Nghiên cứu phát triển các vector nhị thể ứng dụng trong cải biến di truyền một số loài nấm sợi

ĐẠI HỌC QUỐC GIA HÀ NỘI TRƢỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN VŨ XUÂN TẠO NGHIÊN CỨU PHÁT TRIỂN CÁC VECTOR NHỊ THỂ ỨNG DỤNG TRONG CẢI BIẾN DI TRUYỀN MỘT SỐ LOÀI NẤM SỢI LUẬN ÁN TIẾN SĨ SINH HỌC HÀ NỘI - 2019 ĐẠI HỌC QUỐC GIA HÀ NỘI TRƢỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN VŨ XUÂN TẠO NGHIÊN CỨU PHÁT TRIỂN CÁC VECTOR NHỊ THỂ ỨNG DỤNG TRONG CẢI BIẾN DI TRUYỀN MỘT SỐ LOÀI NẤM SỢI CHUYÊN NGÀNH: VI SINH VẬT HỌC MÃ SỐ : 9420101.07 LUẬN ÁN TIẾN SĨ SINH HỌC Ngƣời hƣớng dẫn khoa học: TS TRẦN VĂN TUẤN PGS.TS NGUYỄN QUANG HUY HÀ NỘI – 2019 LỜI CAM ĐOAN Tơi xin cam đoan cơng trình nghiên cứu dƣới hƣớng dẫn TS Trần Văn Tuấn PGS.TS Nguyễn Quang Huy Các số liệu, kết trình bày luận án trung thực, phần đƣợc cơng bố Tạp chí Khoa học, phần cịn lại chƣa đƣợc cơng bố cơng trình khác Hà Nội, ngày tháng 12 năm 2019 Tác giả luận án Vũ Xuân Tạo LỜI CẢM ƠN Để hoàn thành luận án này, lời tơi xin bày tỏ lịng biết ơn chân thành sâu sắc tới TS Trần Văn Tuấn, Trƣởng Bộ môn Vi sinh vật học, Khoa Sinh học, Trƣờng Đại học Khoa học Tự nhiên, ĐHQGHN ngƣời thầy cho hội đƣợc thực luận án, tận tình hƣớng dẫn, động viên khích lệ tạo điều kiện giúp đỡ suốt thời gian thực hồn thành luận án Tơi xin bày tỏ lòng cảm ơn tới PGS.TS Nguyễn Quang Huy, Trƣởng Khoa Sinh học, Trƣờng Đại học Khoa học Tự nhiên, ĐHQGHN, ngƣời thầy tận tình truyền đạt nhiều kinh nghiệm quý báu, ủng hộ giúp đỡ q trình thực luận án Tơi xin chân thành cảm ơn thầy cô Bộ môn Vi sinh vật học, Khoa Sinh học, Trƣờng Đại học Khoa học Tự nhiên, ĐHQGHN, đặc biệt PGS.TS Bùi Thị Việt Hà TS Phạm Thế Hải giảng dạy, truyền đạt cho kiến thức luôn tạo điều kiện để tơi hồn thành đƣợc khóa học Tơi xin gửi lời cảm ơn tới GS.TS Phan Tuấn Nghĩa, Giám đốc Phịng thí nghiệm Trọng điểm Công nghệ Enzym Protein tập thể cán Phịng Genomic, Phịng thí nghiệm Trọng điểm Công nghệ Enzym Protein, Trƣờng Đại học Khoa học Tự nhiên, ĐHQGHN tạo điều kiện để hoàn thành nghiên cứu Để hoàn thành luận án này, nhận đƣợc hỗ trợ từ đề tài NAFOSTED (mã số: 106-NN.04-2014.75 106.04-2018.36) Tôi trân trọng hỗ trợ Tôi xin cảm ơn Ban Giám hiệu, Phòng Sau đại học Khoa Sinh học, Trƣờng Đại học Khoa học Tự nhiên, ĐHQGHN tạo điều kiện thuận lợi cho suốt trình học tập nghiên cứu Trƣờng Cuối tơi xin gửi lịng biết ơn sâu sắc tới gia đình, bạn bè, ngƣời thân ngƣời ln ln bên, ủng hộ, động viên, giúp đỡ trình học tập nghiên cứu Hà Nội, ngày tháng 12 năm 2019 Tác giả luận án Vũ Xuân Tạo MỤC LỤC MỞ ĐẦU Chƣơng TỔNG QUAN CÁC VẤN ĐỀ NGHIÊN CỨU .4 1.1 GIỚI THIỆU CHUNG VỀ NẤM Aspergillus niger, Penicillium chrysogenum VÀ Penicillium digitatum………………………………………………………… 1.1.1 Sơ lƣợc chi nấm Aspergillus 1.1.2 Nấm Aspergillus niger 1.1.3 Sơ lƣợc chi nấm Penicillium 1.1.4 Nấm Penicillium chrysogenum .9 1.1.5 Nấm Penicillium digitatum 12 1.2 PHƢƠNG PHÁP CHUYỂN GEN VÀO NẤM SỢI THÔNG QUA VI KHUẨN A tumefaciens (ATMT) 15 1.2.1 Giới thiệu vi khuẩn A tumefaciens .16 1.2.2 Cơ chế chuyển gen vi khuẩn A tumefaciens 17 1.2.3 Các yếu tố ảnh hƣởng đến hiệu chuyển gen vào nấm sợi thông qua vi khuẩn A tumefaciens 19 1.2.4 Vector dùng chuyển gen vi khuẩn A tumefaciens 20 1.2.5 Marker chọn lọc cho chuyển gen vào nấm thông qua vi khuẩn A tumefaciens 23 1.2.6 Promoter điều hòa biểu gen nấm sợi 25 1.2.7 Gen thị DsRed GFP dùng chuyển gen nấm sợi 26 1.2.8 Ƣu điểm phƣơng pháp chuyển gen thông qua vi khuẩn A tumefaciens .28 1.2.9 Sử dụng phƣơng pháp ATMT nghiên cứu chuyển gen nấm sợi 29 1.3 TÌNH HÌNH NGHIÊN CỨU VỀ GEN laeA Ở NẤM SỢI 32 1.3.1 Protein LaeA tƣơng tác phức hệ protein velvet nấm sợi 32 1.3.2 Nghiên cứu vai trò gen laeA nấm sợi 33 Chƣơng VẬT LIỆU VÀ PHƢƠNG PHÁP NGHIÊN CỨU .36 2.1 VẬT LIỆU 36 2.1.1 Chủng vi sinh vật 36 2.1.2 Các vector sử dụng nghiên cứu 37 2.1.3 Thiết bị hóa chất .38 2.2 PHƢƠNG PHÁP NGHIÊN CỨU 38 2.2.1 Thu bào tử hệ sợi nấm 39 2.2.2 Đánh giá khả mẫn cảm kháng sinh 40 2.2.3 Tách chiết DNA, RNA tổng hợp cDNA 40 2.2.4 Tạo vector nhị thể dùng cho chuyển gen 41 2.2.5 Tối ƣu quy trình chuyển gen nấm A niger, P digitatum P chrysogenum thông qua vi khuẩn A tumefaciens 49 2.2.6 Xóa phục hồi gen laeA nấm sợi nghiên cứu 50 2.2.7 Sàng lọc xác nhận thể chuyển gen 51 2.2.8 Điều tra vai trị gen laeA lồi nấm sợi nghiên cứu 52 Chƣơng KẾT QUẢ VÀ THẢO LUẬN 55 3.1 PHÁT TRIỂN HỆ THỐNG CHUYỂN GEN HIỆU QUẢ CAO SỬ DỤNG MARKER KHÁNG KHÁNG SINH 55 3.1.1 Nghiên cứu chuyển gen vào nấm A niger sử dụng phƣơng pháp ATMT marker gen kháng kháng sinh 55 3.1.2 Phát triển hệ thống chuyển gen hiệu cao nấm P chrysogenum P digitatum sử dụng marker kháng kháng sinh 57 3.2 PHÁT TRIỂN HỆ THỐNG CHUYỂN GEN SỬ DỤNG MARKER TRỢ DƢỠNG pyrG Ở A niger VÀ P chrysogenum 68 3.2.1 Tạo thành công chủng A niger N402 P chrysogenum VTCC-F1172 đột biến khuyết dƣỡng uridine/uracil 68 3.2.2 Xây dựng quy trình chuyển gen tối ƣu vào A niger P chrysogenum khuyết dƣỡng uridine/uracil 74 3.3 NGHIÊN CỨU VAI TRÒ CỦA GEN laeA Ở A niger, P chrysogenum VÀ P digitatum 80 3.3.1 Nghiên cứu vai trò gen laeA A niger 80 3.3.2 Nghiên cứu vai trò gen laeA P chrysogenum 94 3.3.3 Nghiên cứu vai trò gen laeA P digitatum .106 KẾT LUẬN 117 KIẾN NGHỊ 119 DANH MỤC CƠNG TRÌNH KHOA HỌC CỦA TÁC GIẢ LIÊN QUAN ĐẾN LUẬN ÁN .120 TÀI LIỆU THAM KHẢO 121 DANH MỤC CÁC TỪ VIẾT TẮT 5-FOA 5-fluoroorotic acid A nidulans Aspergillus nidulans A niger Aspergillus niger A oryzae Aspergillus oryzae A tumefaciens Agrobacterium tumefaciens AS Acetosyringone ATMT Agrobacterium tumefaciens-mediated transformation (Chuyển gen nhờ vi khuẩn Agrobacterium tumefaciens) CD Czapek-Dox cDNA Complementary deoxyribonucleic acid (ADN bổ trợ) DNA Deoxyribonucleic acid DsRed Red fluorescent protein (Protein huỳnh quang đỏ) EDTA Ethylenediaminetetraacetic acid E coli Escherichia coli FDA Food and Drug Administration (Cục Quản lý thực phẩm dƣợc phẩm Hoa Kỳ) GFP Green fluorescent protein (Protein huỳnh quang xanh) HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid IM Induction medium (môi trƣờng cảm ứng) ITS Internal transcribed spacer kb Kilobase pair LaeA Loss of aflR-expression A (Mất biểu aflR) LB Luria- Bertani (môi trƣờng LB) / Left Border (biên trái) MES 2-(N-morpholino)ethanesulfonic acid MM Minimal medium (môi trƣờng tối thiểu) ORF Open reading frame (khung đọc mở) P chrysogenum Penicillium chrysogenum P digitatum Penicillium digitatum PCR Polymerase chain reaction (Phản ứng chuỗi polymerase) PDA Potato dextrose agar PEG Polyethylen glycol RB Right Border (biên phải) RNA Ribonucleic acid SDS Sodium dodecyl sulfate S aureus Staphylococcus aureus SM Synthetic medium (Môi trƣờng tổng hợp) ura Uracil uri Uridine vir Virulence gene (gen gây bệnh /gen độc lực) WT Wild type (chủng tự nhiên) DANH MỤC BẢNG Trang Bảng 2.1 Danh sách chủng vi sinh vật sử dụng nghiên cứu 36 Bảng 2.2 Các vector sử dụng nghiên cứu 37 Bảng 3.1 Kết xác định số gen laeA chủng A niger 92 biểu mức gen laeA pGreen2 (gpdA-HPH): 11,804 bp AGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAA ACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGC GTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCC GCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACG CGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGAT GCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGAC CTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACA CCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCAT CGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACC CCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGC TTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCG GCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAG GAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGAT GATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGT TTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAA GGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACA AATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCG CAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAG TGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGAC CGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTG TGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGC CGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCG CGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTG AGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACC CGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAG GTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGC AACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACC AAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTAC CAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAG AACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTG GGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATC CGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAG CGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAAT CCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTT CGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCG AAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGC CGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAA CCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTC TGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACG TTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAG CCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTAC CGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCA TCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGAT CTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAAT GACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACC TGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGA AAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGG AACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGA AAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATA ACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCC GCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAG GGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCG GTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG GAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGT AGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCG 187 GTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAG AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG CGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGAC CGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG CCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA CGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGT AGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG TTAAGGGATTTTGGTCATGCAGGATCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCA ATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACC GAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCT ATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGA ATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACT CGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAAT CAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGG AGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCT GTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATC GATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTT GGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTT ATGTAAGCAGACAGTTTTATTGTTCATGATCTGGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACA TGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTA ACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATC GAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTA AACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTGC TCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCC CGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTA CACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATG ACATGATTACGAATTCTTAATTAAGATATCGAGCTCTGTACAGTGACCGGTGACTCTTTCTGGCATGCGGAGA GACGGACGGACGCAGAGAGAAGGGCTGAGTAATAAGCCACTGGCCAGACAGCTCTGGCGGCTCTGAGGTGCAG TGGATGATTATTAATCCGGGACCGGCCGCCCCTCCGCCCCGAAGTGGAAAGGCTGGTGTGCCCCTCGTTGACC AAGAATCTATTGCATCATCGGAGAATATGGAGCTTCATCGAATCACCGGCAGTAAGCGAAGGAGAATGTGAAG CCAGGGGTGTATAGCCGTCGGCGAAATAGCATGCCATTAACCTAGGTACAGAAGTCCAATTGCTTCCGATCTG GTAAAAGATTCACGAGATAGTACCTTCTCCGAAGTAGGTAGAGCGAGTACCCGGCGCGTAAGCTCCCTAATTG GCCCATCCGGCATCTGTAGGGCGTCCAAATATCGTGCCTCTCCTGCTTTGCCCGGTGTATGAAACCGGAAAGG CCGCTCAGGAGCTGGCCAGCGGCGCAGACCGGGAACACAAGCTGGCAGTCGACCCATCCGGTGCTCTGCACTC GACCTGCTGAGGTCCCTCAGTCCCTGGTAGGCAGCTTTGCCCCGTCTGTCCGCCCGGTGTGTCGGCGGGGTTG ACAAGGTCGTTGCGTCAGTCCAACATTTGTTGCCATATTTTCCTGCTCTCCCCACCAGCTGCTCTTTTCTTTT CTCTTTCTTTTCCCATCTTCAGTATATTCATCTTCCCATCCAAGAACCTTTATTTCCCCTAAGTAAGTACTTT GCTACATCCATACTCCATCCTTCCCATCCCTTATTCCTTTGAACCTTTCAGTTCGAGCTTTCCCACTTCATCG CAGCTTGACTAACAGCTACCCCGCTTGAGCAGACATCACCATGCCTGAACTCACCGCGACGTCTGTCGAGAAG TTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCA GCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTA TGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGC CTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTG TTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCC ATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTG TATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTT GGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAA TGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATC TTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTG CAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGG CAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGG CGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAA 188 ACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGAGTAGATGCCGACCGCGGGATCCACTTAACGTTAC TGAAATCATCAAACAGCTTGACGAATCTGGATATAAGATCGTTGGTGTCGATGTCAGCTCCGGAGTTGAGACA AATGGTGTTCAGGATCTCGATAAGATACGTTCATTTGTCCAAGCAGCAAAGAGTGCCTTCTAGTGATTTAATA GCTCCATGTCAACAAGAATAAAACGCGTTTTCGGGTTTACCTCTTCCAGATACAGCTCATCTGCAATGCATTA ATGCATTGACTGCAACCTAGTAACGCCTTNCAGGCTCCGGCGAAGAGAAGAATAGCTTAGCAGAGCTATTTTC ATTTTCGGGAGACGAGATCAAGCAGATCAACGGTCGTCAAGAGACCTACGAGACTGAGGAATCCGCTCTTGGC TCCACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTCTTCTTTACTCTGATAGCTTGACTAT GAAAATTCCGTCACCAGCNCCTGGGTTCGCAAAGATAATTGCATGTTTCTTCCTTGAACTCTCAAGCCTACAG GACACACATTCATCGTAGGTATAAACCTCGAAATCANTTCCTACTAAGATGGTATACAATAGTAACCATGCAT GGTTGCCTAGTGAATGCTCCGTAACACCCAATACGCCGGCCGAAACTTTTTTACAACTCTCCTATGAGTCGTT TACCCAGAATGCACAGGTACACTTGTTTAGAGGTAATCCTTCTTTCTAGCACTAGTGTGACCGGTGACTCTTT CTGGCATGCGGAGAGACGGACGGACGCAGAGAGAAGGGCTGAGTAATAAGCCACTGGCCAGACAGCTCTGGCG GCTCTGAGGTGCAGTGGATGATTATTAATCCGGGACCGGCCGCCCCTCCGCCCCGAAGTGGAAAGGCTGGTGT GCCCCTCGTTGACCAAGAATCTATTGCATCATCGGAGAATATGGAGCTTCATCGAATCACCGGCAGTAAGCGA AGGAGAATGTGAAGCCAGGGGTGTATAGCCGTCGGCGAAATAGCATGCCATTAACCTAGGTACAGAAGTCCAA TTGCTTCCGATCTGGTAAAAGATTCACGAGATAGTACCTTCTCCGAAGTAGGTAGAGCGAGTACCCGGCGCGT AAGCTCCCTAATTGGCCCATCCGGCATCTGTAGGGCGTCCAAATATCGTGCCTCTCCTGCTTTGCCCGGTGTA TGAAACCGGAAAGGCCGCTCAGGAGCTGGCCAGCGGCGCAGACCGGGAACACAAGCTGGCAGTCGACCCATCC GGTGCTCTGCACTCGACCTGCTGAGGTCCCTCAGTCCCTGGTAGGCAGCTTTGCCCCGTCTGTCCGCCCGGTG TGTCGGCGGGGTTGACAAGGTCGTTGCGTCAGTCCAACATTTGTTGCCATATTTTCCTGCTCTCCCCACCAGC TGCTCTTTTCTTTTCTCTTTCTTTTCCCATCTTCAGTATATTCATCTTCCCATCCAAGAACCTTTATTTCCCC TAAGTAAGTACTTTGCTACATCCATACTCCATCCTTCCCATCCCTTATTCCTTTGAACCTTTCAGTTCGAGCT TTCCCACTTCATCGCAGCTTGACTAACAGCTACCCCGCTTGAGCAGACATCACCCTCGAGTACGTAGGTACCA TGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGG CCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGCACCTGAAGTTCATCTGC ACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCC GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCAC CATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC CGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCTTGGGGCACAAGCTGGAGTACAACTACA ACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAA CATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTG CTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGG TCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAGTAAAGCGGCCGCTC TAGTGGATCCACTTAACGTTACTGAAATCATCAAACAGCTTGACGAATCTGGATATAAGATCGTTGGTGTCGA TGTCAGCTCCGGAGTTGAGACAAATGGTGTTCAGGATCTCGATAAGATACGTTCATTTGTCCAAGCAGCAAAG AGTGCCTTCTAGTGATTTAATAGCTCCATGTCAACAAGAATAAAACGCGTTTTCGGGTTTACCTCTTCCAGAT ACAGCTCATCTGCAATGCATTAATGCATTGACTGCAACCTAGTAACGCCTTNCAGGCTCCGGCGAAGAGAAGA ATAGCTTAGCAGAGCTATTTTCATTTTCGGGAGACGAGATCAAGCAGATCAACGGTCGTCAAGAGACCTACGA GACTGAGGAATCCGCTCTTGGCTCCACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTCTTC TTTACTCTGATAGCTTGACTATGAAAATTCCGTCACCAGCNCCTGGGTTCGCAAAGATAATTGCATGTTTCTT CCTTGAACTCTCAAGCCTACAGGACACACATTCATCGTAGGTATAAACCTCGAAATCANTTCCTACTAAGATG GTATACAATAGTAACCATGCATGGTTGCCTAGTGAATGCTCCGTAACACCCAATACGCCGGCCGAAACTTTTT TACAACTCTCCTATGAGTCGTTTACCCAGAATGCACAGGTACACTTGTTTAGAGGTAATCCTTCTTTCTAGAA GTCCTCGTGTACTGTGTAAGCGCCCACTCCACATCTCCACTCGACCTGCAGGCATGCAAGCTTGGCACTGGCC GTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTT TCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGA ATGAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATT GGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCC GTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAA 189 pEX2 (pyrG,RED): 10,926 kb AGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAA ACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGC GTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCC GCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACG CGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGAT GCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGAC CTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACA CCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCAT CGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACC CCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGC TTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCG GCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAG GAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGAT GATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGT TTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAA GGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACA AATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCG CAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAG TGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGAC CGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTG TGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGC CGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCG CGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTG AGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACC CGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAG GTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGC AACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACC AAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTAC CAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAG AACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTG GGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATC CGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAG CGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAAT CCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTT CGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCG AAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGC CGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAA CCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTC TGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACG TTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAG CCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTAC CGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCA TCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGAT CTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAAT GACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACC TGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGA AAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGG AACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGA AAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATA ACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCC GCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAG GGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCG GTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG GAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGT AGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCG 190 GTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAG AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG CGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGAC CGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG CCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA CGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGT AGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG TTAAGGGATTTTGGTCATGCAGGATCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCA ATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACC GAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCT ATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGA ATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACT CGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAAT CAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGG AGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCT GTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATC GATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTT GGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTT ATGTAAGCAGACAGTTTTATTGTTCATGATCTGGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACA TGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTA ACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATC GAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTA AACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTGC TCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCC CGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTA CACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATG ACATGATTACGAATTCATGCGAAGGTAAGTGCTTCTATATTGATCCTTAGTGCTTTCAAACTGTGATGTAGAA GTTGCTCGGTAGCTGATTAAATATTCTAGACCCAAGCCGCTGCTGGAATTGACATTATTATGGCCGATAGGGT TGGGCTTATTGCTATGTCCCTGAAAGGATATCAAAAGCAGGCAAAAAGCCAGGCATAATCCCCGCGTGGACGG TACCCTAAGGATAGGCCCTAATCTTATCTACATGTGACTGCATCGATGTGTTTGGTCAAAATGAGGCATGTGG CTCACCCCACAGGCGGAGAAACGTGTGGCTAGTGCATGACAGTCCCCTCCATAGATTCAATTTAATTTTTCGC GGCAATTGTCGTGCAGTTTGTATCTACATTTCATTCCATATATCAAGAGTTAGTAGTTGGACATCCTGATTAT TTTGTCTAATTACTGAAAACTCGAAGTACTAACCTACTAATAAGCCAGTTTCAACCACTAAGTGCTCATTTAT ACAATATTTGCAGAACCCCGCGCTACCCCTCCATCGCCAACATGTCTTCCAAGTCGCAATTGACCTACAGCGC ACGCGCTAGCAAGCACCCCAATGCGCTCGTAAAGAAGCTCTTCGAGGTTGCCGAGGCCAAGAAAACCAATGTC ACCGTTTCCGCCGACGTGACAACCACCAAAGAGCTGCTGGATTTGGCTGACCGTATGCGCACCGGGGATGCCA CTTACATGTGATCTAGTAATGGTTAATGGTGGATTATATAACAGGACTCGGTCCGTACATTGCCGTGATCAAA ACTCACATCGATATCCTCTCCGATTTCAGCGAAGAAACCATCACCGGTCTGAAGGCCCTTGCAGAGAAGCACA ATTTCCTCATCTTCGAAGATCGCAAGTTCATCGATATCGGAAACACAGTCCAAAAGCAGTACCATGGCGGCAC TCTGCGTATCTCTGAGTGGGCCCACATCATCAACTGCAGTATTCTGCCCGGTGAGGGTATCGTCGAGGCTCTG GCCCAGACTGCTTCGGCCGAGGACTTCCCCTACGGCTCCGAGAGGGGCCTTTTGATCCTTGCGGAGATGACCT CCAAGGGATCTTTGGCTACCGGTCAATATACTACTTCTTCTGTTGACTATGCTCGGAAGTATAAGAAGTTTGT GATGGGATTCGTCTCGACACGTCACCTTGGCGAGGTTCAGTCTGAAGTTAGCTCGCCTTCGGAGGAGGAAGAT TTTGTCGTCTTCACGACAGGTGTCAACCTCTCCTCGAAGGGTGACAAGCTGGGACAGCAGTACCAAACTCCTG AGTCGGCTGTTGGACGCGGTGCCGACTTTATTATTGCTGGCCGTGGAATTTATGCTGCTCCTGATCCCGTGGA GGCGGCGAAGCAGTACCAGAAGGAGGGATGGGATGCATACCTGAAGCGTGTTGGTGCGCAATAAGTAGTGGTG GATACGTACTCCTTTTATGGCAGTATGTCGCAAGTATGATGCGATTTATAAATTCAGCACTCGAAATGACTAC TACTATGTGTCTACGACAGATACCCTCTCCGTACGAATAAGACACCTGCCTCGATATATGGACAAATTCAAAA TCAGGGTCAAGGGTCATGTTTCAAAGTCACAACAATCTCCAACATAGACGAGAATTTGTACCGGAGTGTCTGA AGGTGCAGCTGGAGATTGGTCTATTTTCTTAGAGTGGGGTATCACTAATGTACAGTCGGTCACTATCGTACAA ACAATCACAATTATATACAAGATTTCCCACCACCCCCTACTCTAACACGGCACAATTATCCATCGAGTCAGAG CCTAGCCAACTAGTGTGACCGGTGACTCTTTCTGGCATGCGGAGAGACGGACGGACGCAGAGAGAAGGGCTGA 191 GTAATAAGCCACTGGCCAGACAGCTCTGGCGGCTCTGAGGTGCAGTGGATGATTATTAATCCGGGACCGGCCG CCCCTCCGCCCCGAAGTGGAAAGGCTGGTGTGCCCCTCGTTGACCAAGAATCTATTGCATCATCGGAGAATAT GGAGCTTCATCGAATCACCGGCAGTAAGCGAAGGAGAATGTGAAGCCAGGGGTGTATAGCCGTCGGCGAAATA GCATGCCATTAACCTAGGTACAGAAGTCCAATTGCTTCCGATCTGGTAAAAGATTCACGAGATAGTACCTTCT CCGAAGTAGGTAGAGCGAGTACCCGGCGCGTAAGCTCCCTAATTGGCCCATCCGGCATCTGTAGGGCGTCCAA ATATCGTGCCTCTCCTGCTTTGCCCGGTGTATGAAACCGGAAAGGCCGCTCAGGAGCTGGCCAGCGGCGCAGA CCGGGAACACAAGCTGGCAGTCGACCCATCCGGTGCTCTGCACTCGACCTGCTGAGGTCCCTCAGTCCCTGGT AGGCAGCTTTGCCCCGTCTGTCCGCCCGGTGTGTCGGCGGGGTTGACAAGGTCGTTGCGTCAGTCCAACATTT GTTGCCATATTTTCCTGCTCTCCCCACCAGCTGCTCTTTTCTTTTCTCTTTCTTTTCCCATCTTCAGTATATT CATCTTCCCATCCAAGAACCTTTATTTCCCCTAAGTAAGTACTTTGCTACATCCATACTCCATCCTTCCCATC CCTTATTCCTTTGAACCTTTCAGTTCGAGCTTTCCCACTTCATCGCAGCTTGACTAACAGCTACCCCGCTTGA GCAGACATCACCCTCGAGCACGTGCTTAAGGATATCATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGC GCTTCAAGGTGCGCATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCC CTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTG TCCCCCCAGTTCCAGTACGGCTCCAAGGTGTACGTGAAGCACCCCGCCGACATCCCCGACTACAAGAAGCTGT CCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGA CTCCTCCCTGCAGGACGGCTCCTTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCC GTAATGCAGAAGAAGACTATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGG GCGAGATCCACAAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGTCCATCTACATGGC CAAGAAGCCCGTGCAGCTGCCCGGCTACTACTACGTGGACTCCAAGCTGGACATCACCTCCCACAACGAGGAC TACACCATCGTGGAGCAGTACGAGCGCGCCGAGGGCCGCCACCACCTGTTCCTGTAGGATATCGAGCTCCCGC GGGGATCCACTTAACGTTACTGAAATCATCAAACAGCTTGACGAATCTGGATATAAGATCGTTGGTGTCGATG TCAGCTCCGGAGTTGAGACAAATGGTGTTCAGGATCTCGATAAGATACGTTCATTTGTCCAAGCAGCAAAGAG TGCCTTCTAGTGATTTAATAGCTCCATGTCAACAAGAATAAAACGCGTTTTCGGGTTTACCTCTTCCAGATAC AGCTCATCTGCAATGCATTAATGCATTGACTGCAACCTAGTAACGCCTTNCAGGCTCCGGCGAAGAGAAGAAT AGCTTAGCAGAGCTATTTTCATTTTCGGGAGACGAGATCAAGCAGATCAACGGTCGTCAAGAGACCTACGAGA CTGAGGAATCCGCTCTTGGCTCCACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTCTTCTT TACTCTGATAGCTTGACTATGAAAATTCCGTCACCAGCNCCTGGGTTCGCAAAGATAATTGCATGTTTCTTCC TTGAACTCTCAAGCCTACAGGACACACATTCATCGTAGGTATAAACCTCGAAATCANTTCCTACTAAGATGGT ATACAATAGTAACCATGCATGGTTGCCTAGTGAATGCTCCGTAACACCCAATACGCCGGCCGAAACTTTTTTA CAACTCTCCTATGAGTCGTTTACCCAGAATGCACAGGTACACTTGTTTAGAGGTAATCCTTCTTTCTAGAAGT CCTCGTGTACTGTGTAAGCGCCCACTCCACATCTCCACTCGACCTGCAGGCATGCAAGCTTGGCACTGGCCGT CGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTC GCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAAT GAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGG CGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGT TCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAA 192 pKO2.1.1: 7,789 kb AGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAA ACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGC GTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCC GCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACG CGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGAT GCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGAC CTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACA CCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCAT CGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACC CCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGC TTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCG GCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAG GAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGAT GATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGT TTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAA GGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACA AATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCG CAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAG TGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGAC CGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTG TGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGC CGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCG CGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTG AGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACC CGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAG GTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGC AACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACC AAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTAC CAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAG AACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTG GGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATC CGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAG CGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAAT CCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTT CGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCG AAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGC CGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAA CCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTC TGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACG TTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAG CCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTAC CGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCA TCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGAT CTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAAT GACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACC TGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGA AAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGG AACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGA AAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATA ACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCC GCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAG GGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCG GTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG GAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGT AGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCG 193 GTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAG AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG CGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGAC CGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG CCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA CGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGT AGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG TTAAGGGATTTTGGTCATGCAGGATCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCA ATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACC GAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCT ATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGA ATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACT CGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAAT CAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGG AGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCT GTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATC GATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTT GGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTT ATGTAAGCAGACAGTTTTATTGTTCATGATCTGGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACA TGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTA ACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATC GAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTA AACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTGC TCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCC CGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTA CACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATG ACATGATTACGAATTCTTAATTAAGATATCGAGCTCACTAGTGGTACCAGCGCTAGATCTCAACTGATATTGA AGGAGCATTTTTTGGGCTTGGCTGGAGCTAGTGGAGGTCAACAATGAATGCCTATTTTGGTTTAGTCGTCCAG GCGGTGAGCACAAAATTTGTGTCGTTTGACAAGATGGTTCATTTAGGCAACTGGTCAGATCAGCCCCACTTGT AGCAGTAGCGGCGGCGCTCGAAGTGTGACTCTTATTAGCAGACAGGAACGAGGACATTATTATCATCTGCTGC TTGGTGCACGATAACTTGGTGCGTTTGTCAAGCAAGGTAAGTGGACGACCCGGTCATACCTTCTTAAGTTCGC CCTTCCTCCCTTTATTTCAGATTCAATCTGACTTACCTATTCTACCTAAGCATCTCATGGCCACCCTCGACGA CACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGGTCCTTCACC ACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGACCCGCCCC TGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGACGGGGAGGACGGCGACCCGGACTCCCG GACGTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCTGGAACCGC CGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTTGATGGGGC TCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTCAACGCACCGGC GATCCACGCGTACCGGCGGATGGGGTTCACCCTCTGCGGCCTGGACACCGCCCTGTACGACGGCACCGCCTCG GACGGCGAGCAGGCGCTCTACATGAGCATGCCCTGCCCCTGAGGATCCGGGCCCCTCGAGAGGCCTTCTAGAA GTCCTCGTGTACTGTGTAAGCGCCCACTCCACATCTCCACTCGACCTGCAGGCATGCAAGCTTGGCACTGGCC GTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTT TCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGA ATGAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATT GGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCC GTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAA 194 pKO2.0.1: 8,308 kb AGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAA ACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGC GTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCC GCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACG CGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGAT GCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGAC CTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACA CCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCAT CGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACC CCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGC TTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCG GCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAG GAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGAT GATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGT TTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAA GGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACA AATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCG CAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAG TGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGAC CGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTG TGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGC CGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCG CGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTG AGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACC CGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAG GTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGC AACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACC AAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTAC CAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAG AACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTG GGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATC CGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAG CGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAAT CCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTT CGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCG AAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGC CGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAA CCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTC TGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACG TTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAG CCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTAC CGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCA TCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGAT CTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAAT GACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACC TGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGA AAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGG AACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGA AAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATA ACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCC GCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAG GGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCG GTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG GAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGT AGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCG 195 GTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAG AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG CGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGAC CGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG CCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA CGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGT AGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG TTAAGGGATTTTGGTCATGCAGGATCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCA ATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACC GAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCT ATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGA ATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACT CGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAAT CAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGG AGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCT GTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATC GATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTT GGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTT ATGTAAGCAGACAGTTTTATTGTTCATGATCTGGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACA TGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTA ACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATC GAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTA AACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTGC TCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCC CGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTA CACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATG ACATGATTACGAATTCTTAATTAAGATATCGAGCTCACTAGTGACGTCAGCGCTAGATCTTGTACAGTGACCG GTGACTCTTTCTGGCATGCGGAGAGACGGACGGACGCAGAGAGAAGGGCTGAGTAATAAGCCACTGGCCAGAC AGCTCTGGCGGCTCTGAGGTGCAGTGGATGATTATTAATCCGGGACCGGCCGCCCCTCCGCCCCGAAGTGGAA AGGCTGGTGTGCCCCTCGTTGACCAAGAATCTATTGCATCATCGGAGAATATGGAGCTTCATCGAATCACCGG CAGTAAGCGAAGGAGAATGTGAAGCCAGGGGTGTATAGCCGTCGGCGAAATAGCATGCCATTAACCTAGGTAC AGAAGTCCAATTGCTTCCGATCTGGTAAAAGATTCACGAGATAGTACCTTCTCCGAAGTAGGTAGAGCGAGTA CCCGGCGCGTAAGCTCCCTAATTGGCCCATCCGGCATCTGTAGGGCGTCCAAATATCGTGCCTCTCCTGCTTT GCCCGGTGTATGAAACCGGAAAGGCCGCTCAGGAGCTGGCCAGCGGCGCAGACCGGGAACACAAGCTGGCAGT CGACCCATCCGGTGCTCTGCACTCGACCTGCTGAGGTCCCTCAGTCCCTGGTAGGCAGCTTTGCCCCGTCTGT CCGCCCGGTGTGTCGGCGGGGTTGACAAGGTCGTTGCGTCAGTCCAACATTTGTTGCCATATTTTCCTGCTCT CCCCACCAGCTGCTCTTTTCTTTTCTCTTTCTTTTCCCATCTTCAGTATATTCATCTTCCCATCCAAGAACCT TTATTTCCCCTAAGTAAGTACTTTGCTACATCCATACTCCATCCTTCCCATCCCTTATTCCTTTGAACCTTTC AGTTCGAGCTTTCCCACTTCATCGCAGCTTGACTAACAGCTACCCCGCTTGAGCAGACATCACCATGGCCACC CTCGACGACACGGCTTACCGGTACCGCACCAGTGTCCCGGGGGACGCCGAGGCCATCGAGGCACTGGATGGGT CCTTCACCACCGACACCGTCTTCCGCGTCACCGCCACCGGGGACGGCTTCACCCTGCGGGAGGTGCCGGTGGA CCCGCCCCTGACCAAGGTGTTCCCCGACGACGAATCGGACGACGAATCGGACGACGGGGAGGACGGCGACCCG GACTCCCGGACGTTCGTCGCGTACGGGGACGACGGCGACCTGGCGGGCTTCGTGGTCGTCTCGTACTCCGGCT GGAACCGCCGGCTGACCGTCGAGGACATCGAGGTCGCCCCGGAGCACCGGGGGCACGGGGTCGGGCGCGCGTT GATGGGGCTCGCGACGGAGTTCGCCCGCGAGCGGGGCGCCGGGCACCTCTGGCTGGAGGTCACCAACGTCAAC GCACCGGCGATCCACGCGTACCGGCGGATGGGGTTCACCCTCTGCGGCCTGGACACCGCCCTGTACGACGGCA CCGCCTCGGACGGCGAGCAGGCGCTCTACATGAGCATGCCCTGCCCCTGAGGATCCGGGCCCCTCGAGAGGCC TTCTAGAAGTCCTCGTGTACTGTGTAAGCGCCCACTCCACATCTCCACTCGACCTGCAGGCATGCAAGCTTGG CACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACA TCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTG 196 AATGGCGAATGAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAG GATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAG GTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAA pEX2B : 10,831 kb AGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAA ACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGC GTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCC GCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACG CGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGAT GCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGAC CTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACA CCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCAT CGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACC CCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGC TTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCG GCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAG GAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGAT GATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGT TTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAA GGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACA AATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCG CAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAG TGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGAC CGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTG TGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGC CGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCG CGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTG AGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACC CGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAG GTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGC AACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACC AAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTAC CAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAG AACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTG GGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATC CGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAG CGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAAT CCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTT CGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCG AAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGC CGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAA CCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTC TGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACG TTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAG CCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTAC CGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCA TCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGAT CTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAAT GACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACC TGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGA AAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGG AACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGA AAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATA 197 ACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCC GCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAG GGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCG GTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG GAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGT AGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCG GTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAG AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGG CGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGAC CGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAG CCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA CGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGT AGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCA GAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG TTAAGGGATTTTGGTCATGCAGGATCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCA ATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACC GAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCT ATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGA ATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACT CGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAAT CAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGG AGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCT GTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATC GATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTT GGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTT ATGTAAGCAGACAGTTTTATTGTTCATGATCTGGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACA TGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTA ACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATC GAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTA AACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTGC TCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCC CGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTA CACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATG ACATGATTACGAATTCATGCGAAGGTAAGTGCTTCTATATTGATCCTTAGTGCTTTCAAACTGTGATGTAGAA GTTGCTCGGTAGCTGATTAAATATTCTAGACCCAAGCCGCTGCTGGAATTGACATTATTATGGCCGATAGGGT TGGGCTTATTGCTATGTCCCTGAAAGGATATCAAAAGCAGGCAAAAAGCCAGGCATAATCCCCGCGTGGACGG TACCCTAAGGATAGGCCCTAATCTTATCTACATGTGACTGCATCGATGTGTTTGGTCAAAATGAGGCATGTGG CTCACCCCACAGGCGGAGAAACGTGTGGCTAGTGCATGACAGTCCCCTCCATAGATTCAATTTAATTTTTCGC GGCAATTGTCGTGCAGTTTGTATCTACATTTCATTCCATATATCAAGAGTTAGTAGTTGGACATCCTGATTAT TTTGTCTAATTACTGAAAACTCGAAGTACTAACCTACTAATAAGCCAGTTTCAACCACTAAGTGCTCATTTAT ACAATATTTGCAGAACCCCGCGCTACCCCTCCATCGCCAACATGTCTTCCAAGTCGCAATTGACCTACAGCGC ACGCGCTAGCAAGCACCCCAATGCGCTCGTAAAGAAGCTCTTCGAGGTTGCCGAGGCCAAGAAAACCAATGTC ACCGTTTCCGCCGACGTGACAACCACCAAAGAGCTGCTGGATTTGGCTGACCGTATGCGCACCGGGGATGCCA CTTACATGTGATCTAGTAATGGTTAATGGTGGATTATATAACAGGACTCGGTCCGTACATTGCCGTGATCAAA ACTCACATCGATATCCTCTCCGATTTCAGCGAAGAAACCATCACCGGTCTGAAGGCCCTTGCAGAGAAGCACA ATTTCCTCATCTTCGAAGATCGCAAGTTCATCGATATCGGAAACACAGTCCAAAAGCAGTACCATGGCGGCAC TCTGCGTATCTCTGAGTGGGCCCACATCATCAACTGCAGTATTCTGCCCGGTGAGGGTATCGTCGAGGCTCTG GCCCAGACTGCTTCGGCCGAGGACTTCCCCTACGGCTCCGAGAGGGGCCTTTTGATCCTTGCGGAGATGACCT CCAAGGGATCTTTGGCTACCGGTCAATATACTACTTCTTCTGTTGACTATGCTCGGAAGTATAAGAAGTTTGT GATGGGATTCGTCTCGACACGTCACCTTGGCGAGGTTCAGTCTGAAGTTAGCTCGCCTTCGGAGGAGGAAGAT TTTGTCGTCTTCACGACAGGTGTCAACCTCTCCTCGAAGGGTGACAAGCTGGGACAGCAGTACCAAACTCCTG AGTCGGCTGTTGGACGCGGTGCCGACTTTATTATTGCTGGCCGTGGAATTTATGCTGCTCCTGATCCCGTGGA GGCGGCGAAGCAGTACCAGAAGGAGGGATGGGATGCATACCTGAAGCGTGTTGGTGCGCAATAAGTAGTGGTG 198 GATACGTACTCCTTTTATGGCAGTATGTCGCAAGTATGATGCGATTTATAAATTCAGCACTCGAAATGACTAC TACTATGTGTCTACGACAGATACCCTCTCCGTACGAATAAGACACCTGCCTCGATATATGGACAAATTCAAAA TCAGGGTCAAGGGTCATGTTTCAAAGTCACAACAATCTCCAACATAGACGAGAATTTGTACCGGAGTGTCTGA AGGTGCAGCTGGAGATTGGTCTATTTTCTTAGAGTGGGGTATCACTAATGTACAGTCGGTCACTATCGTACAA ACAATCACAATTATATACAAGATTTCCCACCACCCCCTACTCTAACACGGCACAATTATCCATCGAGTCAGAG CCTAGCCAACTAGTTATTAAGCGCCGAAATCAGGCAGATAAAGCCATACAGGCAGATAGACCTCTACCTATTA AATCGGCTTCTAGGCGCGCTCCATCTAAATGTTCTGGCTGTGGTGTACAGGGGCATAAAATTACGCACTACCC GAATCGATAGAACTACTCATTTTTATATAGAAGTCAGAATTCATGGTGTTTTGATCATTTTAAATTTTTATAT GGCGGGTGGTGGGCAACTCGCTTGCGCGGGCAACTCGCTTACCGATTACGTTAGGGCTGATATTTACGTAAAA ATCGTCAAGGGATGCAAGACCAAAGTAGTAAAACCCCGGAGTCAACAGCATCCAAGCCCAAGTCCTTCACGGA GAAACCCCAGCGTCCACATCACGAGCGAAGGACCACCTCTAGGCATCGGACGCACCATCCAATTAGAAGCAGC AAAGCGAAACAGCCCAAGAAAAAGGTCGGCCCGTCGGCCTTTTCTGCAACGCTGATCACGGGCAGCGATCCAA CCAACACCCTCCAGAGTGACTAGGGGCGGAAATTTAAAGGGATTAATTTCCACTCAACCACAAATCACAGTCG TCCCCGGTATTGTCCTGCAGAATGCAATTTAAACTCTTCTGCGAATCGCTTGGATTCCCCGCCCCTGGCCGTA GAGCTTAAAGTATGTCCCTTGTCGATGCGATGTATCACAACATATAAATACTAGCAAGGGATGCCATGCTTGG AGGATAGCAACCGACAACATCACATCAAGCTCTCCCTTCTCTGAACAATAAACCCCACAGAAGGCATTTCACG TGCTTAAGGATATCATGGCCTCCTCCGAGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGG CTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCC AAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCCAGTACGGCT CCAAGGTGTACGTGAAGCACCCCGCCGACATCCCCGACTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTG GGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCTCC TTCATCTACAAGGTGAAGTTCATCGGCGTGAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACTATGG GCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACAAGGCCCTGAA GCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGTCCATCTACATGGCCAAGAAGCCCGTGCAGCTGCCC GGCTACTACTACGTGGACTCCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGTACG AGCGCGCCGAGGGCCGCCACCACCTGTTCCTGTAGGATATCGAGCTCCCGCGGGGATCCACTTAACGTTACTG AAATCATCAAACAGCTTGACGAATCTGGATATAAGATCGTTGGTGTCGATGTCAGCTCCGGAGTTGAGACAAA TGGTGTTCAGGATCTCGATAAGATACGTTCATTTGTCCAAGCAGCAAAGAGTGCCTTCTAGTGATTTAATAGC TCCATGTCAACAAGAATAAAACGCGTTTTCGGGTTTACCTCTTCCAGATACAGCTCATCTGCAATGCATTAAT GCATTGACTGCAACCTAGTAACGCCTTNCAGGCTCCGGCGAAGAGAAGAATAGCTTAGCAGAGCTATTTTCAT TTTCGGGAGACGAGATCAAGCAGATCAACGGTCGTCAAGAGACCTACGAGACTGAGGAATCCGCTCTTGGCTC CACGCGACTATATATTTGTCTCTAATTGTACTTTGACATGCTCCTCTTCTTTACTCTGATAGCTTGACTATGA AAATTCCGTCACCAGCNCCTGGGTTCGCAAAGATAATTGCATGTTTCTTCCTTGAACTCTCAAGCCTACAGGA CACACATTCATCGTAGGTATAAACCTCGAAATCANTTCCTACTAAGATGGTATACAATAGTAACCATGCATGG TTGCCTAGTGAATGCTCCGTAACACCCAATACGCCGGCCGAAACTTTTTTACAACTCTCCTATGAGTCGTTTA CCCAGAATGCACAGGTACACTTGTTTAGAGGTAATCCTTCTTTCTAGAAGTCCTCGTGTACTGTGTAAGCGCC CACTCCACATCTCCACTCGACCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGG GAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAG AGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGAGCTTGAGCTTGGATCAGATT GTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGA GCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGC CAACCACAGGGTTCCCCTCGGGATCAA 199 Phụ lục 30 Kết biến nạp vector vào A tumefaciens AGL1 A, B, C, D, E, F, G, H, I, J, K, L, M: kết biến nạp vào A tumefaciens AGL1và xác nhận PCR vector tương ứng pPK2-Red, pGreen3, pPK2-Red2, pKO2AnpyrG, pKO2ΔpcpyrG, pEX2A, pKO2ΔAnlaeA, pGB-AnlaeA, pKO2ΔpclaeA, pPK2-PclaeA, pEX2B-PclaeA, pKO2ΔPdlaeA, pG-PdlaeA 200 Phụ lục 31 Trình tự gen pyrG A niger ACTGCTATGCGACCGTAGAGCAGTGCAAGACCAACGTAAGCCAACCTCACACAAACAGGATTCCTCGAGCTAA CATACATTCCGAACCGTGCAGCCCAAGGCCGAGCAGTTCAACTGCGCTCAGCGCGCTCATGCCAACTTCCTTG AGAACTCCAGCCAAACTATGCTCTTCCTCCTGGTAGCTGGACTGAAGTACCCCCAGTTGGCGACTGGCCTCGG AAGCATCTGGGTCCTCGGTCGCTCACTGTTCCTTTACGGATATGTGTACTCCGGCAAGCCGCGGGGTCGCGGT CGTTTGTACGGCAGCTTCTACTTGCTTGCACAGGGAGCTCTCTGGGGCTTGACGTCTTTTGGAGTTGCGAGGG AGTTGATTTCCTACTTCTAAGTTTGGACTGAATCCGTGGTGTGATTGAGGTGATTGGCGATGTTTGGCTATAC CAGCTATATGTAATAATCTCTACTGTATACTACTATTCAACGCATTTTACTATGCGTGCTGCTAGGGTCGGCA ATGACAATGGCAATCTGACTGACGTGGTCTATTTCTCCATGTGCAGCAGGGAATACGAGCTCCAATGGACCTC GGGAGTGGCACAGTCAATGGCAAGGAAACTCCGCCTTTGCAGGTGTGGCTGAACCCCACGGGTCGGAGGCGGA GCAATCCACCCCCGATGTGGCTGGTGCGTGGAGGGGCTCGCGATGATTTTACTGAGCTTGCTTTTCTTGTCGA CATTGAACATTGTCCTTGGTCTTCCTTCAGATTTAAGGGTCAGTCACTGCTACATTTTTCAGTAGTATCCGCG CACGTCTCTGGATTTACGAATCAGGGTCCACCAGTCGAAACTTCGAACTACTCTCATTATACAATCCTCTTTC CATTCCCGCATTAACCCCTCCATCAACACCATGTCCTCCAAGTCGCGATTGACCTACACTGCCCGTGCCAGCA AGCATCCCAATGCTCTGGCGAAGAGGCTGTTCGAGATTGCCGAGGCCAAGAAGACCAATGTGACTGTCTCGGC TGACGTTACCACCACTAAGGAGCTACTAGATCTTGCTGACCGTAGGCCGACCCGCTACTCTGCCTGATTATGC TGCATGCAAACTTATTAACGGTGATACCGGACTGCAGGTCTCGGTCCCTACATTGCCGTGATCAAAACCCACA TCGATATCCTCTCTGATTTCAGCAACGAGACCATTGAGGGACTTAAGGCTCTCGCGCAGAAGCACAACTTTCT CATCTTCGAGGACCGCAAGTTCATTGACATCGGCAACACGGTCCAGAAGCAATACCACGGCGGTACCCTCCGT ATCTCGGAATGGGCCCACATCATCAACTGCAGCATTCTCCCTGGTGAGGGTATCGTCGAGGCTCTCGCTCAGA CGGCGTCTGCACCGGACTTCGCCTACGGCCCCGAACGCGGTCTGTTGATCTTGGCAGAGATGACCTCTAAGGG CTCCTTGGCTACCGGCCAGTACACTACTTCCTCGGTCGATTATGCCCGGAAATACAAGAACTTCGTTATGGGA TTCGTGTCGACGCGCGCGTTGGGTGAGGTGCAGTCGGAAGTCAGCTCTCCTTCGGATGAGGAGGACTTTGTGG TCTTCACGACTGGTGTGAACATTTCTTCCAAGGGAGATAAGCTTGGTCAGCAGTACCAGACGCCCGGATCGGC TATCGGCCGGGGTGCTGACTTCATTATCGCGGGTCGCGGTATCTACGCCGCGCCGGATCCGGTGCAGGCTGCG CAACAGTATCAGAAGGAGGGGTGGGAAGCCTACCTGGCCCGTGTCGGCGGAAAACTAATACTATAAAAGGAGG ATCGAAGTTCTGATGGTTATGAATGATATAGAAATGCAACTTGCCGCAACGGATACGGAAGCGGAAACGGACC AATGTCGAGCACGGGTAGTCAGACTGCGGCATCGGATGTCCAAACGGTATTGATCCTGCAGGCTACTATGGTG TGGCACAAGGATCAATGCGGTACGACGATTTGATGCAGATAAGCAGGCTGCGAAGTAGTAACTCTTGCGTAGA GAAAATGGCGACGGGTGGCTGATAAGGGCGGTGATAAGCTTAATTGTCATCGCAGATAAGCACTGCTGTCTTG CATCCAAGTCAGCGTCAGCAGAAATACGGGACTTCCGAAAGTATATGGCAAAATTAAAGAACTTGACTCTCCA GCAATGTTTTGCCCTGACCGTCGCTAAAACGTTACTACCCCTATACCCGTCTGTTTGTCCCAGCCCGAGGCAT TAGGTCTGACTGACAGCACGGCGCCATGCGGGCTTGGGACGCCATGTCCGTCGCGTGATAAGGGTTGATCCAT GCAGCTACTATCCTTCCATCGTTCCATTCCCATCCTTGTCCTATCTCCATCCTTGAAACTTTACTAGTTTAGT TGGATGCTCGAGCTTGCTCTCGGCTACTCCGTCCAATGGATAAGACCCCGATGCCGGTCCTCATTGGTCTCCA GCTGGTATCGCCCCAACCTTCGTGTGATCGCCTCTCTGCTTCCCCTCATCATCATTACTAACTAGTACATCCA AAAGCCATCCCAGTGCTTCCCCTCACCCTTGCCCAAGACATTCCAAGTGGGCCTTCGGCTGGAAAACATGGAC CCATTGGTTCCATCGATAAGCTAGCTCCTCGTCCGTTACCCCAGATTGATACCAGATAACATTGACCAGCGGC TTATCACCGAGGTCTGCGGGTGAGACCCCCCCTGCGACAAGTTAGATAAAAGAAACTCGCCTCATTGTGCTTC CGATG 201 ... HỌC KHOA HỌC TỰ NHIÊN VŨ XUÂN TẠO NGHIÊN CỨU PHÁT TRIỂN CÁC VECTOR NHỊ THỂ ỨNG DỤNG TRONG CẢI BIẾN DI TRUYỀN MỘT SỐ LOÀI NẤM SỢI CHUYÊN NGÀNH: VI SINH VẬT HỌC MÃ SỐ : 9420101.07 LUẬN ÁN TIẾN SĨ... vi nấm Từ lý trên, đề tài luận án đƣợc tiến hành với tiêu đề: ? ?Nghiên cứu phát triển vector nhị thể ứng dụng cải biến di truyền số loài nấm sợi? ?? MỤC TIÊU CỦA ĐỀ TÀI - Tạo đƣợc vector nhị thể. .. sử dụng nghiên cứu 36 Bảng 2.2 Các vector sử dụng nghiên cứu 37 Bảng 3.1 Kết xác định số gen laeA chủng A niger 92 biểu mức gen laeA DANH MỤC HÌNH Trang Hình 1.1 Một số đại di? ??n thuộc chi nấm