pJL2E1/OR 6565 25 EcoRI 161 XbaI 190 NcoI 196 BssHII 718 AatI 1517 EcoRV 1599 XbaI 1681 NruI 1718 EcoRI 1766 NcoI 2336 NarI 2374 BsmI 2510 Sse8387 3138 NotI 3299 XmaIII 3300 KpnI 3312 Esp3I 3463 NaeI 3485 NheI 3522 NcoI 3746 AvrII 3966 HindIII 4224 ScaI 4718 PvuI 4829 EcoRI 6445 8 tac promoter-TEtac promoter and transl. enhancer 31 161 cw N hP450 2E1hP450 2E1 cDNA sequence 198 1677 cw N ompA signal pepompA signal peptide 1702 1764 cw N reductaserat reductase cDNA 1765 3832 cw N 5S5S RNA 4312 4423 cw N rrnB T1 T2rrnB transcription terminators 4425 4648 cw N Ampb-lactamase 4659 5274 cw N pUC ORIorigin of replication 5913 6088 cw N 1 T7 gene 10 translational enhancer (TE) 166 S sequence AATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGAATTCTGCAGTTTAACTTTAGTAAGGAGTCTAGACCATGGCTGCCCTCGGAGTGACCGTGGCCCTGCTGGTGTGGGCGGCCTTCCTCCTGCTGGTGTCCATGTGGAGGCAGGTGCACAGCAGCTGGAATCTGCCCCCAGGCCCTTTCCCGCTTCCCATCATCGGGAACCTCTTCCAGTTGGAATTGAAGAATATTCCCAAGTCCTTCACCCGGTTGGCCCAGCGCTTCGGGCCGGTGTTCACGCTGTACGTGGGCTCGCAGCGCATGGTGGTGATGCACGGCTACAAGGCGGTGAAGGAAGCGCTGCTGGACTACAAGGACGAGTTCTCGGGCAGAGGCGACCTCCCCGCGTTCCATGCGCACAGGGACAGGGGAATCATTTTTAATAATGGACCTACCTGGAAGGACATCCGGCGGTTTTCCCTGACCACCCTCCGGAACTATGGGATGGGGAAACAGGGCAATGAGAGCCGGATCCAGAGGGAGGCCCACTTCCTGCTGGAAGCACTCAGGAAGACCCAAGGCCAGCCTTTTGACCCCACCTTCCTCATCGGCTGCGCGCCCTGCAACGTCATAGCCGACATCCTCTTCCGCAAGCATTTTGACTACAATGATGAGAAGTTTCTAAGGCTGATGTATTTGTTTAATGAGAACTTCCACCTACTCAGCACTCCCTGGCTCCAGCTTTACAATAATTTTCCCAGCTTTCTACACTACTTGCCTGGAAGCCACAGAAAAGTCATAAAAAATGTGGCTGAAGTAAAAGAGTATGTGTCTGAAAGGGTGAAGGAGCACCATCAATCTCTGGACCCCAACTGTCCCCGGGACCTCACCGACTGCCTGCTCGTGGAAATGGAGAAGGAAAAGCACAGTGCAGAGCGCTTGTACACAATGGACGGTATCACCGTGACTGTGGCCGACCTGTTCTTTGCGGGGACAGAGACCACCAGCACAACTCTGAGATATGGGCTCCTGATTCTCATGAAATACCCTGAGATCGAAGAGAAGCTCCATGAAGAAATTGACAGGGTGATTGGGCCAAGCCGAATCCCTGCCATCAAGGATAGGCAAGAGATGCCCTACATGGATGCTGTGGTGCATGAGATTCAGCGGTTCATCACCCTCGTGCCCTCCAACCTGCCCCATGAAGCAACCCGAGACACCATTTTCAGAGGATACCTCATCCCCAAGGGCACAGTCGTAGTGCCAACTCTGGACTCTGTTTTGTATGACAACCAAGAATTTCCTGATCCAGAAAAGTTTAAGCCAGAACACTTCCTGAATGAAAATGGAAAGTTCAAGTACAGTGACTATTTCAAGCCATTTTCCACAGGAAAACGAGTGTGTGCTGGAGAAGGCCTGGCTCGCATGGAGTTGTTTCTTTTGTTGTGTGCCATTTTGCAGCATTTTAATTTGAAGCCTCTCGTTGACCCAAAGGATATCGACCTCAGCCCTATACATATTGGGTTTGGCTGTATCCCACCACGTTACAAACTCTGTGTCATTCCCCGCTCATGAGTCTAGATAACGAGGGCAAAAAATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCGGAATTCCCGGGGATCCGACCAACATGGGGGACTCTCACGAAGACACCAGTGCCACCATGCCTGAGGCCGTGGCTGAAGAAGTGTCTCTATTCAGCACGACGGACATGGTTCTGTTTTCTCTCATCGTGGGGGTCCTGACCTACTGGTTCATCTTTAGAAAGAAGAAAGAAGAGATACCGGAGTTCAGCAAGATCCAAACAACGGCCCCACCCGTCAAAGAGAGCAGCTTCGTGGAAAAGATGAAGAAAACGGGAAGGAACATTATCGTATTCTATGGCTCCCAGACGGGAACCGCTGAGGAGTTTGCCAACCGGCTGTCCAAGGATGCCCACCGCTACGGGATGCGGGGCATGTCCGCAGACCCTGAAGAGTATGACTTGGCCGACCTGAGCAGCCTGCCTGAGATCGACAAGTCCCTGGTAGTCTTCTGCATGGCCACATACGGAGAGGGCGACCCCACGGACAATGCGCAGGACTTCTATGACTGGCTGCAGGAGACTGACGTGGACCTCACTGGGGTCAAGTTTGCTGTATTTGGTCTTGGGAACAAGACCTATGAGCACTTCAATGCCATGGGCAAGTATGTGGACCAGAGGCTGGAGCAGCTTGGCGCCCAGCGCATCTTTGAGTTGGGCCTTGGTGATGATGACGGGAACTTGGAAGAGGATTTCATCACGTGGAGGGAGCAGTTCTGGCCAGCTGTGTGCGAGTTCTTTGGGGTAGAAGCCACTGGGGAGGAGTCGAGCATTCGCCAGTATGAGCTCGTGGTCCACGAAGACATGGACGTAGCCAAGGTGTACACGGGTGAGATGGGCCGTCTGAAGAGCTACGAGAACCAGAAACCCCCCTTCGATGCTAAGAATCCATTCCTGGCTGCTGTCACCGCCAACCGGAAGCTGAACCAAGGCACTGAGCGGCATCTAATGCACCTGGAGTTGGACATCTCAGACTCCAAGATCAGGTATGAATCTGGAGATCACGTGGCTGTGTACCCAGCCAATGACTCAGCCCTGGTCAACCAGATTGGGGAGATCCTGGGAGCTGACCTGGATGTCATCATGTCTCTAAACAATCTCGATGAGGAGTCAAACAAGAAGCATCCGTTCCCCTGCCCCACCACCTACCGCACGGCCCTCACCTACTACCTGGACATCACTAACCCGCCACGCACCAATGTGCTCTACGAACTGGCACAGTACGCCTCAGAGCCCTCGGAGCAGGAGCACCTGCACAAGATGGCGTCATCCTCAGGCGAGGGCAAGGAGCTGTACCTGAGCTGGGTGGTGGAAGCCCGGAGGCACATCCTAGCCATCCTCCAAGACTACCCATCACTGCGGCCACCCATCGACCACCTGTGTGAGCTGCTGCCACGCCTGCAGGCCCGATACTACTCCATTGCCTCATCCTCCAAGGTCCACCCCAACTCCGTGCACATCTGTGCCGTGGCCGTGGAGTACGAAGCGAAGTCTGGCCGAGTGAACAAGGGGGTGGCCACTAGCTGGCTTCGGGCCAAGGAACCAGCAGGCGAGAATGGCGGCCGCGCCCTGGTACCCATGTTCGTGCGCAAATCTCAGTTCCGCTTGCCTTTCAAGTCCACCACACCTGTCATCATGGTGGGCCCCGGCACTGGGATTGCCCCTTTCATGGGCTTCATCCAGGAACGAGCTTGGCTTCGAGAGCAAGGCAAGGAGGTGGGAGAGACGCTGCTATACTATGGCTGCCGGCGCTCGGATGAGGACTATCTGTACCGTGAAGAGCTAGCCCGCTTCCACAAGGACGGTGCCCTCACGCAGCTTAATGTGGCCTTTTCCCGGGAGCAGGCCCACAAGGTCTATGTCCAGCACCTTCTGAAGAGAGACAGGGAACACCTGTGGAAGCTGATCCACGAGGGCGGTGCCCACATCTATGTGTGCGGGGATGCTCGAAATATGGCCAAAGATGTGCAAAACACATTCTATGACATTGTGGCTGAGTTCGGGCCCATGGAGCACACCCAGGCTGTGGACTATGTTAAGAAGCTGATGACCAAGGGCCGCTACTCACTAGATGTGTGGAGCTAGGAGCTACCACCCTCCCACCCCTCGCTCCCTGTAATCACCTAACTTCTGCCGACCTCCACCTCTGGTGGTTCCTGCCTGGCCTGGACACAGGGAGGCCCAGGGACTGACTCCTCCTGGCCTGAGTGGTGCCCTCCTGGGCCCCTAGGCAGAGCCCGGTCCATTGTATCAGGCAGCCCAGCCCCAGGGCACATGGCAAGAGGGACTGGACCCACCTTTGGGTGATGGGTGCCTTAGGTCCTCTGCAGATCCCGCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGTGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTGACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGCCCGGGGATCGATCCGGAGCTTATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCAT description Human P450 2E1 and rat reductase in pJL2 annotations ; Constructed by Jinsheng Dong from pJL/h2E1 [Nco I-Xba I] and ;+ pINOR [Xba I-Hind III] and cloned into pJL2 [Nco I-Hind III ;+]. 23 bp separate 2E1 termination codon and ATG of ompA sig ;+nal peptide, which is fused to the N-terminus of the reducta ;+se cDNA. This includes a second in-frame termination codon ;+and a ribosome binding site 12 bp upstream of ompA ATG. Aft ;+er signal peptide cleavage, 8 amino acids remain attached to ;+ reductase N-terminus. ; ; (This contains the corrected PCR-generated 2E1 sequence)