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ATGGCAAAAGATCGTCGCAGTAATGAAGCTGAAAATATCGCTGTTGTTGAAAAACCGCTGAAATCGGACCGCTTCTTTA

AtISA1. ATGGCAAAAGATCGTCGCAGTAATGAAGCTGAAAATATCGCTGTTGTTGAAAAACCGCTGAAATCGGACCGCTTCTTTA TCTCTGATGGTCTGCCGAGTCCGTTCGGCCCGACCGTTCGTGATGACGGTGTGAATTTTTCTGTTTATAGTACCAACTCT GTCAGTGCGACGATTTGCCTGATCTCCCTGTCAGACCTGCGCCAGAATAAAGTGACCGAAGAAATTCAACTGGACCCGAG

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ATGGCAAAAGATCGTCGCAGTAATGAAGCTGAAAATATCGCTGTTGTTGAAAAACCGCTGAAATCGGACCGCTTCTTTA

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  1. AtISA1 ATGGCAAAAGATCGTCGCAGTAATGAAGCTGAAAATATCGCTGTTGTTGAAAAACCGCTGAAATCGGACCGCTTCTTTA TCTCTGATGGTCTGCCGAGTCCGTTCGGCCCGACCGTTCGTGATGACGGTGTGAATTTTTCTGTTTATAGTACCAACTCT GTCAGTGCGACGATTTGCCTGATCTCCCTGTCAGACCTGCGCCAGAATAAAGTGACCGAAGAAATTCAACTGGACCCGAG CCGTAACCGCACGGGCCATGTGTGGCACGTTTTTCTGCGTGGTGACTTCAAAGATATGCTGTACGGTTATCGCTTTGATG GCAAATTCAGTCCGGAAGAAGGCCATTATTACGACAGCTCTAATATCCTGCTGGACCCGTATGCAAAAGCTATTATCTCC CGTGATGAATTCGGCGTGCTGGGTCCGGATGACAACTGCTGGCCGCAGATGGCGTGTATGGTTCCGACCCGTGAAGAAGA ATTTGACTGGGAAGGTGATATGCACCTGAAACTGCCGCAAAAAGACCTGGTTATTTACGAAATGCATGTCCGTGGCTTTA CCCGCCACGAAAGTTCCAAAATCGAATTCCCGGGCACGTATCAGGGCGTGGCCGAAAAACTGGATCATCTGAAAGAACTG GGTATTAATTGCATCGAACTGATGCCGTGTCACGAATTCAACGAACTGGAATACTACAGTTACAACACCATTCTGGGCGA TCATCGTGTTAACTTTTGGGGTTACTCGACGATTGGCTTTTTCAGCCCGATGATCCGTTATGCGTCTGCCTCATCGAACA ATTTTGCAGGCCGCGCTATCAACGAATTCAAAATCCTGGTTAAAGAAGCGCATAAACGTGGTATTGAAGTCATCATGGAT GTGGTTCTGAATCACACCGCCGAGGGTAACGAAAAAGGCCCGATCTTTTCTTTCCGCGGCGTCGATAACAGTGTGTATTA CATGCTGGCGCCGAAGGGTGAATTTTACAATTATAGCGGCTGCGGTAACACCTTCAATTGTAACCATCCGGTCGTGCGTC AGTTTATTCTGGATTGCCTGCGCTACTGGGTGACGGAAATGCACGTTGACGGTTTTCGTTTCGATCTGGGCTCCATCATG TCACGCAGCTCTAGTCTGTGGGATGCGGCCAATGTCTATGGTGCGGACGTGGAAGGCGATCTGCTGACCACGGGTACCCC GATTAGTTGTCCGCCGGTCATTGACATGATCTCCAACGATCCGATTCTGCGTGGCGTGAAACTGATCGCAGAAGCGTGGG ATGCCGGCGGTCTGTACCAAGTGGGCATGTTTCCGCATTGGGGCATTTGGAGCGAATGGAATGGTAAATTTCGTGACGTT GTCCGCCAATTCATCAAAGGCACCGATGGTTTTTCAGGCGCGTTCGCCGAATGCCTGTGTGGTTCGCCGAATCTGTATCA GGGCGGTCGCAAACCGTGGCATAGCATTAACTTTATCTGCGCACACGACGGCTTCACCCTGGCTGATCTGGTGACGTACA ACAATAAAAACAATCTGGCAAATGGTGAAGAAAACAATGACGGCGAAAATCACAACTATAGCTGGAACTGTGGTGAAGAA GGCGATTTTGCTTCGATTAGCGTTAAACGTCTGCGCAAACGTCAGATGCGTAACTTTTTCGTTAGCCTGATGGTCTCTCA AGGCGTGCCGATGATCTACATGGGTGACGAATATGGCCATACCAAAGGCGGTAACAATAACACGTACTGCCACGATAACT ACATGAACTACTTCCGCTGGGACAAAAAAGAAGAAGCGCATAGTGATTTCTTTCGCTTTTGCCGTATTCTGATCAAATTC CGTGACGAATGTGAATCCCTGGGCCTGAATGATTTTCCGACCGCGAAACGTCTGCAGTGGCATGGTCTGGCACCGGAAAT TCCGAACTGGTCGGAAACGAGCCGTTTTGTGGCCTTCAGTCTGGTCGATTCCGTGAAAAAAGAAATCTACGTGGCGTTCA ATACCTCCCATCTGGCCACGCTGGTTTCACTGCCGAACCGTCCGGGTTACCGTTGGGAACCGTTTGTGGATACCTCTAAA CCGAGTCCGTATGACTGTATTACCCCGGATCTGCCGGAACGTGAAACGGCAATGAAACAGTATCGCCACTTTCTGGATGC TAATGTTTACCCGATGCTGTCATATTCCTCAATTATCCTGCTGCTGTCGCCGATTAAAGATCCGA

  2. AtISA2 ATGGCACGTCTGTTTACCGGCCGCAAAAACAAAATCAAACTGGGTTCGGAAACCCTGATGTTTACGCGCAAACGTTTCA TGGGTGATCTGGTGACCTCAGCGCTGCAGTCGTACCAATTTAGCAAAATTTGCGCGTCCAAAACGTCAATCGAACTGCGT GAAGCCCTGTCGAGCCGTCGCGCAGAAGCTGATGACCTGAAAAAAGTTACCTCTTACAGTTTCCGTACGAAAGCGGGCGC CCTGGTCAAAGTGAAAGTTGAGAAAAAACGTGAAAAATACAGCATTCTGGTCTATGTGTCTAGTCTGGAACTGTCCGGCG ATGACAAATCACGTCTGGTTATGGTCTGGGGTGTGTACCGCTCTGATTCCTCATGTTTTCTGCCGCTGGACTTCGAAAAT TCGAGCCAGGATAGTCAAACCCATACCACGGAAACCACGTTCGTTAAATCTAGTCTGTCCGAACTGATGCTGGGTCTGGA ATTTGATGGCAAAGAATCGCCGTTTTATCTGAGCTTCCACCTGAAACTGGTTAGCGGTCGTGACCCGGATGGCCAGGAAA TGCTGACCCATCGCGACACGGATTTTTGCATTCCGGTTGGTTTTACCGCCGGTCACCCGCTGCCGCTGGGTCTGTCCTCA GGCCCGGATGACGATTCCTGGAATTTTTCGTTTTTCAGCCGTTCGAGCACGAACGTGGTTCTGTGTCTGTACGACGATTC AACCACGGACAAACCGGCGCTGGAACTGGATCTGGACCCGTATGTGAATCGCACCGGCGATGTTTGGCATGCCTCAGTCG ACAACACGTGGGATTTTGTTCGTTACGGTTATCGCTGCAAAGAAACCGCACACTCGAAAGAAGACGTGGATGTTGAAGGC GAACCGATTGTTCTGGACCCGTACGCTACGGTCGTGGGCAAATCCGTCTCACAGAAATATCTGGGTTCGCTGAGCAAATC TCCGAGTTTTGACTGGGGTGAAGATGTGAGCCCGAATATCCCGCTGGAAAAACTGCTGGTGTATCGTCTGAACGTTAAAG GCTTCACCCAACATCGCTCTAGTAAACTGCCGTCGAATGTGGCAGGCACGTTTAGCGGCGTTGCTGAAAAAGTCTCTCAC CTGAAAACCCTGGGTACGAACGCCGTTCTGCTGGAACCGATTTTTAGCTTCTCAGAACAGAAAGGCCCGTACTTTCCGTT CCATTTCTTTAGCCCGATGGATATCTATGGTCCGTCGAATAGCCTGGAATCTGCAGTCAACAGTATGAAAGTCATGGTGA AAAAACTGCATAGCGAAGGTATTGAAGTTCTGCTGGAAGTTGTCTTTACCCACACGGCAGATTCTGGTGCTCTGCGTGGC ATCGACGATTCCTCATATTACTATAAAGGCCGCGCAAATGACCTGGATTCTAAAAGTTACCTGAATTGCAACTATCCGGT GGTTCAGCAACTGGTGCTGGAAAGCCTGCGTTATTGGGTCACCGAATTCCATGTGGATGGCTTTTGTTTCATTAATGCGA GCAGCCTGCTGCGTGGTGTTCATGGTGAACAGCTGTCTCGCCCGCCGCTGGTTGAAGCAATTGCTTTTGATCCGCTGCTG GCCGAAACCAAACTGATCGCCGACTGTTGGGACCCGCTGGAAATGATGCCGAAAGAAGTGCGTTTCCCGCATTGGAAACG CTGGGCGGAACTGAACACCCGCTACTGCCGTAATGTCCGCAACTTTCTGCGTGGTCGTGGTGTGCTGAGCGATCTGGCAA CCCGCATTTGTGGCTCTGGTGACGTTTTCACGGATGGCCGTGGTCCGGCTTTTAGCTTCAATTATATTTCTCGCAACAGT GGCCTGTCCCTGGTTGATATCGTCTCCTTTTCAGGTCCGGAACTGGCCTCAGAACTGTCGTGGAATTGCGGTGAAGAAGG CGCGACCAACAAATCTGCCGTGCTGCAGCGTCGCCTGAAACAAATCCGTAATTTTCTGTTCATCCAGTACATCAGTCTGG GCGTTCCGGTCCTGAACATGGGTGACGAATGTGGCATCTCCACCCGTGGTTCACCGCTGCTGGAAAGCCGCAAACCGTTT GATTGGAATCTGCTGGCGTCTGCCTTCGGCACCCAGATTACGCAATTTATCTCGTTCATGACCAGCGTGCGTGCCCGTCG CAGCGACGTTTTTCAGCGTCGCGATTTCCTGAAACCGGAAAATATTGTGTGGTATGCCAACGACCAAACCACGCCGAAAT GGGAAGATCCGGCATCTAAATTTCTGGCTCTGGAAATCAAATCGGAAAGCGAAGAAGAAGAAACCGCGAGTCTGGCCGAA CCGAATGAACCGAAATCCAACGACCTGTTTATTGGTTTCAACGCGTCTGATCATCCGGAAAGTGTCGTGCTGCCGTCCCT GCCGGATGGCTCAAAATGGCGTCGCCTGGTGGATACCGCACTGCCGTTTCCGGGCTTTTTCAGCGTTGAAGGCGAAACGG TTGTCGCTGAAGAACCGCTGCAGCAACTGGTGGTTTATGAAATGAAACCGTACTCTTGCACCCTGTTCGAAACCATCAAT ACCACCGCCC ZmISA1 ATGGCTGAAGCAGTCCAAGCAGAAGAAGACGATGATGATGATGATGAAGAAGTGGCTGAAGAACGTTTTGCCCTGGGTG GTGCGTGCCGTGTGCTGGCGGGTATGCCGGCACCGCTGGGTGCAACCGCACTGCGCGGCGGTGTCAACTTCGCTGTGTAT AGCTCTGGTGCGTCCGCGGCCTCACTGTGTCTGTTTGCCCCGGGCGACCTGAAAGCAGATCGTGTTACCGAAGAAGTCCC GCTGGACCCGCTGCTGAATCGTACCGGTAACGTTTGGCATGTCTTTATTCACGGCGACCAGCTGCACGGTATGCTGTATG GCTACCGCTTTGATGGTGTGTTCGCCCCGGAACGTGGCCAATATTACGACGTTAGCAATGTGGTTGTCGATCCGTATGCT AAAGCGGTGGTTTCGCGCGGTGAATATGGTGTTCCGGCTCCGGGCGGTAGCTGCTGGCCGCAGATGGCGGGTATGATCCC GCTGCCGTACAACAAATTCGACTGGCAGGGTGATCTGCCGCTGGGCTATCATCAAAAAGACCTGGTGATTTACGAAATGC ATCTGCGTGGTTTTACCAAACACAATAGTTCCAAAACCAAACATCCGGGCACGTATATTGGCGCGGTCAGCAAACTGGAT CACCTGAAAGAACTGGGCGTGAATTGCATCGAACTGATGCCGTGTCATGAATTCAACGAACTGGAATACTTCTCATCGAG CTCTAAAATGAACTTTTGGGGTTACTCGACCATTAACTTTTTCAGCCCGATGGCACGCTATAGTTCCTCAGGCATTCGTG ATAGCGGTTGTGGCGCGATCAACGAATTTAAAGCCTTCGTCCGCGAAGCACACAAACGTGGCATTGAAGTGATCATGGAT GTCGTGTTCAATCATACGGCCGAGGGTAACGAAAAAGGCCCGATTCTGTCGTTTCGCGGTATCGATAATAGCACCTATTA CATGCTGGCACCGAAAGGCGAATTTTATAATTACTCTGGTTGCGGCAACACGTTCAATTGTAACCACCCGGTTGTCCGCG AATTTATTGTGGATTGCCTGCGTTACTGGGTGACCGAAATGCATGTTGATGGTTTTCGCTTCGACCTGGCGTCTATCCTG ACGCGTGGCTGTAGTCTGTGGGACCCGGTTAACGTCTATGGTTCGCCGATGGAAGGCGATATGATTACCACCGGTACCCC GCTGGTTGCGCCGCCGCTGATTGATATGATCAGTAATGACCCGATTCTGGGCAACGTGAAACTGATCGCCGAAGCATGGG ATGCCGGCGGTCTGTACCAGGTTGGTCAATTCCCGCATTGGAATGTCTGGAGCGAATGGAACGGCAAATATCGCGACACC GTGCGTCAGTTTATCAAGGGTACGGATGGCTTTGCTGGTGCGTTCGCCGAATGCCTGTGTGGTTCGCCGCAGCTGTATCA AGCAGGCGGTCGTAAACCGTGGCACAGCATTAATTTTGTTTGCGCCCATGACGGCTTCACCCTGGCGGATCTGGTGACGT ATAATTCGAAATACAATCTGAGCAACGGTGAAGACAACCGCGATGGCGAAAATCACAACCTGTCTTGGAATTGCGGCGAA GAAGGTGAATTTGCCTCTCTGAGTGTGCGTCGCCTGCGTAAACGCCAGATGCGTAACTTTTTCGTGTGTCTGATGGTTAG CCAAGGTGTCCCGATGTTCTATATGGGTGACGAATACGGCCATACCAAAGGCGGTAACAATAACACGTACTGTCATGATC ACTATGTGAATTACTTTCGCTGGGACAAAAAAGAAGAACAGTCGAGCGATCTGTATCGCTTTTGCCGTCTGATGACCAAA TTCCGCAAAGAATGTGAATCGCTGGGTCTGGAAGATTTTCCGACGAGCGAACGTCTGAAATGGCATGGCCACCAACCGGA AAAACCGGACTGGTCGGAAGCTAGCCGTTTTGTTGCGTTCACCATGAAAGATGAAACGAAAGGCGAAATTTATGTGGCGT TTAACACCTCTCACCTGCCGGTGGTTGTCGGTCTGCCGGAACGCAGTGGCTTCCGTTGGGAACCGGTGGTTGATACGGGT AAAGAAGCACCGTACGACTTTCTGACCGATGGTCTGCCGGACCGTGCTGTGACCGTTTACCAGTTCTCTCATTTCCTGAA CAGTAACCTGTATCCGATGCTGAGCTACTCTAGTATTATCCTGGTCCTGCGTCCGGATGTGA

  3. ZmISA2 ATGGCAGCTGCGCGCAGTTATCGTTACCGCTTTCGTACCGATGACGATGGCGTCGTGGATGTCGCAGTGGCTGGCAAAG ACGGTGATGCGGGCTATGTTGTCGCAATTGAAGCTCCGACCCACGGTCAGCGCGGCGGTCTGGTTCTGCGCCCGGCAGGT TCTGGTGAAGGTGTTCCGCTGGCACCGGCGGCACCGGGCGGTGCGCTGGTCGCCGAACTGTCCTATGACGTGGCGCGTGC CCCGTTTCACGTTTCATTCACCCTGGCAGATGCTATGGGTGCGGAAATTCGCACCCATCGTGGCACGTCCTTTCGTGTTC CGGTTGGTGTGGGTCGTGGTTGTCCGTCACCGCTGGGTCTGTCCCAGTCAAAAGATGGCGCTGCGAATTTCGCTGTGTAC AGCAAAATTGCGAAGGGTATGGTTCTGTGTCTGTTTGGCGGTGGCGGTGGCGATGGTCCGGCACTGGAAATCGAACTGGA CCCGTATGTTCACCGCACCGGTGATGTGTGGCATGTTTCTATGGAAAGTGTGGAAGGCTATGCCCGCTACGGTTTCCGTT CCGGCCTGTTTGCAATGTTCGGCATTGATCGTCCGCTGCTGGACCCGTATGCTAAAGTTATCGGTGATTTTGTCGCGGGC GACAGCGTGGACGAAGATGGTCTGGCGGTGCCGTCCATTCGTTGCCTGGCCTCACTGAAAAATGCACCGAACTACGATTG GGGCCGCGACAAACATCCGTGTCTGCCGCTGCAGAAACTGGTGGTTTATCGTGCAAACGTTGCTCTGTTTACCAAAGACC GCTCCTCACGTCTGGCTGATAATGCCGCAGGCACGTTCTCCGGCATGTCAGCGAAAGTGGAACATTTTCGCCACCTGGGT GTCAACGCCGTGCTGCTGGAACCGGTGTTTCCGTTCCACCAAGTTAAAGGCCCGTATTTTCCGTACCATTTCTTTAGCCC GATGAGCCTGTATTCGAGCGAATGCTCTAGTGTGTCCGCGATCAAATCAATGAAAGATATGGTTAAAACCATGCACCGCA ATGGTATCGAAGTCCTGCTGGAAGTCGTGTTTACCCATACGGCAGAAGGTGGCGCTGAATGTCAGATGATTAGCCTGCGT GGTATCGACGGCTCCTCATATTACATTGCGGATGGTATCGCCGGCTGCAAAGCATCGGTCCTGAATTGTAACCACCCGGT GACCCAAAAACTGATTCTGGATAGCCTGCGCCATTGGGTTCTGGATTTCCACGTCGACGGCTTTTGCTTCATTAATGCAC CGTTTCTGGTGCGTGGTCCGGGTGGCGAAGGTCTGTCTCGTCCGCCGCTGCTGGAAGCCATTGCCTTTGATCCGGTTCTG AGTAAAACCAAAATTATCGCAGACCCGTGGTCCCCGCTGGATATCTCAAACGTGCAGTTTCCGTTCCCGCATTGGAAACG TTGGGCGGAAATGAATACCCGCTTTTCTATGGATGTTCGTAAATTCCTGAAAGGTGAAGCCCTGATTTCTGACCTGGCAA CGCGCCTGTGTGGTAGTGGCGACCTGTTTAGCAGCCGTGGCCCGGCCTTTTCTTTCAATTACGTGTCTCGCAACAGTGGC CTGACCCTGGTTGACCTGGTCAGTTTTTCTAGTGATGAACTGGCCTCCGAATTCTCATGGAACTATGGTGAAGAAGGCCC GAGTGAAAATAACGCAGTTTTCCAGACCCGCCTGCGTCAAATCCATAATTTTCTGTTCATCCTGTTCATCTCCCTGGGCA TCCCGGTGCTGAATATGGGTGACGAATGCGGCAACTCAGCTGCGGGTTCGACCAGCTATAAAGATCGTGGCCTGCTGAAC TGGAAAGCTCTGAAAACCGCGTTTGTGAAAGAAGTTACGGGTTTTATCTCGTTCCTGAGCGCTCTGCGCAGCCGTCGCGC CGATATTTTTCAGCGTTGTGAATTCCTGAAACTGGAAAATATCCACTGGTACGGCTCTGACCTGAGTGAACCGTGCTGGG AAGATCCGACCTCCAATTTCCTGTGTCTGCATATTAACGCGGAACTGGATGAAAAACTGCCGGACTCAACGGGTGGCGAT CTGTATATTTGCTTTAATGCCAACGAAGAATCTGCAAGTGCTACCCTGCCGGCGATCGCCGAAGGTAGCATGTGGCTGCG TCTGGTTGATACGTCCCTGGCGTTCCCGGGCTTTTTCAGCCGTGGCAGCAGCCATGAAACCCACCAGGTGCTGGGCTTTT CGAGCTATCAAGTCAAAGCCCATAGCTGCGTCCTGTTCGAAAGCAAACGTGTCCTGAGCC Supplemental Figure S1: Top panel : this picture exemplifies the construction of the vectors designed for expression in E. coli. ISA1 and ISA2 genes (from maize or Arabidopsis) optimized for expression in E. coli (see below) were synthesized in the form of an operon including START and STOP codons for each ORF and integrated in vector pUC57. From this vector, several constructions were made by transferring ISA1 or ISA2 or ISA1 and ISA2 into the destination vector pBE1343. ISA1 was transferred after digestion with BglII and HindIII and ISA2 was transferred after the digestion with BamH1 and XhoI. In both cases, the corresponding proteins were tagged with the 8xHis sequence (the STOP codon being placed downstream of the tag). The transfer of both ISA1 and ISA2 as a operon into pBE1343 was achieved by the digestion with both BglII and XhoI. In that case, only ISA2 was tagged with the 8xHis sequence. Sequences of maize (Zm) and Arabidopsis (At) ISA1 and ISA2 structural genes optimized for expression in E. coliare shown from the start codon to the last codon that specifies the protein. The stop codon is not shown here, as it is included in the expression vector.

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