pET-28a-c(+) Vectors TB074 12/98 Cat. No. pET-28a DNA pET-28b DNA pET-28c DNA 69864- 3 69865- 3 69866- 3 The pET-28a-c( + ) vectors carry an N-terminal His,Tag®/thrombin/T7,Tag® configuration 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 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). pET-28a(+) sequence landmarks T7 promoter 370-386 T7 transcription start 369 His'Tag coding sequence 270-287 T7-Tag coding sequence 207-239 Multiple cloning sites (BamHI-Xho I) 158-203 His*Tag coding sequence 140-157 T7 terminator 26-72 lad coding sequence 773-1852 pBR322 origin 3286 Kan coding sequence 3995-4807 fl origin 4903-5358 The maps for pET-28b(+) and pET-28c(+) are the same as pET-28a( + ) (shown) with the following exceptions: pET-28b(+) is a 5368bp plasmid; subtract lbp from each site beyond BamH I at 198. pET-28c(+) is a 5367bp plasmid; subtract 2bp from each site beyondfiamHIat 198. PVU 1(4426) Sgf 1(4426) Sma 1(4300) Cla 1(4117) Nru 1(4083) EC057 1(3772) AlwN 1(3640) Bpu1102 l(80) Dra 111(5127) BssS 1(3397) BspLU11 1(3224) Sap 1(31 Bst1107 1(2995) Tth1 11 1(2969) Xho 1(158) Not 1(166) Eag 1(166) Hind 111(173) Sal 1(179) Sac 1(190) EcoR 1(192) BamH 1(198) Nhe 1(231) Nde 1(238) Nco 1(296) Xba 1(335) Bgl 11(401) SgrA 1(442) Sph 1(598) Mlu 1(1123) Bel 1(1137) BstE 11(1304) Apa 1(1334) BssH 11(1534) EcoR V(1573) Hpa 1(1629) PshA 1(1968) Bgl 1(2187) Fsp 1(2205) Psp5 11(2230) T7 promoter primer #69348-3 pET upstream primer #69214-3 T7 nrnmnter i ._»■ Bgl II l / promoter ^ _lac operator_ xba I rbs AGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA Afcol His-Tag_ Nde I Nhe\_ T7-Tag_ TATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAA rle-tGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHisrle-tAlaSerrle-tThrGlyGlyGlnGIn £ag\ 'thrombi n BamH I EcoR I Sac I Salt Hind III A/of I Xho I__His-Tag ATGGGTCGCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28a( MetGlyArgGIySerGIuPheGIuLeuArgArgGInAIaCysGIyArgThrArgAIaProProProProProLeuArgSerGIyCysEnd ...GGTCGGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28b( ...GIyArgAspProAsnSerSerSerVaIAspLysLeuAIaAIaAIaLeuGluHisHisHisHi sHi sHi sEnd ...GGTCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28c( ...GlyArglIeArglIeArgAIaProSerThrSerLeuArgProHi sSerSerThrThrThrThrThrThrGIuI IeArgLeuLeuThrLysPro.. ._ Bpin 102 1 _T7 terminator_ GAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG T7 terminator primer #69337-3 pET-28a-c(+) cloning/expression region Novagen • ordering 800-526-7319 • technical support 800-207-0144 pET-28a(+ ) Restriction Sites T Enzyme # Sites Locations Enzyme # Sites Locations Enzyme # Sites Locations Accl 2 180 2994 BstEII 1 1304 Pfl 11081 1 2010 Acelll 7 890 1618 1949 2733 2874 BstXI 3 925 1054 1177 PflMI 2 705 4689 3176 4967 BstYl 9 132 198 401 687 1899 Plel 9 384 672 759 1555 Acil 77 2416 3865 3876 4675 3603 4658 5062 5070 Afllll 2 1123 3224 Cac8l 40 PshAI 1 1968 Alul 22 Cjel 26 Psp5ll 1 2230 Alwl 13 CjePI 30 Psp1406l 4 785 2153 2549 4912 Alw21l 7 159 190 623 1107 2218 Clal 1 4117 Pvul 1 4426 3042 3542 CviJI 86 Pvull 3 1723 1816 2815 Alw44l 3 1103 3038 3538 CviRI 22 Real 3 521 3944 4819 AlwNI 1 3640 Ddel 11 Rsal 3 1270 3030 4261 Apal 1 1334 Dpnl 21 Sad 1 190 ApaBI 1 807 Dralll 1 5127 Sail 1 179 Apol 6 192 1398 4039 4223 4929 Drdl 3 2917 3332 5082 Sapl 1 3108 4940 Drdll 2 846 5132 Sau96l 14 Aval 2 158 4298 Dsal 3 296 560 2196 Sau3AI 21 Avail 5 1675 2051 2139 2230 2509 Eael 4 166 431 563 1797 ScrFI 22 Bam HI 1 198 Eagl 1 166 SfaNI 23 Ban I 9 253 445 466 580 1043 Earl 3 741 3108 4239 Sfd 4 369 3489 3680 5346 1762 1892 2018 5164 Ed I 3 900 3298 3444 Sgfl 1 4426 Ban 11 6 190 507 521 1334 4081 Eco47lll 3 528 2029 2478 SgrAI 1 442 5202 Eco57l 1 3772 Smal 1 4300 Bbsl 4 1269 1608 1982 2342 EcoNI 2 658 4338 Sphl 1 598 Bbvl 27 EcoO109l 3 53 556 2230 Sspl 2 4351 4919 Bed 14 EcoRI 1 192 Styl 2 57 296 Bce83l 6 21 1937 2107 3315 3613 EcoRII 10 256 846 1161 1701 1758 Taql 15 3854 3250 3371 3384 4314 4671 Taqll 6 1031 1249 1922 3126 Bcefl 6 642 983 1610 3726 4745 EcoRV 1 1573 5031 5153 Faul 17 Tfil 9 1802 2104 2274 2778 Bcgl 9 160 194 228 1415 1449 Fokl 9 1169 1178 2443 2505 2583 4337 4393 4565 4656 1949 1983 2801 2835 2769 2910 4064 4670 Thai 38 Bell 1 1137 Fspl 1 2205 Tsel 27 Bfal 7 70 232 336 2238 3719 Gdill 4 166 431 563 1797 Tsp45l 7 1304 2132 2663 2876 4026 5278 Hael 6 851 2172 3239 3250 3702 4573 5300 Bgll 1 2187 4513 Tsp509l 20 Bglll 1 401 Haell 14 Tth111l 1 2969 Bmgl 1 1332 Haelll 24 Tth111ll 8 962 1655 2685 3814 Bpml 4 961 1450 2084 2751 Hgal 11 3853 4262 4389 BpulOI 2 2330 4443 HgiEII 2 721 3810 UbaJI 21 Bpu11021 1 80 Hhal 47 Vspl 5 384 1808 1867 4625 BsaAl 2 2976 5127 Hin4l 3 1022 4112 4654 Xbal 1 335 BsaBI 3 400 406 2421 Hindi 2 181 1629 Xcml 3 979 1495 1513 BsaHl 5 446 467 581 1080 1763 Hindlll 1 173 Xhol 1 158 BsaJI 10 57 296 560 566 1758 Hinfl 18 Xmnl 2 2782 4815 2196 3384 4297 4298 4699 Hpal 1 1629 BsaWI 7 2 1442 1945 2413 3430 Hphl 16 Enzymes that do not cut pET28a(+): 3577 4561 Maell 14 Aatll Aflll Agel Asd Avrll BsaXI 2 1782 5075 Maelll 16 Bael Bsal BseR Bsp Ml BsrGI Bsbl 2 2940 5034 Mboll 12 Bsu36l Dral Eam11051 Fsel Kpnl BscGI 11 Mlul 1 1123 Msel Muni NspV Pad Pmel Bsgl 3 974 1174 2384 Mmel 7 3439 3623 4068 4262 4624 Pmll Pstl RleA Rsrll Sadl Bsll 1 3397 4633 5104 Seal SexAl Stil SnaBI Spel BsiEl 5 169 1908 3140 3564 4426 Mnll 25 Srfl Sse8387 Stul Sunl Swal Bsll 23 Msel 25 Bsml 2 4310 4387 Msll 6 1175 1463 1493 2211 2406 BsmAI 6 820 1225 1351 1738 2865 2797 4442 Mspl 29 BsmBI 3 1738 2865 4442 MspAII 9 84 264 1153 1723 1816 BsmFI 4 584 2125 2495 5342 2815 2934 3566 3811 BsoFI 48 Mwol 39 Bsp24l 12 Narl 4 446 467 581 1763 Bsp1286l 12 Neil 12 BspEI 2 2 2413 Ncol 1 296 BspGI 1 2750 Ndel 1 238 BspLUHI 1 3224 NgoAIV 4 433 2021 2181 5228 Bsrl 22 Nhel 1 231 BsrBI 4 356 3157 4825 5271 Nlalll 26 BsrDI 2 1170 1536 NlalV 22 BsrFI 7 433 442 809 2021 2181 Notl 1 166 4380 5228 Nrul 1 4083 BssHII 1 1534 Nsil 2 4276 4542 Bst1107l 1 2995 Nspl 4 598 2569 2861 3228 TB074 12/98 Novagen • fax 608-238-1388 • e-mail novatech@novagen.com