KR20230127663A - 살모넬라　사멸능을 갖는 박테리오파지 및 이의 용도 - Google Patents

살모넬라　사멸능을 갖는 박테리오파지 및 이의 용도 Download PDF

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KR20230127663A
KR20230127663A KR1020220025218A KR20220025218A KR20230127663A KR 20230127663 A KR20230127663 A KR 20230127663A KR 1020220025218 A KR1020220025218 A KR 1020220025218A KR 20220025218 A KR20220025218 A KR 20220025218A KR 20230127663 A KR20230127663 A KR 20230127663A
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salmonella
phage
bacteriophage
hypothetical proteins
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김지형
박선영
권혜민
한지은
이남경
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Abstract

본 발명은 살모넬라　사멸능을 갖는 박테리오파지 및 이의 용도에 관한 것으로, 보다 상세하게는 살모넬라(Salmonella) 속 균주에 특이적인 사멸능을 갖는, 서열번호 1로 표시되는 염기서열로 이루어진, 또는 수탁번호가 KCTC 14746BP인, 박테리오파지; 이를 포함하는 살모넬라균에 의해 유발되는 감염성 질병의 예방 또는 치료용 조성물; 항생제; 가축용 사료 또는 음용수; 및 소독제 또는 세척제에 관한 것이다.

Description

살모넬라　사멸능을 갖는 박테리오파지 및 이의 용도{A bacteriophage having pathogen Salmonella specific antibacterial activity and use thereof}
본 발명은 병원성　살모넬라, 특히 살모넬라　엔테리티디스 균 특이적인 사멸능을 나타내는 신규　박테리오파지　및 이를 포함하는 조성물에 관한 것이다. 또한 본 발명은 상기　박테리오파지　또는 상기 조성물을 이용하여 감염성 질병 예방용 조성물, 항생제, 소독제 등 다양하게 활용하는 방법에 관한 것이다.
살모넬라(Salmonella spp.)는 장내세균과(Enterobacteriaceae)의 살모넬라속(Salmonella)에 속하는 감마프로테오박테리아(Gammaproteobacteria)의 일종으로 주로 사람이나 동물의 소화관에 서식한다. 살모넬라균은 사람뿐만 아니라 돼지, 소, 닭 등을 비롯한 다양한 가축에 감염되어 질병을 일으키며, 식중독, 위장염, 패혈증, 장염,　살모넬라증 또는 장티푸스를 일으키는 병원성 미생물이며, 더 심한 경우에는 폐렴, 관절염, 유산 등도 발생할 수 있다.　살모넬라　속 균은 현재까지 2,500여종 이상의 혈청형이 보고되었으며, 동물에 따른 숙주 특이성이 있는 균종, 예를 들면 사람에서의　살모넬라　타이피(Salmonella　Typhi), 소에서　살모넬라　두블린(Salmonella　Dublin), 돼지에서의　살모넬라　콜레라수이스(Salmonella　Choleraesuis), 닭에서의　살모넬라　플로럼(Salmonella　Pullorum)과　살모넬라　갈리나륨(Salmonella　Gallinarum))으로 숙주 특이성이 없는 균종, 예를 들면 살모넬라　티피뮤리움(Salmonella　Typhimurim),　살모넬라　엔테리티디스(Salmonella　Enteritidis) 등으로 크게 구분 지을 수 있는데, 이 중 사람의 식중독을 일으키는 혈청형은 후자에 해당한다.
이러한　살모넬라균은 동물에 감염 시 세포 내에 침투하여 증식하고 감염하는 경우가 많아 항생제나 약제, 기타 생균제가 침투하여 작용하기 어려우며, 분리되는　살모넬라균 또한 약제에 내성을 가지고 있는 경우가 많아 항생제나 약제로 예방하기는 어렵다. 특히,　살모넬라균은 오염된 개체들을 통해 수직 또는 수평 감염이 원인이 되므로 계사나 돈사 내 감염 시 높은 폐사율을 나타냄으로서 농가에 심각한 경제적 손실을 초래할 수 있다.
현재　살모넬라　엔테리티디스 감염증을 예방 및 치료하기 위해 항생제를 주로 사용하고 있으나 최근 많은 분야에서 항생제 내성에 대한 문제가 발생되고 있어 이에 대한 원인을 항생제 오남용으로 규명하고 항생제를 충분히 사용하지 못하게 되었다. 이에 따라 산업동물의 배합 사료 내에 성장촉진제로 사용하는 항생제를 금지하기 시작하였고, 또한 항생제가 없는 안전한 먹거리에 대한 소비자의 요구가 증대되고 있어 항생제를 대체할 수 있는 친환경 소재를 기반으로 한 제어법에 대한 연구 및 요구가 더욱 증가할 것으로 기대되고 있다. 이러한 연구 중 하나가 자연계 환경 도처에 존재하면서 특정 세균만을 죽이는 바이러스인　박테리오파지를 활용하는 것이다.
박테리오파지(bacteriophage)는 특정 세균만을 타겟으로 감염시켜 감염된 세균의 성장을 억제하고 저해하는 세균 특이적인 바이러스를 의미한다.　박테리오파지는 박테리아에 감염(infection)한 후 박테리아 세포 내부에서 증식을 하고, 증식 후 자손　박테리오파지들이 박테리아 밖으로 나올 때 숙주인 박테리아의 세포벽을 파괴하는 방식으로 박테리아를 사멸시키는 능력을 갖고 있다.　박테리오파지의 박테리아 감염 방식은 매우 특이성이 높아서 특정　박테리오파지는 특정 박테리아만을 사멸시키고 다른 박테리아에는 영향을 주지 않는 것이 장점이다. 박테리오파지를 이용한 연구는 최근 항생제의 오남용으로 인한 다재내성(Multidrug-resistant)을 지닌 슈퍼박테리아 출현 등 기존 항생제의 많은 문제점들이 부각되면서, 항생제를 대체할 수 있는 친환경 소재로서 각광받고 있다. 특히, 항생제 대비 개발시간이 짧고 개발비용이 적게 소요되며, 높은 선택성, 지속성 및 부작용 등의 측면에서 우위가 인정된다.
예를 들어 한국등록특허 제 10-2041117호는 살모넬라 티피뮤리움을 사멸시킬 수 있는 박테리오파지를 개시하고 있으나, 나아가 양계 및 축산업에 막대한 경제적 피해를 일으키는　살모넬라　엔테리티디스를 포함하는 복수의 살모넬라 균에 의한 감염을 예방 또는 치료하기 위한 신규한　박테리오파지에 대한 연구가 필요하다.
본 발명의 한 측면은 살모넬라(Salmonella) 속 균주에 특이적인 사멸능을 갖는 신규 박테리오파지를 제공하는 것이다.
본 발명의 다른 측면은 상기 박테리오파지를 유효성분으로 포함하는 살모넬라균에 의해 유발되는 감염성 질병의 예방 또는 치료용 조성물을 제공하는 것이다.
본 발명의 또 다른 다른 측면은 상기 박테리오파지를 유효성분으로 포함하는 가축용 사료및 음용수를 제공하는 것이다.
본 발명의 또 다른 다른 측면은 상기 박테리오파지를 유효성분으로 포함하는 소독제 및 세척제를 제공하는 것이다.
본 발명의 일 견지에 의하면, 살모넬라(Salmonella) 속 균주에 특이적인 사멸능을 갖는, 서열번호 1로 표시되는 염기서열로 이루어진, 박테리오파지가 제공된다.
본 발명의 다른 견지에 의하면, 살모넬라(Salmonella) 속 균주에 특이적인 사멸능을 갖는, 수탁번호가 KCTC 14746BP인, 박테리오파지가 제공된다.
본 발명의 다른 견지에 의하면, 상기 박테리오파지를 포함하는, 살모넬라균에 의해 유발되는 감염성 질병의 예방 또는 치료용 조성물이 제공된다.
본 발명의 다른 견지에 의하면, 상기 박테리오파지를 유효성분으로 포함하는 항생제가 제공된다.
본 발명의 다른 견지에 의하면, 상기 박테리오파지를 유효성분으로 포함하는 가축용 사료 또는 음용수가 제공된다.
본 발명의 다른 견지에 의하면, 소독제 또는 세척제가 제공된다.
본 발명의　박테리오파지는 기존의 항생제 등의 화학물질에 비해　살모넬라　균, 특히　살모넬라　엔테라이티디스에 대한 특이성이 매우 높으므로, 항생제 내성균 문제를 해소할 수 있다. 이에 본 발명의　박테리오파지는 살모넬라　균에 의한 감염성 질병의 예방 또는 치료, 항생용 조성물, 사료 첨가용 조성물, 사료, 소독제, 또는 세척제 분야에서 다양하게 사용될 수 있다.
도 1은 본 연구개발 세부모식도를 도식적으로 나타낸 것이다.
도 2는 본 발명의 박테리오파지(φ) 'vB_SenM-pSJ21'의 플라그(plaque) 관찰 결과(좌) 및 TEM 검경 결과(우)를 나타낸 것이다.
도 3은 본 발명의 박테리오파지 'vB_SenM-pSJ21'의 환경안정성 검증 결과를 나타낸 것이다.
도 4는 본 발명의 박테리오파지 'vB_SenM-pSJ21'의 숙주세균 흡착능 분석 결과를 나타낸 것이다.
도 5는 본 발명의 박테리오파지(φ) 'SJ'의 단회 증식계수 분석 결과를 나타낸 것이다.
도 6은 본 발명의 박테리오파지 'vB_SenM-pSJ21'의 MOI별 SE 세균 제어능 분석 결과를 나타낸 것이다.
도 7은 본 발명의 박테리오파지 'vB_SenM-pSJ21'의 농도 별 생물막 억제능 분석 결과를 나타낸 것이다.
도 8은 본 발명의 박테리오파지 'vB_SenM-pSJ21'의 계통수 분석 결과를 나타낸 것이다.
도 9는 본 발명의 박테리오파지 'vB_SenM-pSJ21'의 비교유전체 분석 결과를 나타낸 것이다.
이하, 첨부된 도면을 참조하여 본 발명의 바람직한 실시 형태를 설명한다. 그러나, 본 발명의 실시 형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시 형태로 한정되는 것은 아니다.
본 발명은　살모넬라(Salmonella) 속 균주에 특이적인 사멸능을 갖는 신규　박테리오파지에 관한 것이다.
본 발명은 상기 박테리오파지는 살모넬라(Salmonella) 속 균주에 특이적인 사멸능을 갖는, 수탁번호가 KCTC 14746BP인, 박테리오파지인 것일 수 있다.
상기 살모넬라(Salmonella) 속 균주는 예를 들어 살모넬라 엔테리티디스(Salmonellaenteritidis), 살모넬라 아고나(Salmonella Agona), 살모넬라 바레일리(Salmonella Bareily), 살모넬라 인판티스(Salmonella Infantis), 살모넬라 센프텐베르그(Salmonella Senftenberg), 살모넬라 몬테비데오(Salmonella Montevideo), 및 살모넬라 티피뮤리움(Salmonellatyphimurium)으로 이루어지는 그룹으로부터 선택되는 적어도 하나일 수 있으며, 바람직하게는 살모넬라 엔테리티디스(Salmonellaenteritidis) 및 살모넬라 티피뮤리움(Salmonellatyphimurium)인 것이다.
구체적으로 다양한 종계장, 양계장, 도계장 환경 및 도계육 등으로부터 예를 들어하수 등의 시료를 채취하여, 시료에서 숙주 세포인 SE를 용균하는　본 발명의 박테리오파지를 분리하고, 이들이 SE 균주의 SW, GS, HC, IGJ, SE, GD, GB, GM, IS, CH 및 스탠다드(standard) 스트레인에 대한 광숙주역을 보유한 것을 확인하였다(표 1).
분리된　본 발명의 박테리오파지의 전장유전체 분석 결과, 약 87 kbp의 크기를 갖는 것을 확인하였다. 또한, 이들의 유전적 특성을 분석한 결과, 분리된　박테리오파지는 펠릭소우나바이러스 속(Felixounavirus genus)에 속하는 것으로 확인하였다(도 8).
또한, 다양한 pH 범위 및 온도에서 본 발명의 박테리오파지의 안정성을 조사한 결과, 선발된 박테리오파지는 pH 5 내지 9까지 안정하였으며, 또한, 온도의 경우, 4 내지 37℃까지 높은 안정성을 나타내는 것으로 판정되었다.
또한, 분리된　박테리오파지에는 인테그라아제(integrase), 항생제 내성 및 숙주세균 병원성 관련 유전자는 검출되지 않았으며, SE 숙주세균에 대한 흡착능(absorption) 분석 결과, 본 발명의 SJ 파지는 15분 안에 80% 이상의 흡착율이 관찰되었다(도 4).
상기한 특성을 갖는 신규　박테리오파지를 살모넬라 바이러스(Salmonella virus)'vB_SenM-pSJ21'로 명명하고, 부다페스트 조약에 따라 2021년 10월 28일에 한국생명공학연구원에 기탁번호 KCTC 14746BP로 기탁하였다. 상기 본 발명의 박테리오파지는 이하 박테리오파지(φ) 'vB_SenM-pSJ21', '선발된'파지 또는 'SJ' 파지로 상호호환적으로 지칭할 수 있다.
다른 측면으로 본 발명은 상기　박테리오파지를 유효성분으로 포함하는　살모넬라균에 의해 유발되는 감염성 질병의 예방 또는 치료용 조성물에 관한 것이다.
상기 살모넬라(Salmonella) 속 균주는 예를 들어 살모넬라 엔테리티디스(Salmonella enteritidis), 살모넬라 바레일리(Salmonella Bareily), 살모넬라 인판티스(Salmonella Infantis), 살모넬라 센프텐베르그(Salmonella Senftenberg), 살모넬라 몬테비데오(Salmonella Montevideo), 및 살모넬라 티피뮤리움(Salmonellatyphimurium)으로 이루어지는 그룹으로부터 선택되는 적어도 하나일 수 있으며, 바람직하게는 살모넬라 엔테리티디스(Salmonellaenteritidis) 및 살모넬라 티피뮤리움(Salmonellatyphimurium)인 것이다.
본 발명의　박테리오파지는　살모넬라 엔테리티디스(Salmonellaenteritidis), 살모넬라 바레일리(Salmonella Bareily), 살모넬라 인판티스(Salmonella Infantis), 살모넬라 센프텐베르그(Salmonella Senftenberg), 살모넬라 몬테비데오(Salmonella Montevideo), 및 살모넬라 티피뮤리움(Salmonellatyphimurium)으로부터 선택된 살모넬라 균주를 특이적으로 사멸시킬 수 있는 항균 활성을 가지므로, 이들 균들에 의해 유발되는 질병을 예방하거나 치료하기 위한 목적으로 이용될 수 있다. 예를 들어,　살모넬라　엔테리티디스 또는　살모넬라　티피뮤리움에 의한 질병으로는　살모넬라증 또는　살모넬라균 식중독 등을 예로 들 수 있지만, 이에 제한되는 것은 아니다.
본 발명에서 용어 "살모넬라증"은　살모넬라균 감염에 의해 발열, 두통, 설사, 구토 등을 수반하는 증상을 총칭한다.　살모넬라증은 장티푸스와 같은 증세를 나타내는 패혈증형과 식중독 증세를 나타내는 급성위장염형으로 대별되며, 장염, 식중독, 급성 균혈증 등이 포함된다.
본 발명에서 용어 "예방"이란 조성물의 투여로 질병을 억제시키거나 발병을 지연시키는 모든 행위를 의미하는 것이며, "치료"란 조성물의 투여로 상기 질병의 증세가 호전되거나 상기 질병의 억제 또는 경감 및 이롭게 변경되는 모든 행위를 의미하는 것이다.
상기 조성물은 1×102　내지 1×1012　pfu/㎖, 바람직하게는 1×104　내지 1×107　pfu/㎖의 농도로 본 발명에 따른　박테리오파지를 유효성분으로 포함할 수 있다.
본 발명의 조성물은 약학적으로 허용가능한 담체를 추가로 포함할 수 있으며, 담체와 함께 제제화되어 식품, 의약품 및 사료첨가제로 제공될 수 있다.
본 발명에서 용어, "약학적으로 허용가능한 담체"란 생물체를 자극하지 않고 유효성분의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 말한다. 액상 용액으로 제제화되는 조성물에 있어서 허용되는 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사 용액, 덱스트로스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 하나 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다.
본 발명의 예방 또는 치료용 조성물은 질환 부위에의 도포 또는 분무하는 방법으로 이용할 수 있으며, 그 밖에 경구 투여 또는 비경구 투여를 통해 투여할 수도 있으며, 비경구 투여의 경우 정맥 내 투여, 복강내 투여, 근육 내 투여, 피하 투여 또는 국부 투여를 이용하여 투여할 수도 있다.
본 발명에 따른 조성물의 적합한 도포, 분무 및 투여량은 제제화 방법, 투여 방식, 대상이 되는 동물 및 환자의 연령, 체중, 성별, 질병 증상의 정도, 음식, 투여 시간, 투여경로, 배설 속도 및 반응 감음성과 같은 요인들에 의해 다양하며, 통상 숙련된 의사나 수의사는 목적하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다.
본 발명의 조성물을 유효성분으로 포함하는 경구 투여용 제형으로는, 예를 들어 정제, 트로키제, 로렌지, 수용성 또는 유성 현탁액, 조제 분말 또는 과립, 에멀젼, 경질 또는 연질 캡슐, 시럽 또는 엘릭시르제로 제제화할 수 있다. 정제 및 캡슐 등의 제형으로 제제화하기 위해, 락토오스, 사카로오스, 솔비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴과 같은 결합제, 디칼슘 포스페이트와 같은 부형제, 옥수수 전분 또는 고구마 전분과 같은 붕괴제, 스테아르산 마스네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌글리콜 왁스와 같은 윤활유를 포함할 수 있으며, 캡슐 제형의 경우에는 상기 언급한 물질 외에도 지방유와 같은 액체담체를 더 함유할 수 있다.
본 발명의 조성물을 유효성분으로 포함하는 비경구 투여용 제형으로는, 예를 들어 피하주사, 정맥주사 또는 근육 내 주사 등의 주사용 형태, 좌제 주입 또는 호흡기를 통하여 흡입이 가능하도록 하는 에어로졸제 등 스프레이용으로 제제화할 수 있다. 주사용 제형으로 제제화하기 위해서는 본 발명의 조성물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알의 단위 투여용으로 제제화할 수 있다. 에어로졸제 등의 스프레이용으로 제형화하는 경우, 수분산된 농축물 또는 습윤 분말이 분산되도록 추진제 등이 첨가제와 함께 배합될 수 있다.
본 발명의 다른 견지에 의하면, 상기 본 발명의 박테리오파지를 유효성분으로 포함하는 항생제가 제공되며, 이는 항균용, 생물막 억제용 등으로 사용될 수 있다. 상기 조성물은 1×102　내지 1×1012　pfu/㎖, 바람직하게는 1×104　내지 1×107　pfu/㎖의 농도로 본 발명에 따른　박테리오파지를 유효성분으로 포함할 수 있다.
본 발명에서 용어 "항생제"는 약제 형태로 동물에게 제공되어 균을 사멸시킬 수 있는 제제를 의미하며, 방부제, 살균제 및 항균제를 총칭하는 것이다. 상기 동물은 인간을 포함한 포유동물이다. 본 발명의　박테리오파지는 기존 항생제에 비하여　살모넬라균에 대한 특이성이 매우 높으므로, 익균은 죽이지 않으면서 특정 병원균만 사멸시킬 수 있고, 약물 내성 내지 저항성을 유도하지 않으므로 기존의 항생제에 비하여 제품수명(life cycling)이 긴 신규 항생제로서 이용될 수 있다.
본 발명의 다른 견지에 의하면, 상기 본 발명의 박테리오파지를 유효성분으로 포함하는 가축용 사료 또는 음용수에 관한 것이다.
축산 및 수산업에서 사용되는 사료 첨가용 항생제는 예방 목적으로 사용되고 있는데, 예방 목적의 항생제 투여는 내성균 발생 가능성을 높이고 가축에 잔류하는 항생제가 인간에게 전달될 수 있다는 문제점이 있다. 항생제가 육류를 통해 인체에 흡수되면 항생제 내성을 유발해 질병의 확산을 초래할 수도 있다. 또한, 사료에 섞여 먹이는 항생제의 종류가 많고 이는 다제 내성균의 발생 확률을 높이기 때문에, 자연친화적이면서도 기존의 항생제의 사용에서 발생한 문제를 해결할 새로운 사료첨가제용 항생물질로서 본 발명의 상기　박테리오파지를 이용할 수 있는 것이다.
본 발명의 가축용 사료는　박테리오파지를 사료첨가제 형태로 따로 제조하여 사료에 혼합하거나, 사료 제조 시 직접 첨가하여 제조할 수 있다. 본 발명의 사료 내　박테리오파지는 액상 또는 건조 상태일 수 있으며, 바람직하게는 건조된 분말 형태이다. 건조방법은 통풍건조, 자연건조, 분무건조 및 동결건조가 가능하지만, 이에 제한되는 것은 아니다. 본 발명의　박테리오파지는 분말 형태로 사료 중량의 0.05 내지 10 중량%, 바람직하게는 0.1 내지 2 중량%의 성분비로 혼합될 수 있다. 또한, 상기 가축용 사료는 본 발명의　박테리오파지　외에 사료의 보존성을 높일 수 있는 통상의 첨가제들을 추가로 포함할 수 있다.
본 발명의 사료첨가제에는 비병원성의 다른 미생물이 추가로 첨가될 수 있다. 첨가될 수 있는 미생물로는 단백질 분해효소, 지질 분해효소 및 당 전환 효소를 생산할 수 있는 바실러스 서브틸리스(Bacillus subtilis)와 같은 고초균, 소의 위와 같은 혐기적 조건에서 생리적 활성 및 유기물 분해능이 있는 락토바실러스 균주(Lactobacillus　sp.), 가축의 체중을 증가시키며 우유의 산유량을 늘리고 사료의 소화 흡수율을 높이는 효과를 보여주는 아스퍼질러스 오리자에(Aspergillus　oryzae)와 같은 사상균 및 사카로미세스 세레비지에(Saccharomycescerevisiae)와 같은 효모로 구성된 군으로부터 선택될 수 있다.
본 발명의 사료에는 식물성으로 곡물류, 근과류, 식품가공 부산물류, 조류, 섬유질유, 제약 부산물류, 유지류, 전분류, 박류, 곡물 부산물류 등이 있으며, 동물성으로 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단밸질, 동물성 플랑크톤류, 남은 음식물 등이 있으며, 이에 한정된 것은 아니다.
본 발명의 사료첨가제에는 품질 저하를 방지하기 위해 첨가되는 결착제, 유화제, 보존제 등이 있고, 효용 증대를 위하여 첨가되는 아미노산제, 비타민제, 효소제, 생균제, 향미제, 비단백태 질소화합물, 규산염제, 완충제, 착색제, 추출제, 올리고당 등이 있으며, 그 외에도 사료 혼합제 등을 추가로 포함할 수 있으며, 이에 한정된 것은 아니다.
또한, 본 발명의 사료는 음용수에 혼합하여 공급함으로써 지속적으로 장내　살모넬라균의 숫자를 감소시킬 수 있으며,　살모넬라　청정 가축 생산을 모색할 수 있다.
상기 조성물은 1×102　내지 1×1012　pfu/㎖, 바람직하게는 1×104　내지 1×107　pfu/㎖의 농도로 본 발명에 따른　박테리오파지를 유효성분으로 포함할 수 있다.
본 발명의 상기 조성물은 약학적 제제의 형태로 동물에게 투여되거나, 가축의 사료 또는 음용수에 혼합하여 이를 섭식시키는 방법을 통해 투여될 수 있으며, 바람직하게는 사료 첨가제의 형태로 사료에 혼합되어 투여될 수 있다.
본 발명의 조성물의 적합한 총 1일 사용량은 특정 대상에 대하여 달성하고자 하는 반응의 종류와 정도, 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다.
이하, 구체적인 실시예를 통해 본 발명을 보다 구체적으로 설명한다. 하기 실시예는 본 발명의 이해를 돕기 위한 예시에 불과하며, 본 발명의 범위가 이에 한정되는 것은 아니다.
실시예
1. 신규 박테리오파지 분리를 위한 국내 살모넬라 균주 확보
국내 양계장 및 도계장에서 출현하는 다양한 혈청형의 살모넬라 엔테리티디스(Salmonella Enteritidis, SE), 살모넬라 아고나(Salmonella Agona), 살모넬라 바레일리(Salmonella Bareily), 살모넬라 인판티스(Salmonella Infantis), 살모넬라 센프텐베르그(Salmonella Senftenberg), 살모넬라 몬테비데오(Salmonella Montevideo), 살모넬라 티피뮤리움(Salmonella Typhimurium) 분리주를 확보하였다(표 1).
본 연구에서 확보된 살모넬라 균주
년도 학명 혈청형 Strain 분리장소 분리원
2020 Salmonella enterica Enteritidis SW 세척실 환경시료
2020 Salmonella enterica Enteritidis GS 부화실 환경시료
2020 Salmonella enterica Enteritidis HC 세척실 환경시료
2020 Salmonella enterica Enteritidis IGJ 도계장 계육
2020 Salmonella enterica Enteritidis SE 도계장 계육
2021 Salmonella enterica Enteritidis Standard 도계장 환경시료
2021 Salmonella enterica Enteritidis GD 부화장 난
2021 Salmonella enterica Enteritidis GB 부화장 난
2021 Salmonella enterica Enteritidis GM 부화장 난
2021 Salmonella enterica Enteritidis IS 종계장 환경시료
2021 Salmonella enterica Enteritidis CH 도계장 계육
2021 Salmonella enterica Agona K1 도계장 계육
2021 Salmonella enterica Bareily K2 도계장 계육
2021 Salmonella enterica Infantis K3 도계장 계육
2021 Salmonella enterica Senftenberg K4 도계장 계육
2021 Salmonella enterica Montevideo K5 도계장 계육
2021 Salmonella enterica Typhimurium K6 도계장 계육
상기의 살모넬라 균주들은 국내 다양한 종계장, 양계장, 도계장 환경 및 도계육 등의 시료를 활용하여, 기존 XLD agar(Xylose Lysine Deoxycholate agar) 등의 선택 배지에 기반하여 분리 및 동정을 진행하였다. 해당 분리주의 동정은 16S rRNA 염기서열 분석 및 MALDI-TOF(matrix-assisted laser desorption/ionization time-of-flight)를 통해 진행되었다. 살모넬라로 확인된 분리주의 혈청형 분석은 multuplex-PCR 기반 분석법(Beaubrun 등, 2017)을 통하여 검증되었다. 이렇게 확보된 SE 외 다양한 혈청형의 살모넬라 균주 중 SE 균주를 중심으로 신규 박테리오파지 확보를 위한 연구를 진행하였다.
2. 살모넬라 엔테리티디스(Salmonella Enteritidis, SE) 감염 파지 확보
(1) 국내 양계산업 유래 SE를 특이적으로 감염하는 파지 확보
양계산업 유래 SE 균주를 특이적으로 감염하는 파지 분리 및 특성 분석을 위하여 도 1과 같은 연구 추진 방법으로 진행하였다.
상기 기 확보된 살모넬라 균주(SE) 중 3주(SW, Standard 및 HC)를 숙주세균으로 이용하여 파지 분리를 진행하였으며, 해당 숙주세균은 TSA(tryptic soy agar)에서 37˚C O/N 배양조건으로 준비되었다. 파지 분리를 위한 시료 샘플링은 국내 양계장 검체 및 양계장 주변 하수 및 토양 등이며, 샘플링 장소, 날짜를 표기한 후 파지 분리에 사용하였다. 각 샘플링 시료는 0.45㎛ 필터로 여과하여 TSB(tryptic soy broth)에서 선별된 SE 숙주 세균 3주와 진탕 혼합 배양한 후, 상층액을 이중한천평판법(Double-layered agar method, DLA)을 이용하여 파지 유래 용균환(plaque) 유무를 확인하여 파지를 확보하였다. 확보된 SE 특이 파지는 3회 이상의 순수 분리 작업을 통하여 순수 분리주를 확보하며, 용해물(lysate) 형태로 냉장보관하였다.
파지 분리 결과, SE를 감염하는 신규 파지가 분리되었으며, 관련 세부 정보는 아래와 같다(표 2, 도 2)
본 연구에서 확보된 SE 특이 파지 세부정보
년도 숙주세균 파지 strain명 분리장소 분리원 plaque 특성
2020 SE(SW+Standard+HC) vB_SenM-pSJ21 하수처리장 하수 투명한 용균환
(2) 확보된 SE 특이 파지의 특성 분석 및 선발
확보된 SE 파지 vB_SenM-pSJ21의 형태학적 분류를 위하여 투과전자현미경(transmission electron microscopy, TEM) 검경을 진행하였으며, 세부 사항은 아래와 같다. 각각의 파지 용해물(lysate) 시료는 글로우-디스차지드 카본-코티드 구리 그리드(glow-discharged carbon-coated copper grids)에 최소 2분간 흡착된 후 수분을 제거하였으며, 2% (w/v) 초산우라닐(uranyl acetate)로 1분간 염색되었다. TEM 검경은 Bio-High voltage EM system (JEM-1400 Plus (120 kV) 및 JEM-1000 BEF
(1000 kV), JEOL Ltd., Tokyo, Japan)를 이용하여 한국기초과학지원연구원에서 진행하였다. 투과전자현미경 관찰 결과, vB_SenM-pSJ21 파지는 Myoviridae로 확인되었다.(도 2)
확보된 SE 특이 파지 vB_SenM-pSJ21의 살모넬라 균주에 대한 숙주역을 검증하였으며, 세부 사항은 아래와 같다. 분리된 파지는 앞서 확보된 11주의 SE 균주 및 6주의 타 혈청형 살모넬라에 대한 각각의 숙주역 특성을 검증하였으며, TSA에서 37˚C O/N 배양된 SE 균주를 TSB에 진탕 배양하여 DLA 방법으로 파지 유래 용균환(plaque) 유무를 확인하여 진행하였다.
숙주역 분석 결과, 파지 vB_SenM-pSJ21는 SE 균주 11주(100%)를 용해하여 넓은 숙주저해 특성을 가짐을 확인하였으며, 타 혈청형을 용해하는 것을 통해 광숙주역 파지임을 확인하였다(표 3).
SE 파지 vB_SenM-pSJ21 숙주역 특성 분석결과
Strain vB_SenM-pSJ21 감염능 특이사항(Serotype)
SW ++ Enteritidis(SE)
GS ++ Enteritidis(SE)
HC ++ Enteritidis(SE)
IGJ + Enteritidis(SE)
SE ++ Enteritidis(SE)
Standard ++ Enteritidis(SE)
GD ++ Enteritidis(SE)
GB ++ Enteritidis(SE)
GM ++ Enteritidis(SE)
IS ++ Enteritidis(SE)
CH ++ Enteritidis(SE)
K1 - Agona
K2 - Bareily
K3 - Infantis
K4 - Senftenberg
K5 - Montevideo
K6 ++ Typhimurium
* 파지의 숙주역 결과는 ++(Clear plaque), +(Turbid plaque) 및 -(No plaque)로 제시되었음
상기 숙주역 분석 결과 확보된 vB_SenM-pSJ21 파지는 2가지 이상의 살모넬라 혈청형(Enteritidis, Typhimurium) 균주에 대한 숙주역을 보유하였으며, 다양한 SE 균주를 용해할 수 있는 것으로 확인되었다. 이에 해당 파지를 선발하여 파지의 환경안정성 및 감염 특성에 대한 분석을 진행하였다. 파지의 환경안정성은 다양한 온도 및 pH 조건에 파지 용해물(lysate)을 노출하여 노출 전/후의 PFU(plaque-forming unit)/ml을 비교하여 결과를 취합하였으며, PFU 계수를 위한 배양 조건은 선행 실험과 같은 조건으로 진행하였으며 검증 결과 선발된 vB_SenM-pSJ21 파지는 pH 5~9까지 안정하였다(도 3, 좌측). 상기 vB_SenM-pSJ21 파지는 이하 박테리오파지 '(φ)vB_SenM-pSJ21', '선발된 파지'등으로 지칭하였다.
또한, 온도의 경우, 선발된 파지는 4~37˚C까지 안정성이 확인되었으며, 이는 통상적인 계란의 부화 조건인 37˚C에 부합하여 활용될 수 있는 것으로 판정되었다(도 3, 우측).
나아가, 선발된 파지의 SE 숙주세균에 대한 흡착능(absorption), 단회 증식계수(one-step growth) 및 농도별 SE 세균 제어능(cell lysis efficacy) 특성에 대한 분석을 진행하였다. SE 숙주세균에 대한 흡착능 분석 결과, 선발된 파지는 15분 안에 80% 이상의 흡착율이 관찰되었다(도 4).
SE 숙주세균에 대한 단회 증식계수 결과, vB_SenM-pSJ21 파지는 잠복기(latent period)는 약 20분 정도였으며, 방출량(burst size)은 약 32.07PFU/ml로 관찰되었다(도 5).
농도별 SE 세균 제어능 분석 결과, vB_SenM-pSJ21 파지는 대부분의 MOI(Multiplicity of infection) 조건에서 MOI별 큰 차이가 없는 것으로 보아 적은 수의 파지로도 많은 양에 유사한 정도의 세균 제어 효과를 나타내는 것으로 확인되었다(도 6).
선발된 SE 파지의 생물막(Biofilm) 억제능 및 분해능 분석을 진행하였으며, 세부사항은 다음과 같다. SE에 대한 vB_SenM-pSJ21 파지의 생물막 억제능 분석을 위해 96 웰 플레이트(well plate)에 숙주(host)균 (10^9CFU/ml) 1%와 각각의 농도 별 (10^4, 10^5, 10^6, 10^7PFU/ml) 파지를 처리하여 37˚C 24시간 반응 후 흡광도 595nm와 CFU(colony-forming uint)/ml을 측정하였다. SE의 생물막 억제능 분석 결과 vB_SenM-pSJ21 파지는 10^6CFU/ml까지 감소시킴을 확인하였다. 선발된 파지는 SE 숙주세균의 생물막 제어 효과가 있음이 검증되었다(도 7a).
SE에 대한 vB_SenM-pSJ21 파지의 생물막 분해능 분석을 위해 96 웰 플레이트(well plate)에 숙주(host) 균 (10^9CFU/ml) 1%를 접종하여 생물막을 형성시킨 후, 각각의 농도 별 (10^4, 10^5, 10^6, 10^7PFU/ml)로 파지를 처리하여 37˚C 24시간 반응 후 흡광도 595nm와 CFU(colony-forming uint)/ml을 측정하였다. SE의 생물막 분해능 분석 결과 vB_SenM-pSJ21 파지는 10^7 CFU/ml까지 감소시킴을 확인하였다(도 7b).
이와 같이 선발된 파지는 SE 숙주세균의 생물막에 대한 분해 효과가 있음을 검증하고, 광숙주역 SE 파지로 선발되었으며, 선발 파지는 환경안정성, 감염 특성 및 생물막 제어능에 대한 분석이 진행되어 활용성이 검증되었다.
3. 선발된 광숙주역 SE 특이 파지의 전장유전체 분석
(1) vB_SenM-pSJ21 파지의 전장유전체 확보
앞서 선발된 양계산업 유래 SE 균주에 대한 광숙주역을 보유한 파지(vB_SenM-pSJ21 파지)의 전장유전체 확보 및 특성분석을 진행하였다.
선발된 파지는 숏-리드 NGS 플랫폼(short-read NGS platform, Illumina) 기반 염기서열 분석을 통하여 전장유전체 분석을 진행하였으며, 확보된 결과는 하기 표 4에 제시되었다.
본 연구에서 선발된 vB_SenM-pSJ21 파지의 전장유전체 분석 결과
Gene product Putative function
[Conserved domain]		 Best match virus
(E-value) 		Identity(%) Predicted TMH
and signal peptide
ORF
No.		 Range Strand Length (aa) TMHHM SignalP
1 1-1116 - 372 Tail fiber protein
[PF03335; Phage_fiber; phage tail fiber repeat]		 Salmonella phage BPS17L1
(0.0)		 100 0 N
2 1119-1421 - 100 Hypothetical protein Salmonella phage vB Seyj1-1
(9e-67)		 99 0 N
3 1421-2278 - 285 Hypothetical protein
[PF11041; DUF2612; protein of unknown function (DUF2612)]		 Salmonella phage FSL SP-010
(0.0)		 98.25 0 N
4 2281-3750 - 489 Baseplate component
[PF04865; Baseplate_J; baseplate J-like protein]		 Salmonella phage BPS17L1 (0.0) 99.80 0 N
5 3750-4169 - 139 Hypothetical protein Escherichia phage vB_EcoM_Alf5 (2e-97) 99.28 0 N
6 4169-4792 - 207 Baseplate assembly protein
[PF18352; Gp138_N; phage protein Gp138 N-terminal domain]		 Escherichia phage vB_EcoM-VpaE1 (2e-153) 99.52 0 N
7 4792-5769 - 325 Hypothetical protein
[PF07569; Hira; TUP1-like enhancer of split]		 Escherichia phage vB_EcoM_AYO145A (0.0) 100 0 N
8 5769-6110 - 113 Hypothetical protein Salmonella phage BPS17W1 (1e-77) 99.12 0 N
9 6110-6907 - 265 Hypothetical protein Salmonella phage S19cd (0.0) 97.74 0 N
10 6907-9135 - 742 Tape measure protein
[PF20155; TMP_3; tape measure protein]		 Salmonella phage Meda (0.0) 98.65 0 N
11 9135-9374 - 79 Tape measure chaperone Salmonella phage Meda (1e-48) 100 0 N
12 9377-9775 - 132 Tape measure chaperone
[PF17721; DUF5566; family of unknown function (DUF5566)]		 Salmonella phage Meda
(3e-90)		 100 0 N
13 9848-10294 - 148 Hypothetical protein
[PF11681; DUF3277; protein of unknown function (DUF3277)]		 Escherichia phage wV8 (6e-105) 100 0 N
14 10310-11662 - 450 Tail sheath protein
[PF11863; DUF3383; protein of unknown function (DUF3383)]		 Salmonella phage Meda (0.0) 96.89 0 N
15 11663-12262 - 199 Hypothetical protein
[PF09349; OHCU_decarbox; OHCU decarboxylase]		 Escherichia phage dune (6e-143) 99.50 0 N
16 12237-12638 - 133 Hypothetical protein Salmonella phage FSL SP-010
(5e-94)		 99.25 0 N
17 12635-13117 - 160 Hypothetical protein Escherichia phage wV8 (2e-112) 99.38 0 N
18 13117-13566 - 149 Hypothetical protein Escherichia phage mio (6e-105) 99.33 0 N
19 13587-14693 - 368 Major capsid protein
[PF03864; Phage_cap_E; phage major capsid protein E]		 Escherichia phage JH2
(0.0)		 99.46 0 N
20 14727-15104 - 125 Hypothetical protein Salmonella phage Meda
(2e-87)		 100 0 N
21 15116-16462 - 448 Capsid maturation protease
[PF01343; Peptidase_S49; peptidase family S49]		 Salmonella phage Meda
(0.0)		 100 0 N
22 16474-16806 - 110 Hypothetical protein Escherichia phage vB_EcoM-VpaE1 (8e-73) 99.09
23 16806-17306 - 166 Hypothetical protein Escherichia phage JH2 (2e-117) 100 0 N
24 17306-18772 - 488 Hypothetical protein Escherichia phage wV8 (0.0) 99.80 0 N
25 18789-20390 - 533 Terminase large subunit
[PF03237; Terminase_6N; terminase large subunit, T4likevirus-type, N-terminal]		 Salmonella phage FelixO1
(0.0)		 99.81 0 N
26 20412-20612 - 66 Hypothetical protein
[PF19111; DUF5798; Family of unknown function (DUF5798)]		 Salmonella phage FelixO1
(7e-38)		 100 0 N
27 21010-21744 - 244 Hypothetical protein
[PF00015; MCPsignal; Methyl-accepting chemotaxis protein (MCP) signalling domain]		 Salmonella phage Meda (3e-172) 98.36 0 N
28 22196-22753 - 185 Hypothetical protein Escherichia phage skuden
(2e-131)		 98.38 2 Y
29 23430-23882 - 150 Hypothetical protein
[PF01844; HNH; HNH endonuclease]		 Escherichia phage SP22 (5e-104) 98 2 N
30 25552-25980 - 142 HNH homing endonuclease
[PF01771; Viral_alk_exo; Viral alkaline exonuclease]		 Escherichia phage radambza
(7e-92)		 88.73 0 N
31 26748-28163 - 471 vWFA protein
[PF11443; DUF2828; Domain of unknown function (DUF2828)]		 Escherichia phage humlepung (0.0) 97.88 0 N
32 28245-28616 - 123 Hypothetical protein
[PF18932; DUF5681; Family of unknown function (DUF5681)]		 Escherichia phage wV8 (1e-82) 100 0 N
33 28866-29027 + 53 Hypothetical protein Salmonella phage FelixO1
(1e-30)		 100 0 N
34 29009-29242 + 77 Hypothetical protein Salmonella phage FelixO1 (2e-49) 100 0 N
35 29236-29826 + 196 Hypothetical protein
[PF09648; YycI; YycH protein]		 Escherichia phage vB_EcoM_AYO145A (6e-139) 99.49 0 N
36 29874-30245 + 123 Hypothetical protein Enterobacteria phage vB_EcoM_IME338 (1e-85) 98.37 0 N
37 30242-30874 + 210 Tail protein
[PF13927; Ig_3; Immunoglobulin domain]		 Salmonella phage Meda (2e-149) 97.62 0 N
38 30874-31338 + 154 Endolysin glycoside hydrolase
[PF00959; Phage_lysozyme; Phage lysozyme]		 Salmonella phage Meda
(4e-109)		 99.35 0 N
39 31389-31787 + 132 Hypothetical protein Salmonella phage BPSELC-1
(3e-92)		 99.24 0 N
40 31780-32187 + 135 Hypothetical protein
[PF10181; PIG-H; GPI-GlcNAc transferase complex, PIG-H component]		 Salmonella phage Meda
(6e-88)		 95.56 0 N
41 32187-32480 + 97 Hypothetical protein Escherichia phage skuden
(2e-64)		 100 0 N
42 32473-32817 + 114 Hypothetical protein Salmonella phage vB_SPuM_SP116 (1e-76) 99.12 0 N
43 32817-33407 + 196 Hypothetical protein
[PF18744; SNAD1; Secreted Novel AID/APOBEC-like Deaminase 1]		 Salmonella phage FSL SP-010 (5e-142) 97.45 0 N
44 33481-33699 + 72 Hypothetical protein Salmonella phage vB_SPuM_SP116 (2e-44) 100 0 N
45 33696-34199 + 167 Hypothetical protein
[PF12046; CCB1; Cofactor assembly of complex C subunit B]		 Salmonella phage Meda
(2e-119)		 98.8 0 N
46 34276-34500 + 74 Hypothetical protein Escherichia phage vB_EcoM-VpaE1 (4e-43) 100 2 N
47 34553-35104 + 183 Hypothetical protein
[PF12288; CsoS2_M; Carboxysome shell peptide mid-region]		 Salmonella virus VSe11
(3e-126)		 95.08 0 N
48 36274-36420 - 48 Hypothetical protein Salmonella phage Meda
(8e-10)		 100 0 N
49 36824-37072 - 82 Hypothetical protein Salmonella phage BPS17L1 (3e-53) 100 0 N
50 37152-37682 - 176 Hypothetical protein
[PF10364; NKWYS; Putative capsular polysaccharide synthesis protein]		 Salmonella phage Meda
(7e-126)		 100 0 N
51 37907-38248 - 113 Hypothetical protein Salmonella phage Meda
(2e-78)		 99.12 0 N
52 38340-38573 - 77 Hypothetical protein Salmonella phage BPS17L1 (2e-48) 100 0 N
53 38640-39182 - 180 Hypothetical protein
[PF17900; Peptidase_M1_N; Peptidase M1 N-terminal domain]		 Salmonella phage BPS17L1
(9e-129)		 100 0 Y
54 39270-39473 - 67 Hypothetical protein Salmonella phage BPS17L1
(2e-40)		 100 0 N
55 39574-39978 - 134 Hypothetical protein Salmonella phage BPS17L1 (1e-92) 100 0 N
56 40064-40336 - 90 Hypothetical protein Salmonella phage BPS17L1 (2e-60) 100 0 N
57 40424-40756 - 110 Hypothetical protein
[PF11337; DUF3139; Protein of unknown function (DUF3139)]		 Salmonella phage Meda
(2e-71)		 100 0 N
58 40750-41046 - 98 Hypothetical protein
[PF10412; TrwB_AAD_bind; Type IV secretion-system coupling protein DNA-binding domain]		 Salmonella phage BPS17L1
(1e-64)		 100 0 N
59 41139-41651 - 170 Hypothetical protein Salmonella phage BPS17L1
(3e-122)		 100 0 N
60 41736-42029 - 97 Hypothetical protein Salmonella phage BPS17L1 (1e-59) 97.94 0 N
61 42116-42499 - 127 Hypothetical protein Salmonella phage BPS17L1 (9e-87) 100 0 N
62 42988-43767 - 259 Hypothetical protein
[PF01661; Macro; Macro domain]		 Salmonella phage BPS17L1 (0.0) 99.61 0 N
63 43768-43968 - 66 Hypothetical protein Salmonella phage D1-2 (7e-40) 100 0 N
64 43981-44208 - 75 Hypothetical protein Escherichia phage EC6 (1e-45) 98.67 0 N
65 44186-44509 - 107 Hypothetical protein Escherichia phage VpaE1_ev035 (1e-72) 100 0 N
66 44512-44781 - 89 Hypothetical protein Salmonella phage Mushroom (7e-60) 98.88 0 N
67 44778-44975 - 65 o-spanin
[PF12971; NAGLU_N; Alpha-N-acetyl glucosaminidase (NAGLU) N-terminal domain]		 Salmonella phage vB_SflM_004
(5e-39)		 98.46 0 N
68 44941-45288 - 115 i-spanin Salmonella phage BPS17L1
(3e-78)		 99.13 0 N
69 45341-45805 - 154 Hypothetical protein
[PF10902; WYL_2; WYL_2, Sm-like SH3 beta-barrel fold]		 Salmonella phage Meda
(8e-105)		 100 1 N
70 45816-46511 - 231 Polynucleotide kinase Salmonella phage BPS17L1
(7e-168)		 97.40 0 N
71 46489-47037 - 182 Putative lysin
[PF01464; SLT; Transglycosylase SLT domain]		 Escherichia phage tootiki
(3e-129)		 99.45 0 N
72 47138-48247 - 369 RIIB protein
[PF13542; HTH_Tnp_ISL3; Helix-turn-helix domain of transposase family ISL3]		 Salmonella phage SA1 (0.0) 100 0 Y
73 48327-50693 - 788 RIIA protein
[PF02518; HATPase_c; Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase]		 Salmonella phage SA1 (0.0) 98.10 0 N
74 50722-50898 - 58 Hypothetical protein
[PF13597; NRDD; Anaerobic ribonucleoside-triphosphate reductase]		 Salmonella phage D1-2 (6e-34) 100 0 N
75 50880-51215 - 111 Hypothetical protein Salmonella phage BPS17L1
(4e-74)		 98.20 1 N
76 51269-53050 - 593 Nicotinate phosphoribosyl transferase
[PF04095; NAPRTase; Nicotinate phosphoribosyltransferase (NAPRTase) family]		 Salmonella phage BPS17L1
(0.0)		 97.81 0 N
77 53096-53977 - 293 Ribose-phosphate pyrophosphokinase
[PF00156; Pribosyltran; Phosphoribosyl transferase domain]		 Salmonella phage Meda (0.0) 99.32 0 N
78 53989-54267 - 92 Hypothetical protein Salmonella phage Meda
(3e-62)		 100 0 N
79 54260-54775 - 171 Hypothetical protein Salmonella phage Meda (3e-126) 100 0 N
80 54827-55138 - 103 Putative HNH endonuclease Enterobacteria phage vB_EcoM_IME338 (5e-69) 100 0 N
81 55150-55407 - 85 Hypothetical protein
[PF02288; Dehydratase_MU; Dehydratase medium subunit]		 Enterobacteria phage vB_EcoM_IME338 (8e-51) 100 0 N
82 55661-56146 - 161 Ribonucleotide reductase of class III (anaerobic) activating protein
[PF13353; Fer4_12; 4Fe-4S single cluster domain]		 Escherichia phage XTG1 (6e-118) 100 0 Y
83 56156-56455 - 99 Hypothetical protein
[PF17603; DUF5499; Family of unknown function (DUF5499)]		 Salmonella phage BPS17L1 (1e-67) 100 0 N
84 56452-56847 - 131 Hypothetical protein Escherichia phage skuden
(7e-21)		 100 0 N
85 57047-57382 - 111 Hypothetical protein
[PF08019; EptA_B_N; Phosphoethanolamine transferase EptA/EptB]		 Salmonella phage Meda
(2e-75)		 100 0 N
86 57445-58311 - 288 Anaerobic nucleoside diphosphate reductase large subunit
[PF13597; NRDD; Anaerobic ribonucleoside-triphosphate reductase]		 Salmonella phage Meda
(0.0)		 99.65 2 N
87 58555-59019 - 154 HNH endonuclease
[PF13392; HNH_3; HNH endonuclease]		 Salmonella phage Meda
(3e-109)		 100 0 N
88 59132-60331 - 399 Anaerobic nucleoside diphosphate reductase large subunit
[PF13597; NRDD; Anaerobic ribonucleoside-triphosphate reductase]		 Salmonella phage Meda
(0.0)		 99.75 0 N
89 60380-60586 - 68 Membrane protein Salmonella phage BPS17L1
(1e-37)		 97.06 0 N
90 60579-60821 - 80 Putative glutaredoxin
[PF00462; Glutaredoxin; Glutaredoxin]		 Salmonella phage FSL SP-010 (3e-53) 98.75 2 N
91 60821-61894 - 357 Ribonucleoside triphosphate reductase beta subunit
[PF00268; Ribonuc_red_sm; Ribonucleotide reductase, small chain]		 Escherichia phage vB_EcoM-VpaE1 (0.0) 100 0 N
92 61891-62148 - 85 Hypothetical protein
[PF07866; DUF1653; Protein of unknown function (DUF1653)]		 Salmonella phage Meda
(7e-56)		 100 0 N
93 62204-64438 - 744 Ribonucleoside triphosphate reductase alpha subunit
[PF02867; Ribonuc_red_lgC; Ribonucleotide reductase, barrel domain]		 Salmonella phage Meda
(0.0)		 100 0 N
94 64485-64823 - 112 Hypothetical protein Salmonella phage Meda
(8e-75)		 99.09 0 N
95 64816-65139 - 107 Hypothetical protein Salmonella phage FelixO1 (2e-74) 100. 0 N
96 65120-65875 - 251 Hypothetical protein
[PF01503; PRA-PH; Phosphoribosyl-ATP pyrophosphohydrolase]		 Enterobacteria phage KhF3
(0.0)		 100 0 N
97 65851-66099 - 82 Hypothetical protein
[PF16043; DUF4795; Domain of unknown function (DUF4795)]		 Salmonella phage FelixO1 (8e-50) 100 0 N
98 66121-66618 - 165 Hypothetical protein
[PF19835; SegE_GIY-YIG; Putative endonuclease segE, GIY-YIG domain]		 Salmonella phage Meda (9e-118) 99.39 0 N
99 66608-67648 - 346 Exodeoxyribonuclease
[PF01367; 5_3_exonuc; 5'-3' exonuclease, C-terminal SAM fold]		 Salmonella phage Meda (0.0) 99.71 0 N
100 67711-68568 - 285 Hypothetical protein
[PF03153; TFIIA; Transcription factor IIA, alpha/beta subunit]		 Enterobacteria phage UAB_Phi87 (0.0) 100 0 N
101 68641-68790 - 49 Hypothetical protein Salmonella phage FelixO1
(6e-26)		 100 0 N
102 69043-71028 - 661 DNA primase/hilecase
[PF03796; DnaB_C; DnaB-like helicase C terminal domain]		 Escherichia phage skuden
(0.0)		 99.85 0 N
103 71021-71215 - 64 Hypothetical protein
[PF09371; Tex_N; Tex-like protein N-terminal domain]		 Escherichia phage EC6 (2e-35) 100 0 N
104 71230-71973 - 247 Deoxynucleotide monophosphate kinase
[PF12286; DUF3622; Protein of unknown function (DUF3622)]		 Escherichia phage vB_EcoM_AYO145A (5e-180) 99.19 0 N
105 72035-72835 - 266 Minor tail protein Salmonella phage DaR-2019b
(0.0)		 98.87 0 N
106 72837-73259 - 140 Hypothetical protein
[PF18595; DHR10; Designed helical repeat protein 10 domain]		 Salmonella phage KhF2
(7e-98)		 100 0 N
107 73517-75700 + 727 Putative DNA polymerase
[PF00476; DNA_pol_A; DNA polymerase family A]		 Escherichia phage HY02 (0.0) 99.44 0 N
108 75930-76484 + 184 DNA polymerase
[PF00476; DNA_pol_A; DNA polymerase family A]		 Salmonella phage BPS17W1 (2e-132) 99.46 0 N
109 76488-77021 + 177 Homing endonuclease
[PF13392; HNH_3; HNH endonuclease]		 Escherichia phage bumzen
(5e-127)		 99.44 0 N
110 77073-77282 + 69 Hypothetical protein
[PF00949; Peptidase_S7; Peptidase S7, Flavivirus NS3 serine protease]		 Escherichia phage vB_EcM-Ro111lw (1e-42) 100 0 N
111 77279-77497 + 72 Hypothetical protein Escherichia phage vB_EcoM_Alf5 (7e-44) 98.61 0 N
112 77494-77640 + 48 Hypothetical protein Salmonella phage FelixO1 (1e-26) 100 0 N
113 77630-77887 + 85 Hypothetical protein Escherichia phage VpaE1_ev035 (3e-44) 93.24 0 N
114 77898-78104 + 68 Hypothetical protein Escherichia phage humlepung (7e-42) 100 0 Y
115 78101-78304 + 67 DNA ligase Salmonella phage BPS17W1 (7e-38) 92.54 0 N
116 78301-78555 + 84 Hypothetical protein
[PF14026; DUF4242; Protein of unknown function (DUF4242)]		 Escherichia phage EC6
(3e-47)		 91.46 0 N
117 78629-78820 + 63 Hypothetical protein Escherichia phage wV8
(3e-40)		 100 0 N
118 78822-79925 + 367 DNA ligase
[PF01068; DNA_ligase_A_M; ATP dependent DNA ligase domain]		 Escherichia phage SME50
(0.0)		 99.73 0 N
119 79976-80125 - 49 Hypothetical protein Salmonella virus VSe11
(5e-25)		 97.96 0 N
120 80358-80741 + 127 Hypothetical protein Salmonella phage FelixO1 (3e-88) 99.21 0 N
121 80743-80955 + 70 Hypothetical protein
[PF04175; DUF406; Protein of unknown function (DUF406)]		 Escherichia phage wV8
(2e-42)		 100 0 Y
122 80948-81247 + 99 Putative transcriptional regulatory protein
[PF01381; HTH_3; Helix-turn-helix]		 Escherichia phage wV8
(2e-67)		 97.98 0 N
123 81249-81608 + 119 Hypothetical protein
[PF15246; NCKAP5; Nck-associated protein 5, Peripheral clock protein]		 Escherichia phage vB_EcoM-VpaE1 (3e-82) 99.16 0 N
124 81622-82137 + 171 Hypothetical protein
[PF09252; Feld-I_B; Feld-I_B (PF09252)]		 Salmonella phage DaR-2019b
(5e-118)		 98.25 0 N
125 82138-82398 + 86 Hypothetical protein
[PF14690; zf-ISL3; zinc-finger of transposase IS204/IS1001/IS1096/IS1165]		 Escherichia phage KhF1
(7e-57)		 98.84 0 N
126 82395-82940 + 181 Dihydrofolate reductase
[PF00186; DHFR_1; Dihydrofolate reductase]		 Enterobacteria phage UAB_Phi87 (1e-129) 99.45 0 N
127 82942-83841 + 299 Thymidylate synthase
[PF00303; Thymidylat_synt; Thymidylate synthase]		 Salmonella phage Meda
(0.0)		 99.67 0 N
128 83886-84251 - 121 Hypothetical protein
[PF07889; DUF1664; Protein of unknown function (DUF1664)]		 Salmonella phage FelixO1
(2e-81)		 100 0 N
129 84252-84446 - 64 Hypothetical protein Escherichia phage vB_EcoM_AYO145A (3e-37) 100 1 N
130 84526-87048 - 840 Tail fiber protein
[PF12604; gp37_C; Tail fiber protein gp37 C terminal]		 Salmonella phage BPS17L1
(0.0)		 94.64 0 N
131 87100-87273 - 57 Tail fiber protein
[PF03335; Phage_fiber; Phage tail fiber repeat]		 Salmonella phage BPS17L1 (1e-28) 100 0 N
선발된 파지는 SE 확보주 중 스탠다드(standard) 균주에서 증식이 되었으며, 108PFU/ml 이상 고농도로 증식된 파지 용해물(lysate)에서 전체 지노믹(total genomic) DNA를 추출하여 전장유전체 분석 후 숙주세균의 지놈(genome) 정보를 트리밍(trimming)하는 방식으로 분석을 진행하였다. 전장 유전체 확보를 위한 로-리드 어셈블리(raw-read assembly)는 SPAdes 3.13.0으로 진행하였으며, 유전자 특성 분석을 위한 주석달기(annotation)은 온라인(online) 분석 툴(tool)인 RAST(Rapid Annotations using Subsystems Technology, Aziz 등 2008)에서 우선적으로 분석 후 수동 오류 정정(manual error correction) 방식을 통하여 수행하였다.
(2) 선발된 파지의 유전체 기반 신규성 검증 및 안전성 분석
1) 선발된 vB_SenM-pSJ21 파지의 신규성을 검증하기 위하여 확보된 전장유전체 기반 계통수 및 비교유전체 분석을 진행하였다.
파지의 전장유전체의 계통수 분석은 VICTOR(https://ggdc.dsmz.de/victor.php, Meier-Kolthoff 및 Goker, 2017)를 이용하여 수행하였고, 비교유전체 분석은 파지를 Blastn 결과 가장 유사한 파지와 Easyfig(10.1093/bioinformatics/btr039, Sullivan 등, 2011)을 이용하여 수행하였다.
오우나비리나에 서브패밀리(Ounavirinae subfamily)로 분류되는 파지들 25종을 이용하여 vB_SenM-pSJ21 파지 계통수 분석한 결과 펠릭소우나바이러스 속(Felixounavirus genus)에 속하는 것을 확인하였다(도 8).
파지 전장유전체를 블라스트(blast) 비교분석한 결과, vB_SenM-pSJ21 파지는 살모넬라 바이러스(Salmonella virus) BPS17W1(NC_042097)과 Query coverage 95%, Identity 96.12% 유사함을 확인하여 파지의 주요 특징을 갖는 단백질들을 비교하였다(도 9).
vB_SenM-pSJ21 파지의 안전성을 검증하기 위하여 확보된 전장유전체 기반 위해인자(integrase, 항생제 내성 및 숙주세균 병원성 관련 유전자)의 존재유무를 확인하였다. 파지 유래 잠재적 위해인자의 존재 유무는 1)blast search(https://blast.ncbi.nlm.nih.gov/Blast.cgi), 2)The Comprehensive Antibiotic Resistance Database(https://card.mcmaster.ca/) 및 3)VFDB database(http://www.mgc.ac.cn/VFs/)에 기반한 분석을 통하여 수행되었다.
광숙주역 SE 파지 vB_SenM-pSJ21 의 전장유전체 기반 위해인자 검증 결과, 인테그라아제(integrase), 항생제 내성 및 숙주세균 병원성 관련 유전자는 검출되지 않았다.
상기 결과를 토대로, 선발된 광숙주역 vB_SenM-pSJ21 파지 전장유전체 분석을 통하여 신규함을 확인하였으며, 잠재적 위해인자를 검증하여 최종 파지를 선정하였다.
이상에서 본 발명의 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고, 청구범위에 기재된 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 다양한 수정 및 변형이 가능하다는 것은 당 기술분야의 통상의 지식을 가진 자에게는 자명할 것이다.
<110> Korea Research Institute of Bioscience and Biotechnology <120> A bacteriophage having pathogen Salmonella specific antibacterial activity and use thereof <130> P2022009KR <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 87273 <212> DNA <213> Artificial Sequence <220> <223> vB_SenM-pSJ21 <400> 1 gaatgcttga ccagcacctg tagtacctgt agtacctgag tgagtatgct cagatgactg 60 caaagttact gtgcctttat gggtatgagc accaattgca acagtatgcg tgtgagcacc 120 aattgcaaca gtgtgttggt gattgcctgt ggtatttgtt gaaccgctaa tagtgtgtga 180 gtggtcacca gctacactgg aaacctgttc tgtaccatat acctgaacac gtggtttacc 240 accgatagag tcacctacat aatgaccacc aacagtatgt gtgtgattac ctgtggtatt 300 tgttgaaccg ctcactgagt gggtgtgagc accagtagtg ttagtggttt tagtaccata 360 atcgaaactg tttgtgctct tattgccgta atcaaagcca tcgatagtaa tagcagcacc 420 atgagtatgt ccaccaccag ttaggtagat tgaatggcta tgtgatggta ggttgttact 480 agataagcta acactacttg agccaaagtt actaccaact ggcctagaat cactatcata 540 acctacaagt gctcttcctc ttgaaactaa ctcccaagta cccccacaaa ttaaatatgt 600 agaagggttt gcaggattca tagagagatg gatagtacct actggataag aagcctgaac 660 agccttgtac aagttattta ctgctcttgc tgtagcgtac ttatctgcat cttcattata 720 aagattagat gttgtccagt tctggacatt gctcaaacca acctgtgcct tagttgtatt 780 gtgtgggtta ctcttatcat taatatgttg ttgaacaaga ctattaacct cttctgaaga 840 cataatctgt agatttgctc ttgcctcatc aacattagta atgtctgata aattatttgc 900 agcaactaac tgaagagcat tgataacgtt atctaaacca atttgtgttt tagtaacacc 960 gtgggggtta tttcttaggc ttgagtgtgg agcaagtaat tgctcaagag tacatctctt 1020 atcctcaata ccctgcttaa gatggaagat atcactaagg tcaattggta aggctgcttg 1080 aggcaaggca ctaatttgaa tttcacctac tgccattatt aagctcctac aaattcataa 1140 gtgtaaaggt tttttgtact tgttgatgca gtacctgttt cggtcttaat aagactccag 1200 ccattagcta ctaaatctgg ttgctcagtg gcaaaagacc ttacttcacc aacagttccg 1260 tttgctttct tcaggagata gtttaagata tagttaaacc attgtcgtcc cataggctca 1320 ccccttaata aaccagtggc ctgaatttct ggtggtggta atacttttag ctggttacca 1380 tcagcatcta cttcatctgt agaccaattt aaaaatgcca ttaggaactt ccttcttgct 1440 gtgatttatc ttttctgcct ttgatgattt gagctacttc agccataaca ccataatcac 1500 cacctgctac agtttcttta ccaacgatat agttatctgt agcgttataa tttttattaa 1560 cctttaagta atctgtggaa ccactattag ccgttctgtc aatcttgaaa taagcatctc 1620 ttacgccagc atctgccaaa ctacctagta agttcttatc taaagaacca cctgtgttgt 1680 tagtaacaag acccttgtta gcatctgtaa cgaaccagtt atctttatca tcaacaattg 1740 ctaaagctga atcagcaacc tctacaggtg tccaagcagt accattcaga gttacgtctc 1800 ttagaataac tgctgagcca atagttgttg cagcaatttt tgctaatgtg tacgctgtat 1860 caatgacgtt atttcttgta ttaaccctga ctacaatacc agcagtcaca ggagtaatat 1920 gctcaaaaat ctgtgaaaat gttgcattat acagagtcat gatagcattc tgtaaaaatg 1980 ttggagttgt atcagagcgt cttaggaaaa tctggatata caacattgct ctatatgttt 2040 catcatcagc accaagtggt cgtggcactt taattaatgc accgatatta tcaagttgct 2100 ttccagtagc ctttctaata ttcctttcgg tgtgcatttg ccatgaaaca tcttccaatg 2160 tctgcaactc atcagtgaca gctttcagta aacttgtata gatgaattta tctttaaact 2220 gtgtaacagt tctttcatca agagttttat aataaacatt atcaattttc tgaaacatta 2280 ttactcctta gcaatggtgt actggctact ttcccataca gtatattggt caccatcaac 2340 agtaattctt gctgtagtat actgtccatc actaggaggc actgactggt tatttgaaag 2400 tgctactttg atttcattaa tctcaatacc tttaatgaca tcataaatat acccatagat 2460 tctgttaggg ataacatcat taccaacttt cagagttctc ccgtaagcat taataccttg 2520 aacaatactg tctctgatat cttcttctgg gattgttaag ctctcttcat catacaaaga 2580 ataagatact ttgacaaaag catacttggg tgttggtctg ctaaaataga cattatgagc 2640 taaattacct aagtcataag ctgtaccaaa gatagctcca taagccctga taccagcagg 2700 tttggtgtcc cagattgctt gagcaacatt atcattttga ccaccaacta caacaatctt 2760 gaaagatttt ggtggcagtc cttctgaact tgtctcttca gtatcgtttt caacacctga 2820 agcatctgat acgccctgaa ctcttttaac agcagctacg attgcatcga gagttcctac 2880 accagtaact gccaaagatt ctaaatatct ctgccgaagt tctgtatcgg tttcttcatt 2940 cctacctgtt gtcaggtcat atcggttgta tacactgtca agaccatcta cagttgtttc 3000 aatttcgata agtgttccag ctaatgcagg gattgcacca acttcctcag caacaacatc 3060 attgatagtt gtaatttttg tgaatgtaag gaacgtcgta gcagtcacca ccatagggtt 3120 ggttctggca ataatgtcac cttcatcttt ataaactcgt aatgctgaac cgtcattgat 3180 aacttcggct tttgctacga taccaccatt gattgcatcg gcaagttcag ttaatagtac 3240 tgtgattgtg tctgaagatt ttggctgata agagaaaata acgttatcaa taataataac 3300 ataatttgca tcagttcgta aagagttaac ttcaagaaca gcttcaacac aatatgaagg 3360 tgttaatgta atgtttgaaa ctggatagaa gatattacca gcagtgcttc ttagtctagt 3420 tgtagacgga attgttgcac ccgttgttcc agtaaactct acttggcctc ttgtagcttg 3480 agccacatat ctgtatacgg cgtttaaagc tgtaatatca tcaaggttaa aaccctcagc 3540 tttatcaatt gtccccccat cataaatttc tgacaggact tcatgggtgt ctgctaaaga 3600 ccttgcaatt gaagccagaa agagacctaa ttgactatct tcagaaacgt caaggtttgg 3660 tgaaatatct ctaagaagtc ttgatttaat attatcaaaa atttcctgat atcttagagt 3720 ttgtaatcct gttgtagtta atcctgccat tagatattaa cctcttgcgt aatgtctgtt 3780 aaaatatctg ttgtagtagt tgcatcaaaa ttaacagtta cttttctttg agcattatcc 3840 attgaagatg agtagttata gatattagat acatctcttg tttcaacaag gtaagctttc 3900 atataattat caaagataga agttttctgt ttaaatttgg caagttgtaa atatgggaat 3960 ccagcagatg tgttaaagaa gacttcacca gcccttaaaa ggcatctgat atgaagtctt 4020 tgagcaacct gagtagcttt atcatcctct gggataattc taatttggtt accagtaatc 4080 tttaaatcac catgagccac atacactgaa tctgaaccta aagtggcaac atagtcacca 4140 cctagattta atgcaaaatc tgttttcatt attgtgcctc tgtagtatca gcctcaccag 4200 cagggtctgt ccagtaataa tggtgcgtgt gctcattaaa gctcacacca gtcgtgtcac 4260 tgataaaatc tgaaccgtgt acttcttctg ttacgtacaa gtttttagaa atatgtacat 4320 caccttcaaa ataaaaatta ccatcatctg taactcttaa cacagagtta ccaaaatgaa 4380 gtctgactgc tgttgggtct ggtttaaaat tctgtgttct tgtacagatg cctacgaaag 4440 ctacacagtc tgaaatatcg tgtgtccttc tcatatttgt ttccatctga acattcttgt 4500 cgttgacaac gaagtcatct aaaggtaaca ttgagaaagc taaccagcat ctgtcattag 4560 tttttacagg gaatgttaaa gatgcgccac caccacttgg aaattgaaca ggaacaccag 4620 taatctctgg cataggtaac ccattaatag agtaaagtgg cttaacagtg gctgtttgag 4680 tctttgaatc aaaagactgg ataatagctg gtaacccagt atacagttct tttctaaatt 4740 catcgagaca ttctgaaaca tacccagaca ttctagtaac tggtgatttc attattccac 4800 cttctctaaa tctagttcag ttgtccaagc accaccagta aagtcaagat tatgagaaag 4860 accttttact cgatactgac cttcaaaatc ttcactttcc ctaatagtga cgttatcacc 4920 catcttaatt ctcccatcta aatggatttt gcaacgaact ccagtcttaa ctttaataac 4980 tgtcttattc tcttttttca gaacctttct agttcttctg tagtaacctt gcaaagagtc 5040 aataacgttg tatgggtaaa tttcccaaga aagttgtcta gccttagcgt taaaaggtac 5100 tactcggatt tgcttattaa atgtatacca acgtagactg ctttcttcac aaacctttgt 5160 aagtgcctct gcaacacttc cccagacact aaaaccgttc ttgtaagtgt aaccatcaat 5220 gcttgagagg tcttcatcaa taagtgagaa gcctaaccta ttgactaaat ctctaattac 5280 attcttacgt gttgttcctg ctttataaga aattgatgtc ttaatcgtgg ttctttccat 5340 tttatcattg gaacagataa cctttgtaat catatcaaca ccacgcttat atgtataagc 5400 atattcgata gttcctagat aaattaatgg aaggttgtca tattcaataa taaggtcacc 5460 atttgcatct cttttaaatc cagtagtgta acctgccctg agcataactg ttgcaccaac 5520 tgttttaaat ttggctctca tctctttatt taggttgtag atttcaaaag tggtgtcatc 5580 ggaggttact ttatttttct gagatgtata agacacgtta caagtgaatt gcaagttgtc 5640 gaaataatcc atttgcatag aatctttagc atggcttgta ggtttatcat taaaggctgt 5700 agtttcacta cctacagcca attgatagca cctaaaagaa gccccagcag tgctatcttt 5760 tacagacatt ataaattctc cattaatctc atatcttctt gagtgtaata attaagctca 5820 aatgcctttt ctcttccgaa gttatttcta gtaggttgta aatcagtacc atacattcgt 5880 tcaacaaaaa gctctccagc taatgaagga attacatagc gtcctgtgat tgactggtct 5940 gcaaggcatt tcttttcaga taataataca ttaccatcaa catcagatag cgtcaagaac 6000 catctgtcga gtctctcttt gtactttaac tcaattacaa agacagtgcc atccagagtt 6060 acagtttggg tagaccattc tgtatcagga acaggaatgt attgtgacat tagtaagtct 6120 ccttcttatt cggattcact gagtactttt gcaatgcttt tccagttcca gcagtcctat 6180 ttaatgctgc tctagcattt ctctcagcct cgtcactaaa tgtggtaaca cctttgctgg 6240 tctttgcaga cattgagcat ttaccaagtg ccctgtcttc atcactagta agttctctta 6300 caccattagc attcaggtca aatagcaact ggcagtttaa tttaccatta cctaagcttg 6360 ttgtagtgtt acctgtattc ttcttcgagg ttgtgcctcc atcattcgtg gtagcggtct 6420 ttcctgtcgc agccgaaata tcagtttgcc ctacgatagc tttaaagtta atttcctgaa 6480 aagttagctg gactcttaga ccatttgaaa taccaacatc tttagaggct ttaaagcttg 6540 taataatgga atcatcaatt ttaattccgt ctttacagat gaccgaaata atctgcttct 6600 ggtcacgcca gttttccaga gtgtcgatga agttctctac taattgaccc tgacgagtta 6660 ataataaact tccttcatag ccaactacaa cgacaccact aatagtaatt gttctgggtg 6720 ctctttgcac attatctgtg acagtttgcc ctgattgcat gttctgtgta gtcacctgca 6780 tagggctgtc aaattccatg ttttcagttg ctgataaggt taagaaggca tctacattat 6840 ctcttaagtg gaaatagatg ccatctttgc cactatattt gatttgcata ttagaatccc 6900 ataacattat tcttcctctg gatagcttga acttagaaga atgtcttctt gattcttgtc 6960 agtaatatcc accatcttag tagcaatttg tttaccatca agattgaaag taacattcag 7020 ggtttgttta gtctgcatag gtaaaccaga aggtgtcatc atcattggtg tctggttaaa 7080 cttattggca aaattatcaa tggatgttga tagcttctcc atgataatct cccaattaga 7140 taggccattg tcaattaact ttctgttgcc ttcaacatct tgagtgtact gcgcaaactg 7200 aagttgacca ttctcatcaa agaacattgg tctttttggg tttgtaatat ttgcaacagc 7260 attttcaaat ggttttggta atgtaacttc actgtagttt ttagcagcat ttgggtctgt 7320 agaacctctt agcattaatg ccgaaccaac agtaccaagt gccattctcg ttgctgtaac 7380 accacctgct gctgcggctg cttctcctgc tgctgctgta cctgctgcgc taacaccaag 7440 tctctgtaag attttaccaa agataccacc accaaccaaa ccacttagga gcttgactgc 7500 ttttgatact acaccaactg tgcctccaat tgtgacaact gttcctagaa attcaccagc 7560 acttttgaca agcttttgct gactgctatc aaggtcttta taccaagctc ttgcatagta 7620 gtataatgca gatgttctgt acatgaagtc tgtaacgaaa tcaagtagat taccagcacc 7680 ttttaaaaga ttcccaatca caagacctaa agcttgtgta gtccccatag aaccttgcaa 7740 gaacattgca acggagttag ataactgtga aataccctca ctagagccgt taaacagtgc 7800 tacaagtgtg ttatcccaca tagccttagc ctgacccatt gatgtagcgg tctgcttaga 7860 tacagcattc ataccacctg cttgcttgac aagttcagcc attctttcag atactttagg 7920 gagaacgtct tgagcaagaa gtttaccgtc ttgcatcagc ttatcaagtt cttgtggggt 7980 cttaccaatg gcatcagcga ataactgaac agcacctgct aaacggtcac ctaactgtcc 8040 acgaagttct tcagcttgaa ttttaccttt tgatgccatc tgctggaatg caaccatgat 8100 acctttcaag tcttcatcag tagcacccct gatacgggca aacattgcag cattcttata 8160 gaactcttga gtaccctgaa aaccaagtgt tggttgagca ccagcagcaa agtttgagta 8220 ctgcttcata gtgtctgtgt agttctgacc aatctggtgt gcgaatgatg cagcaaacat 8280 tctagcttgt tgtgtatctg ctccaaagat agctgtagag gctaactgtg cagactgtct 8340 ttttacacca gcttcaatag tcttttgaga tagctccagt aaagtataag ctgaaacaaa 8400 tccaccaact aattgacgta atgatgcgtt agctctatcc tgtagccaag cggattcttt 8460 aactgatttt agtctagcat tttctgcaat aacccaacgt ttggttacgt cgatgagctt 8520 tttaacttcc atctcatact caccaacttt accagtacct ttatatctgt tatagatatt 8580 ttgcaagctc cctctgaaag aggatgccat ttggttacct tgaccaccaa ttgtttccag 8640 tctacgggtt aaccctgagt agaagttatt attaaacatc ctttccattt gtctctgagc 8700 aacatctact ctcggacctc tgggtgctcc accaccaaca ggagggatat tctgtccacc 8760 tcttccccta cctgtcttga tagtgatttt accgtcaacc ttcatagcat ctcttaagga 8820 cttgttaata ccttttgcag ccttttttgc ttgagtttca agtttcttaa gagatttaac 8880 accttgtgaa tcaaggttca aggaactgtt gagtgcttta ttgattctgc ccgaagcaga 8940 ctgagcattt tttacaattc tattaagtgc ttcctgagag cttttattag gtttcacatc 9000 aaaggcttta ttaatatttc gctcaatacg ttgagcagct tgcatagaca tcttctcaac 9060 tctttgcaag cccttaacaa ccttttcact gaaaccaagt tccacaatga agctatcaac 9120 tgtatattgt gccattacat ttttcctgct cttctaagtt cgttgtaagc aatttcctct 9180 ttatacgacc tctgaatttc aagaaattgt ctcaatgata ataaatcaga gaatgtcata 9240 gcaaagagtt ggtcaagtgt ttctttacac ccttccatac cataaatagc aagcacaaat 9300 ttcatctcgt ctgcttcttc ataggttgcc tctacagcag catcagttag tggtgtctgt 9360 agagtgttac ccatgtttac tgagaagtta ggcttttgaa aatgcttgct tcgaaaaaac 9420 ttccgaagtt tgcctccagc gcaaatgcta agtaatcaat aaattcacca tagtttgctt 9480 ggaagtatgt atcaatattg agtgggaagt catcaacagt tgccccttga aataacaggg 9540 tagccatttc ttcaaggtta atttcttcaa ttctgtcaaa acaagcttca acaagttctt 9600 taaatggaac cattggagct tctttcttac ccttgtcagt cagacttgat agcatctgtg 9660 caaaggttgg gacaacaatc ttacccaact tcatagacat cttaatacca tctcttgccc 9720 caagcagaat gatatttact ttcttaccat taattactct aaattctgtt ttcattgtga 9780 ttccttaata cttttaaaga aacaaaaaag gggaagacct tttaaagtct cccccttata 9840 ggatttatta aacacttgac gctggaattg tagaagtgta gtctagcttc tcacaaccaa 9900 aaatccaagt cttagagttc tggtcacgac caagttcaat ctgtggtagt tcctgcaacc 9960 aagcattaat accagttgcc agaacagagc ctgatgggtc gtagattacg aagttagaag 10020 agatatcttc ttcaagttcc atattgtctt gtttagcttg aattgcagaa agcatctggt 10080 tagagagaga agtctgcatt agctcaatct caatagtacc tgtcttgtct gcatttcttg 10140 tcagagcaac ttgaccacct gcaccaacaa ctggtgtcac aagtggtgat gttctctgta 10200 gacgtaagaa tgagtctggg gcaaagcctt caatggcaat accattccag ctacatacaa 10260 cgtctttagg ggaatattgc tgatacatag ccattccaat ttacctctat tattcgtaag 10320 ccactgtacc tttcaagtca acatccaaga tagcccctgc taagacacct gcgaaggtaa 10380 catctttcag gatacgtgct ttcttgtctg ctaaagcaac ttgagaggct ttaggaacat 10440 taactgtgta agatgacagg aagtttctgt tgactgctct ttgcagagag gtttcaatga 10500 cttgacgaat acgggtaata ccagtatcat cataagtaat cttaccaccc ttctgattaa 10560 ttagcaagtc tctcagagaa gttttcaggt ctgattctaa ccagtcaaca ccacggatga 10620 tatcaatcca ttccccacca gaagtaatcc ctctacgaac cactggaaca ccaccatcga 10680 ggtcaataaa gttacagtga cgtgcatcta aagctgactt ctgaatactt gtcagaggtc 10740 tcttattagc tagctgtaga gaagcagcta caccagttag ctgtgcattg ccccaagcaa 10800 tcgaacctgc atcatatgga gcaccataag caatgtatgc catttctgga tagtcttctg 10860 ctgccgtgtg atgccacaag caaacggtac gagtgtactg actcttagca agctgtgctg 10920 gaacatcatt tgcactagct aattctgtac cttgcagtgc tgttacatca gagttagctg 10980 taaagaaaat cttcttacga gcttgaatct cagaagccat tgctaagaca aactgttgag 11040 ttctgtcttc tgctgcaatg aaataccagt cagttgaata agcttcaata gctgccagag 11100 cagttgatgc tgtatcagca gttgtgcttg caataaacac agtttgtgct gtagtggtta 11160 ccttcacaaa gtcattatca ccagctttag taataatcat tgtggcagaa ccagcactac 11220 cagttacatt cacagtgacc ttatctttaa ttgttgggtc agcttcaatc tgtgttttga 11280 actgctgtag cactttctca gcggtatctg atgtctgtgc tgtatactga tatggttgag 11340 aaattccacc gccaacagct acagtaattg agtagtctgt actttcggta acggcatcag 11400 gaattgatac cgtgtactgc atagcacgtc taccaatata aagctgagtt actttaggag 11460 tctgactcca aagttgttta gcagccttat atgcagcaga cctttcatcg aaatcttcag 11520 caacttcagt taaggaagtg tagccacgca ccctttcttc aaaattatct gttgaagcta 11580 aaaatagtgg cagcccaaaa ccttctcttg tagttcctgc ggtgttcaat gtaatatcta 11640 cattaacaat tggattccac atttatttta cccctttgga gtctacatct agatggatag 11700 tatattctgg tggctcttgt ccttcttcgt aaaccaattc accatcaaca atgacacgct 11760 caataatact tccacgttca tctttcagga ctgaattttt tacaagagtt acaacaagag 11820 gtgcagaatt ttcgaaatct gtattaagat aagtatagtc atttgggata gctccagtat 11880 ctagaactgt agctcctgtc tcttcaagaa ttaaatctct gactgaactc atctctaatc 11940 gttgtttaag ctcaagcatg atgctgtgag cacctttacc attcaccgta attagtacag 12000 gaatctgaaa agcaattctg tagcaaacta catcatcctc aacaaactta tcaagaaccc 12060 aaccataagg ggttgcagca tcttgacaat atacggtaat aaatggctgg tcaggtttta 12120 aacctttgtc atttgagtta tcagaagggt aagctctaat tacatttggt ctattatttt 12180 tatcacgagc tagtctgtga ccaataacat ccactaaggt tctaactaga cctttttcaa 12240 gttctgctgt ttctaactgc attcatttta tcccttctaa taatgatata ttcataatgg 12300 gacgtatggg ctaattgctg tgaccaatcc atagtcataa atacttcata ctcatgacca 12360 tcaatcataa caatatcaga ctcattccac tctacatcat ctgaagtcct aagtttatat 12420 gtagtataca ggattcttgt atcggtaagc ctgatacctt ccggtaaagc aatctgtgta 12480 ccattcttta cagaaccttt gatatatggc tggatattac ctttacagtt aacctctaca 12540 atatcttgtg aagctaccca atcaccatca tcattataat aaccgtcttc agagacttta 12600 cgctttacta caaagctgtg tctgtttaag agtctcattt cttaataccc ttcttagtag 12660 aaattttata agcaaggtta tctcttaagt cacctgtctc aacaagaggt gcgttaaatc 12720 cttttttctt gactgtggaa ggtgcgttag gagggaggat agcagagtta ccaaaacccc 12780 ttttaattgc cttttgagca ttctttgcaa atgcttctaa ggtatttgaa gggtctgtgt 12840 tgagactgct aagttgctta tatagattct tcttagtctg ctctaacaag gtctgtttgt 12900 ttagcatcat agtgatttca aacaacctac gatatacttt accagaagct gaaggaaccc 12960 caataacttc ttgtaaatac attaaagcag gataagaaaa accagagcta tgttgacctt 13020 gttcttgaaa atacccaact tgagcattag ccgtttgcaa gttcttcata gccccgacta 13080 attttgctct agcggggtga ataacccttt taaccattat tcatctcgct cgataataaa 13140 tacgccatta acacggttgc agtaatctct gccttcgtat ctggcagcat caccatattc 13200 tgtgtatttc ttgacagagc aaggattttg acgacgcata tcaatgtcgc actgattaat 13260 accacctgca tagggtagtc ctgaaacaga gcttttaaca aggtcgtcat aaacagcttt 13320 cagagatttg aatctggaag agttacgtaa ataaacacca ccaaccttct catctcccat 13380 cttggctact tgaaagagta aatatttaag agctttgatg gctgctttct tttcatcctt 13440 tccagattct agatagaacc actctagcac agactgctca ataagaattt catcattatt 13500 agtgtctgtg cagaggattc ttactctatc aagagggtta ttggctgggt cgcctgtata 13560 acacattcat aaccctccta agtttattag ccaccttgag cgtcagcgcg aacatctacc 13620 agcaactgag gacgagtaca gtatggcagc atgtaagagt gagcttcgaa gtcaatacct 13680 tcgtcacggt ctttttcgta ttcaaatacg tacagttcct gaccaagtgt atttgcataa 13740 cccatcttag gacatgggcc atatgccact tcgaagatgt tgttagcttc acccagcata 13800 gatacgttag ggaaggcatg accaacacca acagttgctg ctacatcacc aatgcccacc 13860 agagtgtgaa ccttaccacg cttgtctttg aacttaccgt tgtactggac aaacttaaca 13920 ccaccgtagt agaaggtgtt catatgagcc tgaacgccgt cagtaccacc agttctcaga 13980 gaaccagtaa tctgttgcca agccagcgga gtctgctgtg caagataagc gtcacgaagc 14040 ttaggatgct taaccagttt gctgaagaat acacggtcaa caactacgtg aatttcttcg 14100 ccgttgatta cagtgccagt cttagcttcg tcttccatgt gcatacgcag ttcttcgata 14160 gaagcgtcga tgtcagcatt agggttggca aggtcgaagt aaacagtctt cttctcaacg 14220 tcgaactgct tgtacaggtc agcgtacaga gtaccacgag catcaacaac tttacccttc 14280 agagcttgca taaacaggaa ctcacgagta atatcgaact tggtacgaat cttcatcaac 14340 ttcttagcac gtactacagc ttcagtagtc agttcgtttg cagtgcctgg ctgacgtaca 14400 ccctgaatct catcaggagt aatgctttca acttctttga agtacatcat tgggaagctg 14460 atttgacgaa cacgttcagg tgcgctagtc tctgctttac ggctatcacg gtctaccgca 14520 tcaagcaagc taacatccca atcagtcaag tccataagga aagtggtttg ggtgattggc 14580 gctgaacgga acagacctaa gttggaaata tacccataag tatttgggat agactggact 14640 tcaccagtca ggtcagcaag gaaaaatctg cttttttcag aattagtcaa cattgtaaaa 14700 ttctccagaa tgtcttatta ttgttattac aggccagttg gtacgaaatc aataccttta 14760 gcagccagag ctttcttgac gttctcagca ttaacaccgg attcaagttt aagcatgtct 14820 ttcagttctg catcacggtg gataccaacc actttcaact gaccatgata agacagttgc 14880 aggtctgcat aaaagttaac gatacataca gaatcagcct gagattcttc tcctgctgca 14940 actttagagc catctgcttt cagaacttca cctacacggt attctgtaga agcttttgga 15000 gtatactctt tacgagagtg gcctgttggg gtaacctgtt cccaaagaat gatatcattc 15060 agaggttctc tgttacctaa cttagtaaaa ccttgatatg ccattgttgt gttccttatt 15120 tgataagaga ttttagagca ttctggagag ccagtttgcg ttgttcagcg gtgtcttcgg 15180 aagcattctt agctggttct ttttcttctt caaccaaatc agcttcgccg tcattaccca 15240 cttcttccat agcgttggaa tgttcaagaa cagcaccagc agtttctttc agcttggtaa 15300 tttctgattc tttttcttcc attgcagaag catgggatgc gatagcttca ttaagtttct 15360 ggttagcacc ttccatagca ttcataaaca gaacactcag agggttatca agaccagcac 15420 ctaaaatagt cgttgcagct tcttttgcat caaatccaaa agcttcagca gaagcagaaa 15480 tcttattagt caaatctgac aaagcagctt cctgttcttt agctttcatt tgagcaacct 15540 gaagacgcaa agcttctagt tcttgctttt cttgttcagt catttcttca cctgaattgt 15600 taacgtttaa acttacagga gcctcttcag aaccttgtaa gtaatttaag aaatcatctt 15660 gagacatgat tgagttaatt aaaccaagtt caagagcttc ctgagcagaa taaacattcg 15720 cctcagtatt ctttacagct tcttcagaga gattacgaga ttcggctaca aaacctgtaa 15780 aggttgcgta ggtatcatta attctctttt gaagtctttc tttgctttct tctgagagtg 15840 cttggaatgg tgaacccata cctttaaact caccagcttt gatgacgtta attgttacgc 15900 cattcttttc aaatgcctta gttaattcct gatgaaccat aattacacca atagaaccaa 15960 catctgcatc tggtgatgca ataatttctt cagcagaaga tgcaagagcg tatgcagcgg 16020 aacaagcgaa ctcatctaca taagcaataa ttttcttttg gcctcttgaa gccataatgt 16080 gacgtgctaa ttcaaagcaa cctgaagctt caccaccacc agaatcaatg tgcagaacaa 16140 tagtcttgat tgactcatct gctaaagctt cgtcaaagcc tctacgcaaa ccttcataag 16200 agcttaaacc acctgtacac attgcatcaa tgaatgtcat acgatgggtt aaaccaccca 16260 taatagggat aatagcaatg tcatctttca cttttaaaag acttcttgct tcacctttag 16320 gtttatcaaa gtttactgct gcttggacat cacccagcaa tctgttattc acataagttg 16380 ctgctgagtg agctaatgat tcagtggcta gtaaaggttg gttgaataat ctatcagcaa 16440 gtctgaagat attcgaactc atttttactc accctatttg tttaaatcta cagaaactga 16500 agagataaca tagatacctt cttcagcaaa gaattgaggt ttgctaagag caccagttgc 16560 cacgcattta tcgttagcat cccacaagct atagtgtgag accgttgcag aagctggaac 16620 agtaatgtta actgtgtctt ctgaggcaat taagccatta tccggttcag aaaaataaat 16680 agccactggc tgagtaacct tatttgctgt agggtctgcc gtcgggtcta cattgtgtaa 16740 aataatagtc gttggggtta gcgtggcgag gattttattc ttaccatcaa tagttaatgt 16800 tcccattaat taaaccttac tttttgttta aggactgttg aatggttacc agattcatct 16860 accacattta caatcatatc atatactttg cctttgacaa gtactttgta atcattttgt 16920 gaaaaaatgt actcaagtct gtttgtttct ttattaactg tcattggaga ttgaaatgca 16980 gtgtcgaggg taatgaaagc ggcttcaatg ctattaacat tgatacgttt attctcgcag 17040 ttatataact ttacacccaa aaggcatgaa ctgtcaaatg gaattttaac aatttcacta 17100 caatttcctg aaataaacgg ttttccactc attggagcat caagtaacct gcaaatagtg 17160 aaaacgtcag aggttccacc atcacttaca taacctgaaa ggctgaccct tgagccagcc 17220 tctacagata atttgtcagt gataataaga acacctctat acgaatgcac tcgtgtagca 17280 tttgaaatag agataacttc agccattatt tatttgcctt atttgctgtg cttgggtctt 17340 ttgctgaagg tgtctttgca gtaccttctc cagcggtctt ataaccatct cctgaacggc 17400 tttggctatt tggagaaagc ttttcagata ctggctgaga ctcatcagca ggaggaagac 17460 caatatgctc tctaagcttg ttagatagct ctttgtcaac ttccaaagca cctactgcaa 17520 cagttttctg aatataagaa ccaattgctt caaggtctgg agtttcgata tcatcatatg 17580 tgatttgtac atgttcttca tcgtcccaca tattaagagc ataagtctgt gcaactaaat 17640 cacggttaat tacgttctta acttgcttca gcagaatatc tactgacatt gctagtaagc 17700 ttgtcttaga atctgcaaga gagaatgaac catattttga ctgacccata gcaagaacat 17760 ctgacataaa tgccatcata atctgcttgg aatatctgtc aataatagaa cctgtgtcgt 17820 atgctttagc accctgtcta gagactaatg agaactcgaa aatatcctct ttagtatctg 17880 ggtcgatata tctaggccag attaaacctg ctctgtcatt tgcaatcata tcattaacaa 17940 cagttttgca gtattgtacg aaagctttct tttcaggttc tgcattttca tccagataat 18000 ctggtggtaa accaatcttt ggcataccta ccaagtctct tgaaacacca acagcttcat 18060 actcttcaat ctgtacttta tacttccacg gtacataggc attaagtaat ggtgaacgac 18120 cttctgggtt tccatattcg tcatcatact taaacagcat gaatttagct cgtggaagtt 18180 tccttgttag tggtctttct ccaagattaa ttgctccagc aatatgtgaa acatttctca 18240 gattctgtct gacaccagta acttttctaa agtcttcgtc aaaataccac ttatcaagtg 18300 ttgattggtt tctgattggt aatttagccc atccaattag accatcatca aattttgact 18360 ggtactttcc ttttttaccc tgtcgcttct tataaacttt ctcgttaaca cagaacccat 18420 aagtgcagaa tgacattaca gaattaataa aatctgccca atcatgctcc atgtcatcca 18480 ttaaagaatt aaagaagtct gctctttcaa gcattttagg gtcttgctct tttccctttg 18540 gaggtacgaa tctccagttg acttttctga caaacatctt aataatattt acagatgctg 18600 ctacagcagg gtcacgcatc attaattgga aagttttaat actttcaggg aaccttagtg 18660 cctgacgagg ttcttcatag attcttccat tcttaacctt cagacccaaa gaacctactt 18720 cacccattct aaatggtggt aagctttctt gtgtttctgt aatatctgcc attcttctca 18780 cctagctatc aacgtctaag cccctcatat gggtttcctc tcactaagtc tgtgtgagca 18840 cccattgatg gtggcttgaa taatttaact tcgttaagac tgttgaaagc atcactggta 18900 gcatccactt ggtcatcttt agttttacca tcaccacaaa agccttcaag ctcttgaaaa 18960 taagcttcat tccaactacc tctcaagact tttacaagtc cagcttcaga agcagcagaa 19020 aaccccgcaa atcgggtaac tttatcttta tttgttggtt tggctcttgc tcgataacct 19080 ttctcggcaa gtttcctgat gagtgatgtt gcgtaggatt taccagcagc gcctgggtct 19140 tgagggataa aaataccagt tcgcttaccg tcactttcag cagtcaaatt aatttgtgtt 19200 tcgactccag agggtctatc tctaaatctt actacatcaa tgatataata gcaaccgtct 19260 tttttagatt tacccatctt aacacctgct gtccagtctg ggttagggtt aatctcagac 19320 ggtaaagttg ctgctaagtc ccatgctctg acatcgaata catcttctgg gagtgaatca 19380 acaatttcac accattgtct ttgccaatag tttgaacctt ctgcacgagc cttccagtta 19440 ccgaaacgaa gtcttgcaac gtttacaggg gtgttgtttt ccaacttacc acgatattta 19500 ggttctaaga agtcaagaat tgggttatca tcaatcgtac cagagatgaa tgtatatgtt 19560 tgaggaatct caagaggaaa catttcaaga atcttgtctc tctcccaatc agaaaccata 19620 acaccatcat tcattacata ccaacgaata cgaccacact tttcagggtc tgggtagcct 19680 tcttcatcta agaatggttc tacccaatcg taaataaaat ggtctctgtc tgggttcata 19740 gaaatcttca tgtatgaatc accttcagcc ccagaacgta gacgagtctg taggtatgaa 19800 atctgtgaag cagagaagtg cgtaccttcg tcaaagtaaa tagctgagta ttcaataccc 19860 tgatgacctt cagcgtgctt ttcaagttct agataggtaa acttgatagt tgccccagaa 19920 gggaatgtga tagtcatctt ctgctcgtga ggaaccccac caaacttacc aaatagtttc 19980 tttgcagcag gccataaacc accttgtaac tgtgttgtat ttcgacggaa atatacagca 20040 ttatagttag ggtcttcaat aaaccttaaa gagtccatta acaatgcagc agttttacca 20100 gcaccagcag caccaccata taaaaccaag tcagcattag tatttaaaaa cacctcttga 20160 gaacccggtt gaggggctac ataattctta tcagtcatca atttgaagat aagtctaact 20220 tggtctggtg tgtatcttaa taaagtcaga atttgagttg gaaggaattt agaggggtct 20280 ttaccgaatg atttgataat ttcttttact tcatcagaaa gccccaactc tccagctagg 20340 actttcctaa catcttccac tcgcttctgc ttaacagcat ttaaatccat taagcacctc 20400 cgcaaataga attattcaga gtctgtagca gtctcttctt tcttaacttc gaactttgca 20460 tcaacagcat caagaagtgc atccatagat gcttctttaa tatcaatacc tgttgctaca 20520 gatagtctgt ctgcaattgc catgatagta ttctgcataa actctagctt cttgttagct 20580 tcataaagtt ctttataaac gttctcgctc attatgtttt ctccaaataa ttaatattgt 20640 ttccccttaa aaaagacggc acagagaccg tcaaggagaa accacaatgt acgtcagaga 20700 catactgtat aagtcttcct ataaagtata actctgtaaa aaagccctgt ctaaaaatac 20760 tcttcggtag cgaaaggaag aatacttata gcagggcatt attattattt taattgagag 20820 agagtaaaat aaattggaga atccgaaggg actcgaaccc ttataaacct gttttgcaga 20880 caggcacata gccatttctg tcacggattc aaattggagg aagataccag acttgaactg 20940 gtacaccgat ttctcagcta ctggcagttt agcaaactgc tcccttacct tttagggtta 21000 atcttccatt tatttctgta atgcgctctt catttctggt gttgcaatag tgtcaatcac 21060 accagtttta caggcatcat caaaccagtc tggtaagata cctgctaaga acccattgat 21120 attcgtttta aggtactctg caataatggc agcttcttct tcaatcatct gtacaacttc 21180 atctgcataa gcggtaatct ttgagatacc ttcattaatc ttactgacag cttggttagc 21240 taaatcataa acagtatcaa gacccttatc aatagcatct tgtaagtcac ttaacacacc 21300 attaacacta tctagtgtac tgttcatcat gtcaatggct ttttgaccat attctgtagc 21360 aacacccata ataccactga atggtgtaca accaacttgt tctcctgctg cacccataac 21420 attggagtat ccctttgcta cctgcatacg tgatgaaaac tcatcaatag acttttgacc 21480 atagtctgtt agggtcttag tagttgctgt agtgcttgtc aggcttgttg taaagctgtt 21540 tagaaggatt gttgtaagtc cagcagcaac aagtttctcc ttcattgtag ggtctgttac 21600 agagctaata gagcttacaa gtgatgtaga ggcagctacg gttccaccga gaactgctgc 21660 accagtaata aggggattag aaaaccctct accggctttt aagagattaa aaatctcctt 21720 accttgttct gtcatctctt tcattaagca cctttgaaat tggtagtcca gtgggatttg 21780 aaccctcttc tcatgttttt cagacacgcg ctttaaccat ataagctact tggactataa 21840 attggggtga cctacgggat ttgaacccgt atagaccatg ttcacagcat gggtcattac 21900 catttatgat aaggccacat ttaagaactc tcgtaagaat ccttagaagt ggcagcggca 21960 taaggatttg aaccttaata agacagcttc agagactgct gcattgccag ttatgctata 22020 ccgctaaatt tggtactcca tatcggattc gaaccgatac ataacacaga ttttaagtct 22080 ggcctctctg ccaattggag taatggagca ttggcggggg atgttggaat tgaaccaact 22140 tcttcgattt caaagaccga ggttttaacc ttgtaaacta atccccttta aatctttact 22200 ttctcagtgg atgaataaag aatgccaaca taactctttc ccttgagaat gctctttctt 22260 ctgggacaac actttttagt ttccatccaa tataaattct ccagtaaaat tgtttaccaa 22320 agattttaat tgatgggata aaagcgaata atccccaagc attactgttc cacattagga 22380 gataacctgt ctgattatct tcggggtcag aacttacgtt gatattacct ttccacttag 22440 taacatcttt tacatctctt cctaatacat ggtaagagaa gttataagct ttgtttctcc 22500 agagccatcc gactctctgc atgtagacac ccagcctacc aatctttcta atattagccc 22560 atcttttaac gtggccttca tcaccatcaa tcgggttgtc ataggtctcc atccatctga 22620 atccaaaagg tagatgtcct ttcttctcac tgtagaatgg aactacgaaa ggtgctaaga 22680 tgactgctag ggttgctgca aatggttcta gtaaagctag gaaaatccat gaagcatact 22740 tcaaatatct catctaaata gcctcttaaa attggtgttc caagacggat tcgaaccgtc 22800 actagtacaa ggtttgagct tgcatcctct gccaattggg atactggaac atggtactca 22860 ctaaagtact tgaacctttt tcttcatctt gtaagggtga tgttttacca tataaactaa 22920 gcgagtatga aagaggtctg gagttgtgcc gctaactcaa ctccgtggaa tttgttacgg 22980 tcttcagttg actcaaagcg gtctttcact tgacctctga attggtgctg ctcacaggac 23040 tcgaacctgc atttccatcc ttacaaggga tgtgtaatag ccaattatac gaaagcagca 23100 ttgtttggag catccagagg gaatcgaacc ctcaacctca gtttggaaga ctgtaatttt 23160 cccgtttaaa ctatggatgc actaattggt ggagaagcag ggaattgaac cctgtactta 23220 agtttgcaaa acttatgttt taaccatgta aactacatcc ccattgtttg gtacaggtgg 23280 agggaattga acccatcgtc ttactgatta agagtcagcc gcataaccat tttgctacac 23340 ctgcattaat ttggtacacc tagagagatt cgaactcaca ctgagcagat tctaagtctg 23400 ctgcctctac cagttgggct ataggtgcat taaatcaaat gtcttgagaa ttgactgtgg 23460 caatttggac acaagaaccg aagattctct cttctatcat ctaaccagtt ctcgttaatg 23520 tggtcaacat caagagttag agggttatcc aaccaatacg gtaactgacc acatttagag 23580 catttgtatt cgacaccaga ggctattaaa gctcttctta gttgtgaaga cctagctctt 23640 tcaccagatt cacgtagaat tagtatttct tcaggctttc tttttggtac agttttaatc 23700 ttaccagcct tttgtccgag gaaatgagaa gtgtctattt caaacttctc tataacacgc 23760 tttagatgtg actgagaacc acctgctggt tttctcccaa gcttaataag tacttctctg 23820 aatgatactg aatcctttac taaaggctca agaatatctt tagtcaaact gctacgtgcc 23880 atgtgtcaat ctcccattaa atctttttag agaacttcta agaacctaag caacaatgtt 23940 ccacaacaag ctgtaataac cttagaagcc ctctaaaaag acctaaatag gtctttgcat 24000 agtcttgttt aatccggtga ctagaacctt tggagagctt gatattatct cttctccgtg 24060 acacctacta ggtgttttat cagtgtgtac gacaatacac gagggatacc aaagcaacat 24120 ccgaatgaag cttggcttta aaatcttatt aggcggtaag taaaggagtc gaaccctcac 24180 cgtatctcta cagtggcaac tgttttcaag acagtttggc taccattagc cgctacctac 24240 ccctaataag actttggcat gggacggagg agttgaaccc ctttgaaacg gtgttggaag 24300 ccgttgctca tgccttagag tcttaacatc gtcccatact ataattcgaa gcttaccgta 24360 ttatctcaac actgtaaagc cccttctcag attatttaca tgttctggaa aacatgagat 24420 acagaccacc tccctacagg aagacccgct taggtcgtct aagtaatctg ctaactattt 24480 ggcacaccct acaggattcg aacctgtaac tcaatgatta gaagtcattt gctctgtcca 24540 gttgaactaa gggtgcatta atctttaaag actctcttag aaagccctta aagatgccca 24600 ccttattaat cataccgtgg gcgagtacgc caaattcttt gatgagggat tggaagacct 24660 cactggtgtt tagcctatca agctactgcc agaaaaacat tgtcgtttgc atttatttta 24720 aatttgcaaa atagacgcta cgcaacgaaa actaaggtta ctataaagta gtcacatctc 24780 aatgtcaaca actttattga aattggtact gtgagaagga ttcgaacctt ctgcctttcg 24840 gttatcagcc gaatgcacat ccatatgtgc tttcacagca ttggaggttc agatgggaat 24900 cgaacccaca ttcatagggc ttatgagacc cttgcattac cttatctgcg actgaaccat 24960 attggtagaa gtggagggat tcgaacccat cgcctgtcag attaaaagtc tgccgcatca 25020 ccattctgct acacttccat taaattatct ttgcagtaac ctacgcagac cttgctaaag 25080 ggtctgtaga gtcatttcct attaagctaa tctagaatca tgctaaggaa attcatcggc 25140 actcatggga cgatataatt agccacataa gctactgcaa agataattac cagaccgttg 25200 tttatcatct taagtgctac cattacacca actcgacatc tgccgagtga aggaatcgaa 25260 ccttcgcctt ttctttaccg gagaaataga ttgtttaagt tttgctgtaa acggtcttct 25320 gtaaatttgt tgagccagac caagttttaa atttttcaaa attaaattgg cttgaaattt 25380 tcatacgttc ttgctgtatt tggtcttctg ctcctttaaa ggatactcgt aagaaccctt 25440 taagggaggg gctaaatagc cctctcaaaa ttggcgaggt atgcgagaat cgaactcgcg 25500 gctcaggaga gacaatcccg tattttaacc gtctaaacta ataccccaaa actatgtgcc 25560 atttcttaaa tacttcttcc tacgcctttc tgggctatag ttcttcctgc tcgcagcatt 25620 cttagccaac ctacactcat cacacctgca acctttaaag tagctatgtg cagtgccgtg 25680 tttaattggt ttaggctttc tatcacctat ctgtgctggt ctggctgctt tcatgttgca 25740 gcgcagatgg ctataagaaa tgttatctag tgaaaagaaa agttcttttg cattttcttt 25800 gtgtctccac ggggtcttat gctcaacaga gaaatcctcg cgggacattt cttttccgca 25860 ctgaaaacac ttgttttgat ttgttttaca tacaaaatca aataacaagt cttttactag 25920 cctattatgc gcagtgccta aactcatacc aagttgtgat ttatatactt catctcgcat 25980 aactagatat ctcataaagt gtttggtgtg ccgtgtagga gtcgaaccta ccgagtctca 26040 atgacaaggg atttacagtc cccaccgcta ccatctacgg gataacgaca catttaattt 26100 tgcggttagt cagggattcg aaccctgtgc cattcgctta acaggcgacc gcacatacct 26160 tatgtgcttc ctaaccttaa tacattgcca gaccgtgttt tttcttttat ccgcaaaaag 26220 tattttgttg ctgaatacgg tcttctgtaa atttggaggc gggtgcagga gtcgaacctg 26280 ccgataccat gctaatgaga catgtgagac gccctttctc tatacccgca attcttaatg 26340 accagaccaa atatcttctt tgtccgatta actttcagat gagtaggtta agtttgctgt 26400 aattggtctt ctgtcaaaac tggcgttcca gaagggattt gaaccctcag atatccactt 26460 tgaaagagtg gtgactatac cattttgtct actggaacat taatttggtc tctgttggag 26520 gacttgaacc tccggcctta ccgccccaaa cggaacgctc taccaagctg agctaaacag 26580 agataaactt ttcaaactct atgtaaccac tttaacatta ttttttagtg attgtcaaga 26640 acttttttaa aatatttttc agtatcttag aaaagctctc atctcgtttc tatgtagaac 26700 atattaaagg gtattaaaca cattgtcaat acccttttta aaacttttta ccagatataa 26760 cggtcaatca ttactgcttt gagcattacg ctgattgggt caaacttccc accaccaagc 26820 agagctttta aagtggctgg agagaaacca gataccattg ctacaccgtt atccttaaca 26880 gacacttcgc aagtaccatt acggtttgct aagtaccaga atactagttg tggcatttcg 26940 tatccagctt ttttgtactt actctgaatt gcttcaaagt ttgtacgacc atttgcacca 27000 tcaacctgat taaactccat atcagagaag ataataagct tacttggcat atctttctga 27060 gtcaagttgt ttcttttacc tgtctcaaga atacggtcaa aagctccttg taagtaagtt 27120 gagccatatt caacgtgctg catcacctga cgatgacggt ttcgtaaatc accactcagt 27180 tcaatgaagt gagggtttgt tgaataaacc atcaacttat ccttaaagca acctgtattg 27240 cgttctgcta catacaaggc aagtgatacg ccaatatcaa gagcagtgat tgaaccaaaa 27300 ttcacccaag acattgaact tgatacatca gacatacaca agatgttttc accttctgcc 27360 atccagtttg gtagtgcttt ccactgctca ttagcaacat ctgcattacc atacttgaca 27420 gatttaatca catcgtatgg gtaaacagca ccagcgttaa tcttagtctc accttttgat 27480 aaggactcga tgtaagcttt gtaacgctct ccatctttac ggttaaacag tttttggtaa 27540 cgtgcagcag caagtgaagg aatcttgctg tagtcaatct taccaaactc attagcagag 27600 attttttgct caactgtatc agatagcgca gacaacagtg tgcggtactc tttctcactc 27660 aagttggcaa acttgcagaa acgttttaca aactgcttat gacgtggttt gactcgtggt 27720 aaccacttag ccgccaaacc tgctgttgca gggtctagta atgctgcttc taaatgtttg 27780 aaggcatctg cctcgaaacg agtacctacg aagattttga agtcatcaaa acgaccaagt 27840 tctgcaatct tatccataat gcgaagaacc tgcgtaggct ctaaaacttt gtcttcaatt 27900 gcttgaagca aaacagttct gaaggcttta cgctcaccca tgccttctcg tacatctcgc 27960 atatgcagta aaatacgaac tgcaacatca acatcctcac gaaaagcttt gtagaacaag 28020 tctggtaaga tttctacatt gctacggctt gaaccagcag ctttgtaaaa gtctacaaga 28080 gcagacattg atgaagtatg gtttacagca ccattttcag ttcgacctgc atgaaggtgc 28140 gcatgtttaa ataattcgct catattttac tctcttctct catttgtttg ttgatgtgac 28200 agactttaga gcaactttgt agagtctgtc aacacctttt aaaattattt tttaagcgtt 28260 gctacagcag agaacgttgg tttgttatct gtctgagact gaccgccgtt atcaggagtc 28320 ttcttagtgt ctgctgcaat ggctttcatc tcacctgctg agtgagtcat gatagtttta 28380 ccgacctcaa tcatgctctt gatggttacg tcgtctgtgt ttaacccgaa ctctgcaagc 28440 ttagcagcgt cccttgtaac aattgcttca aacaactttg cagcaaactc agcagaatta 28500 tcaatggtca gttgagcttt tacgagtgaa cttttgttac gagagccttt tggtcttcca 28560 gatgggtttc cagactggcc ttttttaaac tggcctttgt ttgttctgtt tttcattggt 28620 atgcctctta taagacctct taaaaggctt ttaagataaa agatagatat ccagaaagaa 28680 atctatttaa atagcctttt tagaggaacc ttttaagtta tcttctaagt atttataagc 28740 ctataccttg tcaagtactt tgtcaacaac ttttttaact tgcaatagtt cttgacttgt 28800 tgtatggatt actgtaccat cttacttaaa gctgtaggtc tgccttgttc tacaaaggag 28860 acttaatgag aaaatcaaat aaaccgaaga aaggtaagaa taccaatcac tgtaaggaat 28920 caaaaagggt agaattaatc tactattcat cctctgaaat tggtctgtac ctgttctttc 28980 aaaattacag aagacaagag gattatctat gtgtagttcc caattagaaa tcgcagatat 29040 tatagattta tatcaaactg caaagagtca tggctatata acctcaattg gaaagaatag 29100 tcactatgat gctttgactg gaatgtattt cagggcaatg gctcaatcta gtgaacaaca 29160 cttagtggtt tcttccagtg agtttacttc gtttctctat tgcagcaaaa ttataaatcg 29220 caggagaact gaaaaatgtt aacagtaagt tttaattaca atagtgatgg ttctgtatca 29280 attaactcac catatgcaaa tgacctactt aaagagctag tcaatcagtg tgatagaggt 29340 cttcactatg ttccaaattc ttttaaacag aagactattg ctaataactt gatgcgtgta 29400 acagtgacga catcgaatcc aaactatgac ttcgatagtg aaagtccgta ctcattagtt 29460 gcacttggag agagcagcca attcaaactt gtttgccacg actcagaaac atttcttaag 29520 gtattttcca accttatcca taataataag tacagctacg ttgatggtag tgtaaatttc 29580 tatccagcaa actatacatg tttgttggtt gataatatga gaaaaagtaa gcaagagcct 29640 acagaaattt catttgatgt gaactctagt ccagacgcag aaacaagtaa gaactttgat 29700 atgagttacg cattgtcact aagtaagaaa tccgagttca ttgattatgt caatggattt 29760 ggttttaagt ttgatgagag tatgaatctt aaaaaactta agaacctact taagaccaaa 29820 gcttaagtgt ggataggggc atcagcccct ttaactattt ataaggatat ttaatgaaag 29880 ctaagagtgc aaaagacttt tattgcttcc tacaatccta tatcaattcc gtgcaaaatg 29940 gtgaaaggta caaccttgat gatgttatcc caacaccttt gacatggaga ctcagcaaat 30000 ggcctgaaga agatattaca ccaacaatcg aacagtgtac ctacaaacct gaaatcaata 30060 ttccagactc tgagaaccta ttatatccaa tgttccatat tgttggactt ggaagatttc 30120 ttatggatat ccagtatgta attggtaaag gttataaggt taaaggtatt gttgttggtg 30180 atgtatctcc gcatcacaaa ggttatttta gattaaacgc acatttagag gctaaaaaga 30240 aatgattaaa gcaaagactt acccagactt caaagagttt gtgaagggct tcattgcaaa 30300 cgtaaaggct ggtaagagat atgattttag aacatatcaa gaggctattt taccacttac 30360 ctatagctcg tattggcctg aagctgatat tgcagaagtt gagaagtttg actataaacc 30420 agactataaa gttccattta gtgatgagtt gctttatagc attggtgctc aaatgagaac 30480 ttctgacttc ttcatggact tacaatatgc aattatcaat ggtaaagacg ttgatacagt 30540 ttattgtgaa tggcttgcaa gagttaaacc attctcaatg ttgaatgcta agttgaagga 30600 tgctatcaag ccaccagcaa tcactcagca accaaccaat caaacagtaa atgaaggtgg 30660 cacacttacc ctaagcgtta ttgcaaccaa tgccactggc tatcaatgga agaaagatgg 30720 ctctgacatc ccttcagcta caaatgctac ctatagcaag tctggtgttc tacaagcaga 30780 tgcaggaagc tacacctgtg tagttactgg tgcaggtggc tctgtaacat ccaatgcagc 30840 aacagttaca gttaacccag cagtggaggc gtaatgcaac tctcaagaaa aggtttagaa 30900 gctattaagt tctttgaagg tctgaagtta gaggcttacg aagactctgc cggaatccca 30960 acaatcgggt atggtacaat ccgtattgct ggtaatcctg ttaagatggg tatgaagatt 31020 accgctgaac aagctgaaca gtatcttctc gcagatgttg aaaagttcgt tgcagcagtg 31080 aacaaagcta tcaaggttcc aactacccag aacgagttcg atgcacttgt aagtgaaaca 31140 tataacatcg gtatcacagc tatgcaggac tctaccttta tcaagcgcca caacgctggt 31200 aataaggtag gttgtgcaga agctatgcag tggtggaaca aggttacagt caaaggtaag 31260 aaggtcactt caaacggcct gaaaaacaga cgtagaatgg aagctgagat ttatcttgac 31320 agtgtatatc caaagtaata tcttcatagg ctccttcggg agccttttta ttttctaagg 31380 agaaaactat gaaactttgg gctagtgact ttgggacttt taagtacact cgtaatggtt 31440 cgcttgtacg catcattgga agtaacattg ttacagttgg tgatagggtg catacaatgt 31500 tcactgttga gttggttgaa ctatcaccta ttgagtctgc taataatggt ttattcaagt 31560 ttgaaactta tcatgtaaat gagcatgggc aatttaatac tctaggtgaa agtggtcttg 31620 atattatttc agagaaccca ttgacaagag agcaacttgc aggttactat aaaaccattc 31680 ttgaaagaac tttggcgaca catgaacatg aagccagata tcacattgaa cggtgtgaag 31740 ttctaaaagc gaaaatcgaa caagcagaga gaggttttta tgaataaaga tgttagagat 31800 atccaagtaa aggttgacac atttggtcat gatggtcgag aagacattgg ttttgccgaa 31860 gcagtaatat tcaagtgcaa tggagattta ttccttcgtt ttacagggtc tggaagcacc 31920 cttcatcgaa tgcgagatga tgaagatggc tgccctgtat ttggttggtt tgacagggag 31980 ttccctctct acgcaatcca ctacccagat ggcggtgaag actggacaac ccaatcaatc 32040 tttgatgagc ttaatggtgt tccagttgag caagaagaag aagaagcgga agaacctgtt 32100 gaacttacct tcacttttat caaagaagag aaagtaggtg acttgtcagt aactgaagca 32160 attcaagtct cacaggtgat tcgttaatga gtaaagtaca ggttattttc cctatttgtg 32220 acttctcact agagcgtgaa cttgacctgt acgaagaaat tactgacgaa attatctggt 32280 ctgttgtaga agaggctatc aagaaactgt atggtggcct cttaaaccca tcaagtaaaa 32340 agctcagtac taagcaagta gctgatacgt acacttcata tgatgcctac aacaagcctt 32400 ttgagaacac ctgctttgac cttatggtag gtaataataa agtcaactat ttcttcgtca 32460 gagagtttaa cgatgagtaa gctccatgtc acagtttaca aaaacttctc agatatcaaa 32520 gagtctttaa caaataagct tgacatgcaa cggaaaagac tctttttaat gtacgacata 32580 gacaactacg accatcctaa agagtttaac tacaaagatg gtacaaaggt cgttgaattt 32640 gaagattctg taacggtgta tgtcaagcat gacttaccag caaagtacat agggatgtta 32700 gagtactaca tattcaaaca tacaggtatg cgtggtgaag ctgttaagat atcttctata 32760 gaagtttttg agaaacctaa cacacaactt aaaaagtatt taatgaggaa actgtaatgt 32820 ccgaagagca acagaatatt actccacaag taacactggt acaacatttt ggaaatattg 32880 aagggtgtat tgcacttttt caaccaagta tcaactctcc tgcaaaagtt tgcaagttga 32940 ctatgaatgt taacaatatc agtgtttgcc ttgttgaaga ggttcagtat ttcaagttca 33000 atgacagaga ggttgatgct gcactattga agtatcgagc aaacctcgaa aaagatatcg 33060 accacaaaga acttgtaaca ctgtttggtg accttcacaa acttcttgaa aaggttatga 33120 agcgcacata ttacatgaac aacggttcaa tcatcaccac tttgatttca ccatgtattt 33180 cagagccaat tttaactgat gacggtggat actacgtggt agcatcagca gattctgatt 33240 ggtggatgaa gaacacagca cttaaaacag tgattgaggc tatccgcgaa catatccctt 33300 cattcagccc gtggaaaggc aaaggtgatg actttatcgc attgttgagt gaagagagta 33360 gcaaacgtag tgcattaatg cctaagaaat actcttgccg agtataaaat attaactata 33420 atgggagcta ttgagaggta gttccttttt tagtttctgg agaagatgtg tggtgaaaga 33480 atgaaaaagc taactacagt tgaagattac tacaggctgt ctctattaga acaataccgt 33540 agaagtcaga atattagaaa atgctatggt aaacacgctg aaggtgactt ttaccgttgc 33600 tacgatgcag atttaaaagg tgtaacacct agagggaaag tcctgcaaag acttgttgac 33660 cttgagtggg acaaacgatt gagaggggtt ggaaaatgat ttacgaagaa aggtataaga 33720 tagattatca ggatactcgt catcatactt ctttgagagt aaccaaacca aatggagata 33780 ccggtattat agcacatttt ggtggtgatt attggtacgg tacaggttgc tttgaaggct 33840 acaataaaga ttacttgaaa gctttctaca gagactttgc aaatgactac aacagagttg 33900 tttacgaaaa gaataagtgt attaagcatg aacaccatgc cagaggatgc ctaagtgttg 33960 tcatggtact ggcgttcttc ttagcaatgt tactggcagt atcagcaatc agttgtatag 34020 ctcaagactt gaccattaca cagattactg caaaggtaca tgatgtttgg tacttgtatg 34080 ctgttccttt agctggtatc atcatctcac taataagatt cagggttcat aagaaacgac 34140 ttaaggactc tgaggttaaa cttgaagagg taagtaaaga atgcaaccta caattatagc 34200 tgtatgtgtt cgttttgcaa tcgctgaaat gattaacaag gcaatcttaa aagatgccta 34260 cggagagact aagtaatgat taagacacct gtaccaattt ttggatttcc ttctatagaa 34320 gagtttaaag tttatcttga caaaaacttc tacaacgagc agcctgtaac acttctgaag 34380 agtgacttat cagagcttct tgatatggtt atcaaggcaa ctcctgagaa ggaacctgaa 34440 cagaaagctg agaagaagac tagtaagaaa tccgataaga agactgaaaa gtctgagtaa 34500 tagcttaggg gttacttgat agcccctttg tagaaactta gaaggtagcg atatgcaagt 34560 ttttaaagat gtaaaagaag ttgaaggtaa ctatctagtt cttgattgga gtgatgttcg 34620 agaagcactc tccaaagaaa gtcttggcat actagagcaa cttattttct cagtacgaca 34680 tatcagagaa gttagaaatg gtaaagagcc acttgaagga gtctttgtag agaagtctta 34740 tccgttctat gaagacactt tacagaaagt taagatgtac tttaaacaga agaaccgtaa 34800 agtggtcact atggtatctc ttggtggaca gagtatgtct attatggaag atatcaaccc 34860 taagagaccg agtaaaggtg tctgtataaa aatcacagga catgaagagt atatctcagc 34920 aagtgatttc agtgtgttct gtaatggttc tgctgtcttt aatgggtttc actatgatat 34980 ccgagtgtac ccaatcaagg gagatgatat catgatgcag atattcgata acaacaatgg 35040 attcacccat ttttatcaga caaccaagtc atcattaaaa acagttctgg aaacactgat 35100 ttaaaatatc ttaagagcct ccttaattgg gggcttttta atttttataa aattttatcg 35160 gttgtcccaa cctctgggtc accatcatat gtgaacaaaa tatctatcct tcttaacaac 35220 cttcttaatc ttactctcta tatagttata tagtactata tagtatctta gtaactatct 35280 agtggtctta atagccctct gtataaccct tctaacaagc cctctaaata ctcttctaac 35340 aagccttctt aacaacctat acaatcacct taaccatctc tctatacact cttagaagct 35400 cctatacacc tcttaatctc tatgtcagct atgttaccct ctataggtgt tcactaggta 35460 ttactaagcc tctatataga ctgttttaac aaacttaaca ggaatatctt tacagacctt 35520 atctcacaag gtctaacacg gtactaacaa ggtatcttat aagcctgttc ctacctgtat 35580 ttttaaaaga ctgtataggt aattattcag aatcttaaaa tttttataaa ataggactat 35640 caagtcattt tttagggtct ttgaaggaac tgaaaaatat ctgtctagga tgtatatctg 35700 cttggtagcc cttaacatcc ctgtaacatc ccctttgcag aattttaaaa tatccttaac 35760 aatctcttaa caactctctc aacctcttaa catctcttca gaggtcttaa cagactctta 35820 acagaactaa caaggctgta acaagttctt aacagtcaat acatggtctt tatagtctct 35880 taacagatga ctaacaagag ctaacaagtt aatttttaag agttattttg ttagggattg 35940 gtaacaatac caattagata attaacaagt ttctaacaag tgtctaatct ttttaacaag 36000 ctattaacat ggtcactact cttttcttat cgttcccttt cctttcttag tgattcctta 36060 aacatacctc ttaaaagctc tatataaccc cttgtaagcc cttttaagtc tcttcctagt 36120 aactacagcc caacttgata gatagctctt tatagggctt tacaggggct ttaaaggcta 36180 tgtcaaaggt tcttacaagg cttttacatg tttgtaacag agatttaaca aggggctaac 36240 aagttgctaa caagcataac tatgtagggg ttgttaatgg attgttaaag aaatgttaaa 36300 ggggctagca tttacgtaac tttgaagtaa ctttataacc tcttttacgt aactttgaag 36360 ttttacgtaa ctttgaagta actttataac ctcttttacg taactttgaa gtgacttcat 36420 agtccccttt ttagttattc acaagaatac taacgagata ataaagctta taaccttata 36480 agattctctt aagcctctat aaagccctgt aaagagcttt ttaaggtgag ttaatgcaat 36540 cccttaagtt aggttgataa ggctgttata gagctttata gagcgtttta gtggttgccc 36600 ttccctttcg ggttatgggc ttatatttaa gttgatttga taaaaggcta tagagtagac 36660 tatagagtta ttgcttttta aggtatgaga taggtctatc gaattagtgg gatagattct 36720 agaaaggaaa gggaaaggga ttgtataagg attcgataga cgtaaaaagc cccatttaag 36780 gggctgtaaa ggtatctggg gctatttagc cccgttgcgg cgtttagtta gtgccttctt 36840 tgtcgtagta cagagagaca attttttcac cccagtagtg gtatgaataa ccctctttga 36900 gagcttcctg ttttgtttct ttccagaatt catcagaggg gttgtagtca ctgatggtct 36960 gactgataag gcctttcacc atttcaaagg cgttatcagt acctacaagg tcaaatttaa 37020 cgtccagaga gtagaagcca aattcgttaa cagttgcgcg gatagtagcc attttagaat 37080 ccccagttta ggaagttgat taagggtttt atttagtggg gaaggtcatt ccctccccgt 37140 tgattgacaa tttacagact tttgattgtt tcgtcaatat ctttttcaat atcttttaaa 37200 gttttttcta agttcttgat attgaaagat aagccttctc ccagtgtctt agagtaaccg 37260 ctaggacgtg agtcatgcag tttgatttct tccacttcga agaggctaac caactcaccg 37320 cttacagtgt aagccattga gacagtattt acaggtgctt tataaaactt gtttaaggac 37380 ttgatagaac gttcataagc cattttataa ccgtttaaaa ggtttttaac gtggttacga 37440 acgtttttac actcatctag ataaatctct aagaataacg gtaataattc cgctacagct 37500 tgcataaagt gagagactac aacggttgca acattacgac gatggatata ataagccccg 37560 ttttctgatt tggtgatggt tgcaaagtgt accccattac aacggaactc atatgtgtct 37620 ttaacaggct tagacattac agcatttgaa cgaatgaagt tgatagcttt aaacttgttc 37680 attttgactc tcctagttta aggactttat ttagtgggga aggtcattcc ccccccccgt 37740 tgattgataa tttacttgct ttgatagtta attgcaagca attttttaag cttatctata 37800 aagaatcttt taagtgaatc ccttttagat aaaccccctc gaacaaagtt accaatgtca 37860 aggggtattg tcaacacttt tttaaaagtt ttttgtaagt tgttgattaa ctgatgaata 37920 ttttttcagg ttttacatag ttgatacgtt ggttttttag caggtcatcc cagattaacg 37980 cggcgatatg ggtataaccg gattcacgta gaagcttcat tgctttactt ctcgtttcaa 38040 agcggcttgc atagtactca ctatttaaaa ccgttttaat cccttcgttg tctgtatgga 38100 taatctgcat cacttgaaag gggcttgttt cctcgccttt attccactgc tcaaagtatt 38160 ttaggcactg cataggtgac attttatcgc ttacttgagt tgaccaagat gagccagctt 38220 tatcggtaat tacggttaca taagccattt ttgaatctcc tagtttaagg gttttattta 38280 gtggggaagg tcattccctc cccgttgatt gacaatttac agacttttat ccggcttgtc 38340 tagtcttttt caataataat ttcatattga ccatcaacac tatcgaaata gattccatcg 38400 tcatcttctg ttttaaagta gcattgaata tcatcacttg cgggatagtc ttgtcttgct 38460 atctggcaaa cctgaatagc attttcttta ctccctgtaa actccccaag ctcttcgaaa 38520 tcacatgaat gaccattaca aatagaaagg atgaggtaaa cggttgtaag catgatttaa 38580 gctccttaaa agcccctaca aggggctgaa tagtgtttat aaggtgattg cataggtttt 38640 tataaaaagc tcttcaaatc actttctatg atgttttctg tcagtgtgtc catgatatca 38700 caaaaaacct gaatactgtc ggactgttga acacttttag cgagaagttc cagagactca 38760 tcaagatagt ttaaattcgg gttatccagt gaagggagac aaaaaggcga aaacatagca 38820 ttaaattcag tgatgtaaac ctttttaatc caatttaaat cgaagtattc tgtaaaactt 38880 gctacgccag ccatattatc acaagaataa tcattattta gctcatcttc tggaatatca 38940 agttccaaga taacaatgtt ttgagttttc atctggaaag ctgcggtgat agcggcattg 39000 cctaacgctt gctggattcc ttcagatgct atctcttccg catcgtcgcc acagatttta 39060 ttttctggat aaacgtaaaa attcccatcc atatccgaac aattccatgc acctgatggc 39120 ttatcgccgt tattaatcag gttgatgaag ttttcttgag tagtaccgtg aaagcatttc 39180 atttttgaat ctcctagttt aaggattgag tggtaaagct ctttatctgg ggcttaagat 39240 atcagctttt aagccccctg taaagtactt tatcggttat ttttgacaac ttttaaaact 39300 gcgcgatagc ctaagcaata agaatcaccg aagagagacc ggcccatgaa agagtcttcc 39360 gtgatatcct tttcagtaac agttctttct acgccgtgaa aaaggactat atcaccaact 39420 ttaatatctt tgatgtgagt agtttcaatg tttaagccgt taataatagc cattttagaa 39480 tctcctagtt taaggacttt atttagtagg gaggtcatcc ctcccgttga ttagtaattt 39540 acttgtttta aaaaccaaaa tcaagagaaa tttttatagt tctgcgtaaa ttgcgtcaca 39600 gatatcacga aaatcgtgat ttgttaaatt acgttgcaac catgttgcaa aaaagtcagc 39660 gattgcaaaa tttgcgtcat actcgacaga tagccacact ttaacagcaa aacgaaacat 39720 tttcaaatct aactccctat taattacctt agatttgcct aacatgtcgg tcatttcata 39780 ttgtgcaccg tctagcgtga tacctgctgc aacgtcagtc aatgagtttt tcacgttttc 39840 aacgtctaag ataacacttt gaatcttccc aataatgttg tgcaatgttt catctggcgc 39900 tgctgatgtc atcacattaa ttcgattaat ggtcaaacgt gcttttaaga ttgataattc 39960 acggttatta atagccattt ttgaatctcc aatttaggat ttagtggtaa agctctttat 40020 ctggggctta agatatcagc ttttaagccc cctgtaaagt actttatact aaatgcagtt 40080 ttttaccttt taccatgatg taacttgagg cgctattact gtacacatct tgataaattc 40140 tgcgcaaacg tccttcgtag aatactttcc aactagttgg aatctttttc ccatatccgg 40200 tagcagtttt ttggagtcct gcaagttgcc aatccagaat atgttgttca gcccgtaaat 40260 cctcgaagtg cattacaccg ttcacatagt ggcgtagggt tacgtttaag gtgcgaccgt 40320 tgttagtagt atacattttg aatctcctag tttaagggct ttacttagtg gggaggttat 40380 cgcctcccgt tgattagtaa tttacttgtt tttaaaatca aaatcaagag aaattttttc 40440 gtttgctcaa ttcttgaaca attacagcgt aatagtcttt taagtcacta atatttaaag 40500 catttatcag agagttataa tcatccacta actgaaaatc atctttgaag atgtcactta 40560 tctgataggt tttcgctttg tatctgaagc tcatctctag ccgctttatt tgctcatcac 40620 tgaggcgccc tagaaagctt tccacaaggt tattgctaac ctgctgtgtg gttgccttat 40680 ttgctagatt ataggcttta tatgccttat atagcgctat aatggatatg attgcagtaa 40740 tgccagtcat taacatggtg tttcctcttt acatgatacc ttgccattta cagttatctc 40800 ataggattct aataaatcac ttttagactt tacagggctt ttaaaagtat catcataaac 40860 attgacttgc aatacaggtt ttttatactc cctgcaacta ttccataagc aaatcactac 40920 acgacggcct ttgatttttc cgtgtaacca ttccataaca tcatggctat tccttgatgt 40980 ttcaataaaa tcaattttgt taagtaactt gcttagattt tcacggactt tatttgataa 41040 tttcatttta gaatctccta gtttaagggt tgatttgaat aaagctcttt atctggggct 41100 atcttatcag cttgataacc ccgtgtaaag aaatctattt agttcttaaa gatattcatc 41160 actgagtctt tcaacgattc caaacgttct aatacttcag tttctgcgtc tgaattgttc 41220 acctcttctt ttagtctatc ctctaaatca tcgccgtcac gatagcacca atcgaaagca 41280 taaccaagat aaaaactttc taaaaccgtg ttaccgtttg catcgacaac tttcgcggca 41340 aggttgtatt ctgaagcctc taaatctctt tctagttctt tctgtaagct tgcgtaagcc 41400 tcttttgatg gattttcccg gccttgtttt gcatagtcac tagaaagctg tttaaggctg 41460 taatgtaaag ggattgcata tttaaattca ttatgctgac agttaggatt tttaacagtc 41520 acaccacctt gatggtcatc tgtaaacttc caattatcaa gattatggct tgcttcgtag 41580 caatttaccg cttcataaat aataatgaat ttttctttta caacttcaaa agtagcgtct 41640 acatttttca ttttgaatct ccaatttagg atttagtggt aaagctcttt atctggggct 41700 taagatatca acttttaagc cccctgtaaa gtactttatt tgttaatttt cagctttgta 41760 taacctctca ttgtacgaat gggggatgtt aaggctacgc gtctaatata tgcctttttc 41820 gtgtatacat ttgtcttccc aatagtgttt acacatttaa aaaggtttat cggtgaattt 41880 tttaacttca actgcttatc agccttcacg ccccatctta cagcagattg ttttccgttg 41940 tcaaccactt tcccgtctaa ctttttggcc atgatgcgag cgtcatcgcg agtctttgca 42000 gtaattacaa cgtttttgat aatagccatt tttaagttct ccaatttagg atttagtggt 42060 aaagctcttt atctggggct tactttatcg aactataagc cccttgtaaa gtactttatt 42120 tgttaatttt gaagatagtt acataaccat cactaccgat tattttagat actccagctt 42180 tttctacttt atagcccata ttttcaagat gataaacagc atcatcataa ccatatgcgc 42240 caccctgata acgtctctta tcacatttta tcaggttttt accacgtcca gcaagtgcat 42300 gtaaaatggc tttttcactt gaaagagtac catctaacat ggttgccgtg taatggttga 42360 ttctattccc gtttacatca tacttaaaat tgaatgcgtg aacaatgata atgttacctt 42420 taaaattttc atcaagtgac ttctccagtg cgttgcgaaa gttgtctttg ttgatgaatt 42480 taggtgcacg ataagccatt tttgaatctc cagtttagga ctttatttag tggggaaggt 42540 cattccctcc ccgttgatgc aaatattaga taaccacact caaagagtca agaaaaattt 42600 taaaaatatt tttggcaaaa ctgtaatgaa acgggcgcac acgactacca caaaacagaa 42660 cacaaatcaa gaattatttt tcaatccgct acaaattttt ctcttgactt ttagggagag 42720 ggggagtaag agggggagag gggttggtct ggctctctat aaaaacctac ctgcacaatt 42780 ctaaaatgat ttttcttgcc cgacctgagc ggggtctgca cgttttgaaa atgatttttc 42840 tatccggcga tgagattttt tgggatatct gtacagaccc ctacagatac ctgcacaaat 42900 tgaaaatgaa aaaggccgcc ttaccttaaa tttttcaatg ggttaggtag ccttcttaga 42960 tgatttgtag atactatgaa gtgctaatca gcttatgaat ttgaactttt tagaatacag 43020 caagtagtga acttcatcct gatttgttgt gtagatgtat gctgctgggt agttatcaaa 43080 gaacacccct tcatatcttc caaacgggtc aactgggaac atttcactgt tacacatacc 43140 catctcacta accagagggt aactaacctc aaccttgcca tcaacaagct gatagagggt 43200 aacttgatta gtcttcccgt cacttagcac gacaccatca tagaattgtg gtgctgaggt 43260 agcatactta ggggtaacta cacttgccat tgcttcatca tagacgtaga ggtacacatc 43320 aacgttatac ataacactat taatgaggct tttcacagct tgtgcatcta aaccgcctat 43380 acctgcccca atcatgggta aaccaatctt acgaatgttg tgcagccagc aaaatttacg 43440 gagtttttct agtgactcca ctaagtatcg gtatgatgca cacttcccaa tatccaactg 43500 tgtatatagg ttaaaaatgt aaccatactc cgttcgagca tgtgacacct caccaaccat 43560 ctggtatccg atatcatgag cactgtagcc aaacttttta tggaatttcc ggtcagcttc 43620 ttcagctttt gggaaaatct tagcaatttt ttgagctatt ccagcaccca tgaggttacg 43680 acagttgcaa ccatgagcaa ttaactcaat atctgtgttt ttgaaggcag ccagtaagtt 43740 accttttaca acttttacaa tacccattta acatctctcc caagattctt tgtcttcaga 43800 aggttttaca cgttgccctt cgtggtctac ccagatacac ccacagcctt cacagattac 43860 tggcattgca taaccagctt taaagtcatc ttcagtgatg agacctttta agtcacctgt 43920 atcacgtccg aacatttcga tagaacagtc tttgcagaag tcagccatat aatatacacc 43980 ttatagttca gtttctcgta attctttacc tttatcatag aagcatttat agaatgtgtc 44040 tatagagatt ctggcaaagg taaggtcttc aaagtattct tctggagtta tccctgtaaa 44100 actaaaacta tcgaggttgt aaaatggtat cactgtgtcg ttactaacga aaacacatgc 44160 tgatgtgact tctccagtat ctttgtcaat tacctctaga gctttcatag cttacctttt 44220 cggttagttc atataaatca acatgctcaa tactgtcaat gttgatatca atctttttga 44280 aacgctccat cacttcgtta tcagtcatcc atatcagaca ccaatgattc tgaatggatt 44340 cttcaagaag gtcaacgcaa tctttaaaag tttttgtatc acttcttgtc cctactactg 44400 cgattgttcc atatagccac ttaccattgc cataggttac ttgtgctaca ccaacttctt 44460 tatctttctc gtagtaaacg gctacaaagt ctcttccatt aatctgcata gttacttcct 44520 cgttttaaga actgctaaga tttctttaga atctggtaag aaccatgtgt agtgattctc 44580 tttgatttct ggatgagtgt caacctttat tttgcagtct ttaccaacat gcttaagttg 44640 tgctgtgaca ccttgcataa agatatgaca catcacatta tccataatgg ttggtgagaa 44700 atcaacccgc atataagttt cttttggtcg tggtagcagt aagtaccctt ctacatagaa 44760 cgtgtcttca ttctctgtca ttgctgtttg tttcctaata aaatcatatc aataaggtta 44820 ttagctttga ttgtgcactg gtaaaaacca tcacgataaa actcaagggc tgctgccaca 44880 tcactgcctg tgttaccttt gatgatatac cgtgattctt cttgagtgta cttatcactc 44940 tcacttactt gggagttttt gaatgtactg ttcactttgt tcgttgaaca ccctgacata 45000 gttatcagta acacaaacat tataaaactc tggcttagtt ttttcatatg ttagtacctc 45060 ttttgtatgc ttattcttag caagcacatc tttcaaatca tttttgtaat tggtgctcag 45120 tgtgactaaa ccttcctgat aagcatcttt ggcaacactt attagtttct cattatttag 45180 ctcagtttca gcaacttgat agtctctgta agagtatcct ccccagacac ctgccccaac 45240 taagaaaaca attatgaaag tagcttgaca gaactcttta aatgccattt aacaatctcc 45300 tgaaatgaaa aaggctcccg aaggagcctg tatcttaacc ttaaccgagg actttagcaa 45360 ggacatttgc agccatcgaa gaagcttttg cagcagcagc taccccagct ttgacggtgc 45420 tatccttgat agcagatacc gttgcagcat cagtaaagag ataagtagtt gttttagaac 45480 cacttgtaaa ggaaagcata agagcagttt ccagattgaa agagcgatac ggagcacctt 45540 ctttaacgat accttcagca cctaactctg aagccatacc catatcatag attgtcatca 45600 gattttcttt gtgtgctgtc gttgagccag aaccttttac gtgtttctta acattcaaaa 45660 gagcacgata ctctcggatg ttaccgtctg cttttacgtt gacagcacgg aaaattttac 45720 cctcaaagtt gctcttgata atgttacgaa ctacttcaga tttgtttgcc gtaacgtcca 45780 gtgctaccgt aatgatattg ttcataagtt ttctctcaat gtttaactaa catggttttg 45840 atgtgtggaa ggattctgtt caccttctgc acattcttct ctgtgtcttc tataaaacat 45900 actatattaa agtaaggcag aatgcaagac tgaatcatcc tcactttcaa actttctgca 45960 cttatagagt tagtcccaaa accacgcata aaaagcatgt agtcatgctc tgtgttatgc 46020 ctcaagaaca tctcagtagg tattctctga gtttcacctc ttgctgtcaa gagtccgata 46080 gcagcatgat tagcaattgc atcaatgata ttgaaaatat atgttggttt tgctcttaca 46140 gagtcaagaa gattagtgta ctgtgaaaat gacccgtcag tcaactctgt tgagtaatct 46200 tcgtgattaa aatttgtaag aactccatca atatctgcta agatgaggtt acctttttct 46260 acaaggtctt tgttaactat cactaggcta tcacgtctta cacctataga ttgtccggtc 46320 tctttgtcca taacattcac gattggctct ggtgtatgga actcttcaag cgagcataca 46380 acctctcttg cagtatcttc aaaatccctg cactgttctg gagtccatac acaggaatca 46440 agacctaact catgaggatg taagtcataa gcgtgacaca gtacttcatc agactttttt 46500 aattttttca tggtttacct tagaaatgtt tgttgcaaat tcacattgag caatcgcttt 46560 catgttgtta tagacatctt gagaatattt cttagcttta gggattttgt acgaataccc 46620 agcattataa gatgctaaaa ctttttgtaa agtcttcttt gactttggct gaccatgtac 46680 ctttgtccaa aactcaagct ctttgtgtgt ttcctttgca gcatagtcaa aatcctttaa 46740 gagtttcttt ttagccacat taggactgat tttgttacgc tttacaacag tcttcaagtg 46800 attctggaaa ataccatagt catgtgtctt tttattctct accttcagac ctaactctga 46860 ctcttgtaag gctatagcag ctagagtaat accccaacct ttgcccatgt tattctcacc 46920 atactggtaa gcttttaaca tgtttacttt ttggctaatt gatagctctg ggcagtcaac 46980 tgcataagat aagtgtgctg taaacattaa gcacaaacct agaatcaatt tcttcattgg 47040 ttctcctgtt tagtttatcg tgacaacaat tatacagcac tctgcacaaa atacaaataa 47100 aaaaggctgc cgaagcagcc ttttaagaaa ttacttatta gatttcacgt ttgcgttgag 47160 agatgagttc accagcagtg ccaacgatga catgcaaagc ttcagtctta ccatcccatg 47220 ctttcagaac tttacgttga ttatcaacga ttgctgcaac ttcataacgg gatgaacgca 47280 tcttcatatc gttgtaatca gtcggaacag atacaacatc tcgtggatgg acacgaacct 47340 tcaggattgt atcacctgag aagcaacgaa cataatccca agcaccaaca tgaagaccct 47400 gagagcaagt tacgttacgg ttgttatcaa ccatccaacg tggcatttct acaatgttac 47460 ccaaatcatt aggtactttg taggtgtggg agtcaaccag ctttccttca cgagtagaga 47520 ctttcttcca accaatgatg taaccttctt catcaatttc aacatcaagg tgagatacga 47580 atccccaaag ctgttctaca gagtctttag atgggttttc catcagtttt tcgaagaaca 47640 tcacaaggcg ttcgaaacct ttatcaccag tcttcatcat gtgaagaata cggtcaacca 47700 gagtagaacg catctcaaca gcaccgtaga ataacttgtc acctttgatt gtgatagcgc 47760 cctgagtgaa gttttcgata gatttacgaa tgttcatcag ttcgaaagct ttcttgtact 47820 caccacctac aacagccatt acgatttctt tgtagtttgg atgggtagat tcaacgattt 47880 cagaatctgc accgaaagtc atgataacag aatcgccagt aatcatatac tcaatctggt 47940 taccattctg catagcttcg tgtagtttat ttaccggagc cttttgtaca gcctctttaa 48000 caaccttttc aactggtttg ccagctttct ttttaactga cttagcaact tgcttcaaag 48060 tttttgtagc tggtgtagaa gctacctttt tcttgccttt taaggttgct tcatgacgct 48120 ctacagcacg accaactgaa cgggttgaag tattaaattt ttgcgcaata gccgttttag 48180 tcagcttgcc ttctttaacc agtttgtaaa tttctgcgtc aatctgtgct ttagttttag 48240 tagtcatctt gttactctct cttgttagtt aataaatcat tttgtgaggc tattctaagg 48300 ggcttttaaa ccccttgtca aacactttaa tcgtaaattg tagtcccttc tggaacacat 48360 tttacatcaa atcctaaaaa cttgctcact tcaacaggtg acactttatt ccaatctaaa 48420 tgagaaagca agaagttttc ttgttttctt tttgagaagt agtttgtcac cttaatgacc 48480 atacggtcac cagctttctt aattttcttg tagagctttg tatcgttatc aagacacctt 48540 ttcaaatctt gcaacacact aatagtgtaa gtgtatgcaa acggtgcagc aacgtattgg 48600 attttaccaa acattgcctc aacagcatct tcattacctt ctaggaagat agttttgttt 48660 gtatcccgtg aatagcaata accacgagaa atcttcctgt tattgaacgt aaagtttttg 48720 gcaataatcc aacagcttgt aaggttgagg acaccatcca tatagctgcg tgtcatatac 48780 ctgttatgat taatccaatg aacatcagtt aagctgtcgt tcaagagctt ctcatctact 48840 tcaatccagt cttctggtat ctttttccaa tttgcttttc ggaaaacata gactgtcttc 48900 ccaatgacat tggcaactga cttagctaca tcatatggag atgagcaaaa acatttacca 48960 tcaacagtgt cacctactgc cttaatataa agttggggtt cttcaattgt gtctaagtct 49020 tctgacacct ctttgtatga agctgcccct tctgctggaa cagctttcca gagttttaca 49080 acacctctta cagcttcttt acgctgataa tggtgttcct tatcagacat cttcacaatc 49140 tttaacaagc ttttatcaag tttatgcaga ttgattaggt cgtctaactc tttctcagta 49200 gagaatacaa agacaatacc attgtatctg tgaaacagat ttgactcact ggcatagtct 49260 cgacatgcac cacgcaagat ttgattgcgt cctacagtct tctcagtacc gtttttattg 49320 cgacggtcat tgataacaaa caaaaactgt tcaatttggc ttttacgcat tgcaccaaag 49380 atattgaaca tacttgcctc ttgagtgtag gaaagtgctg ttgcacgaat cttactctct 49440 aaagagttga acttgacata agcaactggg tcataaagat aatctacttt aggtatattg 49500 ttcccgttcc tatcaacctt aatattgcct ttaccgtcac gttcataaat gactgagccg 49560 tcttctgcgt agataattcc acgacgaatg tttagcaatt cttcttccag agaatcaagc 49620 ttaacacctc cccactctag ctttggacac acagcattaa acatctctcg tgagttcaaa 49680 cgtaactcag cataagcctg tgcagcatcc atgagtgtag gttggctatt aactttcttg 49740 ataacatcct ttgtaatggc ttcagttatc tttttagtag cctcaatgat aacatttttt 49800 gtcgtgtcat tcatctgcaa tgcttcacga gaagctgcaa tagcaactga accaataggc 49860 atgtagatgt ttacaaggtc tacgctccta cggaaaaact ctggcaagac tttgaagaaa 49920 tcatcaccaa gtaacgcttc catgttcact ggataggcaa tgttacccat caccacatta 49980 aactctgtcc tgttatcact agaacgccag ctgcgcttat gaatcatggc atcataaaca 50040 ccttcttcac gggcaatgac gttcatatct gctaatacat cgtcgtacac aatattactt 50100 tctggtttta cagaaaagta tgagtatacg tgacaagcct cttcaaagaa ctttgagata 50160 cgatggtcag caactgctac acgtacagct aaaccattgg gttcatttgt tgggttggta 50220 gtaagcttag ttacttgagg gataccattc tcaaggtaaa cagagtactt attaacaaca 50280 ccatcaacat agctagatac tgtgaatgac tgagcaattg caaatgggga ttttgagccg 50340 atacccattg caccaatgta atcattggag tcattcttcg tagaagcccc gtagttcaga 50400 tacaaactca ttactttgtc gtgggttaaa ccagttccaa agtcacgtac ttcaaagtaa 50460 ggctcaaatc gagtaggtaa atgcacatgg aatgggatgt tctcttttcc agcttctttc 50520 tgagcatcca ctgcattaca tgacagttca cgaatcactg ccctttcttt gaatgtgtat 50580 acaccagaac tcagaaggct gaacatttca ggtgtcattg taatctgtgc ttgagatgtc 50640 tccaaagaag ttgaactctt aatcacttct gcgtggtcat ttaccatgcg catagtactt 50700 tcctcttagt ttactgttaa attacttatt aaacatttta ccagaaccgc cacatatagg 50760 gcaacaacct gctcctgtat aaccttctcc attacagtag tcacattttt tgtgctctgc 50820 atagtacttg caaagaaggt aaacaaatac tgctacacta ccaagtgaca gtaaaagttc 50880 tatccaatat gcttgcatga ctcacctctc atatttttgt aaaggataaa gcttctgtca 50940 tggacgatac ttgtagacac tgcaaagtta ctctcaagca tctgtttgtt tggattgtag 51000 aagctttctt taaactcgtc aagtgcaaat tggtgattgt ctggaagacc ttttacacag 51060 acctttatta tgattgctgc tgcaaccttg ccagtaagtt cttctttagc tgctactaaa 51120 attttgttaa gaaacttatg gccatcttca ggcttattga tgttctccca acgttcttct 51180 aagacaatgt tgatgttcat ctctttgatt ttcatagtct tctccagaaa ttaaaaaggc 51240 tcccgtagga gcctcttatc atatactttt aaagctgctt gtcaatatta tttcgaatat 51300 caccaagaga tgtgtaaccg tactgctcag agtcactaaa gactaaacgt aaagcacatg 51360 ccggatggtc aagtgcatct gcaaagctct ggaatccata gccatctaca gctttcagct 51420 tatcaccatc ccacataggt gctacaccac caaaggcaga tttcttaaca ccactatctg 51480 ttttagggtc ttttgcaagc ataatctctt tgccaccaat gcttgcaaga gttgctttga 51540 cagcaaatgc aaaggtgtca cgagtcatgt actggtaagt gtaagagcct acaccaaaca 51600 ccacgttaga gcttgcaaac cccatttcat acagacgctt caggatttcg tttgcacgtt 51660 ccaacgtgat agagtcgcca tagataagtc caatgtgctc atccagtacc ttaaaacctt 51720 tagagttgat agttcctcca aagatgttat acagagtctt aatagctcca tcaatctcag 51780 ctacagggcg tgttacaacg tttgcagagc cgttgatgta agcatctttc agcaacactg 51840 tatcggctac ttcaaagtct tctctgtcaa ccaccatttc atagccttct gcacgtaacc 51900 acccagccat aacatagtta atatcttcaa gcttcatgtt cagaacagag ttcagcattg 51960 tatcactggc ttccagcttg cttaggtgct cgtaataagc cttcttagca cattctaaat 52020 ggattgcttt ataacctgtg acaatatgta cagggtctcc agagtcaggg cgaattacca 52080 gtttgccatc acgctccatg atttctttac ggagtgctgg taagatttct gacacagttc 52140 tccagaagtt ataagtatct gaaacaatac ttgcaacacc agttgggtaa gtttctgtta 52200 aaaaacgacg gaaggtttgt aactcacctt taaagcgtct ttcatcttca atcaattcat 52260 taccaccttc ccaagcaatg tttgcacaca ttacagagtg ttcagtggct ggtacagaac 52320 taccaatatc agagattggg taagactgtc catagatgcg tttagccgta tatacagcag 52380 ggaagctatc agtcccttta aagctggtta agtgacctac agcattaaat gcgtcatcag 52440 taaagccgga cataccacgc atagcaaagt catggcactg ataaggtaaa tgtaagtcat 52500 tgtcgcaagt aaggtcagcc cacttcttac agatgcgttt gtaatgtaat gcaatagttg 52560 caatggtgca agccttccaa atctcagcag agaaagcatc ttccagataa cctgctaccc 52620 aatggaaacc tgaaacagtg ttctggaaga caatcattgg gacgcgcatc ggaacaactg 52680 taccttcttc tacagcgtat acttcaactg gtagataacc taagtcgtga agtgcttccc 52740 aatgctctcg accgatagca tctttaccca gaacaccatt catgacttct aagatttcat 52800 caatggcttc ttttttatca cgttcaaaga aagtggcgtt ccagtggtct atcaagtaat 52860 ctttaacgaa acgttgaata ccaaaagcta ctacaccatc aattgctaaa ggactgttaa 52920 accatttatc actacgtggt gtcaggttta acataagata ttgcgtagca tctggatact 52980 gatatatatg tgatgccttg taagcatctg cgtttaaacc tgctggtact gcataaagtg 53040 atttagtcat cttttaatct ctctcaaaat ggggctttta agcccctata aactttcaat 53100 caaggtttgc tacagtaact tgaccgtaat gtgttaagcc acgttcttta gcttcgccta 53160 aagagtttgt agtgtaaata tggtcgatac cattgtcaag aaggttttca acatctttag 53220 agaagatacc atgtgttaca taaagctcta cacgttttgc acctgcttca cgtaggtgtt 53280 tagctgcctc tacgaaggtt cgaccaccat cacagatatc atcaagaatc atgacggttt 53340 tatcggtcaa atcaacatcg tcaaggattt gcataccagt gatttctcca gtcttaagat 53400 ttcgcacctt agacattgtg atatatggtt tatcaacctc tttagcagtc tctgcaatct 53460 tcttagaagc acctgcatct ggggctacta agtaatcaat cagagggtca tttgcgtaat 53520 ggactgcaat ctccttttga ggaatactct ggaagcaatt gaaaaggtta tctggaacgt 53580 aactatgagg gtcaactgca cagactgcat caaaacccat tgcattaacc tgttgtgcaa 53640 acactttcaa agctgctgca tcacctttaa acatgtgacg gtcgtaacgt gcatttggca 53700 aatagtaaaa gatagcagtc ttcatagcaa acttatgagg tactaaatca tcaattgcct 53760 ctttagcaag tgccacagca aataatgtgt ctttgtcata accctttata atcataatca 53820 cgttgttgat ggatgatgca gcataagcag taaaatcaac aaactctggt gaaaaatgtc 53880 caccaatctc accagaagga aattggacga tgttaaactc ttcttcgtga gtccctttag 53940 ttggtgaatg aactgtaaca cggatagatg ttttcatagt tttctctctc aatcaaaagt 54000 tacgtttgtt actgccattg cagatttgca ttttacaggg tctacaacgt ggtaaagtac 54060 actacagtta cctgtgctgt ctggtacagg ctttacccat tgatgcttta tagttggctc 54120 tcctaagtct ttgtaagaac cgtctgtaaa gttgtttacc atctcaagtg caattggttc 54180 tggaacatga ctacgtatca tgtacatatc acaaccgtta ccaccttcac tgactaatgg 54240 taaaatctcc cacttttctt tagccattgt aagttgccct ataaactgtc tcgaatttca 54300 acaggagaga tattgccaaa tctgtatagt tattgtcaag cacatctggc atattatttt 54360 tgatatattt ctcttgctct tcgtaggtca tcttaggggt gcttagaagg ttatcccctt 54420 gcttctggtt atcaagttta gtctgaataa accagtttgc acagtgatgc tcaccaccct 54480 ttgtaacagg tttgtggtgg cctaatgtga aggattgact acctgtgtat ctgcaaagtt 54540 tattgagaac tgtaatggtg ttcttcacaa caaaacattc atacaagtta tctatagtgt 54600 agtgtgggta atagcggaat agtgctcttt cacgagtact ctcacgattc cagagaatca 54660 tgtggtttga gttagaaggg tcgtattggt gggagtcaat gaactcttga cgttcctcaa 54720 agctgagtct tagaactgac attgcagccg aacgtttcag atttgacaga tacatgttat 54780 ctccaaaata aaaagggaac ctttacagtt ccctaatcat atagtcttac tgtgagattt 54840 tcaacacttt gaggaagcct tcttcaccaa tgttgcttat aatgtaattt gtaggtttaa 54900 tagaatggtt tagacataca atcttcttgt ctttcttgtt gtaagttagt ttaagctgtt 54960 ctagctcttt actaccatca agagattcat tgtacatctt cacagctttc ttaaatggga 55020 tgtgttcacc accaaaggtt agaaatcccg taccacaata atgtgcgtta tgttgctcaa 55080 ccataaactg acacagaact gcaatctgaa tcttacgagt gttataaatc tttttcatgt 55140 taactaccct tatttgttac gtttacggtt acgagctttc ttagctgcac gtttaattgc 55200 tgctgcacca gttggtcgat gtgcttgttt tttaccacct tttccacgtc ctacgtggat 55260 gtaaggctgc ttttcaattt ggcttgcaag aacttgtgca actgctgaag catctacacc 55320 aacctgcttt ccagtcatag aaatgattgc tgctgctttt gccagtgcct tcaagaagcc 55380 agccttcata ttacctaaaa ttcccatttt taatctctct cattaaagtt taatacagaa 55440 cttatgaagt tcttcttcag tgatacctat tggagtgata ccttctctga tatttttaac 55500 cctgtaggag tctccaaact tatctgattc atgaatgaca aacgtgacat tctttacaat 55560 cgttgcaaga gcgcataatg aacgtgaacc aaatttttct ctcgtataca tcatccaagc 55620 atctaacttg ttaatgtcgt aagtccactt accagaataa tcattctgct cgccattgac 55680 aatcaccttg ccatccatac tcttttctcc agaggttttg gttatcagaa ccacgataag 55740 gtttaactgt tggtttagaa gaatcatact taccatcaat caccacgtca acatatttca 55800 tcacatcgag gtggattttt tcgtgtaact gaaaacctgt ccaaagccat atagattttt 55860 ctgggtaaac agttttaata cgtttacata tattggtaac ctcttgaatg tttctatcat 55920 ccagaggctc cccaccaagt attgacagcc cactgatagc attatcatcc attaacttga 55980 tgatgccata gaggtttgca taagtaaact ctttaccagc attaaatttc caagactccc 56040 tgttaaagca gccttcacag tgatgtttgc agccagctac gaagaggctt acacgaaccc 56100 cttcaccatt agctgtgtca aatggtctaa tctccatgta attcatctag ttacctcacc 56160 aatacatata agtgctctga gcagctttct ttggagactt gctatcaatg tcaaagtctt 56220 catctaagta ggatgtctta gaaagctgcc ttatttgtgt tcttgtaaga cgtttagtag 56280 ccttcttaat ctctctacaa ggttggcttg taaagtcata agagccacct tttctggaca 56340 tcatatcaga gtggtagtag acctctgaag cattttctct gttacgtacc tcatttctcc 56400 actcataaag tccatgaacg ttctttaaac cttttcttcg aattgtttta ctcattcaaa 56460 caccacctga caaggaacct gtttagtaat aaattcttca cagtgctctt taatgaagtt 56520 ttgtaatggc acacagtctt gtgcacaagt cattagcata atgtactctg gattcataat 56580 attttcaaga ctgtagaaga caacattgtc agatagttcg tgaccatctt cttcactcca 56640 cgttccagta actacagcta atttgtcaag caattcatta ggcaatgcta caccatcttc 56700 tacagaatca cttagaacgt tgtcgtcaat atacaatgtg aagactgtgt gaaatgttga 56760 gtaccttacg tgattctttt cgcaagagcc acactctgag gcataatctt taatccactc 56820 taactgtttt ttgttcaact taatcataat ttcaccttag ttggcaatgc actgaatgct 56880 atagctaacc ctaaaaatgg tttgaagcat gaccataaat ccatctgacc acgatatgag 56940 aaactccatt gtgggtctga aaacccccat accataacac taacaccaaa gaagaagatt 57000 gctatgtgta gtgtgggtgt caataccttt aagagtgttg tcattgttaa ttccaccata 57060 tagttttacc attatcacat gtgtaaagat actctacaga ggtttgtggg acaagtacag 57120 tggtgttccc actaagtaca ctcacaaaat aagtaacctc acgatgttga tgagttttta 57180 cacaattgtt tgccttcatc catatcacag ctttgtatga atagaacacc gccaagacac 57240 agagtgtaat aactccaact atcatggcaa actcaggcaa aaaatctgac tgtatgatgt 57300 ctctaaatcg cattattcca agaataaata agattatcag acatataatc ccacaaactg 57360 ctacataaag ccacaagtcc ataatgtaac tccaaataaa aaggtgatgt aagcctatca 57420 agacccacac caccttgtca agttttacat agagacccta tccttaattt ctgccatctt 57480 agcgtcattc attcgagact gacctttaat ttttgtgaat gacaagtacc cgcacaccct 57540 gttaatcaca gagatatcat gtgaatgaca aacctcacat tcttcaacat cagcctttgg 57600 tctgttacca caatgttcac agattgctaa gtcaaagtta agtccctgat agaaaccttt 57660 caacatccct cttgtaatac aacttttaag tgctgataag ttttctggat tagctaccct 57720 tacatactgg attctgccac ctctacagat atggaagaag ggttcttcta agtcctgctt 57780 ctcaaatggt gtaatatctg ctgcaacatt catatggaaa ctgtttgtaa agtattcttt 57840 atcagaaacg cctttgataa cgccaaacat atctctgaac tgttttagct gagttccaca 57900 aagtgattct gctggagtgc catagactgc atataagaaa ccatcttcat ttttaaactc 57960 ttcagtcttt atgttgatat attccagaac atcatatgca aaactacagc ttccaacttc 58020 atgaagtcgt ttaccttcag caagaacaga tagctcatca agcgcagtaa caccaaaaga 58080 agctgtgaag gacttcacaa tatcccaacc aaccctgtca gtaggcttct tagttccttt 58140 gtacagacca ccttgtgtaa atgcaagagg gttagaactt gctggcatat tagcaatcat 58200 ttcgtaacgt ttcttatgga aactacgaat catctctagg tacttatcaa gctctttcca 58260 gaaatctaaa ccattctctt tagaatactg gtaaatcatt ggtaagttca aagatacagc 58320 accaatgtta gcacgaccta cataaaactc ttcaccatgt tcgttatggt atggtgataa 58380 aaatgctcta cacttgttgt tcaggtaagt tcgctaagct taccccgctt tattcaagct 58440 gcttactgtc gccagtaagt ccagactata tcttctactt atttctaagt agcgtaccat 58500 ttcgagtcgc ttgaccctac accgctacat tcatcacggt tagtcgttcg gcatttagat 58560 gtattcaaac tcatattcga agttattatg ctcttttaca cccttcaata ttgaagagat 58620 ggtcttgcga ttgtgtccaa gtatattgga taactctcgt attgacttag taacaataac 58680 ttcctttgtc actttgtgtg tggctttcac ggcatatgaa cgcttacaaa acttgtagca 58740 accgttctca taaccttctt taacattctc agaatgagtt ccccattcta agttagaagc 58800 atggttgttc agtttgttat cgtccttatg ttttacaatt ggaagattat ttgggttagg 58860 tacatacttc tcagcaacta acctgtggac atacatgtca cgattcttct taccgtaaaa 58920 cctcactttc atatacccag tgttggcttt ccactgcgta agctctacac cattttccct 58980 aaagactttt ccatcttcag tgacgtgata gtttggcatg taatactcct atgttgtata 59040 gtttcttaca tcgtacatca tttagcacag gattgtctac atctgtagag tttccctgtt 59100 taggcacgtt ttagatgagc tatagagctt gttaacccat cggtgaaatt acctttccag 59160 aacgctcaaa agcctctgct acagcaccat gaccagatac acttaagaag tctggataca 59220 tcgctttaga acaacactca atagctttac tgtacaggtg gccttgacag atattctcat 59280 tatgtttcct ctggtcataa atataaacca gtttagggaa tacaacaggt ttcttgctct 59340 taccttgtcc attcatacga acatctagga gagtacttgc aatcatatat tgcagtcggt 59400 tatcttcatt ggacatatct gagtcaagta acccgaatgt caaagttgta aatgcaaaat 59460 ccccacgact acaaggtaca gtgtttagtt tcatttcaag tgactggaaa ccttgagtca 59520 gttcaatctg tagctgctcc atgacataat tgtggtaatg ttctttagga ataccataag 59580 atgctgcttt ctcagcatga tagcgtagag acttcttagc atacggtaca agtaccttat 59640 caatctctgc taaagtaaag ccaccaaatt gctgtgcagt tgctgaaaga actacgtcac 59700 caataacctg taaggctgac agtacagact ttggttcaca atattcaatg ccagacattt 59760 caaagccacc tttcaggact ttaccaatgt caaacaggca acagttgata ccaccaaaga 59820 ttaggtctct taaatcatgg atatagataa accctttttc aatggcttca agttcctctg 59880 gtgttaaatg gtactgttta aagatttctt tagtcaggta accacgaata attgagcctt 59940 ttgtagaaat taaactgctg tcaaagttag cattctctct gtcacctaag aagagtgtat 60000 ctctggtctt ctggtagagt tcatcccaat tttgagcaac ttctttacgg taatttctat 60060 atgttgagta agactcataa atctcgtgat tgacttctgc caaagcaccc tcaacaatac 60120 tgtgaatatc atttacagaa attagcaaat tattctgctt agtggacttt accagaatcc 60180 tcataaatgc tgactcaaga gcttgagtaa catctggtgg aagctcttta taaccaactc 60240 tgttagctga ctttgtgact gctgctaaaa cttttttgat atcaggttct tcaagtgagc 60300 cgttcttttt aataatctgt actttgttca ttattgcccc tttacatgct aaaaaggtct 60360 ccgaagagac cctttcattt taaatctttt tagaaaactt cgcaaagagt tttcttagtt 60420 tttcagttat acctaactgt atctctaata tttcaaagat aacccatagt aacatagctc 60480 caaacaatga accttgttca aagtcagtaa ataggttaac aataagcata ccaatcacaa 60540 tagctggtgc atcaacaacc aaaccttccc aaatgcgttt aagcattatt cacctctgcc 60600 ataaaattct gtagcgtccc aacaggtttt aggttttgtc catcagtttt catgatgaat 60660 ggcatctgac gaactggcat ctgtgcaata tccatcaggt ctgatagttc atagtcctgc 60720 cctaacattc tcacaacatg gtcaatacca cgagcttttg caaaattctt tgcagtctca 60780 cactgagggc atccagtttt ggagtaaatt acataagtca ttaaggaacc tctaaaaatc 60840 cactattcaa atcatctaca acagtattca acaggtacgc accgttctgt tgctcctgat 60900 tagcattctg ctctttatca atttccatct tcttaatcat gtacttcaaa ggtggttctt 60960 taggagctac aaaatctctg ggaatgccaa acatatcata cagtggggca gcattatagt 61020 aaacccactc atgaagaagc tttgtattca agccaactac agcacgtcct tcggagaaga 61080 tatagtacga ccatttctct tcactttcaa ctacttcatc taagattact ttaatctctg 61140 gaagaatttg ttgaaaagct ttctgccact catcatctct taaagtttct tttaaaactt 61200 caatatcaat tttagtgtga aggatttcgt caagcataat tttctggaca gcttgagcaa 61260 taccttgaaa tttatcttga gcatcaagtg caaaagtaca tgcaaaggat gccataaaag 61320 aaatgccttc aagtgcagta actgcaaata gaccttttag aatcacttta tggaagtgta 61380 aagggtcttt atccagaagt gaatcacgaa cataacttag acgatagttt ataccttcat 61440 ccagtaattc ttcaagaaca cgattcacag tttttaatcg gtcttgtaca gcaacattct 61500 ggttaatctc atctaagatt gtttcagggt ttttaataca ttgccttaca atctctgagt 61560 aagtaagagc atgtaggttt tcaatctcag actgcttcat aattgcagtt gcatagatat 61620 catcagagat aaagggtgca aaggcaaatg ccaagctctt agcaacttga gtgtctgctt 61680 cccattgcca cttaagaatc tcaagcatta cacctgacat cgatgctgga acactctcaa 61740 aatcaagacg tgattgttca aaagggaact catcttctga ccagtcttgt gctttttgtt 61800 gtttataaag ttcaaagatt tttgggtagt gtttattaag tgagtcaaaa gtttttctct 61860 caccacctaa aaagattggg tgctggttaa tcatagtgtg atttctcctt tctttatcat 61920 ttctaaggct tgttttctgt ctaaaacttc ccaattttct ttgttaaaca tctcataagg 61980 tcttgcatag attttaccat ctgctgtaga gatgtaagag ataccagcaa cccatgagtc 62040 atcattttgt tttatcatca tgtctgtgct actcagatag tacattgttt tccgaggctt 62100 gtggagcaag tatataggtc gctcatgttt ttctattaaa tctttcatgg tctctccaaa 62160 gttattttta gagggtcttt tacgaccctc atagttgtta aaactaaaca ccacaaccct 62220 cgcaataagc atcttgcagt gcagatttac ctacaccaat gcgactgtta aggtagtaca 62280 tggttttcat acctaccgag ttggcataaa tcatgtactt caaagcttga gccaatgata 62340 ccttcttaga ctttgcgtag tcaacataga aatctgaaga gatagcttga ccagtgaact 62400 tttgaacaat tgcataacaa tcaatcatat caaaggtgtc gatatcccaa gcaatttcat 62460 agacatactt caactcttca taatctggaa caataaacag tacgttacca gttgcagact 62520 ttttagttaa aataaagtca cgaattgggt acaagccatt tgtcgtatta gttgctagtg 62580 aagaactctc attaggcatg taagcttcta atacagagtt tcggattcca ccattttctt 62640 tgatacgttg tgctaagtca tcccaatcat atcgtagttt tgcatcatgc ttttcatcaa 62700 tcttcttgtt agctgtcttt ggaggaaccc atccttcagg atacttagtg aacttcatgt 62760 actctggaac accacgctct ttagcaagtc tcagagaggc ttcatgcaga tagtaagagt 62820 gcatttcagc aagctcatga agcttcgtct tacctgctct tgaagagtag ttcacgtagt 62880 tcttcgcaag gtaatgagcc acatttgtaa ggccaatccc aacagaacga cgcttctgaa 62940 catggttacg catcgacgga tacggataat ccataaggtc aataacggag tcaaccattg 63000 caagagcata ataagcaacg tcagcgtatt catcttctga aattctccct gcaaccaaac 63060 tagctaggaa gcaaagagcc acctcaccat cctctttcac agcatcatct ctgtaaaggt 63120 ctgtctcttt ctcaaaacca tacactggca atacaatttc catacaaaga tttgacatct 63180 tcaatggttc tttaaatggt gtatgtgtgt tcgcattatt tgtgaagaat ggatacacac 63240 gacctgtagc ataacgctgt tgaataaaca gcttagcaat ttcacgagcc ttcacccgtc 63300 tgtgtttaac acctgaatgt actgcgtgac caactgccat agcaaactca tcagcagatg 63360 ctgtgtagaa catgtcatag agctttggtg catccttgta agagaaaagc aaccagtctg 63420 tatcatattg aacacactgc cagaaataat catttgtgcc gaatgagtag tccatctcgt 63480 taatacgttt cgaaggaacc gttgtagggt gcttcaaacg cagtaaatct tcaatctgcg 63540 ggtctagagc agtgtagaag ttgttagctg aaccaccacg gctcttctgt ttgtttgcct 63600 ctacagatga acgtacaagc ttgtaataag gcagcttacc catgtgctca atagtatttt 63660 ggcggatacc atcaccaata gtgcgggtct ccatcagcat cccaatacca gcttgctttg 63720 tagtcatatc ataagcaacc tttgcagcaa taccaagtga ttcagcagtg tcatttgcct 63780 taatcaaaca gcatgacgca tagcctgact tggttgctct taaaccgttt aaataaggtg 63840 taggagcgtt aatcttcagg tcagataggt aagtgtacag cttgataaca tcttgcagtc 63900 tacggtgctt tggttgcttc tcaaaggctt tcatggccat acccataaac ataaattgtg 63960 gtgactcaaa cagtcttccc gttttaatat cacggatacc gtacttatct ctgaactgtt 64020 tcaagactgc ataaccgtaa gagatatctt ttgagtgcac gatgtaacct tgcagatatt 64080 caagctcttc ctgtgaataa tccatcttct cccaaagtcc tgctctctcc atatttttaa 64140 cgaaggtaat cagcgtagga accttagtaa agcctccaaa ggcttctttg tagataatac 64200 ccagaagtag tcgtccagcc atatctgagt actcttgagt ttgcttatca acacaaacat 64260 caatcatggc ttggtgcatc tcttttgtag tgcaaccatc atagacacgt ttcatagctt 64320 ccatagtgac ttctgaccag ataattccac gtttatctgc ccatgatgcc cacttattca 64380 gtctttctgg gtcaaagctt actactgtac cgtttgattt tttaattgtc ttaatcattt 64440 ttgaatccta caggtgaaaa gagcctccga agaggctccg tagtttaaat cttgaaaatt 64500 tgctcattct gccacatatc gtaaaactta tcacttatct ttgtgactga agaggtgtgc 64560 atacaattca gggagaacca tgatggtgta ttatcatcca taagtgtttc aagtttttgt 64620 gtaatattca catccatgtc tgcaacaacg taattgcctc gcatcttgca aacaattctt 64680 ccaaacaaga ttgcacttct gcctcttcgg tcatcacagt acataactgt gtccccgtgc 64740 ttaacatctt gtccgatact atcaacacct aatttagcac ctgagataat gtcatcataa 64800 gatagctttt ttgctttaga cacggtattt ctccaagttt cttttcagat acaaccaacc 64860 atagtcagtc ctttctacac ccataaagag tggagccatc acaactttac cgaaagtgtt 64920 tctgtgttgg cagatttcaa aagaatactc tttctcaatg tcaatcccgt atttgtacag 64980 gactgctttt aaaacttgtt cattatccag aagtgtatct ggagtctcac catacttttc 65040 aagtaacggg tcattcataa acaccgtcat agaaatattg tagtttgaga agtggctctt 65100 ctgagccaca ccctctttat taatgttctg tgaatccatt ataaccaacc ccttttaacc 65160 agcacttcag gttattagca tcataattca gtttcgggta tgaggttaca tggaactgct 65220 caccatcata gtagacaata tctgcaatcc atagtccgtt ctctttacag aaatctttat 65280 ctgctttaga ggtaattgga ttaacttcta caaaaccatc tacacccaaa tcctttaagc 65340 tacctacaat acctttgcag ataacgcatg tctcagatac taccacaaaa agttcttggc 65400 tttcaagttc tacaatgtat tcaccaagct gcactttagg aagattagac tgcttcataa 65460 tcttaccatc ggctgcacga acaatcgcat accactcttt accatcaact actgacattc 65520 tgatgataca ctcttgacca gtacgcttct caatatctgc aagacgtttt aaagcttctt 65580 catagtcgtc tgtgtacttc aggtcattat tatggcaaac agcttccata gcaccattat 65640 ggtcatgttg tgacagataa atcaaaccgt caagaacata ttgcaaatct gcctgagcat 65700 ccagtgtctc aatggggtct ttctcttcaa ttgcttttac aagctctttt gcttcttcaa 65760 gcatacacag agattgagat tttaaagatt cccaatactc attactgtaa ggttgctttt 65820 gagtgtttcc acaacgaagg ttccagtttt ttactgattc tcttgagtta aacattcgga 65880 ctccttaaaa tatttgttaa cctttagctc tcgtgtttca atatattcta gatgctcacg 65940 tagctcttga cgtttaagca ttaggtgctg catttgagac tcaacagctt caacttcttt 66000 aattattgac tctctagcat ttgcaaggat gcgctcaagt tcttcaatct tgctgtcaac 66060 ttttgctaca tgactcacag ctttactttt gttaaacatt ttattctctc ttaatagtta 66120 tcaagcacga tttgctttca ttaccttatg gattcttttt tcagcgtctt cataagtttc 66180 tttagtaata aacttaattg ctgcaatatt ggcattgtag aagagtctga gcttcgagtc 66240 aatccttttt gtcattacat cgaacttatg ttgaaggttt gcttcaccat aaactaggcc 66300 accttttgtg taataggttt ggataatgta aaagtcaaaa gattcctttc caaagctttc 66360 aatatctttt ttaatatact cagaagaggt ttcgtaagtc atccaatcac tctccttagt 66420 gactaccttc ttccgagtct tcccagcaac ttttcttttg gtcacgctat taaactgttt 66480 ccttccgata tagtattgtc cagtcttctt acagtgtact aaatagacaa agccaaaatg 66540 tttagtgggg tcaacttccc cacataaaga tacccaatga ccataggtag ggcaattgcc 66600 aaatccttta atcttcattc atacacggct cccaattata tttcttaaaa gagaaattat 66660 cttttggatt tctctcttga tatgccaccc agaaatgctg ctccattaac tctaacggtg 66720 tctttgtaac ggtctgtcca tcccatgaca cataggtata agaatctttt ttagcataaa 66780 gttcatagat agcatcaaga cactctttgt aggtctcttt accctctaaa gcattcatca 66840 cagctacttt accagcacct ttaaggccaa agtagttgtc tgcattatct ccagcaacag 66900 cctgataaca caagaattta aaacctacac caactgtttt tgcagctttt ggttttgatt 66960 tgataggaca atcccagatg tcaccaacat tattgtcagc aataaagatt agtggtgatt 67020 tttcataagt catatcaata caataagtcc cttcagcttg tcgaaggtct ttgtcaatac 67080 tcataagagc agctttctta cccatctttt cagctttagc aattacgatt gaatcggctt 67140 cgaagcctcc ccttaagagc ttgaactctg gtcttgacag aagatactca cgacaagcaa 67200 ctaagtgtgt tggtgtgaca gcatctttgc ggttaccttg atattggtgc tcaagacctt 67260 taatgtcttt atgtttatgc acacctttct ctgttaaata acctacccaa gttctctctt 67320 taccaacaac cttaagccat tcctgaagca cctgttgagt agccatgata gcttcttttt 67380 cactcttagc ttctttccaa gtctgtcttt cccattcatc ttcatcaaat gtcaggccaa 67440 gctcttccac aaggattctc tggtctgcta accatcttgc agcatctttc gcattatcaa 67500 atggctcaga ttcttctgct gtgagtttat tgacatattt atattttgct ttctcaacta 67560 cacaagcacc tttataggca atactgtcag agtcaataaa gacatgtgta actgaatcgg 67620 gaagttttgt taatgtgtac ttctccattg tggctctcca tatgaaaaag ccccatactg 67680 agtacagggc tttaaagtaa cttttagaga ttagtcttct gtatcgaagt cttcatcctc 67740 ttcgtcatct gggtctggca aatcttcatc atcaccatca tcagaatcgt tcgaaggttt 67800 gtggtctttt gcgtcttctt cagtgatttc accgttatct tcaacaccat caagaccaag 67860 catagccagt tcatcttcat ccagttcagg ttcaccatta gcaccgttac caccagtgta 67920 aggtacaaga gtatcaatga taaactgttc ctgaataggt tttgtcagaa cattgttctc 67980 aaaagtgtag aagtgagtag aaagaatcac actaccaaaa gaaccgttac caactgcgat 68040 atctggatga attacgtcat agttcttgtc atcttcatgt ttatcagatg cttgagcttt 68100 gattttcttc atcggctgct taacagctac acgcttacca tttacttctt caatcagcat 68160 tacagggaat gactgtttag ctgtccatac agcaccatct ttataagctg ctgcacgact 68220 tactttcaag atgtagtaag tatctgcttc aaaaggtggt ttacaaccaa acttctcttc 68280 aaagtcgtct gcatcaactg cttcagtagt gactttatcc cagccttctg gatttttctt 68340 agacttagta aactctttaa acagtttgtt accgtcttct gccagaattg atacgctgta 68400 gttacaatct tttcctggga atttcttgtc aatagattta cctttacccg gacgtggtga 68460 agtgttcagg taataaaacc agacatcttt aagcaggtaa cgcagagttt gacgttcagt 68520 accattgtac ttctctaccg gagctttcat tttaacaact ttagacatta tttaaacctc 68580 tatctcaatt tatgaagagt accaattcta gtttgttgat actctattgt caaatactaa 68640 ttactggaag ttgtgcttat ctttagaagc tgctttacgt gcggtttttc cagttcttac 68700 acgtttttct tcatagtatt ttgctggttt tccagcagtg gcttttaagg aatccccaaa 68760 aactttttca aatgctttag agtgtttcat gatattctcc gatatacttg atattcaaac 68820 acattttctt tcattataaa caaccaacca atattatact gaggcagtgt aatgatgact 68880 gtaatcatac tttgatggaa ctgttgttct atttcaactt ctccatcgca tttcatgtgg 68940 agcattctat agaacctatc aatctttgtc aaacactctt catgaaattt atttaaactt 69000 ttttcttcaa agaagggtgt aaacccctct cttttataac tatcactcag tttcgaatgg 69060 gcagtaatct gcatcctcac ttaatacagg tggaatcgtt gagctttgac gctgctcttc 69120 agtataaact tcaccagtct cacggtcaaa cacttcatcc tcataatgag gtaaagagtc 69180 atcgtaatgg tcttcagctt caccaatacc aaactgttgt cgaatatttt ctgctgcacc 69240 atcaatatca acaccacatc cagaagcttt gataagacga cctgtatctg gattgtacca 69300 agtatgacca gcaatacccg ttgacttacc gtgacgacga catttagtta acttgatttt 69360 tgtcaagttt ttcttaacag ggtctgggtc aaccttgtta cgcattaaca gaatgttgtt 69420 catagaaatc tggaaatacg caccagaacc cttgatatcc tcttcagaga tatccccacc 69480 ttcagagtta gctttctgac cacctgcgct cttacgaacg tgacagacgt tcacttgtgc 69540 atactggtag cgtttgcaac gacgcaatag ctcagacaaa acttcctctt catccgtatc 69600 ggaacgtgac agagccaatg taatagggtc aagaataata atcttacagt ctaaactatt 69660 aacaagatag tcaacaaact ccagcaagtt atcttggtca attgcccctt gatggtctac 69720 gatgtggata cgacgacctt tagatagttc tgcgtgtgct tccttcaatt catcccaatc 69780 acgttcgtca taaggaatct cagaaatctg cttgctcagg tggattgcac aaagcatctc 69840 catcaactct tcataggtat cctctacagg aattacacca atattgtaat cggtatcttt 69900 ccaagcagta taaatcattt cacgagtgta tgcagattta cccactgaag atggtgctgc 69960 aagagttgta atctcaccta aaccgtaacc accataagta agcctgttca aatctccgaa 70020 agattctggg aaaggaatca atggaatctg accacgattc ttcattgcct caaaaccatc 70080 tgcgaagttc ttgataccag cagggcagta acgaggtgcg ttgtagatac gctgtttaaa 70140 tccttccaga actgtgtctt tctctttata gaactttgtc caccattcgt taaggtcttt 70200 tacaccttct ggatactgga acaaacgaac cttctcaata ggtaggatac cagcagcctc 70260 tttggtagct ttagcacctg cttcatcgtt atcaaagcac aagtaaatct catcaaatga 70320 tgtgatgtac tgatagttgt ctttgataga cttaatgttt gcacctgatg gtacagatac 70380 gtgacagtaa ttcttacgac gagacttatc tttaatcgca agagaagtca tatagattgc 70440 tgtcgcacat tccatctcac cttcccagat gaatagacgg ttaccacctt ctggagcaat 70500 ccatgaaccg aacattgcca gttcaccttt aatgtctcca acaccaccag aaaagtcttt 70560 tagcttacca cgtaggtgtt cttttggatg gtcttctggg taacggtgac gaacacgata 70620 accaacatgt tctagcttgc catcttcatt acgtttgtaa gttggataga aatgtgcatc 70680 aatttcaccg tcactatcaa tgtcaacctt gatacctaaa cgttcaagga cttttgcagg 70740 aatcttccta tctttcaagt ccattgcttc taggttttct tttacatcgt ctaaatccat 70800 tccacggaaa gtacggtttt tgttatctga accagtagaa taagtgctca cgatttgtcc 70860 tttatcaaaa tcccactctg ggaaaccttt gttacagcta aagcaagtca tcgaataaga 70920 atcatcgtca tgatgataga ttgaaccagc atctgatgaa ccacaacgtg gacatgcaca 70980 atgaccaata aactgaccag cctctttcaa tttacgacct ttagacatta gcacctcttc 71040 gttgtagttc tgccttcaat ccattttcaa tcttgtccag ttcatgaatt tcatctgcaa 71100 tctctttcct tcgtgattca actctcttaa gacgttcaat cattacctca ttggaaagag 71160 atgagagttc cactaaacgg tggtcaataa ctttaaaatt gtcttttact ctcatcttca 71220 ttctctcttt taaatcttca ggatagctct tagcttactt tcaaggtctg caagagtacc 71280 attattatgg ataatgtcac gcgcatattt ggtagaaatc ccattttctg aaacatgtga 71340 tgaaaccttg tccacattgt ctctttttac ttcaatagtc tggtgtgcaa atctactcag 71400 ccactcagct tcagagtcaa accttaagtc actgattaaa acaacaccct cttgattcat 71460 aagtgaacaa gattcaaaga agcgagccat tcgtttttca aggtctttag cccagaattt 71520 gtcacccata actttacgga tgacttcagt accccaaatc tgttgaattt gccttgatga 71580 gaatttatac tttttactaa accctagacg tgtcaataga gtcggtttag cgaccttctt 71640 aagctccatg actaatcgtc cggttaactc tgacataagc ttaaagtcca tatgataacg 71700 ctcatctctg aaggtgaact cgatagcctc cgtaactttg aacatcagtt cggggtatga 71760 taaatcaaaa acctgtggag tctctttagt ttcaccatac aggtcattcc aagtcaggtc 71820 aaatatcttt gatgcagata gcttaaggtt gtctgcatag gccattactg caacattgta 71880 gccgtactca tcctctaaga tgttcttcac aatagaacac gaggtgtctt ttccagaacg 71940 tgcctttcca gtaaatgcga taatattact cattttatct cccttaatta ctaaaaagcc 72000 cccaactaag gaggcttgta agatttataa aggcttagtg cagttgctca gcagactctt 72060 tttcagccat agctgagatt tgcttcacaa aatcatcacc gaagttaatg cgtagtttct 72120 cttcaatgat tgaagcacca atgttcacca aaacgtcatt gattgcttgc atactaacgc 72180 caccagtcat ctcctcaatg aactcaatac ttgcacccat catctgtgaa agttggatta 72240 gtgcaacgat attcatcatc gtagagatag ctggaatcat cagtgcaact ttcacctgaa 72300 gtggttctat atcagctaca gagtcatttt caacagcttc ttccaaagaa tctttagttt 72360 gctggataat ctctttgatt cgtgggctta attcttctac gccccaatca agcttttcat 72420 atcgtgacag cttttcttcc atacgcttct caagcgcagc cataacagca cttgttgatg 72480 agataagcat gtcaataagg aaatcagtat ccttagtcag taacagttcc ttatcttcgt 72540 ctgacatcct gatatagtca ttcttttgca tttcataaaa aatgaactca gcaagtgcat 72600 caacaccaac cgcaatagat gccagagcaa gtgtagtgtc tgtcaacatc atattaacct 72660 gttctgcaaa agcaccgtta tcagacttgt cttcaggcaa gttatagctt gcaggcataa 72720 actgttctgt gtagttatta ccacgaagaa tcatagccat gctttctacg gcgttgatta 72780 agaatgcttc atcaacacct ttctcttcaa gcatcttttc tacatcgata ttcatattat 72840 ttctctctca gttggttaag tttctttcta gaacatttta agttttcgga tgaagaaaag 72900 tcaagtgtct tttcagaacg tggataatag tgcctatccc aagaagtttc aacatcgtta 72960 atcagtgatg ctagattaca caggtctgta ccactttgca acctcttttt aagagaatct 73020 agtaaagcaa ctgtgtcttt agctttacgt ctcgctgtag caacatcttt catatgctca 73080 aatacagagc attttaagtc atcttcatag tcttcagaca actctatttc atgttgtata 73140 tcaaccattc tcctgtgatg gtgagcatat tcccgttgag cagcaatgtc aagttcttcc 73200 aacttctgca aagctctaac aaagtcagct atgtctgggt gaacaaacat tttctgcatg 73260 taagcctctt ttaagtagct attaagtttt aatcttttat cttaacgtat tctatacgtt 73320 atactttaaa gctttttaaa agctattaaa taatctttta agagttttaa agtatctgta 73380 tagttaaact gttaagtaac cttttaagta aaaacccact tcgcaaagat taacaccttg 73440 tcaagtgcat tgtcaacttg ctttttataa ttttttgcag taatgtatga ggttaatgat 73500 tttagaggga gtaaaaatgg aaaacgtaga ttttaaaaac ttacatttag ttggtgatac 73560 agagactgat ggtttactcc ttgagttcac taaagttcac gtaatggctt ttgcagacta 73620 taaatctgac gatgaagagc cacctgtatg ggtctttaca gatgagccta tcctcggtca 73680 caagtacact aagtacatca agggtggttt acgtgaaggt gttgagtttg cgttaaaggc 73740 aaaacgactt tgtatccata acggcctcgg ttatgactgg tgggtcttca atcacattgc 73800 accggattta tggaactttg ataatccaaa atgtaagccg tggagtaatt tctttcagga 73860 ttctcttatc cagtctcgtg ttcagtggat ggatagacca actccaaagg gttataaagg 73920 tgctcatggt ttggctgcat ggggtgctcg tgttggtgtc cgtaaaccag agattgaaca 73980 ttggggtgtg tggaatgcag aaatcttcac tcgtgttgta gaagatatcc gtattaacgc 74040 caaaactaaa cgggcacttg ataacgagta tctcaagctt aagaagtgtg gtatagacac 74100 ttatgagact tacatgcgag ctaaagagac atctttctgg atgagtcaac aagctattaa 74160 tggctggaaa gctgataaag agcttatgga gttccatgtg aaggaacttg acaaattgac 74220 taatgagctt gcttcagagg ttgaaccgca tctacctcca actattaaga ccaaaggtaa 74280 agtcactgga gaagagtttg caaaagcttg gaatgagtat gttgaaacat ttggtcatgc 74340 agatggactg aagagaatta ccaagtaccc taagacaaag tatcgtcagc aggtacgtaa 74400 cggtgagatg cagacatatg aaatcaagcc atttggtaag ccaactacaa agatttttaa 74460 cattgaaaag cggaattgct atacaccaac caacactgta actggcgaag agtacaagga 74520 aggttttgta gcaatgaagg atgctcgtgc aatttgcaac gagttgaatg caaagattgg 74580 taagaaatgc aaagactgga agccagtaaa aactgttaaa actgtgaagt actataacag 74640 ccatgttgtt aaccactttg aactggagtc aagtcgctac acaggattga ttgatgcacc 74700 atatacacca attgagtttg aagtttctcg tatgactcag gtagcagttg ttaaagacta 74760 cttgaaatca gttggttgga ttccagatga ctggaactac aagaaagact cagacggtcg 74820 ccctgtcaaa gtttgtcgtt tcaaagacaa taaaaagatg attacaaaac accctaagtg 74880 gcaggaaatg gttgagcggt gtggtttgag ctatgttgag catgaaggtg tacagtacat 74940 tgagcataac tggtctgtga agaagtacac agatttgctt gaaccttgct taatccgtac 75000 ttcaccaaaa cttacggaat catcttatga tacgattgaa ggtgagcttg gacagaagat 75060 tgctaaatac tacactttga tgcacagacg cagaactatt gagaactcaa aggatgatga 75120 aaaaggttgg ttgaaccaga ttcgtcctga tggtcgcctt agtgctggtg caatggtgtt 75180 tggtacttca actggacgta tgacacaata tggtattgta aacgtaccgt ctggtgctgc 75240 tgtctatgga gcacctatga gggaggtgtg gatttgtgaa gaggggacta acattgtttc 75300 tgttgaccag aactcagctc agctagttct tctttgcaac tttatgggtg ataaagactt 75360 caccaaagcg gtaactgaag gtaaagagtc agtagagttc actaagcaag atgatggtag 75420 atactactgt aaacatcttg acaagtatct taacccagaa actgacaagt atctgcgtta 75480 tgatgctgag aatgacctct atgaagtcta cacagggact gatgcgcata cactgaacag 75540 tatttacttt ggtctgaaca aggaagaaga tattgttcgt tgtagggaga cacaggatga 75600 ggaacttctc cacgagatta caaaaggtcg taagaaagcc aaaaatggca tttatgcgtc 75660 agtggcgcat gtaaaacgtt gtgaattgct ggaagcctaa gtctgtaaag atatggtaat 75720 cagcagcgaa gcctcttgag aggaacgttc agagactatc gaaaagcata cttaacgtat 75780 gaactgagta gagtagggtg gtaagctaat gaccatcccc aagtgcaacg gtagcaccac 75840 tgctacgtga tatagtccga cactccgtag aaatgcggag aagttcataa gagaactggt 75900 acaccttgcg agtgtattga ccagatgtat tgttcggtgc aggtgatgag aagtttgcca 75960 aaaccattaa ggcagcttct acagaggaag gtgcattgac taaacaaact tactatatgc 76020 gtctgccaaa gattaagaag ttacttgata gcttagaagc cgattttaag gcgtctaaaa 76080 aggctctgga agaggttttt gggaagaatt cttcaattgc taaaggtggt tatatcaggg 76140 tcgctggagc atggatatgg tgcaaatctc cacataaact actgaactac ctcctgatga 76200 gttcggaagc acagattcag aatgaggcca tcaacctagc agcaagaaaa gcctgtgaag 76260 atggcctgac aaaactgaat gctcgaaaac cagctatcgg agcaaggtta cttctggcat 76320 atcacgatga aaatagttgg gaatgtccag agggtatgac acatgatatg aaagcaattg 76380 ctgactggat gtatggacaa gcctcaaaaa acttaggttt gaggaaagaa acacttgtaa 76440 ctggtacagc aaaggttggt aagagctggt atgaggtaca ctgattaatg gatttaattt 76500 tacaaaaacg tttaaaggag ctgctgcact atgaccccga aacaggggtc tttacttggc 76560 ttcgtagaga aggtaaatca agagctgtta gtgtttttaa cagtaattat gcaggaaagg 76620 tggcaggtaa tatccagacc gattcaagtg gtcataaaca gattagtatc tattttgaca 76680 agaaagcaca taaagcacac agactcgcat ggctttacgt gtatggcaag atgccaaaag 76740 gtatcattga ccatatcaac ggagactcac ttgacaacag gattgtaaac ttaagggaag 76800 cagatgactt tcaaagtgct tggaataaag gtaaaccagt caccaataaa tctgggtaca 76860 agggcgtaag tcttaagaag aagtctggta agtgggtcgc acagataagt tataaaggta 76920 agaagatgtt tttaggttac catgacaccc ctgaagaagc ccataaagct tactgtgaag 76980 ctgctatcaa actccacgga gagtttgcaa aactaagttg acaaggcgct catggaagag 77040 taccttatac tatatcttgt ataggagggc ttatgagcca ccgtggaaga acccatgcag 77100 caatgatgaa aggtgcttca aaggaaacta tcaaaaatcg taagcagaag ttgtttgaga 77160 gaatgaacag gttaattgac aattcatctc tttcaggttc tgagaaggtt ttcttgaaag 77220 gtaatctgaa aagcattgca caagaacttg tagatattga atattggaga catcaaaaat 77280 gacacttgca gacgttattc agcaacttca tgataactgc tacactccag agttaattca 77340 ggagatgtcc attgtagtca tgcctagcaa gttcgtaaaa acctttaaca acgacgcttt 77400 aaggtctaca cacatcctta tcgttgacgg taagattgca aaagaccgta ctggagtact 77460 taaaggtgaa cgtattggta ttctggagtt gctatgaaga aaatgtacag tctctgggga 77520 agggttggta aaggttttga ctggacgctt cttcgttcaa acgttaaacg tagtgaatta 77580 ccagaactca tcactcacta tttaaaaaca tacagagagg tagactatcg tgaacaataa 77640 ggttaagagt tgtgtgaaag caatggttgc acttggagta atttttctat ctggctgcaa 77700 cccctcttat gaagataaaa atgcttctta cagcctccca ccagagatgc aagattgcat 77760 agtctatgag ttatatggtg atactgtaag cagagatatt gttgttgtca gatgtccaaa 77820 ctctcaaaca acaacatctt atagctatgg caaaaatggt caatcacata ctacggttat 77880 tgagtgaggt tttcacgatg gaagtcctag taaactatat ctactgttat gatgttgttc 77940 actccactac aacggtagcc caacgtaacc caatcgtccc acgggaaggt gagttggtta 78000 gaatcgaggg ttggacttac actgtggaaa gtatcattca taagtttgat gttgctggtg 78060 atgttcaagt tatcgacgta gagattggtg gtaaaagaaa atgactgtag gagataaatt 78120 gaagaatgca gttcttacag aagagggtga gcttgagacg tttattcttc gtgttgatgg 78180 taaactattc cggtgtcgtt gtgggtcaaa ctgttttcac aaacctgata acacaaagtt 78240 agacatatac gagtgtaata actgtggggt aaggttctta ggtaagatta aggatatgca 78300 atgaagataa ctcaagaaat gaaggaattg tatgagaaaa gtttggtaat ccctccaaag 78360 actaatacta agaaattact atttgactct tggttagata gctccacata tgggaagtac 78420 tacctaaagg ttatttcatt tggtgaaaga ccagaatcac cagaatttta tgagagctac 78480 ataaatgagc aagataaaaa atgtttatgt ttctacgaaa aacacatgga attagtaaat 78540 tcaattaatc actaaagaga gcttagatgg ttgtgcttgg tactacttct actcaaaagg 78600 ttgcatagct tgtgaggcaa gagataaaat gaacaaagaa gataaacata aaaatgctgt 78660 acgaactcct gatggaaagg ttcaatcctt catccttaca gttggtggaa agccatttcg 78720 ttgccactgc ggagcaaact gttttcataa accagataaa aatgacttag agctttacgc 78780 atgtaatgct tgcaatactt ggtatcactc agaggaataa aatgacaata ctatacaaac 78840 agaataaaga tggttctttt aacgtctggt catgcgttgc tgtaggtgac aaagtcatta 78900 caacctacgg taaagaaaat ggcaagatga tgtttgaaga gtatacagca gaacctaaaa 78960 acatcggtaa aaagaatgag cgtaatgctg aacagcaagc tctctttgaa gttgctgcta 79020 agtacaaaaa gcaagttgac cgcaaaggct atgcttacac aaaagagtct gcacagaata 79080 ctgagaaggt aggtgtacag cttgctcatg atgctgcaaa ggttagtcat gcaaagtatc 79140 tgaagttccc tgctgacgct caaccaaaac ttgatggtgt ccgttgtaga atttcaagac 79200 acttaagcat tggcagtgta tttacagcat actctcgtga gaatactgtt tataaaatac 79260 ctactgagct aatacctgac attatggcaa tgttaaggct ccacaaagat gttgatgaat 79320 ttgatggtga gatttatgct catgggtggg acttagaaga tattgtctct atgattaaaa 79380 atgaagacaa tcctgaccgt catctactta agttttactg gtatgatatc tgtgacagta 79440 ctaagacgtg gcctgagcgt agagatatta ttgagacttc accattgaat gactttagag 79500 atggctgtaa agttgtccct gttaagtctc gtcgtgtaaa ttcttgggaa gagtttgatg 79560 aagctcatga taagtgggtt gaagctaagt ttgaaggtgc aatgtaccgt tcaatctctg 79620 aagactcctt ctatgagtgt tgtcaccgtt catacttctt gattaagcac aagaagatgc 79680 acactgaaga gtttaaagtg actggtgtaa agactgataa gcgtggtcat ggcaagttcg 79740 ttgtagagac tcttcctaac gtctttgtag atgtctcatg gaagactact catgagaaga 79800 aacagtatct tgctgagcat cctgaagagt ttatcgggaa acctttgacg gtacagtttc 79860 agaagatgac tcgtaagggt tctttacagt ttcctgttgg gctagttatt agggattatg 79920 agtaagctat gttaagcaca atcattctgt ttgcattagt tgtctttgta ttgtcttaca 79980 taacaagtaa acttttgaac acatcctttg caatgggtgt cttgttcaca atcttctgtt 80040 ttgtaacaca tatagctatc aggactttta tataaaagtt gttgacatag aaaatttggt 80100 gagtatactg agcagcataa accaacaggt actcaccaga atcacttaag agtcttttaa 80160 gagggttttt aagtaatgta ttgcacggca aggcgatgtg aagtctggtt ttgcatggtg 80220 tggtctggta tggaaggatg gcagtagcct tataaaaaat ctgccagata agctataaaa 80280 tctgttgaca agtcaggtgt tatagcttat ttttatactc aatgtaatac aaaatttaat 80340 ataactttga ggtcattatg aaaaagattt tattagctgc tgcaatggtt atggcaatga 80400 atttaccagt caatgcaaca gaactcccaa atgtggactt atcaggtgtt ccagaagaca 80460 cttgtcagat tgttaaaggt gttgctctag ctaatggcga gctacttaaa ccaatctctg 80520 aagagtcttt aacagagatg actgataagg tgactgacta tcaatatcgc gttcttgcag 80580 agtatttcct gcaatctgca aatattaaag agaagcacca tgatgatatt gatgtacagg 80640 ctatgctcaa tcatcgcatc cagtttaaag aagatttgat gcaaaaagct atgtatggtg 80700 ttgagtattt cttagaaaac agaagctgca taggtatttg atatggctct taaaaagtta 80760 catcccagaa gcggttatgg taagataatt gacgatacag acggctttac agtctttaca 80820 gttatctgtc aagatgattc acagattgaa aaggctcttg atgattatct taacgatgaa 80880 cgtgaaaagg ttagagctac aaacatagat tcattaattg acacttcacg caaacggaag 80940 aagaaagatg aatgaagttt ttgaccctta tgctccacaa gatgattggg aggctgacag 81000 agaagctgaa atggagagtt atgtttgtcc aatggatgta gacgaaatga gagacttcgt 81060 tgcacaccgt tttaagaaag agattaaatc cagaggtctt tctcaagagc aagttgctaa 81120 aatttgtggt atctctcagg ctcgcgtatc caacataatc cacctcactg gtaatgtctc 81180 ccttgagtat atgttggaag tatgtgaaaa atttggtgtt aattttaatt taaggttggc 81240 agattaatat gaaacgtgaa aacattatcc actctgaaaa cttcgcatta ggcttttatg 81300 gcgtacccac tcaccttgaa aagtattatg gtgtgaagat tctttccaat ctcattatgg 81360 cttacaaaga tggtaagatt aagcacactg agaagaaacg cgtcatgggt tatatggctg 81420 taggttcagc aatctcgaac atcaagctgg aaacaaccag tagccagatt gtgaaagacc 81480 acttcatcca agagctttac cagaatcttg atggtgtaga tgttcaggct gtttggctgg 81540 atgttgacgg tcataactac acaagttttg ttttcaaaaa cgatgacatc aagtgtctgt 81600 tcccataata ggtgattaac tatgattgat atctacttac aagatgctca tgcagatttc 81660 cttaaagtga tgcttaaaaa gtttatggct tcgcagcatg agaatgaagc atcttttaaa 81720 atagtcacat gtggcgacga agctggtttt gttgagattg aacatgaagg tactggaaag 81780 actgtttgta agttacctga tagcatgttc tctagcacat tcttaacaaa gactagtata 81840 aatgttaagc ttgtgcctca gattgaaaca tactctggta cagattaccc taaaggcttc 81900 aagtcactga tgaaacactt cttagatgac tttgtgagta atcttctaag tgaggtaaca 81960 gaaagccgca caatattaac tgtagagaat atagggaaca ctatcagggt tacttctgac 82020 cgatattcta tgagcctctt cgactttgta cctaagaact ttgacggtat tctggatgaa 82080 gaagatgatt gtgtagactt tatattggtt cttgagccag tttttggggt taaatagatg 82140 aaaattgaac actgctatga gtctgatgga acacctatcc gatgtccaca ttgtgggtgt 82200 acagatttac aaggtgaagt tcatgtgatt gttaatggtc atatagctga agagagtacg 82260 acatgtgttg gttgtggtga gcagttatca ttctgggctt atggttcata ccaacccacg 82320 ccgcacttaa tctaccatag caataaagct gtgaagaatg ttatcaactg gttcattaag 82380 aaaggattta caaaatgatt aaattaatct ttgcaagtgg tgaaaatggg gagtttggta 82440 ctccaactgg tatgccgtgg cctcgacata aacaggacat gcaagagttt aagagactca 82500 ctaaaaataa cttagtagtc atgggtaatg agacttttaa gactctgggg agtaagcctt 82560 taccagaacg tgcaaacctc atcttaacaa attctgtacc atacttaggc atagactttg 82620 gcaaagatga cgtaatgtat gctaaagcca gtaaagagtc atttggagca tttttgaagt 82680 atcttgatag ctctattgat gaagatgtct ttgtaattgg tggtgcaggt gttcttgtca 82740 atgctttgcc gtatgccggt gtagttttcc atacagtttt ccataaagtt actgaagagg 82800 ccactgtgca tttgcctttt gaaaacttct ttgagaagct gtatgataac cgtgtattta 82860 caaagattca gtcaaaacca tcagaggatg gtaaagcaac ttttgaaatt tatgttccac 82920 aagtaaaagg acacttttga tatgtcacaa gcagattcaa gttacaaaaa tatcctgaac 82980 catgttttat cagttggtga actgcgtact acacgaactg gagatgttat ctctgcattt 83040 gctccacctc agtttcgttt tgatatgcga actggtttcc cgctcttaac atctaaacag 83100 gtgtttacac ggcaagttat tggggaagct ttatggttcc tgaatggtga gaataaactt 83160 ggtgaactcc gttaccgcac ttgggatgaa aatgatggag aacgctggac tatctggtca 83220 gatgatttta aacgctggtt aagctctaat tattcttctg aacaagactg gttagatgac 83280 gcaggtggga gaatctacgg ggttcagtgg agaaactttg aaggtcataa tggttgtgtt 83340 gtagaccagt tagagacctt agtaacgaag atgaagggtg atattacaga ccgctacatg 83400 cttgttaatg cttggaatgc agcagatatt gcatccaact cgatggcttt agcaccttgt 83460 catgttctgt ttcagattta tatcactaac gaaggtgaag ttgacttaca atggtatcag 83520 cgttctgtag acaccttttt aggacttcca tttaacattg catcctatgg ttttattctg 83580 gaagttcttt gcaagatgac tggatacact ccacgatact tgataggtgt ctttggagat 83640 actcaaattt atcagaatca tatgaagcag gtttatgaac tgatgaacaa tgaagagttc 83700 catgcaccta cttttgagat tggtgttcag cttaacactc taagtgactt aaaacatctt 83760 actgcaagtg actttattgg cggcattaac aactacaaac atgcaggaaa gattgaagca 83820 cctttatcag taggtaagta aaaacaaaaa aggctccttt ttacgggagc cttaaaattt 83880 tattttcagt gttcttttgt gttcttttca gtaatagctt ggagggctga tactgattga 83940 gccagattgt tcacactgtc agagaattta tcaagagttt tggtaagttt tgcgttttca 84000 cccttaacat tctctaactg gactttctgg ttctccatcc ctagctgaat cagtctcatg 84060 tcagactgta aatcacgaat agctgaatag ttacttttgg aataattatc tagctgctgt 84120 aactttgttg tgacagatac ttcttgctta ccacttgaaa cttgcatagt ggtatacatc 84180 ccaataacac taaaaatacc aactacaatt gcaccaatat tgtttttaaa agcttcctct 84240 agccacttca tttatttctc ccccttaaaa gctttttcta agttatctac gaactcatca 84300 tcaataggtg tgtctgtttt actcgcaaga tatcttgcaa gcctaaagaa cactttctca 84360 atcatgtatt cactcagaag ggataaaatg agtttccaga agaaactacc tagatttttt 84420 agaagaattg ctaggattgt aggcatttaa tcacctcatc agccaagatg gtgagaatac 84480 ccataaagaa tattaacacc atcttaacaa tcagtcaata agggattaag cagttcttac 84540 ccaagccatc agcttataga actggttagt tacactaaat gctgaaccag aacctgtacc 84600 accagtgtta ccactaactg tatgactgtg agcaccaata ccaactgtgt gagcatggtt 84660 tccgttagag tcagcagtac cagacacagt gtgtgagtgg tcaccagcta cactggaaac 84720 ctgttctgta ccatatacct gaacacgtgg tttaccaccg atagagtcac ctacataatg 84780 accaccaaca gtatgtgtgt gattacctgt ggtatttgtt gaaccgctca ctgagtgggt 84840 gtgagcacca gtagtgttag tggttttagt accgtagtca aagctagagg ttgtcgcaga 84900 gaaactatga gtgtgtgaag gtaagtttcc aactgctaac gtaacagaat ctgaacctcc 84960 agttgtagca acatctgaac cattagctgc tgcaaccctg atagttctac ctacaccatt 85020 gttcaggtac gtccaagtta atccaggaag tgcggtatta gggttaacgt tactgttaaa 85080 ccacgttaca ataccaacag ggtagatttt attaaggtct gttgaatcac ttactgcctg 85140 tgcaatcttc tggtcagttt cagctttagt gtatgcaccg atatctgaag gggttggttt 85200 ttgcatactt gtgtagtatc ttgcaattgt accctttaca ataccttgtg gagcatcatc 85260 aaatgtttgt ggggcttcaa acaattgcac acttgcattg ctagaagatt gaactcttga 85320 tgttgtgtca ttttggaatg gaccaactct acagtaaatg tcataagtat caccagatgt 85380 attaacataa ccgatatccg taataatgag attctccgag tttttccaag caacaatatt 85440 taaacctttt gggctattac cccctgctct gagaacaatt tccgttttac tagcttggta 85500 agggaaacca tagttataac ctgacccacc aaacacttca atcaccgcag tagatgcggt 85560 aggcggcatt acaacagtag caattttagc ccacttgtca tcgtttccat tattgaagtt 85620 agtacttctg attctcaggt atctactatc actagcactt tgagtaaagt atctagcatc 85680 taagttagag aaatctgaag gtttaacttg accagtgata tttaaattct tattagaatt 85740 aatacctgct gaatccaatg tcaatgtcgt gccagaggct tcatttgata atccaagaac 85800 atttgaagta ttaaatccaa cataccattt attagttcca gattggtttt ggcctctaac 85860 atagagagag gggctatcag ttgcacttct taacaatagt gcttgaccat ctgctacaat 85920 tgtctgccca gatttaaaag tattttgtgc attatttctt gcaatcgtac ttaatgttgc 85980 tgccggaata tatctagagt caatgttagc ccaatctgaa ggttgaactt gaccaatgat 86040 ttttacaccc ttattaaatg ttacaccaga tgagtctaat ttaatagcag taccaccctt 86100 gtaatcatat aatgtgacat tagcgtctgt atcaccttta ccaatatacc acaaattatt 86160 gtttccactg tctcttgcta ggatgaacaa attatcatta gcagttttat ttttcagaat 86220 aatagcagca gcatcagagt taatgttaac agggttgctg aatgtgttta aaccagtgaa 86280 attattattg ccagctaact gtgcaaatct ctgattagct actgtctgag tgaagtatct 86340 agcatctaag ttagagaaat ctgaaggttg gacttgacca gtgattgcta aagacttatt 86400 aacagagaca ttacttgcaa cagtcaaggc tgagttgtat ttgttattgt acagctttac 86460 ttcgtcagag ccattactac caataccaac ccaccatcta gccgaaccat ctaggtttac 86520 accttcgata tattgtgcat tatttgcatt ttggtttcta aggcgaagta agttagcatc 86580 accttgaata ttttgagttc cagagaatgt gtttgtgata gcaagtcttg cgtatcttgc 86640 atcattctct tcattagttc tcataccaag ctcattagga cttggcttat tcagagtatg 86700 ataaacagtt gctgaccttg aagcatctgc aatagttagt ttaagaccat ttgcgttaag 86760 tttaaaagtc tttaaaacat caccaatagt aatttcagaa gagcctgtag ggtcaaatac 86820 agtcttgcca ccgtaaagta acttgacaat acctaaatcc cccaccatag tgctcccagc 86880 aatctgaaca aacctttcaa gcttaaaaga tgctaagaaa tcttgatagg tcattcgacg 86940 gtcttcatca ccaagcattt caggtctctt tttaaccctg acatgaagaa ggtcatctga 87000 acggattgta tcgattgagt ttaattcact caacttgtaa tctgccatta tttttgtcct 87060 ctagaaagag ggctgtacag cccccctgta aaagaatttt taagcggttc tttgccatac 87120 ataaaccaca aaggatggtt gttcaacgct gaatgcttga ccagcacctg tagtacctgt 87180 agtacctgag tgagtatgct cagatgactg caaagttact gtgcctttat gggtatgagc 87240 accaattgca acagtatgcg tgtgagcacc aat 87273

Claims (8)

  1. 살모넬라(Salmonella) 속 균주에 특이적인 사멸능을 갖는, 서열번호 1로 표시되는 염기서열로 이루어진, 박테리오파지.
  2. 살모넬라(Salmonella) 속 균주에 특이적인 사멸능을 갖는, 수탁번호가 KCTC 14746BP인, 박테리오파지.
  3. 제1항 또는 제2항에 있어서, 상기 살모넬라(Salmonella) 속 균주는 살모넬라 엔테리티디스(Salmonellaenteritidis), 살모넬라 아고나(Salmonella Agona), 살모넬라 바레일리(Salmonella Bareily), 살모넬라 인판티스(Salmonella Infantis), 살모넬라 센프텐베르그(Salmonella Senftenberg), 살모넬라 몬테비데오(Salmonella Montevideo), 및 살모넬라 티피뮤리움(Salmonella Typhimurium)으로 이루어지는 그룹으로부터 선택되는 적어도 하나인, 박테리오파지.
  4. 제1항 또는 제2항의 박테리오파지를 포함하는, 살모넬라균에 의해 유발되는 감염성 질병의 예방 또는 치료용 조성물.
  5. 제4항에 있어서, 상기 살모넬라균에 의해 유발되는 감염성 질병은 살모넬라증 또는 살모넬라 식중독인, 살모넬라균에 의해 유발되는 감염성 질병의 예방 또는 치료용 조성물.
  6. 제1항 또는 제2항의 박테리오파지를 유효성분으로 포함하는 항생제.
  7. 제1항 또는 제2항의 박테리오파지를 유효성분으로 포함하는 가축용 사료 또는 음용수.
  8. 제1항 또는 제2항의 박테리오파지를 유효성분으로 포함하는 소독제 또는 세척제.

KR1020220025218A 2022-02-25 2022-02-25 살모넬라　사멸능을 갖는 박테리오파지 및 이의 용도 KR20230127663A (ko)

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