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1、Features of pBAD-DEST49 6160 nucleotidesArabinose O2 operator: bases 4-19 Arabinose O1 operator: bases 161-182 CAP binding site: bases 203-216 Arabinose I1 and I2 region: bases 213-251 Arabinose minimal promoter: bases 248-276 RBS: bases 329-332 His-Patch Thioredoxin: bases 346-674 Enterokinase reco
2、gnition site: bases 691-705 attR1 recombination site: bases 718-842 Chloramphenicol resistance gene: bases 951-1610 ccdB gene: bases 1952-2257 attR2 recombination site: bases 2298-2422 V5 epitope: bases 2436-2477 6xHis tag: bases 2487-2507 rrnB transcription termination region: bases 2610-2767 bla p
3、romoter: bases 2948-3046 Ampicillin resistance gene (bla): bases 3047-3907 pUC origin: bases 4052-4725 araC gene: bases 5256-6134 (complementary strand)pBAD-DEST49 6160 bpPBADAmpicillinpUCoriaraCStopattR2ccdBCmRattR1V5 6xHisEKHP-thioredoxinGGT CAG TTG AAA GAG TTC CTC GAC GCT AAC CTG GCC GGC TCT GGA
4、TCC GGT CCA GTC AAC TTT CTC AAG GAG CTG CGA TTG GAC CGG CCG AGA CCT AGG CCAHis-Patch ThioredoxinGENE. . . . pro ala phe leu tyr lys val val ile lys leu gluEnterokinase Recognition SiteEnterokinase cleavage siteV5 epitopeGGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG CGT ACC CCA TTG GGA TAG
5、GGA TTG GGA GAG GAG GGT GAG CTA AGA TGC GCA TGGgly lys pro ile pro asn pro leu leu gly leu asp ser thr arg thr6xHis tag69174224367252408640gly his his his his his his * GGT CAT CAT CAC CAT CAC CAT TGA GTT TAA ACG GTC TCC AGC TTG GCT CCA TTG CCA GTA GTA GTG GTA GTG GTA ACT CAA ATT TGC CAG AGG TCG2484
6、gly gln leu lys glu phe leu asp ala asn leu ala gly ser gly ser glyasp asp asp asp lys leu gly ile ile thr ser leu tyr lys lys ala glyCTA CTG CTA CTG TTC GAC CCT TAA TAG TGT TCA AAG ATG TTT TTT CGT CCGGAT GAC GAT GAC AAG CTG GGA ATT ATC ACA AGT TTG TAC AAA AAA GCA GGCANN - - NTG GGT CGA AAG AAC ATG
7、TTT CAC CAC TAG TTC GAA CTTTNN - - NAC CCA GCT TTC TTG TAC AAA GTG GTG ATC AAG CTT GAAattB2attB1General Description DNA pBAD-DEST49Entire molecule length: 6160 bp Restriction Map Enzyme # of cuts Positions AccI 31699 2294 4953 AccIII 21161 5876 AciI 6185 113(c) 238(c) 447(c) 611(c) 844(c) 848 1143 1
8、507 1601(c) 1673(c) 1868(c) 2207 2539(c) 2590(c) 2617(c) 2628(c) 2818 2827(c) 2865(c) 2878 2991 3090(c) 3199(c) 3276(c) 3320 3441(c) 3487 3678(c) 3769(c) 4131 4140(c) 4275 4385(c) 4506(c) 4525(c) 4652(c) 4680(c) 4771 4792 4799 4842(c) 4859(c) 4885(c) 4898 4936 4961 5003 5013 5052 5161(c) 5347 5488(c
9、) 5650 5751(c) 5835(c) 5910(c) 5978 6018(c) 6072 6149 AcsI 31165 2606 5944 AcyI 3194 2874 3295 AflII 2401 5462 AflIII 4136 1612 2476 4723 AgeI 275 2482 AluI 24706 737 937 946 1065 1194 1768 2131 2404 2430 2525 3482 3545 3645 4166 4423 4469 4559 4785 5066 5085 5427 5461 5688 Alw44I 5627 2065 3163 440
10、9 4907 AlwI 19235(c) 248 358 678(c) 691 910(c) 923 1613(c) 1626 2045(c) 3196 3200(c) 3517 3980(c) 3981 4077(c) 4079 4165 6121 AlwNI 21944 4314 AosI 13612 ApaLI 5627 2065 3163 4409 4907 ApoI 31165 2606 5944 AseI 13660 AsnI 13660 Asp700 3653 2553 3235 AspEI 13835 AspHI 7631 2069 3167 3252 4413 4911 54
11、18 AspI 14979 AvaI 3541 2034 5315 AvaII 6116 449 2633 3471 3693 5448 AviII 13612 BamHI 4240 683 915 1618 BanI 2856 3882 BbvI 16573(c) 1748 3401(c) 3792 4095(c) 4301(c) 4304(c) 4394 4813 4831 4975(c) 5072(c) 5143 5436 5697 6117 BcgI 5445(c) 602 3297(c) 5292(c) 5659(c) BclI 12423 BfaI 4302 3642 3977 4
12、230 BfrI 2401 5462 BglI 13717 BmyI 9439 548 631 2069 3167 3252 4413 4911 5418 BpmI 31287 2505(c) 3766 BsaAI 14973 BsaBI 22049 5402 BsaHI 3194 2874 3295 BsaI 52174 2470 2523 2701 3769 BsaJI 12345 541 568 861 1396 1397 1466 2034 2056 2460 4563 5325 BsaWI 975 686 1161 1805 2482 3539 4370 4517 5876 BseA
13、I 21161 5876 BsiEI 10848 3317 3466 4389 4813 5387 5888 5935 6022 6026 BsiHKAI 7631 2069 3167 3252 4413 4911 5418 BsiYI 17560 1397 1860 2041 2179 2461 2861 4245 4524 4690 4708 5079 5713 5788 6021 6081 6108 BslI 17560 1397 1860 2041 2179 2461 2861 4245 4524 4690 4708 5079 5713 5788 6021 6081 6108 BsmA
14、I 91389 2093 2174 2470 2523 2701 2993(c) 3769 5080 BsmFI 4129 1955 2619(c) 5937(c) BsmI 398(c) 1158 1565 Bsp1286I 9439 548 631 2069 3167 3252 4413 4911 5418 BspDI 1531 BspEI 21161 5876 BspHI 22995 4003 BspMI 12280(c) BspWI 2441 63 413 453 671 863 943 1506 1895 2536 2623 3717 4105 4677 4791 4856 4906
15、 5174 5467 5540 5757 5811 5820 5916 BsrBI 32827 2993(c) 4794(c) BsrDI 530(c) 1184 3601 3775(c) 5378(c) BsrFI 675 674 2165 2482 3750 5299 BsrGI 3726 2009 2411 BsrI 2458 446 832(c) 972 1412(c) 1949 2062(c) 2158(c) 2282(c) 2311 2764 3190 3360(c) 3629 3672 3790 4196 4308(c) 4321(c) 4949(c) 4982 5806(c) 5824(c) 5858 BssHII 31659 5146 5831 Bst1107I 21700 4954 BstEII 178 BstNI 15422 671 863 1342 1398 2058 2614 2724 2892 4564 4577 4698 5434 5566 5907 BstUI 24138 1614 1661 2209 2478 2628 3276 3769 4099 4680 5021 5124 5126 5146 5148 5150 5349 5455 5508 5596 5833 5835
