1、pEASYBlunt clonning vector 载体说明书WORD版pEASY-Blunt Complete sequenceAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCA
2、AGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTGCCCTTAAGGGCAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAA
3、TGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTC
4、TTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTA
5、CATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGT
6、CAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCCCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATC
7、GCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCT
8、TCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCAC
9、TTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGA
10、AGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGA
11、TGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTT
12、GCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG
13、ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTC
14、TGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGLacZ fragment: bases 1-548M13 reverse priming site: bases 205-221Multiple cloning site: bases 234-358T7 promoter/priming site: bases 364-381M13 Forward (-20) priming site: bases 389-404f1 origin: bases 546
15、-983Kanamycin resistance ORF: bases 1317-2111Ampicillin resistance ORF: bases 2129-2989pUC origin: bases 3134-3807pEASY-Blunt Restriction Map Enzyme# of cutspositionsAcc65I (1)ggtacc246AceIII (3)cagctc949, 2630, 3870AciI (47)ccgc88, 191, 329, 561, 575, 1014, 1159, 1515, 1579, 1680AclI (2)aacgtt2319,
16、 2692AcuI (4)ctgaag3283, 1576, 2008, 2235AflII (1)cttaag297AflIII (2)acrygt1021, 3810AhdI (1)gacnnnnngtc2922AloI (1)gaacnnnnnntcc835AluI (22)agct58, 122, 217, 239, 251, 477, 691, 948, 1069, 1122AlwI (16)ggatc258, 1700, 2292, 3072, 3169, 257, 1263, 1280, 1310, 1621AlwNI (2)cagnnnctg1073, 3399ApaBI (1
17、)gcannnnntgc1002ApaI (1)gggccc356ApaLI (2)gtgcac2250, 3496ApoI (3)raatty964, 975, 986AseI (3)attaat52, 111, 2746AvaI (1)cycgrg338AvaII (3)ggwcc1966, 2557, 2779BamHI (1)ggatcc258BanI (7)ggyrcc137, 246, 740, 1202, 1450, 1485, 2969BanII (4)grgcyc252, 356, 706, 1812BbeI (2)ggcgcc1202, 1450BbvI (18)gcagc
18、1002, 1410, 1594, 1687, 2500, 3194, 3400, 57, 457, 530BccI (11)ccatc771, 1243, 1269, 1334, 1762, 2470, 2757, 2881, 319, 789BceAI (6)acggc397, 1424, 3324, 270, 737, 1860BcefI (6)acggc397, 1424, 3324, 270, 737, 1860BcgI (1)cgannnnnntgc2401BciVI (2)gtatcc1659, 3601BclI (1)tgatca1292BfaI (7)ctag263, 349
19、, 626, 1150, 2727, 3062, 3315BfrBI (1)atgcat345BglI (2)gccnnnnnggc539, 2803BglII (1)agatct1287Bme1580I (5)gkgcmc356, 1393, 1486, 2250, 3496BmrI (5)actggg420, 1075, 1156, 1390, 2872BpmI (1)ctggag2832BpuEI (5)cttgag1842, 3442, 2297, 3165, 3704BsaAI (3)yacgtr779, 1022, 1753BsaBI (3)gatnnnnatc319, 1286,
20、 1310BsaHI (3)grcgyc1202, 1450, 2381BsaI (1)ggtctc2850BsaJI (7)ccnngg142, 430, 1056, 1260, 1614, 1883, 3650BsaWI (5)wccggw1184, 1482, 2626, 3457, 3604BsaXI (2)acnnnnnctcc833, 28BscAI (11)gcatc1418, 1673, 1889, 2230, 2670, 3722, 346, 1740, 1804, 2079BseMII (4)ctcag2960, 3126, 3535, 2420BseYI (3)cccag
21、c1197, 1955, 3506BsiEI (8)cgrycg272, 332, 508, 1357, 2401, 2550, 3473, 3897BsiHKAI (6)gwgcwc252, 1560, 1750, 2250, 2335, 3496BslI (9)ccnnnnnnngg39, 561, 887, 1431, 1975, 3331, 3610, 3776, 3794BsmAI (2)gtctc1298, 2850BsmFI (1)gggac1587Bsp1286I (11)gdgchc252, 356, 706, 1393, 1486, 1560, 1750, 1812, 22
22、50, 2335Bsp24I (1)gacnnnnnntgg3130BspCNI (4)ctcag2960, 3126, 3535, 2420BspHI (1)tcatga3090BspMI (3)acctgc1118, 1347, 1710BsrBI (5)ccgctc191, 335, 635, 2062, 3879BsrDI (3)gcaatg2863, 1677, 2681BsrFI (4)rccggy676, 1770, 1951, 2837BsrI (20)actgg277, 1032, 2446, 3394, 3407, 81, 326, 393, 419, 864BssHII
23、(1)gcgcgc1848BssSI (3)cacgag2043, 2253, 3637BstAPI (1)gcannnnntgc1002BstF5I (10)ggatg463, 1632, 1059, 1242, 1304, 1761, 1786, 2469, 2756, 2937BstKTI (25)gatc257, 507, 1263, 1280, 1286, 1291, 1310, 1621, 1699, 1780BstNI (6)ccwgg142, 430, 1836, 3649, 3662, 3783BstUI (12)cgcg33, 550, 574, 594, 970, 151
24、4, 1815, 1847, 2360, 2853BstXI (2)ccannnnnntgg284, 326BstYI (11)rgatcy258, 1264, 1287, 1622, 1868, 2275, 2292, 3060, 3072, 3158BtgI (1)ccrygg1883BthCI (34)gcngc57, 273, 330, 457, 530, 562, 576, 598, 1002, 1255BtsI (4)gcagtg2524, 94, 1129, 2496Cac8I (24)gcnngc61, 90, 343, 476, 500, 619, 633, 676, 110
25、1, 1125CdiI (7)catcg1888, 2669, 790, 1736, 1939, 2080, 2265ChaI (25)gatc257, 507, 1263, 1280, 1286, 1291, 1310, 1621, 1699, 1780CjeI (7)ccannnnnngt3228, 3783, 243, 482, 3129, 3229, 3307CjePI (4)ccannnnnnntc3277, 2538, 3010, 3130Csp6I (3)gtac245, 1754, 2438CviAII (14)catg226, 342, 1139, 1320, 1665, 1
26、851, 1882, 1908, 2474, 2510CviJI (78)rgcy41, 58, 122, 144, 165, 217, 239, 251, 271, 331DdeI (4)ctnag2420, 2960, 3126, 3535DpnI (25)gatc257, 507, 1263, 1280, 1286, 1291, 1310, 1621, 1699, 1780DraI (3)tttaaa2342, 3034, 3053DraIII (1)cacnnngtg782DrdI (3)gacnnnnnngtc828, 1478, 3708EaeI (9)yggccr42, 272,
27、 332, 396, 1357, 1531, 1922, 1949, 2529EagI (3)cggccg272, 332, 1357EarI (4)ctcttc494, 1800, 2010, 2128EciI (4)ggcgga1964, 2764, 3592, 3738Eco57MI (5)ctgrag3283, 1576, 2008, 2235, 2832EcoHI (6)ccsgg1056, 1453, 1613, 2384, 2735, 3431EcoICRI (1)gagctc252EcoO109I (1)rggnccy356EcoRV (1)gatatc315EsaBC3I (
28、9)tcga337, 744, 1562, 1718, 1742, 1778, 1940, 2266, 3710FalI (1)aagnnnnnctt1773FatI (14)catg226, 342, 1139, 1320, 1665, 1851, 1882, 1908, 2474, 2510FauI (6)cccgc89, 579, 1580, 32, 500, 633FmuI (8)ggncc355, 498, 786, 1966, 2557, 2779, 2796, 2875Fnu4HI (34)gcngc57, 273, 330, 457, 530, 562, 576, 598, 1
29、002, 1255FokI (10)ggatg463, 1632, 1059, 1242, 1304, 1761, 1786, 2469, 2756, 2937FspI (3)tgcgca528, 1551, 2697GdiII (11)cggccr42, 272, 332, 396, 1357, 1922, 1949, 272, 332, 1357HaeI (4)wggccw1531, 3334, 3786, 3797HaeII (6)rgcgcy7, 622, 630, 1202, 1450, 3566HaeIII (19)ggcc41, 271, 331, 355, 395, 497,
30、786, 928, 1356, 1530HaeIV (2)gaynnnnnrtc1628, 2922HgaI (4)gacgc3130, 3708, 549, 2380HhaI (30)gcgc6, 34, 99, 527, 551, 564, 573, 595, 621, 629Hin4I (8)gaynnnnnvtc257, 346, 1628, 1658, 2848, 2922, 1628, 2922HindIII (1)aagctt240HinfI (10)gantc46, 376, 828, 850, 1935, 2069, 2922, 3439, 3835, 3910HinP1I (30)gcgc6, 34, 99, 527, 551, 564, 573, 595, 621, 629HpaII (21)ccgg163, 675, 1056, 1183, 1354, 1431, 1453, 1481, 1612, 1702HphI (6)ggtga2202, 2443, 768, 2217, 2839, 3066Hpy188I (15)tcnga255, 721, 1266, 1289, 1415, 1759,
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