SUPPORTING INFORMATION

Transcription

1 SUPPORTING INFORMATION Investigation of the Biosynthesis of the Lasso Peptide Chaxapeptin Using an E. coli-based Production System Helena Martin-Gómez, Uwe Linne, Fernando Albericio, Judit Tulla-Puche,* and Julian D. Hegemann* corresponding authors. Julian D. Hegemann: Judit Tulla-Puche: S1

2 Supporting Information Figure S1. UV chromatograms of the media screens. UV peaks corresponding to chaxapeptin are highlighted in green boxes. In the last spectrum, a large UV peak of a different compound (shown in a gray box) overlays with where the mass of chaxapeptin is detected, thus the amount of lasso peptide produced under these conditions could not be quantified. As can be seen, the best yield is obtained when the production plasmid is expressed for 3 d at 20 C in M9 minimal medium. Furthermore, comparison of the chromatograms shows that in M9 minimal medium the background is much lower than for the richer LB and TB media. S2

3 Supporting Information Figure S2. Incubation of chaxapeptin with carboxypeptidase Y for 4 h at 25 C. Mass and retention time remain unchanged, meaning that chaxapeptin is resistant against proteolysis by carboxypeptidase Y. Supporting Information Figure S3. Incubation of chaxapeptin with thermolysin for 2 h at 70 C or 24 h at 37 C. Mass and retention time remain unchanged, showing that thermolysin is unable to degrade chaxapeptin. S3

4 Supporting Information Figure S4. Incubation of chaxapeptin for 1-4 h at 95 C. The constant retention time and lack of formation of a new peak corresponding to an unthreaded branched-cyclic peptide shows that chaxapeptin is a heat stable lasso peptide. S4

5 Supporting Information Table S1. UV-integrals obtained from LC-MS analysis of MeOH pellet extracts of the chaxapeptin production tests in different media. For determination of the relative production, the highest observed UV integral was set to 100%. medium condition UV integral relative production M9 minimal medium 1d, 37 C % M9 minimal medium 3d, 20 C % LB medium 1d, 37 C % LB medium 3d, 20 C % TB medium 1d, 37 C % TB medium 3d, 20 C mass only - Supporting Information Table S2. Oligonucleotide primers used for cloning and optimization of the cpt gene cluster. Overhangs used for Gibson Assembly are underlined. Overhangs used for SLIM are highlighted in bold. name FP_pET41a RP_pET41a FP_cptACBB RP_cptACBB FP_TermProm RP_TermProm FP_cpt-TermProm RP_cpt-TermProm FP_cptC_RBS_cptB1 RP_cptC_RBS_cptB1 FPTail_cptC_RBS_cptB1 RPTail_cptC_RBS_cptB1 FP_cptB1_RBS_cptB2 RP_cptB1_RBS_cptB2 FPTail_cptB1_RBS_cptB2 RPTail_cptB1_RBS_cptB2 sequence AGA TCT GGG TAC CGG TGG TGG CTC CGG ATG TAT ATC TCC TTC TTA AAG TTA AAC AAA ATT ATT TCT AGA GGG TAA CTT TAA GAA GGA GAT ATA CAT ATG GAA CCC CAG ATG ACT GAG CTT CAG CCG GAG GC GAG CCA CCA CCG GTA CCC AGA TCT TCA GGA AAC CGG AGG AAC GGT CAG CAA AGG CCG GGT ACC AGC TTA ATT AGC TGA GCT TGG ACT CC AT GTG ACC TCC TTA AGT TAT GCC TTT ACA CTC AAT TGG AAT AAT AAA ACT TCT GAA TTT ATC CATAACTTAAGGAGGTCACAT ATG AGC ACC GGG TTT CTT ATC CTG CCG GAC TCT GTC G TCAGCTAATTAAGCTGGTACC CTA AAA GAA GTT CAG GCC GAA GCT GTC CAG CGG CTT G CGC CTG CAC CCT GAT ATC GTC ATC GCG GAA ACC G GGG TGC AGG CGC ATC TTG TCT CTT TGG GGT GGT GC TAA AGAGGAGAAATTAACC ATG CGC CTG CAC CCT GAT ATC GTC ATC GCG GAA ACC G CAT GGTTAATTTCTCCTCT TTA GGG TGC AGG CGC ATC TTG TCT CTT TGG GGT GGT GC AGT ACC CCC GCC GCC CTG GTC AAG CGC AGA AAA C CGA GGT GCT CTC CAC TAT CTG GGC CGC GTG TAG TTG TAA AGAGGAGAAATTAACC ATG AGT ACC CCC GCC GCC CTG GTC AAG CGC AGA AAA C CAT GGTTAATTTCTCCTCT TTA CGA GGT GCT CTC CAC TAT CTG GGC CGC GTG TAG TTG Supporting Information Table S3. Recipe for the M9 vitamin mix. component amount choline chloride folic acid pantothenic acid nicotinamide myo-inositol 2.0 g pyridoxal hydrochloride thiamine riboflavin 0.1 g disodium adenosine 5'-triphosphate 0.3 g biotin 0.2 g add 300 ml ddh 2O* *After addition of ddh 2O, 10 M NaOH is slowly added until the solution becomes completely clear (typically at a ph of approximately 12). The cleared vitamin mix is then sterile filtered and stored at 4 C until used. S5

6 Supporting Information Table S4. Oligonucleotide primers used for mutation of the cpta gene in cpta_term λt0 - Prom mcjbcd _cptc_rbs_cptb1_rbs_cptb2 pet41a. SLIM overhangs are underlined and mutated residues are shown in bold. name FP_cptA_-17to-11 RP_ cpta_-17to-11 FPTail_ cpta_y-17a-p-14a sequence GAG GTG GGC GAG TTC TCG GAG GAC ACG CTC GG GGC CTC CGG CTG AAG CTC AGT CAT CTG GGG TTC C GCG GAG GCG GCC TCC CTC ATC GAG GTG GGC GAG TTC TCG GAG GAC ACG CTC GG RPTail_ cpta_y-17a-p-14a GAT GAG GGA GGC CGC CTC CGC GGC CTC CGG CTG AAG CTC AGT CAT CTG GGG TTC C FP_ cpta_-8to-2 CTC GGC TTC GGA AGC AAG CCG CTG GAC AGC RP_ cpta_-8to-2 CAC CTC GAT GAG GGA CGG CGC CTC GTA GGC C FPTail_cptA_G-8A RPTail_cptA_G-8A FPTail_cptA_T-2A RPTail_cptA_T-2A FP_ cpta_1to8 RP_ cpta_1to8 FPTail_cptA_G1A RPTail_cptA_G1A FPTail_cptA_D8E RPTail_cptA_D8E GCG GAG TTC TCG GAG GAC ACG CTC GGC TTC GGA AGC AAG CCG CTG GAC AGC CGT GTC CTC CGA GAA CTC CGC CAC CTC GAT GAG GGA CGG CGC CTC GTA GGC C GGC GAG TTC TCG GAG GAC GCG CTC GGC TTC GGA AGC AAG CCG CTG GAC AGC CGC GTC CTC CGA GAA CTC GCC CAC CTC GAT GAG GGA CGG CGC CTC GTA GGC C AGC TTC GGC CTG AAC TTC TTT TAG GGT ACC AGC TTA ATT AGC TG GAG CGT GTC CTC CGA GAA CTC GCC CAC CTC G GCG TTC GGA AGC AAG CCG CTG GAC AGC TTC GGC CTG AAC TTC TTT TAG GGT ACC AGC TTA ATT AGC TG GTC CAG CGG CTT GCT TCC GAA CGC GAG CGT GTC CTC CGA GAA CTC GCC CAC CTC G GGC TTC GGA AGC AAG CCG CTG GAA AGC TTC GGC CTG AAC TTC TTT TAG GGT ACC AGC TTA ATT AGC TG TTC CAG CGG CTT GCT TCC GAA GCC GAG CGT GTC CTC CGA GAA CTC GCC CAC CTC G Supporting Information Table S5. Waters Synapt G2-Si settings for the IMS measurements. ESI + V resolution mode DC potentials traveling wave parameters m/z range trap collision energy 5 V source wave velocity 300 m/s capillary voltage 2.7 kv transfer collision energy 0 V source wave height 0.2 V source temperature 70 C trap DC entrance 3 V trap wave velocity 300 m/s sampling cone 20 V trap DC bias 40 V trap wave height 5 V extraction cone 5 V trap DC -1 V IMS wave velocity 350 m/s source gas flow 20 ml/min trap DC exit 0 V IMS wave height 20 V desolvation temperature 250 C IMS DC entrance 25 V transfer wave velocity 350 m/s cone gas flow 0 L/hr helium cell DC 35 V transfer wave height 5 V desolvation gas flow 500 L/hr helium exit -5 V trap gas flow 0.4 ml/min IMS bias 0 V helium cell gas flow 180 ml/min IMS DC exit 0 V IMS gas flow 70 ml/min transfer DC entrance 4 V sample infusion flow rate 30 µl/min transfer DC exit 15 V Supporting Information Table S6. UV-integrals obtained from LC-MS analysis of MeOH pellet extracts of the mutational analysis of cpta. name replicate 1 replicate 2 replicate 3 mean deviation relative production WT % 7.0% Y-17A/P-14A mass only mass only mass only G-8A % 1.8% T-2A mass only mass only mass only G1A not detected not detected not detected D8E not detected not detected not detected deviation S6

7 Sequence of the cpta_term λt0 _Prom mcjbcd _cptc_rbs_cptb1_rbs_cptb2 pet41 plasmid. The gene sequence of cpta was highlighted in red, the sequence of cptc in green, the sequence of cptb1 in light yellow, and the sequence of cptb2 in dark yellow. The kanamycin resistance cassette is marked in gray. The Term λt0 -Prom mcjbcd and RBS sequences (including spacer residues up to the start codon) are shown in blue font and underlined. TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTC CCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGT GATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATC TCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTT CAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAA TGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGG TATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAG AATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGC GAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATAT TCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAG CCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTG ATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCT TGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCA GCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTG CTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAG CGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGA GCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGG TATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACC CGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCG AAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCT GGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAG GATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCA ATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTT TACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAG GCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCG AGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG GGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCA CGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCCGCACCAACGCGC AGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGG ACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGC TAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGA AATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCG CTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAG ACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGC AGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCT CTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTT GAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGA GCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCG AGATCGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGG AACCCCAGATGACTGAGCTTCAGCCGGAGGCCTACGAGGCGCCGTCCCTCATCGAGGTGGGCGAGTTCTCGGAGGACACGCTCGGCTTCGGAAGCAAGCCGCTGGACAGCTT CGGCCTGAACTTCTTTTAGGGTACCAGCTTAATTAGCTGAGCTTGGACTCCTGTTGATAGATCCAGTAATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCC GGGCGTTTTTTATTGGTGAGAATCCAAGCTAGCCATCAATTAAGAAAAAAATTTAGCTTGTAGATAAATTCAGAAGTTTTATTATTCCAATTGAGTGTAAAGGCATAACTTAAGGA GGTCACATATGAGCACCGGGTTTCTTATCCTGCCGGACTCTGTCGGTACTGATGCCCTCCGCGACAGGCTTCCGTTGCTGGCGTTACGCACGCTTCGGTACCCATCGGGGCGGCC GTGGATCGTCGGCAGATGGGACGCTGAGGAGATCATGTCCGCGCGTGTCGGCCCGGTGCACGTGGTGGTGATCGGGTTTTGCCCGGTCACCACCACCGAACTGACCTCGTTGA TCAGTCGGATCCACACCGTCTCTGACGTGAATCGTTTGGTTGCCAGTCTGCCTGGAAGCTTCCACCTCGTCGCCGCGGTCGGGCGGCAGTTACGGGTTCAGGGCAGCATCACGG GTCTACGCAGAATCGCTTGGGCAAAGAGCGACGGCCATACCTTCGTCTCCGACCGCACCGACATACTCGCCGACCTGACGAACGCAAAAATCGACGATTCTCTCCTCGCCACGA GAGTGGCATGCGGCGGGATGGTCCCCCCACCGCTGCGTGAACGCAGTATGTGGCTTGGCGTCAACTCTCTGCCGCCCGACCGCTATCTCTTGGTCGAGGGCGGGCAGCGGACG CGCGAGGTGAAATGGTGGCAGCCGCCAGCACCAGAAACGACTCTTGCTGAGGGCGCCTGCAGAGTCAGGGAAGCTCTCGTATCGGCCCTGTACAGCAGGAACCCCGTCCACG GCCGACTCAGCGCGGACCTGTCAGGCGGGCTTGACTCTACCTCGCTGGCCTTTCTCGCCGCCCGGACCACGCCTCATCTACTCACACACCGCTGGGCGGAGGCCGAGGCGGGCA ATGACGACGGCAAGTATGCGGCGATCGCGGCGCGGGAGTTGCCCAATGCTGAACACCTTGTCATTCCGCAATCAGATATACCGCCTGTCTTCTCCGACCCGCACCTGAACGGCG ACCCCGAGCAACCCTATCTTTTCGCGCGGACGGCTGCCCGAGCCGTGTATACGGCTCGTATGCTAGTCGCGCATAGTTCCACGAGACATATAGCCGGCCATGGGGGAGACGAA CTCTTCTACAAGTTCCCTGGATATCTTCACCCTCTGCTGCGTCGGCGTCCACTCACCGCCATCCGGCATATTCGCGCCCATCTGGCGCTCAGCCGGTGGGACAGGAAAGCCACCG TCGCACAATTGCTGGACACCAGTGGTTTCCCGGATTGGTGGAAGCGGCAGGCGGACCAACTTACTGCACCTCCACCAGGCGGACGTTTTCCGCCTTTGGGCTGGGGATTTATGC CTCTGCACGCCCAGCCCTGGGTGACAGGGGAGGCGCTGCACGCGGCACGCGCCACACTCCGCGAGGCGGCGGAAGAGGCCCACCCCCTCGCCCCGGACCGGGGGCAACACCA GTACCTGCTGGCCCTGCGTACCACCGCGCCGGCATACGCACAGCTCGGTCGCCTATTCGAGCGGGCGGGTGTCCGGCTGGACTTGCCATACCTGGATGACCGGGTCGTAGAAG CGGCCCTGTCTGTCCGGCTGGAGGAGCGGGCCCACCCCTGGCGTTACAAACCACTTCTCGCGGAGGCCATGCGGGGTGTCGTCCCGGAGAGCATCGCTACCCGCGTCACCAAG GGAGAGTTCAGTGCCGATTTGAGTCTCGGGTTACGCCGGAACCGCGCGAGCGTGCTGGACTTTCTCGCCGATTCTGCCATCGCCAAGAGGGGGCTGATCGACGTCGACACCCT GCGTCGCCTGCTTCTCGCGCCCCAGGCTGATCACTCCCGAAATATAGCTCTCGAACACCTCGTGGGCTGTGAGACCTGGCTCCGCACCACCCCAAAGAGACAAGATGCGCCTGC ACCCTAAAGAGGAGAAATTAACCATGCGCCTGCACCCTGATATCGTCATCGCGGAAACCGAAGACGGTGCGGTCCTACTACACCAAAGGACCGGCCGCTACTGGCAGCTGAAC ACCACGGGCATGGCGGTCATCGAAAGCCTGACTTCCGGTGAGACCCTCGCCGAGATCACGCACCGTATGGCCAAGCGTTACGGCTTGCCCGCTGAGCAGGTCCGGCAGGACGT CGAAGCCGTCGTCCATCAACTACACGCGGCCCAGATAGTGGAGAGCACCTCGTAAAGAGGAGAAATTAACCATGAGTACCCCCGCCGCCCTGGTCAAGCGCAGAAAACTGCCC TTCCATCGCAGAGTGCTGCCGCTCGTTGCTGTCGGTGTTGCCCGCCTCCTCAGCAGGACAAAGCCTGCACGGCTGCGAGCCATCCTGAGCTACGCACGACGGGGTGCCGCGCCG GCCACGGTGGAACAGGCGATGGCCGCGCGACAAGCGGTCGTCGCCGTCAGCCAACGGTGCGCCGGACAGGCCTGCCTTCAGCGTTCCATCGCGACGGCACTGCTCTGCCGGG CGCGTGGATCCTGGCCGACCTGGTGCACCGGGGTACGGACCAGCCCGTTCGAAGCCCATGCCTGGATCGAGGCCGAAGGTCGGCTCATCGGCGAGCCGTACCCGGACGGTCA CTATCGGCCTTTGCTGACCGTTCCTCCGGTTTCCTGAAGATCTGGGTACCGGTGGTGGCTCCGGTGATGACGACGACAAGAGTCCCATGGGATATCGGGGATCCGAATTCTGTAC AGGCCTTGGCGCGCCTGCAGGCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACCACCACTAATTGATTAATACCTAGGCTGCTAAACAAAGCCC GAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT S7