Novagen ? ORDERING 800-526-7319 ? TECHNICAL SUPPORT 800-207-0144 TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA TATACATATGAGC...315bp...CTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCT MetSer 105aa...LeuAlaGlySerGlySerGlyHisMetHisHisHisHisHisHisSerSerGlyLeuValProArgGlySer GGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAG GlyMetLysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerProAspLeuGlyThrAspAspAspAspLys GCCATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA AlaMetAlaAspIleGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEnd GCCATGGCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32b(+) AlaMetAlaIleSerAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd GCCATGGGATATCTGTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32c(+) AlaMetGlyTyrLeuTrpIleArgIleArgAlaProSerThrSerLeuArgProHisSerSerThrThrThrThrThrThrGluIleArgLeuLeuThr CAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG LysProGluArgLysLeuSerTrpLeuLeuProProLeuSerAsnAsnEnd BamH I Hind III Sac I Nco I Not I Sal I Xho I EcoR I EcoR V Ava I Eag I Trx?Tag T7 terminator Bpu1102 I T7 terminator primer #69337-3 T7 promoter rbs Xba I S?Tag primer #69945-3 His?Tag thrombin S?Tag enterokinase Nsp V lac operator His?Tag Msc I Kpn I Bgl II pET-32a(+) 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 puri?cation. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 con- vention, 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 f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corre- sponds 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, K.L., Schendel, P.F. and McCoy, J.M. (1993) Bio/Technology 11, 187–193. trxA (366-692 ) l a c I ( 1 1 7 1 - 2 2 5 0 ) o r i ( 3 684) A p ( 4 4 4 5 - 5 3 0 2 ) f1 origin (5434-5889) Bpu1102 I(80) Ava I(158) Xho I(158) Eag I(166) Not I(166) Hind III(173) Sal I(179) Sac I(190) EcoR I(192) BamH I(198) EcoR V(206) Nco I(212) Kpn I(238) Bgl II(241) Nsp V(268) Msc I(351) Rsr II(589) Xba I(729) SgrA I(840) Sph I(996) EcoN I(1056) ApaB I(1205) Mlu I(1521) Bcl I(1535) BstE II(1702) Bmg I(1730) Apa I(1732) BssH II(1932) Hpa I(2027) PshA I(2366) Psp5 II(2628) BspG I(3148) Tth111 I(3367) Bst1107 I(3393) Sap I(3506) BspLU11 I(3622) AlwN I(4038) Bsa I(4576) Pst I(4760) Pvu I(4885) Sca I(4995) Dra III(5658) pET-32a-c(+) cloning/expression region TB122 12/98 pET-32a(+) sequence landmarks T7 promoter 764-780 T7 transcription start 763 Trx?Tag coding sequence 366-692 His?Tag coding sequence 327-344 S?Tag coding sequence 249-293 Multiple cloning sites (Nco I - Xho I) 158-217 His?Tag coding sequence 140-157 T7 terminator 26-72 lacI coding sequence 1171-2250 pBR322 origin 3684 bla coding sequence 4445-5302 f1 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 1bp from each site beyond BamH I at 198. pET-32c(+) is a 5901bp plasmid; add 1bp to each site beyond BamH I at 198 except for EcoR V, which cuts at 209. pET-32a-c(+) Vectors pET-32a(+) (5900bp) Eam1105 I(4357)– Cat. No. pET-32a DNA 69015-3 pET-32b DNA 69016-3 pET-32c DNA 69017-3 TB122 12/98 pET-32a(+) Restriction Sites Enzyme # Sites Locations AccI 2 180 3392 AceIII 8 1288 2016 2347 3131 3272 3574 4814 5498 AciI 85 A?III 2 1521 3622 AluI 25 AlwI 17 Alw21I 10 159 190 414 1021 1505 2616 3440 3940 5101 5186 Alw44I 5 410 1501 3436 3936 5182 AlwNI 1 4038 ApaI 1 1732 ApaBI 1 1205 ApoI 5 192 270 1796 5460 5471 AvaI 1 158 AvaII 8 589 2073 2449 2537 2628 2907 4653 4875 BamHI 1 198 BanI 11 BanII 5 190 905 919 1732 5733 BbsI 4 1667 2006 2380 2740 BbvI 29 BccI 14 Bce83I 7 21 2335 2505 3713 4011 4252 5120 BcefI 5 1040 1381 2008 4124 5684 BcgI 14 BclI 1 1535 BfaI 7 70 730 2636 4117 4370 4705 5809 BglI 2 2585 4635 BglII 1 241 BmgI 1 1730 BpmI 5 1359 1848 2482 3149 4585 Bpu10I 1 2728 Bpu1102I 1 80 BsaI 1 4576 BsaAI 2 3374 5658 BsaBI 3 794 804 2819 BsaHI 6 844 865 979 1478 2161 5052 BsaJI 9 57 212 469 496 958 964 2156 2594 3782 BsaWI 7 2 1840 2343 2811 3828 3975 4806 BsaXI 2 2180 5606 BsbI 2 3338 5565 BscGI 14 BsgI 3 1372 1572 2782 BsiI 2 3795 5179 BsiEI 6 169 2306 3538 3962 4885 5034 BslI 25 BsmAI 7 1218 1623 1749 2136 3263 4576 5352 BsmBI 2 2136 3263 BsmFI 4 982 2523 2893 5873 BsoFI 50 Bsp24I 10 811 843 1362 1394 1664 1696 4115 4147 4293 4325 Bsp1286I 14 BspEI 2 2 2811 BspGI 1 3148 BspLU11I 1 3622 BsrI 25 BsrBI 4 750 3555 5356 5802 BsrDI 4 1568 1934 4576 4750 BsrFI 8 363 831 840 1207 2419 2579 4595 5759 BssHII 1 1932 Bst1107I 1 3393 BstEII 1 1702 Enzyme # Sites Locations BstXI 3 1323 1452 1575 BstYI 12 Cac8I 39 CjeI 26 CjePI 18 ClaI 2 508 798 CviJI 90 CviRI 28 DdeI 11 DpnI 32 DraI 3 4381 4400 5092 DraIII 1 5658 DrdI 3 3315 3730 5613 DrdII 3 357 1244 5663 DsaI 4 212 469 958 2594 EaeI 7 166 349 365 829 961 2195 4903 EagI 1 166 Eam1105I 1 4515 EarI 3 1139 3506 5310 EciI 4 1298 3696 3842 4670 Eco47III 3 926 2427 2876 Eco57I 2 4170 5182 EcoNI 1 1056 EcoO109I 3 53 954 2628 EcoRI 1 192 EcoRII 9 367 495 1244 1559 2099 2156 3648 3769 3782 EcoRV 1 206 FauI 17 FokI 13 FspI 2 2603 4737 GdiII 6 166 365 829 961 2195 4903 HaeI 7 217 351 1249 2570 3637 3648 4100 HaeII 14 HaeIII 27 HgaI 13 HgiEII 3 419 1119 4208 HhaI 46 Hin4I 4 203 1420 4514 4588 HincII 2 181 2027 HindIII 1 173 HinfI 16 HpaI 1 2027 HphI 18 KpnI 1 238 MaeII 15 MaeIII 18 MboII 15 MluI 1 1521 MmeI 3 3837 4021 5635 MnlI 28 MscI 1 351 MseI 28 MslI 9 1573 1861 1891 2609 2804 3195 4767 4926 5285 MspI 32 MspA1I 10 84 283 1551 2121 2214 3213 3332 3964 4209 5150 MwoI 41 NarI 4 844 865 979 2161 NciI 12 NcoI 1 212 NdeI 2 346 691 NgoAIV 4 831 2419 2579 5759 NlaIII 26 NlaIV 27 NotI 1 166 NspI 4 996 2967 3259 3626 NspV 1 268 Enzyme # Sites Locations P?1108I 2 2408 4533 P?MI 2 260 1103 PleI 10 466 778 1070 1157 1953 3516 4001 4504 5593 5601 PshAI 1 2366 Psp5II 1 2628 Psp1406I 6 1183 2551 2947 4741 5114 5443 PstI 1 4760 PvuI 1 4885 PvuII 3 2121 2214 3213 RcaI 3 919 4342 5350 RsaI 5 236 642 1668 3428 4995 RsrII 1 589 SacI 1 190 SalI 1 179 SapI 1 3506 Sau96I 19 Sau3AI 32 ScaI 1 4995 ScrFI 21 SfaNI 21 SfcI 5 763 3887 4078 4756 5877 SgrAI 1 840 SphI 1 996 SspI 2 5319 5450 StyI 2 57 212 TaqI 18 TaqII 10 429 1429 1647 2320 3524 4863 5048 5201 5218 5562 T?I 6 566 2200 2502 2672 3176 3597 ThaI 35 TseI 29 Tsp45I 8 1702 2530 3061 3274 3369 4771 4982 5831 Tsp509I 18 Tth111I 1 3367 Tth111II 6 1360 2053 3083 4212 4219 4251 UbaJI 21 VspI 4 778 2206 2265 4687 XbaI 1 729 XcmI 3 1377 1893 1911 XhoI 1 158 XmnI 3 388 3180 5114 Enzymes that do not cut pET-32a(+): AatII A?II AgeI AscI AvrII BaeI BseRI BsmI BspMI BsrGI Bsu36I FseI MunI NheI NruI NsiI PacI PmeI PmlI RleAI SacII SexAI S?I SgfI SmaI SnaBI SpeI SrfI Sse8387I StuI SunI SwaI Novagen ? FAX 608-238-1388 ? E-MAIL novatech@novagen.com
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Novagen ? ORDERING 800-526-7319 ? TECHNICAL SUPPORT 800-207-0144 TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA TATACATATGAGC...315bp...CTGGCCGGTTCTGGTTCTGGCCATATGCACCATCATCATCATCATTCTTCTGGTCTGGTGCCACGCGGTTCT MetSer 105aa...LeuAlaGlySerGlySerGlyHisMetHisHisHisHisHisHisSerSerGlyLeuValProArgGlySer GGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGACGACGACGACAAG GlyMetLysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerProAspLeuGlyThrAspAspAspAspLys GCCATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA AlaMetAlaAspIleGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEnd GCCATGGCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32b(+) AlaMetAlaIleSerAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd GCCATGGGATATCTGTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA pET-32c(+) AlaMetGlyTyrLeuTrpIleArgIleArgAlaProSerThrSerLeuArgProHisSerSerThrThrThrThrThrThrGluIleArgLeuLeuThr CAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG LysProGluArgLysLeuSerTrpLeuLeuProProLeuSerAsnAsnEnd BamH I Hind III Sac I Nco I Not I Sal I Xho I EcoR I EcoR V Ava I Eag I Trx?Tag T7 terminator Bpu1102 I T7 terminator primer #69337-3 T7 promoter rbs Xba I S?Tag primer #69945-3 His?Tag thrombin S?Tag enterokinase Nsp V lac operator His?Tag Msc I Kpn I Bgl II pET-32a(+) 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 puri?cation. Unique sites are shown on the circle map. Note that the sequence is numbered by the pBR322 con- vention, 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 f1 origin is oriented so that infection with helper phage will produce virions containing single-stranded DNA that corre- sponds 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, K.L., Schendel, P.F. and McCoy, J.M. (1993) Bio/Technology 11, 187–193. trxA (366-692 ) l a c I ( 1 1 7 1 - 2 2 5 0 ) o r i ( 3 684) A p ( 4 4 4 5 - 5 3 0 2 ) f1 origin (5434-5889) Bpu1102 I(80) Ava I(158) Xho I(158) Eag I(166) Not I(166) Hind III(173) Sal I(179) Sac I(190) EcoR I(192) BamH I(198) EcoR V(206) Nco I(212) Kpn I(238) Bgl II(241) Nsp V(268) Msc I(351) Rsr II(589) Xba I(729) SgrA I(840) Sph I(996) EcoN I(1056) ApaB I(1205) Mlu I(1521) Bcl I(1535) BstE II(1702) Bmg I(1730) Apa I(1732) BssH II(1932) Hpa I(2027) PshA I(2366) Psp5 II(2628) BspG I(3148) Tth111 I(3367) Bst1107 I(3393) Sap I(3506) BspLU11 I(3622) AlwN I(4038) Bsa I(4576) Pst I(4760) Pvu I(4885) Sca I(4995) Dra III(5658) pET-32a-c(+) cloning/expression region TB122 12/98 pET-32a(+) sequence landmarks T7 promoter 764-780 T7 transcription start 763 Trx?Tag coding sequence 366-692 His?Tag coding sequence 327-344 S?Tag coding sequence 249-293 Multiple cloning sites (Nco I - Xho I) 158-217 His?Tag coding sequence 140-157 T7 terminator 26-72 lacI coding sequence 1171-2250 pBR322 origin 3684 bla coding sequence 4445-5302 f1 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 1bp from each site beyond BamH I at 198. pET-32c(+) is a 5901bp plasmid; add 1bp to each site beyond BamH I at 198 except for EcoR V, which cuts at 209. pET-32a-c(+) Vectors pET-32a(+) (5900bp) Eam1105 I(4357)– Cat. No. pET-32a DNA 69015-3 pET-32b DNA 69016-3 pET-32c DNA 69017-3 TB122 12/98 pET-32a(+) Restriction Sites Enzyme # Sites Locations AccI 2 180 3392 AceIII 8 1288 2016 2347 3131 3272 3574 4814 5498 AciI 85 A?III 2 1521 3622 AluI 25 AlwI 17 Alw21I 10 159 190 414 1021 1505 2616 3440 3940 5101 5186 Alw44I 5 410 1501 3436 3936 5182 AlwNI 1 4038 ApaI 1 1732 ApaBI 1 1205 ApoI 5 192 270 1796 5460 5471 AvaI 1 158 AvaII 8 589 2073 2449 2537 2628 2907 4653 4875 BamHI 1 198 BanI 11 BanII 5 190 905 919 1732 5733 BbsI 4 1667 2006 2380 2740 BbvI 29 BccI 14 Bce83I 7 21 2335 2505 3713 4011 4252 5120 BcefI 5 1040 1381 2008 4124 5684 BcgI 14 BclI 1 1535 BfaI 7 70 730 2636 4117 4370 4705 5809 BglI 2 2585 4635 BglII 1 241 BmgI 1 1730 BpmI 5 1359 1848 2482 3149 4585 Bpu10I 1 2728 Bpu1102I 1 80 BsaI 1 4576 BsaAI 2 3374 5658 BsaBI 3 794 804 2819 BsaHI 6 844 865 979 1478 2161 5052 BsaJI 9 57 212 469 496 958 964 2156 2594 3782 BsaWI 7 2 1840 2343 2811 3828 3975 4806 BsaXI 2 2180 5606 BsbI 2 3338 5565 BscGI 14 BsgI 3 1372 1572 2782 BsiI 2 3795 5179 BsiEI 6 169 2306 3538 3962 4885 5034 BslI 25 BsmAI 7 1218 1623 1749 2136 3263 4576 5352 BsmBI 2 2136 3263 BsmFI 4 982 2523 2893 5873 BsoFI 50 Bsp24I 10 811 843 1362 1394 1664 1696 4115 4147 4293 4325 Bsp1286I 14 BspEI 2 2 2811 BspGI 1 3148 BspLU11I 1 3622 BsrI 25 BsrBI 4 750 3555 5356 5802 BsrDI 4 1568 1934 4576 4750 BsrFI 8 363 831 840 1207 2419 2579 4595 5759 BssHII 1 1932 Bst1107I 1 3393 BstEII 1 1702 Enzyme # Sites Locations BstXI 3 1323 1452 1575 BstYI 12 Cac8I 39 CjeI 26 CjePI 18 ClaI 2 508 798 CviJI 90 CviRI 28 DdeI 11 DpnI 32 DraI 3 4381 4400 5092 DraIII 1 5658 DrdI 3 3315 3730 5613 DrdII 3 357 1244 5663 DsaI 4 212 469 958 2594 EaeI 7 166 349 365 829 961 2195 4903 EagI 1 166 Eam1105I 1 4515 EarI 3 1139 3506 5310 EciI 4 1298 3696 3842 4670 Eco47III 3 926 2427 2876 Eco57I 2 4170 5182 EcoNI 1 1056 EcoO109I 3 53 954 2628 EcoRI 1 192 EcoRII 9 367 495 1244 1559 2099 2156 3648 3769 3782 EcoRV 1 206 FauI 17 FokI 13 FspI 2 2603 4737 GdiII 6 166 365 829 961 2195 4903 HaeI 7 217 351 1249 2570 3637 3648 4100 HaeII 14 HaeIII 27 HgaI 13 HgiEII 3 419 1119 4208 HhaI 46 Hin4I 4 203 1420 4514 4588 HincII 2 181 2027 HindIII 1 173 HinfI 16 HpaI 1 2027 HphI 18 KpnI 1 238 MaeII 15 MaeIII 18 MboII 15 MluI 1 1521 MmeI 3 3837 4021 5635 MnlI 28 MscI 1 351 MseI 28 MslI 9 1573 1861 1891 2609 2804 3195 4767 4926 5285 MspI 32 MspA1I 10 84 283 1551 2121 2214 3213 3332 3964 4209 5150 MwoI 41 NarI 4 844 865 979 2161 NciI 12 NcoI 1 212 NdeI 2 346 691 NgoAIV 4 831 2419 2579 5759 NlaIII 26 NlaIV 27 NotI 1 166 NspI 4 996 2967 3259 3626 NspV 1 268 Enzyme # Sites Locations P?1108I 2 2408 4533 P?MI 2 260 1103 PleI 10 466 778 1070 1157 1953 3516 4001 4504 5593 5601 PshAI 1 2366 Psp5II 1 2628 Psp1406I 6 1183 2551 2947 4741 5114 5443 PstI 1 4760 PvuI 1 4885 PvuII 3 2121 2214 3213 RcaI 3 919 4342 5350 RsaI 5 236 642 1668 3428 4995 RsrII 1 589 SacI 1 190 SalI 1 179 SapI 1 3506 Sau96I 19 Sau3AI 32 ScaI 1 4995 ScrFI 21 SfaNI 21 SfcI 5 763 3887 4078 4756 5877 SgrAI 1 840 SphI 1 996 SspI 2 5319 5450 StyI 2 57 212 TaqI 18 TaqII 10 429 1429 1647 2320 3524 4863 5048 5201 5218 5562 T?I 6 566 2200 2502 2672 3176 3597 ThaI 35 TseI 29 Tsp45I 8 1702 2530 3061 3274 3369 4771 4982 5831 Tsp509I 18 Tth111I 1 3367 Tth111II 6 1360 2053 3083 4212 4219 4251 UbaJI 21 VspI 4 778 2206 2265 4687 XbaI 1 729 XcmI 3 1377 1893 1911 XhoI 1 158 XmnI 3 388 3180 5114 Enzymes that do not cut pET-32a(+): AatII A?II AgeI AscI AvrII BaeI BseRI BsmI BspMI BsrGI Bsu36I FseI MunI NheI NruI NsiI PacI PmeI PmlI RleAI SacII SexAI S?I SgfI SmaI SnaBI SpeI SrfI Sse8387I StuI SunI SwaI Novagen ? FAX 608-238-1388 ? E-MAIL novatech@novagen.com