Novagen ? ORDERING 800-526-7319 ? TECHNICAL SUPPORT 800-207-0144 pET-29a-c(+) cloning/expression region TB076 12/98 AGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA TATACATATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCCTGGTGCCACGCGGTTCC MetLysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerProAspLeuGlyThrLeuValProArgGlySer ATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-29a(+) MetAlaAspIleGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEnd ...GCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-29b(+) ...AlaIleSerAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd ...GGATATCTGTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-29c(+) ...GlyTyrLeuTrpIleArgIleArgAlaProSerThrSerLeuArgProHisSerSerThrThrThrThrThrThrGluIleArgLeuLeuThrLysPro... GAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG Hind III Xba I lac operator Nde I pET upstream primer #69214-3 T7 promoter primer #69348-3 T7 promoter S?Tag Nsp V EcoR I Xho I His?Tag BamH I Sac I Not I Nco I Eag I EcoR V Sal I thrombin Bpu1102 I T7 terminator primer #69337-3 T7 terminator rbs Bgl II Kpn I The pET-29a-c(+) vectors carry an N-terminal S?Tag?/thrombin con?guration plus an 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). pET-29a(+) sequence landmarks T7 promoter 368-384 T7 transcription start 367 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 775-1854 pBR322 origin 3288 Kan coding sequence 3997-4809 f1 origin 4905-5360 The maps for pET-29b(+) and pET-29c(+) are the same as pET-29a(+) (shown) with the following exceptions: pET-29b(+) is a 5370bp plasmid; subtract 1bp from each site beyond BamH I at 198. pET-29c(+) is a 5372bp plasmid; add 1bp to each site beyond BamH I at 198. pET-29a-c(+) Vectors l a c I ( 7 7 5 - 1 8 5 4 ) o r i (3288) K a n ( 3 9 9 7 - 4 8 0 9 ) f1 origin (4905-5360) Bpu1102 I(80) Xba I(333) SgrA I(444) Sph I(600) Mlu I(1125) Bcl I(1139) BstE II(1306) Apa I(1336) BssH II(1536) Hpa I(1631) PshA I(1970) Bfa I(2189) Fsp I(2207) Psp5 II(2232) Tth111 I(2971) Bst1107 I(2997) Sap I(3110) BspLU11 I(3226) BssS I(3399) AlwN I(3642) Eco57 I(3774) Nru I(4085) Sma I(4302) Pvu I(4428) Sgf I(4428) Dra III(5129) Xho I(158) Not I(166) Eag 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) Nde I(295) pET-29a(+) (5371bp) Cat. No. pET-29a DNA 69871-3 pET-29b DNA 69872-3 pET-29c DNA 69873-3 TB076 12/98 Enzyme # Sites Locations AccI 2 180 2996 AceIII 7 892 1620 1951 2735 2876 3178 4969 AciI 75 A?III 2 1125 3226 AluI 22 AlwI 13 Alw21I 7 159 190 625 1109 2220 3044 3544 Alw44I 3 1105 3040 3540 AlwNI 1 3642 ApaI 1 1336 ApaBI 1 809 ApoI 7 192 270 1400 4041 4225 4931 4942 AvaI 2 158 4300 AvaII 5 1677 2053 2141 2232 2511 BaeI 1 233 BamHI 1 198 BanI 10 226 234 447 468 582 1045 1764 1894 2020 5166 BanII 6 190 509 523 1336 4083 5204 BbsI 4 1271 1610 1984 2344 BbvI 25 BccI 13 Bce83I 6 21 1939 2109 3317 3615 3856 BcefI 6 644 985 1612 3728 4747 5155 BcgI 8 160 194 1417 1451 1951 1985 2803 2837 BclI 1 1139 BfaI 1 2189 BglII 1 241 BmgI 1 1334 BpmI 4 963 1452 2086 2753 Bpu10I 2 2332 4445 Bpu1102I 1 80 BsaAI 2 2978 5129 BsaBI 3 398 408 2423 BsaHI 5 448 469 583 1082 1765 BsaJI 11 BsaWI 7 2 1444 1947 2415 3432 3579 4563 BsaXI 2 1784 5077 BsbI 2 2942 5036 BscGI 11 BsgI 3 976 1176 2386 BsiI 1 3399 BsiEI 5 169 1910 3142 3566 4428 BslI 26 BsmI 2 4312 4389 BsmAI 6 822 1227 1353 1740 2867 4444 BsmBI 3 1740 2867 4444 BsmFI 4 586 2127 2497 5344 BsoFI 43 Bsp24I 10 415 447 966 998 1268 1300 3719 3751 3897 3929 Bsp1286I 12 BspEI 2 2 2415 BspGI 1 2752 BspLU11I 1 3226 BsrI 21 BsrBI 4 354 3159 4827 5273 BsrDI 2 1172 1538 BsrFI 7 435 444 811 2023 2183 4382 5230 BssHII 1 1536 Bst1107I 1 2997 BstEII 1 1306 Enzyme # Sites Locations BstXI 3 927 1056 1179 BstYI 9 132 198 241 689 1901 2418 3867 3878 4677 Cac8I 40 CjeI 24 CjePI 18 ClaI 2 402 4119 CviJI 84 CviRI 22 DdeI 11 DpnI 23 DraIII 1 5129 DrdI 3 2919 3334 5084 DrdII 2 848 5134 DsaI 3 212 562 2198 EaeI 4 166 433 565 1799 EagI 1 166 EarI 3 743 3110 4241 EciI 3 902 3300 3446 Eco47III 3 530 2031 2480 Eco57I 1 3774 EcoNI 2 660 4340 EcoO109I 3 53 558 2232 EcoRI 1 192 EcoRII 10 229 848 1163 1703 1760 3252 3373 3386 4316 4673 EcoRV 1 206 FauI 17 FokI 9 1171 1180 2445 2507 2585 2771 2912 4066 4672 FspI 1 2207 GdiII 4 166 433 565 1799 HaeI 6 853 2174 3241 3252 3704 4515 HaeII 14 HaeIII 23 HgaI 11 HgiEII 2 723 3812 HhaI 46 Hin4I 4 203 1024 4114 4656 HincII 2 181 1631 HindIII 1 173 HinfI 18 HpaI 1 1631 HphI 16 KpnI 1 238 MaeII 14 MaeIII 16 MboII 12 MluI 1 1125 MmeI 7 3441 3625 4070 4264 4626 4635 5106 MnlI 25 MseI 25 MslI 6 1177 1465 1495 2213 2408 2799 MspI 29 MspA1I 9 84 283 1155 1725 1818 2817 2936 3568 3813 MwoI 39 NarI 4 448 469 583 1765 NciI 12 NcoI 1 212 NdeI 1 295 NgoAIV 4 435 2023 2183 5230 NlaIII 26 NlaIV 24 NotI 1 166 NruI 1 4085 NsiI 2 4278 4544 NspI 4 600 2571 2863 3230 NspV 1 268 Enzyme # Sites Locations P?1108I 1 2012 P?MI 3 260 707 4691 PleI 9 382 674 761 1557 3120 3605 4660 5064 5072 PshAI 1 1970 Psp5II 1 2232 Psp1406I 4 787 2155 2551 4914 PvuI 1 4428 PvuII 3 1725 1818 2817 RcaI 3 523 3946 4821 RsaI 4 236 1272 3032 4263 SacI 1 190 SalI 1 179 SapI 1 3110 Sau96I 14 Sau3AI 23 ScrFI 22 SfaNI 23 SfcI 4 367 3491 3682 5348 SgfI 1 4428 SgrAI 1 444 SmaI 1 4302 SphI 1 600 SspI 2 4353 4921 StyI 2 57 212 TaqI 17 TaqII 6 1033 1251 1924 3128 4682 5033 T?I 9 1804 2106 2276 2780 3201 4339 4395 4567 4658 ThaI 36 TseI 25 Tsp45I 7 1306 2134 2665 2878 2973 4575 5302 Tsp509I 21 Tth111I 1 2971 Tth111II 8 964 1657 2687 3816 3823 3855 4264 4391 UbaJI 18 VspI 5 382 1810 1869 4627 4816 XbaI 1 333 XcmI 3 981 1497 1515 XhoI 1 158 XmnI 2 2784 4817 Enzymes that do not cut pET-29a(+): AatII A?II AgeI AscI AvrII BsaI BseRI BspMI BsrGI Bsu36I DraI Eam1105I FseI MscI MunI NheI PacI PmeI PmlI PstI RleAI RsrII SacII ScaI SexAI S?I SnaBI SpeI SrfI Sse8387I StuI SunI SwaI pET-29a(+) Restriction Sites Novagen ? FAX 608-238-1388 ? E-MAIL novatech@novagen.com
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Novagen ? ORDERING 800-526-7319 ? TECHNICAL SUPPORT 800-207-0144 pET-29a-c(+) cloning/expression region TB076 12/98 AGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA TATACATATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCCTGGTGCCACGCGGTTCC MetLysGluThrAlaAlaAlaLysPheGluArgGlnHisMetAspSerProAspLeuGlyThrLeuValProArgGlySer ATGGCTGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-29a(+) MetAlaAspIleGlySerGluPheGluLeuArgArgGlnAlaCysGlyArgThrArgAlaProProProProProLeuArgSerGlyCysEnd ...GCGATATCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-29b(+) ...AlaIleSerAspProAsnSerSerSerValAspLysLeuAlaAlaAlaLeuGluHisHisHisHisHisHisEnd ...GGATATCTGTGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-29c(+) ...GlyTyrLeuTrpIleArgIleArgAlaProSerThrSerLeuArgProHisSerSerThrThrThrThrThrThrGluIleArgLeuLeuThrLysPro... GAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG Hind III Xba I lac operator Nde I pET upstream primer #69214-3 T7 promoter primer #69348-3 T7 promoter S?Tag Nsp V EcoR I Xho I His?Tag BamH I Sac I Not I Nco I Eag I EcoR V Sal I thrombin Bpu1102 I T7 terminator primer #69337-3 T7 terminator rbs Bgl II Kpn I The pET-29a-c(+) vectors carry an N-terminal S?Tag?/thrombin con?guration plus an 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). pET-29a(+) sequence landmarks T7 promoter 368-384 T7 transcription start 367 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 775-1854 pBR322 origin 3288 Kan coding sequence 3997-4809 f1 origin 4905-5360 The maps for pET-29b(+) and pET-29c(+) are the same as pET-29a(+) (shown) with the following exceptions: pET-29b(+) is a 5370bp plasmid; subtract 1bp from each site beyond BamH I at 198. pET-29c(+) is a 5372bp plasmid; add 1bp to each site beyond BamH I at 198. pET-29a-c(+) Vectors l a c I ( 7 7 5 - 1 8 5 4 ) o r i (3288) K a n ( 3 9 9 7 - 4 8 0 9 ) f1 origin (4905-5360) Bpu1102 I(80) Xba I(333) SgrA I(444) Sph I(600) Mlu I(1125) Bcl I(1139) BstE II(1306) Apa I(1336) BssH II(1536) Hpa I(1631) PshA I(1970) Bfa I(2189) Fsp I(2207) Psp5 II(2232) Tth111 I(2971) Bst1107 I(2997) Sap I(3110) BspLU11 I(3226) BssS I(3399) AlwN I(3642) Eco57 I(3774) Nru I(4085) Sma I(4302) Pvu I(4428) Sgf I(4428) Dra III(5129) Xho I(158) Not I(166) Eag 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) Nde I(295) pET-29a(+) (5371bp) Cat. No. pET-29a DNA 69871-3 pET-29b DNA 69872-3 pET-29c DNA 69873-3 TB076 12/98 Enzyme # Sites Locations AccI 2 180 2996 AceIII 7 892 1620 1951 2735 2876 3178 4969 AciI 75 A?III 2 1125 3226 AluI 22 AlwI 13 Alw21I 7 159 190 625 1109 2220 3044 3544 Alw44I 3 1105 3040 3540 AlwNI 1 3642 ApaI 1 1336 ApaBI 1 809 ApoI 7 192 270 1400 4041 4225 4931 4942 AvaI 2 158 4300 AvaII 5 1677 2053 2141 2232 2511 BaeI 1 233 BamHI 1 198 BanI 10 226 234 447 468 582 1045 1764 1894 2020 5166 BanII 6 190 509 523 1336 4083 5204 BbsI 4 1271 1610 1984 2344 BbvI 25 BccI 13 Bce83I 6 21 1939 2109 3317 3615 3856 BcefI 6 644 985 1612 3728 4747 5155 BcgI 8 160 194 1417 1451 1951 1985 2803 2837 BclI 1 1139 BfaI 1 2189 BglII 1 241 BmgI 1 1334 BpmI 4 963 1452 2086 2753 Bpu10I 2 2332 4445 Bpu1102I 1 80 BsaAI 2 2978 5129 BsaBI 3 398 408 2423 BsaHI 5 448 469 583 1082 1765 BsaJI 11 BsaWI 7 2 1444 1947 2415 3432 3579 4563 BsaXI 2 1784 5077 BsbI 2 2942 5036 BscGI 11 BsgI 3 976 1176 2386 BsiI 1 3399 BsiEI 5 169 1910 3142 3566 4428 BslI 26 BsmI 2 4312 4389 BsmAI 6 822 1227 1353 1740 2867 4444 BsmBI 3 1740 2867 4444 BsmFI 4 586 2127 2497 5344 BsoFI 43 Bsp24I 10 415 447 966 998 1268 1300 3719 3751 3897 3929 Bsp1286I 12 BspEI 2 2 2415 BspGI 1 2752 BspLU11I 1 3226 BsrI 21 BsrBI 4 354 3159 4827 5273 BsrDI 2 1172 1538 BsrFI 7 435 444 811 2023 2183 4382 5230 BssHII 1 1536 Bst1107I 1 2997 BstEII 1 1306 Enzyme # Sites Locations BstXI 3 927 1056 1179 BstYI 9 132 198 241 689 1901 2418 3867 3878 4677 Cac8I 40 CjeI 24 CjePI 18 ClaI 2 402 4119 CviJI 84 CviRI 22 DdeI 11 DpnI 23 DraIII 1 5129 DrdI 3 2919 3334 5084 DrdII 2 848 5134 DsaI 3 212 562 2198 EaeI 4 166 433 565 1799 EagI 1 166 EarI 3 743 3110 4241 EciI 3 902 3300 3446 Eco47III 3 530 2031 2480 Eco57I 1 3774 EcoNI 2 660 4340 EcoO109I 3 53 558 2232 EcoRI 1 192 EcoRII 10 229 848 1163 1703 1760 3252 3373 3386 4316 4673 EcoRV 1 206 FauI 17 FokI 9 1171 1180 2445 2507 2585 2771 2912 4066 4672 FspI 1 2207 GdiII 4 166 433 565 1799 HaeI 6 853 2174 3241 3252 3704 4515 HaeII 14 HaeIII 23 HgaI 11 HgiEII 2 723 3812 HhaI 46 Hin4I 4 203 1024 4114 4656 HincII 2 181 1631 HindIII 1 173 HinfI 18 HpaI 1 1631 HphI 16 KpnI 1 238 MaeII 14 MaeIII 16 MboII 12 MluI 1 1125 MmeI 7 3441 3625 4070 4264 4626 4635 5106 MnlI 25 MseI 25 MslI 6 1177 1465 1495 2213 2408 2799 MspI 29 MspA1I 9 84 283 1155 1725 1818 2817 2936 3568 3813 MwoI 39 NarI 4 448 469 583 1765 NciI 12 NcoI 1 212 NdeI 1 295 NgoAIV 4 435 2023 2183 5230 NlaIII 26 NlaIV 24 NotI 1 166 NruI 1 4085 NsiI 2 4278 4544 NspI 4 600 2571 2863 3230 NspV 1 268 Enzyme # Sites Locations P?1108I 1 2012 P?MI 3 260 707 4691 PleI 9 382 674 761 1557 3120 3605 4660 5064 5072 PshAI 1 1970 Psp5II 1 2232 Psp1406I 4 787 2155 2551 4914 PvuI 1 4428 PvuII 3 1725 1818 2817 RcaI 3 523 3946 4821 RsaI 4 236 1272 3032 4263 SacI 1 190 SalI 1 179 SapI 1 3110 Sau96I 14 Sau3AI 23 ScrFI 22 SfaNI 23 SfcI 4 367 3491 3682 5348 SgfI 1 4428 SgrAI 1 444 SmaI 1 4302 SphI 1 600 SspI 2 4353 4921 StyI 2 57 212 TaqI 17 TaqII 6 1033 1251 1924 3128 4682 5033 T?I 9 1804 2106 2276 2780 3201 4339 4395 4567 4658 ThaI 36 TseI 25 Tsp45I 7 1306 2134 2665 2878 2973 4575 5302 Tsp509I 21 Tth111I 1 2971 Tth111II 8 964 1657 2687 3816 3823 3855 4264 4391 UbaJI 18 VspI 5 382 1810 1869 4627 4816 XbaI 1 333 XcmI 3 981 1497 1515 XhoI 1 158 XmnI 2 2784 4817 Enzymes that do not cut pET-29a(+): AatII A?II AgeI AscI AvrII BsaI BseRI BspMI BsrGI Bsu36I DraI Eam1105I FseI MscI MunI NheI PacI PmeI PmlI PstI RleAI RsrII SacII ScaI SexAI S?I SnaBI SpeI SrfI Sse8387I StuI SunI SwaI pET-29a(+) Restriction Sites Novagen ? FAX 608-238-1388 ? E-MAIL novatech@novagen.com