIaAIaAIaLysPheGIuArgGInH i sMetAspSerProAspLeuGIyThrAspAspAspAspLys pET-32a(+) Nco I EcoR V BamH I EcoR I Sac I Sal I Hind I Eag I Not I Ava I Xho\ ~l enterokinase His-Tag GCCATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA A I aMetA I aAspI I eG I ySerG I uPheG I uLeuArgArgG I nA I aCysG I yArgThrArgA I aProProProProProLeuArgSerG I yCysEnd GCCATGGCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32b(-i A IaMetAIalIeSerAspProAsnSerSerSerVaIAspLysLeuAIaAIaAIaLeuGIuH i sH i sH i sH i sH i sH i sEnd GCCATGGGATATCTGTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32c(+ A I aMetG I yTyrLeuTrpI I eArgl I eArgA I aProSerThrSerLeuArgProH i sSerSerThrThrThrThrThrThrG lull eArgLeuLeuThr_ BPO1102 I T7 terminator CAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG LysProG I uArgLysLeuSerTrpLeuLeuProProLeuSerAsnAsnEnd T7 terminator primer #69337-3 pET-32a-c(+) cloning/expression region Novagen • ordering 800-526-7319 • technical support 800-207-0144 pET-32a(+) Ffestr iction Sites TB122 12/98 Enzyme # Sites Locations Enzyme # Sites Locations Accl 2 180 3392 BstXI 3 1323 1452 1575 Acelll 8 1288 2016 2347 3131 3272 BstYl 12 3574 4814 5498 Cac8l 39 Acil 85 Cjel 26 Af 1 III 2 1521 3622 CjePI 18 Alul 25 Clal 2 508 798 Alwl 17 CviJI 90 Alw21l 10 159 190 414 1021 1505 CviRI 28 2616 3440 3940 5101 5186 Ddel 11 Alw44l 5 410 1501 3436 3936 5182 Dpnl 32 AlwNI 1 4038 Dral 3 4381 4400 5092 Apal 1 1732 Dralll 1 5658 ApaBI 1 1205 Drdl 3 3315 3730 5613 Apol 5 192 270 1796 5460 5471 Drdll 3 357 1244 5663 Aval 1 158 Dsal 4 212 469 958 2594 Avail 8 589 2073 2449 2537 2628 Eael 7 166 349 365 829 2907 4653 4875 2195 4903 BamHI 1 198 Eagl 1 166 Banl 11 Eam1105l 1 4515 Banll 5 190 905 919 1732 5733 Earl 3 1139 3506 5310 Bbsl 4 1667 2006 2380 2740 Ecil 4 1298 3696 3842 4670 Bbvl 29 EČ047III 3 926 2427 2876 Bed 14 EC057I 2 4170 5182 Bce83l 7 21 2335 2505 3713 4011 EcoNI 1 1056 4252 5120 EcoO109l 3 53 954 2628 Bcefl 5 1040 1381 2008 4124 5684 EcoPJ 1 192 Bcgl 14 EcoPJI 9 367 495 1244 1559 Bell 1 1535 2156 3648 3769 3782 Hal 7 70 730 2636 4117 4370 EcoHV 1 206 4705 5809 Faul 17 Bgll 2 2585 4635 Fokl 13 Bglll 1 241 Fspl 2 2603 4737 Bmgl 1 1730 Gdill 6 166 365 829 961 Bpml 5 1359 1848 2482 3149 4585 4903 BpulOI 1 2728 Hael 7 217 351 1249 2570 Bpu1102l 1 80 3648 4100 Bsal 1 4576 Haell 14 BsaAl 2 3374 5658 Haelll 27 BsaBI 3 794 804 2819 Hgal 13 BsaHl 6 844 865 979 1478 2161 HgiBI 3 419 1119 4208 5052 Hhal 46 BsaJI 9 57 212 469 496 958 Hin4l 4 203 1420 4514 4588 964 2156 2594 3782 Hindi 2 181 2027 BsaWI 7 2 1840 2343 2811 3828 Hindlll 1 173 3975 4806 Hinfl 16 BsaXI 2 2180 5606 Hpal 1 2027 Bsbl 2 3338 5565 Hphl 18 BscGI 14 Kpnl 1 238 Bsgl 3 1372 1572 2782 Maell 15 Bsil 2 3795 5179 Maelll 18 BsiB 6 169 2306 3538 3962 4885 Mboll 15 5034 Mini 1 1521 Bsll 25 M mel 3 3837 4021 5635 BsmAI 7 1218 1623 1749 2136 3263 Mnll 28 4576 5352 Msd 1 351 BsmBI 2 2136 3263 M sel 28 BsmFI 4 982 2523 2893 5873 Msll 9 1573 1861 1891 2609 BsoFI 50 3195 4767 4926 5285 Bsp24l 10 811 843 1362 1394 1664 Mspl 32 1696 4115 4147 4293 4325 MspAII 10 84 283 1551 2121 Bsp1286l 14 3213 3332 3964 4209 BspB 2 2 2811 Mwol 41 BspGI 1 3148 Narl 4 844 865 979 2161 BspLUHI 1 3622 Ndl 12 Bsrl 25 Ncol 1 212 BsrBI 4 750 3555 5356 5802 Ndel 2 346 691 BsrDI 4 1568 1934 4576 4750 NgoAIV 4 831 2419 2579 5759 BsrFI 8 363 831 840 1207 2419 Nlalll 26 2579 4595 5759 NlalV 27 BssHII 1 1932 Natl 1 166 Bst1107l 1 3393 Nspl 4 996 2967 3259 3626 BstBI 1 1702 NspV 1 268 Enzyme # Sites Locations Pfl 11081 PflMI Plel PshAI Psp5ll Psp1406l Pstl Pvul Pvull Peal Fteal Fterll Sad Sail Sapl Sau96l Sau3AI Seal ScrFI SfaNI Sfcl SgrAI Sphl Sspl Styl Taql Taqll Tfil Thai Tsel Tsp45l Tsp509l Tth111l Tth111ll 2 2408 4533 2 260 1103 10 466 778 1070 3516 4001 4504 1 2366 1 2628 6 1183 2551 2947 5443 1 4760 1 4885 3 2121 2214 3213 3 919 4342 5350 5 236 642 1668 1 589 1 190 1 179 1 3506 1953 5601 5114 3428 4995 19 32 1 21 21 5 1 1 2 2 18 10 35 29 18 1 4995 763 3887 4078 4756 5877 840 996 5319 5450 57 212 429 1429 1647 2320 3524 4863 5048 5201 5218 5562 566 2200 2502 2672 3176 3597 1702 2530 4771 4982 3367 1360 2053 3061 3274 5831 3369 3083 4212 4219 4251 UbaJI 21 Vspl 4 778 2206 2265 Xbal 1 729 Xcml 3 1377 1893 1911 Xhol 1 158 Xmnl 3 388 3180 5114 Enzymes that do not cut pET-32a(+): Aatll Aflll Agel Asd Bael BseRI Bsml BspMI Bsu36l Fsel Muni Nhel Nsil Pad Pmel Pmll Sadl SexAl Sfil Sgfl Avrll BsrGI Nrul PJeAl Smal SnaBI Sunl Spel Swal Srfl Sse8387l Stul Novagen • fax 608-238-1388 • e-mail novatech@novagen.com