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 beyond .BamH I at 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 HetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySerHisHetAlaSerHetThrGlyGlyGlnGIn £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( .GIyArgAspProAsnSerSerSerVaIAspLysLeuAIaAIaAIaLeuGluHisHisHisHisHisHisEnd .GGTCGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCC pET-28c( .GIyArgI IeArglIeArgAIaProSerThrSerLeuArgProHi sSerSerThrThrThrThrThrThrGlulleArgLeuLeuThrLysPro. . ._ BpiA 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 Enzyme # Sites Locations Accl 2 180 2994 Acelll 7 890 1618 3176 4967 Acil 77 Afllll 2 1123 3224 Alul 22 Alwl 13 Alw21l 7 159 190 3042 3542 Alw44l 3 1103 3038 AlwNI 1 3640 Apal 1 1334 ApaBI 1 807 Apol 6 192 1398 4940 Aval 2 158 4298 Avail 5 1675 2051 BamHI 1 198 Ban I 9 253 445 1762 1892 Banll 6 190 507 5202 Bbsl 4 1269 1608 Bbvl 27 Bed 14 Bce83l 6 21 1937 3854 Bcefl 6 642 983 5153 Bcgl 9 160 194 1949 1983 Bell 1 1137 Bfal 7 70 232 4026 5278 Bgll 1 2187 Bglll 1 401 Bmgl 1 1332 Bpml 4 961 1450 BpulOI 2 2330 4443 Bpu11021 1 80 BsaAl 2 2976 5127 BsaBI 3 400 406 BsaHl 5 446 467 BsaJI 10 57 296 2196 3384 BsaWI 7 2 1442 3577 4561 BsaXI 2 1782 5075 Bsbl 2 2940 5034 BscGI 11 Bsgl 3 974 1174 Bsll 1 3397 BsiB 5 169 1908 Bsll 23 Bsml 2 4310 4387 BsmAI 6 820 1225 4442 BsmBI 3 1738 2865 BsmFI 4 584 2125 BsoFI 48 Bsp24l 12 Bsp1286l 12 BspB 2 2 2413 BspGI 1 2750 BspLUHI 1 3224 Bsrl 22 BsrBI 4 356 3157 BsrDI 2 1170 1536 BsrFI 7 433 442 4380 5228 BssHII 1 1534 Bst1107l 1 2995 1949 2733 2874 623 1107 2218 3538 4039 4223 4929 2139 2230 2509 466 580 1043 2018 5164 521 1334 4081 2107 3315 3613 1610 3726 4745 228 1415 1449 2801 2835 336 2238 3719 2084 2751 2421 581 1080 1763 560 566 1758 4297 4298 4699 1945 2413 3430 2384 3140 3564 4426 1351 1738 2865 4442 2495 5342 4825 5271 809 2021 2181 TB074 12/98 Enzyme # Sites Locations BstEII BstXI BstYl Cjel CjePI Clal CviJI CviRI Ddel Dpnl Dralll Drdl Drdll Dsal Biel Eagl Earl Ecil Eco47lll Eco57l EcoNI EcoO109l EcoRI EcoRII EcoRV Faul Fokl Fspl Gdill Hael Haell Hael 11 Hgal HgiEII Hhal Hin4l Hindi Hindlll Hinfl Hpal Hphl Maell Maelll Mboll MLiI Mmel Mnll Msel Msll Mspl MspAII 1 3 9 40 26 30 I 86 22 II 21 1 3 2 3 4 1 3 3 3 1 2 3 1 10 1 17 9 1 4 6 14 24 11 2 47 3 2 1 18 1 16 14 16 12 1 7 25 25 6 29 9 1304 925 132 2416 5127 2917 846 296 166 166 741 900 528 3772 658 53 192 256 3250 1573 1169 2769 2205 166 851 4513 721 1022 181 173 1054 198 3865 1177 401 3876 687 4675 3332 5132 560 431 2029 4338 556 846 3371 1178 2910 431 2172 3810 4112 1629 2196 563 1797 4239 3444 2478 1161 1701 1758 3384 4314 4671 2443 2505 2583 4064 4670 563 1797 3239 3250 3702 1123 3439 3623 4633 5104 1175 1463 2797 84 2815 264 2934 4068 4262 4624 1493 2211 2406 1153 1723 1816 3566 3811 Mwol 39 Narl 4 446 467 581 1763 Neil 12 Ncol 1 296 Ndel 1 238 NgoAIV 4 433 2021 2181 5228 Nhel 1 231 Nlalll 26 NlalV 22 Notl 1 166 Nrul 1 4083 Nsil 2 4276 4542 Nspl 4 598 2569 2861 3228 Enzyme # Sites Locations Pfl 11081 1 2010 PflMI 2 705 4689 Plel 9 384 672 759 1555 3603 4658 5062 5070 PshAI 1 1968 Psp5ll 1 2230 Psp1406l 4 785 2153 2549 4912 Pvul 1 4426 Pvull 3 1723 1816 2815 Real 3 521 3944 4819 Rsal 3 1270 3030 4261 Sad 1 190 Sail 1 179 Sapl 1 3108 Sau96l 14 Sau3AI 21 ScrFI 22 SfaNI 23 Sfd 4 369 3489 3680 5346 Sgfl 1 4426 SgrAI 1 442 Smal 1 4300 Sphl 1 598 Sspl 2 4351 4919 Styl 2 57 296 Taql 15 Taqll 6 1031 1249 1922 3126 5031 Tfil 9 1802 2104 2274 2778 4337 4393 4565 4656 Thai 38 Tsel 27 Tsp45l 7 1304 2132 2663 2876 4573 5300 Tsp509l 20 Tth111l 1 2969 Tth111ll 8 962 1655 2685 3814 3853 4262 4389 UbaJI 21 Vspl 5 384 1808 1867 4625 Xbal 1 335 Xcml 3 979 1495 1513 Xhol 1 158 Xmnl 2 2782 4815 Enzymes that do not cut pET28a(+): Aatll Aflll Agel Asel Avrll Bael Bsal BseR Bsp Ml BsrGl Bsu36l Dral Eam1105l Fsel Kpnl Msd Muni NspV Pad Pmel Pmll Pstl RleAl Rsrll Sadl Seal SexAl Stil SnaBI Spei Srfl Sse8387 Stul Sunl Swal Novagen • fax 608-238-1388 • e-mail novatech@novagen.com