KR101896512B1 - Novel Citrobacter freundii specific bacteriophage CF1 and antibacterial composition comprising the same - Google Patents
Novel Citrobacter freundii specific bacteriophage CF1 and antibacterial composition comprising the same Download PDFInfo
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Abstract
본 발명은 사이트로박터 프렌디에 특이적인 사멸능을 갖는 신규 박테리오파지 CF1, 상기 박테리오파지를 포함하는 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료용 약학적 조성물, 항생용 조성물, 사료 첨가용 조성물, 사료, 소독제 또는 세척제 및 상기 박테리오파지를 개체에 투여하는 단계를 포함하는 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료 방법에 관한 것이다. 본 발명의 박테리오파지 CF1을 이용한 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료 방법은 기존의 항생제 등의 화학물질에 비해 사이트로박터 프렌디에 대한 특이성이 매우 높고, 상기 박테리오파지 CF1은 사이트로박터 프렌디에 감염하여 사이트로박터 프렌디 내에서 100배 이상 증식함으로써 용균 활성이 우수하고 물리화학적 자극에 강하다는 장점을 가지고 있다. 또한 본 발명의 박테리오파지 CF1은 사람, 동물, 식물 등 세균 외의 다른 숙주에는 감염되지 않으므로, 항생제의 오남용으로 인한 항생제 내성균의 문제점, 식품 내의 항생제의 잔류 문제, 광범위한 숙주범위의 문제점을 해결할 수 있는 장점이 있다. 따라서, 본 발명의 박테리오파지 CF1은 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료, 항생용 조성물, 사료 첨가용 조성물, 사료, 소독제, 또는 세척제 분야에서 다양하게 사용될 수 있다.The present invention relates to a novel bacteriophage CF1 having bacteriorhodia-specific killing ability as a site, a pharmaceutical composition for preventing or treating infectious diseases caused by bacteriophage in a site containing the bacteriophage, an antibiotic composition, , A feed, a disinfectant or detergent, and a method for preventing or treating infectious diseases caused by bacteriopterin, which comprises the step of administering the bacteriophage to a subject. The method for preventing or treating infectious diseases caused by bacteriophage using the bacteriophage CF1 of the present invention has a very high specificity to the site-bacteriophage as compared with a chemical substance such as existing antibiotics, It has an advantage that it is excellent against lytic activity and strong against physicochemical stimulation by proliferating more than 100 times in bacterifide by infecting with prandy. In addition, since the bacteriophage CF1 of the present invention is not infected with any host other than bacteria such as human, animal, and plant, it can solve problems of antibiotic resistant bacteria caused by misuse of antibiotics, residual antibiotics in food, have. Therefore, the bacteriophage CF1 of the present invention can be used variously in the field of prevention or treatment of infectious diseases caused by bacteremia, antibacterial composition, feed composition, feed, disinfectant, or detergent.
Description
본 발명은 사이트로박터 프렌디(Citrobacter freundii)에 특이적인 사멸능을 갖는 신규 박테리오파지 CF1, 상기 박테리오파지를 포함하는 사이트로박터 프렌디 에 의한 감염성 질병의 예방 또는 치료용 약학적 조성물, 항생용 조성물, 사료 첨가용 조성물, 사료, 소독제 또는 세척제 및 상기 박테리오파지를 개체에 투여하는 단계를 포함하는 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료 방법에 관한 것이다.The present invention relates to a method for the production of Citrobacter The present invention relates to a novel bacteriophage CF1 having a specific killing effect on freundii , a bacteriophage-containing site, a pharmaceutical composition for preventing or treating infectious diseases caused by bacteriorbide, an antibiotic composition, a composition for feed addition, And a method for preventing or treating infectious diseases caused by bacteriopterin, which comprises the step of administering the bacteriophage to an individual.
사이트로박터 프렌디( Citrobacter freundii )는 장내세균과 (Family Enterobacteriaceae)에 속하는 그람 음성의 간균으로, 구연산염을 이용하며 보통 한천배지에서 잘 생육하고 주모성 편모를 이용하여 운동성을 가지는 통성혐기성 또는 호기성 세균이다. 사이트로박터 프렌디는 토양, 물, 식품, 그리고 온혈 동물의 장관계 등 다양한 환경에 분포하고 있다. 사이트로박터 프렌디는 사람이나 온혈동물에 감염되어 주요한 질병을 일으키는 기회감염균으로 알려져 있다. 또한 최근에는 다양한 연구에서 분리, 보고되고 있으며, 항생제 내성을 나타내는 대표적 세균으로 확인되고 있다. 이전에는 비교적 병원성이 높지는 않았으나 최근의 연구 결과에 따르면 특히 요로감염, 중이염, 농양, 설사 등을 일으키며, 쇠약한 환자에게 있어서 이차 감염이나 중증 패혈증의 원인이 되기도 한다. 또한 베타 락탐 분해효소(ß-lactamase)를 생성하여 베타-락탐 계열의 항생제에 내성을 나타내는 경우가 발견되고 있어 공중보건에 있어서 중요한 세균으로 대두되고 있다고 할 수 있다. 이러한 이유로 사람이나 동물의 건강과 관련하여 사이트로박터 프렌디 감염의 치료는 항생제를 대신할 수 있는 약제의 사용이 필요하다고 할 수 있지만, 사이트로박터 프렌디는 다른 병원성 세균에 비해 상대적으로 연구가 많이 되어 있지 않고 있다. 또한 최근 사람 뿐 아니라 개나 고양이 같은 반려동물에서 급성 또는 만성의 방광염이 많이 발생하고 있으며, 반려동물에서 사이트로박터 프렌디가 감염하여 질병을 일으킨다는 보고가 있다. 특히 대장균, 스태필로코커스 아우레우스(Staphylococcus aureus), 엔테로박터 종(Enterobacter spp.), 프로테우스 미라빌리스(Proteus mirabilis) 등과 복합적으로 감염을 일으켜 병원성을 나타내기도 하므로, 이에 대한 근본적인 세균 제어가 필요한 실정이다.The site Friendster bakteo di (Citrobacter freundii) is an enteric bacteria and (in Gram-negative bacilli of belonging to Family Enterobacteriaceae), use of citrate and usually resistant anaerobic or aerobic bacteria having motility using the well and the growth state resistance flagellum in agar medium. As a site, Betterfriendy is distributed in a variety of environments including soil, water, food, and warm-blooded animals. As a site, Better Frenzy is known as an opportunistic bacterium that infects people and warm-blooded animals and causes major diseases. Recently, it has been isolated and reported in various studies and it has been confirmed as a representative bacterium showing antibiotic resistance. Previous pathologies were not relatively pathogenic, but recent studies have shown that they cause urinary tract infections, otitis media, abscesses and diarrhea, and can cause secondary infections or severe sepsis in debilitated patients. In addition, beta-lactamase (β-lactamase) has been found to be resistant to beta-lactam antibiotics, and it has been found to be an important germ in public health. For this reason, regarding the health of humans and animals, the use of antibiotics as a substitute for antibiotics may be necessary for the treatment of bacterifende infection in the site. However, as compared to other pathogenic bacteria, It is not much. Recently, it has been reported that acute or chronic cystitis occurs in many animals such as dogs and cats as well as humans, and that bacteremia is caused by infections in companion animals. Especially, the bacteria are infected with E. coli, Staphylococcus aureus , Enterobacter spp., Proteus mirabilis , and the like, and therefore, essential bacterial control is required It is true.
한편, 박테리오파지(bacteriophage)는 특정 세균을 감염시켜 감염된 세균의 성장을 억제하고 저해하는 세균 특이적 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 박테리아에 감염할 수 있는 박테리오파지의 종류는 일부로 한정된다. 즉, 특정 박테리오파지는 특정 범주의 박테리아에만 감염할 수 있고 이로 인하여 특정 박테리오파지는 특정 박테리아만을 사멸시키며 다른 박테리아에는 영향을 주지 않는다. 따라서 최근 세균성 질환의 대처 방안으로 박테리오파지(bacteriophage)의 활용이 크게 주목을 받고 있다. 자연 친화적 방식의 선호로 인하여 박테리오파지에 대한 관심은 어느 때보다 높다고 할 수 있다. 박테리오파지는 1900년대 초반 처음 발견된 이후 세균을 제어할 수 있는 수단으로 사용되었으나 항생제 개발이 시작된 1940년대부터 수년 전까지는 적용이 미미하였다. 그러나 최근 항생제에 내성을 나타내는 세균들이 많이 발견되고 있으며, 항생제 오남용에 대한 각성과 약제에 대한 규제로 인해 기존 항생제의 한계성이 나타나고, 기존 항생제의 대체 물질로의 개발 가능성이 부각되면서 다시 박테리오파지가 항-박테리아제로 주목을 받고 있다.On the other hand, bacteriophage refers to a bacterium-specific virus that infects a specific bacterium to inhibit and inhibit the growth of the infected bacterium. Bacteriophages have the ability to kill bacteria by infecting bacteria, multiplying inside bacterial cells, and destroying the cell walls of the host bacteria when the progeny bacteriophages come out of the bacteria after proliferation. The bacteriophage infection method of bacteriophage is highly specific, so the type of bacteriophage that can infect a specific bacterium is limited to some. That is, certain bacteriophages can infect only a specific category of bacteria, which causes certain bacteriophages to kill only certain bacteria and not other bacteria. Recently, the use of bacteriophage has attracted much attention as a countermeasure against bacterial diseases. Interest in bacteriophages is higher than ever because of the preference of nature-friendly methods. Bacteriophage was used as a means of controlling bacteria after it was first discovered in the early 1900s, but was not applied until several decades from the 1940s when the development of antibiotics began. However, recently, many antibiotic-resistant bacteria have been found, and the limitations of conventional antibiotics due to antibiotic abuse abuse and regulation of pharmaceuticals have appeared, and the possibility of developing antibiotics as a substitute for existing antibiotics has been highlighted, It is attracting attention as a bactericide.
그러나, 중요한 문제가 되고 있는 다양한 질병을 일으킬 수 있는 사이트로박터 프렌디에 의한 감염증을 예방 또는 치료하기 위한 신규한 박테리오파지에 대한 연구는 여전히 부족한 실정이다. 따라서 사이트로박터 프렌디에 특이적인 용균활성을 갖는 박테리오파지 및 이의 응용에 대한 기술 개발이 필요하다.However, research on novel bacteriophages to prevent or treat infections caused by bacteriorbital as a site that can cause various diseases that are important problems is still lacking. Therefore, it is necessary to develop a technique for bacteriophage having bactericidal activity specific to bacteriorhodi and its application to the site.
이에 본 발명자들은 사이트로박터 프렌디에 특이적인 용균활성을 갖는 박테리오파지를 분리하고 분리된 박테리오파지의 형태적 및 유전적 특성을 분석하여 상기 박테리오파지가 사이트로박터 프렌디를 선택적으로 사멸시킬 수 있음을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors have isolated bacteriophages having lytic activity specific to streptavidin, and analyzed the morphological and genetic characteristics of the isolated bacteriophages to confirm that the bacteriophages can selectively kill bacteriorbide as a site Thereby completing the present invention.
따라서 본 발명의 목적은 사이트로박터 프렌디에 특이적인 사멸능을 갖는, 서열번호 1로 표시되는 염기서열로 이루어진 박테리오파지 CF1을 제공하는 것이다.Accordingly, an object of the present invention is to provide a bacteriophage CF1 comprising the nucleotide sequence shown in SEQ ID NO: 1, which has a killing activity specific for bacteriorbione as a site.
본 발명의 또 다른 목적은 상기 박테리오파지 CF1을 포함하는, 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating infectious diseases caused by saterobacterium, which comprises the bacteriophage CF1.
본 발명의 또 다른 목적은 상기 박테리오파지 CF1을 포함하는, 항생용 조성물, 사료 첨가용 조성물, 사료, 소독제 또는 세척제를 제공하는 것이다.It is still another object of the present invention to provide an antibiotic composition, composition for feed addition, feed, disinfectant or detergent comprising the bacteriophage CF1.
본 발명의 또 다른 목적은 상기 박테리오파지 CF1을 개체에 투여하는 단계를 포함하는, 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료 방법을 제공하는 것이다.It is a further object of the present invention to provide a method for the prevention or treatment of infectious diseases caused by streptococci, comprising the step of administering said bacteriophage CF1 to a subject.
상기 목적을 달성하기 위하여, 본 발명은 사이트로박터 프렌디에 특이적인 사멸능을 갖는, 서열번호 1로 표시되는 염기서열로 이루어진 박테리오파지 CF1을 제공한다.In order to attain the above object, the present invention provides a bacteriophage CF1 comprising the nucleotide sequence shown in SEQ ID NO: 1, which has an apoptosis activity specific for bacteriorhodi into the site.
또한, 본 발명은 상기 박테리오파지 CF1을 포함하는, 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating infectious diseases caused by streptococcal bacteria comprising the bacteriophage CF1.
또한, 본 발명은 상기 박테리오파지 CF1을 포함하는, 항생용 조성물을 제공한다.The present invention also provides an antibiotic composition comprising the bacteriophage CF1.
또한, 본 발명은 상기 박테리오파지 CF1을 포함하는, 사료 첨가용 조성물을 제공한다.The present invention also provides a composition for feed addition comprising the bacteriophage CF1.
또한, 본 발명은 상기 사료 첨가용 조성물을 포함하는, 사료를 제공한다.In addition, the present invention provides a feed comprising the feed composition.
또한, 본 발명은 상기 박테리오파지 CF1을 포함하는, 소독제를 제공한다.The present invention also provides a disinfectant comprising the bacteriophage CF1.
또한, 본 발명은 상기 박테리오파지 CF1을 포함하는, 세척제를 제공한다.The present invention also provides a detergent comprising the bacteriophage CF1.
또한, 본 발명은 상기 박테리오파지 CF1을 개체에 투여하는 단계를 포함하는, 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating infectious diseases caused by streptococci, comprising administering the bacteriophage CF1 to a subject.
본 발명의 박테리오파지 CF1을 이용한 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료 방법은 기존의 항생제 등의 화학물질에 비해 사이트로박터 프렌디에 대한 특이성이 매우 높고, 상기 박테리오파지 CF1은 사이트로박터 프렌디에 감염하여 사이트로박터 프렌디 내에서 증식함으로써 용균 활성이 우수하고 물리화학적 자극에 강하다는 장점을 가지고 있다. 또한 본 발명의 박테리오파지 CF1은 사람, 동물, 식물 등 세균 외의 다른 숙주에는 감염되지 않으므로, 항생제의 오남용으로 인한 항생제 내성균의 문제점, 식품 내의 항생제의 잔류 문제, 광범위한 숙주범위의 문제점을 해결할 수 있는 장점이 있다. 따라서, 본 발명의 박테리오파지 CF1은 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료, 항생용 조성물, 사료 첨가용 조성물, 사료, 소독제, 또는 세척제 분야에서 다양하게 사용될 수 있다.The method for preventing or treating infectious diseases caused by bacteriophage using the bacteriophage CF1 of the present invention has a very high specificity to the site-bacteriophage as compared with a chemical substance such as existing antibiotics, It has an advantage that it is excellent in lytic activity and strong against physicochemical stimulation by proliferation in bacteriorbide as a site infected with fren. In addition, since the bacteriophage CF1 of the present invention is not infected with any host other than bacteria such as human, animal, and plant, it can solve problems of antibiotic resistant bacteria caused by misuse of antibiotics, residual antibiotics in food, have. Therefore, the bacteriophage CF1 of the present invention can be used variously in the field of prevention or treatment of infectious diseases caused by bacteremia, antibacterial composition, feed composition, feed, disinfectant, or detergent.
도 1은 박테리오파지 CF1의 사이트로박터 프렌디에 대한 반점 분석법 수행 결과를 나타낸 도이다.
도 2는 박테리오파지 CF1의 사이트로박터 프렌디에 대한 용균반 형성 활성을 확인한 결과를 나타낸 도이다.
도 3은 박테리오파지 CF1의 전체 유전자를 분석한 결과를 나타낸 도이다.
도 4는 전자 현미경을 통해 박테리오파지 CF1의 형태학적 특성을 확인한 결과를 나타낸 도이다.
도 5는 박테리오파지 CF1의 단백질 패턴을 SDS-PAGE를 통해 확인한 결과를 나타낸 도이다.
도 6은 박테리오파지 CF1의 pH 안정성을 확인한 결과를 나타낸 도이다.
도 7은 박테리오파지 CF1의 열 안정성을 확인한 결과를 나타낸 도이다.Brief Description of the Drawings Fig. 1 is a diagram showing the results of performing spot analysis on bacteriophage as a site of bacteriophage CF1.
Fig. 2 is a diagram showing the result of confirming the activity of the bacterial generators to bacteriorbene as the site of bacteriophage CF1.
FIG. 3 is a diagram showing the result of analysis of whole genes of bacteriophage CF1.
FIG. 4 is a graph showing the morphological characteristics of the bacteriophage CF1 through an electron microscope.
FIG. 5 is a graph showing the result of SDS-PAGE of the protein pattern of bacteriophage CF1.
6 is a graph showing the result of confirming the pH stability of bacteriophage CF1.
7 is a graph showing the result of checking the thermal stability of the bacteriophage CF1.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 사이트로박터 프렌디(Citrobacter freundii)에 특이적인 사멸능을 갖는, 서열번호 1로 표시되는 염기서열로 이루어진 박테리오파지 CF1을 제공한다.The present invention relates to a process for the preparation ofCitrobacter freundii), Which is a bacteriophage CF1 comprising the nucleotide sequence shown in SEQ ID NO: 1.
본 발명에서, 용어 “박테리오파지(bacteriophage)”는 특정 세균에 감염하여 당해 세균의 성장을 억제하고 저해하는 세균 특이적 바이러스로, 단일 혹은 이중 사슬의 DNA 또는 RNA를 유전 물질로 포함하는 바이러스를 의미한다.In the present invention, the term " bacteriophage " means a bacterium-specific virus that infects a specific bacterium and inhibits and inhibits the growth of the bacterium, and includes a single or double-stranded DNA or RNA as a genetic material .
본 발명의 박테리오파지 CF1은 사이트로박터 프렌디에 대해 용균활성이 탁월하다. 상기 박테리오파지 CF1은 200-220 nm의 크기로, 정이십면체의 머리를 가지며 수축성 꼬리가 있는 형태인 마이오비리데 (Myoviridae)에 속함을 확인하였으며, 단백질 패턴 분석 결과 80 kDa, 65 kDa, 35 kDa, 33 kDa 그리고 16 kDa 크기의 주요 단백질을 포함하고, 전체 염기서열 분석 결과 50,339 bp의 크기를 갖고 GC 함량이 43%, 전체 ORFs의 수는 85개임을 확인하였으며 그 전체 염기서열을 서열번호 1로 표시하였다. 또한 상기 박테리오파지 CF1은 열 및 pH에 대한 안정성이 우수한 특성을 가지고 있다.The bacteriophage CF1 of the present invention has an excellent lytic activity against bacteriorbide as a site. The bacteriophage CF1 was found to belong to Myoviridae, which has a size of 200-220 nm and has a regular twenty-sided head and a shrinkable tail. Protein pattern analysis showed that 80 kDa, 65 kDa, 35 kDa, 33 kDa and 16 kDa, and the total nucleotide sequence was 50,339 bp. The GC content was 43% and the total number of ORFs was 85, and the entire nucleotide sequence was represented by SEQ ID NO: 1 Respectively. The bacteriophage CF1 has excellent stability against heat and pH.
본 발명에서 상기 사이트로박터 프렌디는 베타락탐계 항생제에 내성을 갖는 사이트로박터 프렌디를 포함한다. 상기 베타락탐계 항생제(β-lactam antibiotics)는 페니실린, 세팔로스포린, 모노박탐, 카바페넴, 베타-락탐 분해효소 저해제를 총칭하는 단어로, 모두 베타락탐 핵을 분자 구조 내에 포함하고 있다. 베타락탐계 항생제는 예컨대 메티실린(메탐피실린), 디클록사신, 옥사실린, 플루클록사신, 아목시실린, 암피실린, 피페라실린, 티카실린, 아즐로실린, 카르베니실린과 같은 페니실린계, 세팔렉신, 세팔로틴, 세파졸린, 세프테졸, 세파제돈, 세파클러, 세푸록심, 세파맨돌, 세포테탄, 세폭시틴, 세프트리악손, 세프탁심, 세프타지딤, 세파핌, 세프피롬과 같은 세팔로스포린계, 이미페넴, 메로페넴, 에르타페넴, 파로페넴과 같은 카바페넴계, 아즈트레오남과 같은 모노박탐계 항생제를 포함하나 이에 제한되는 것은 아니다. In the present invention, the bacteriorbide as the site includes bacteriorbide as a site resistant to betalactam antibiotics. The beta-lactam antibiotics are collectively referred to as penicillin, cephalosporin, monobactam, carbapenem, and beta-lactamase inhibitors, all of which contain the beta lactam nucleus in its molecular structure. Beta-lactam antibiotics include, for example, penicillins such as methicillin (methamphetamine), dicloxacin, oxacillin, flucloxacin, amoxicillin, ampicillin, piperacillin, ticacillin, azulocillin, carbenicillin, , Cephalosporins such as cephalotin, cephazoline, ceftizole, cephadone, separac, cefuroxime, cephamandol, cytetan, cepoxitin, ceftriaxone, ceftazim, ceftazidime, But are not limited to, carbapenems such as spearin, imipenem, meropenem, erthapenem, and paropenem, and monovavirus antibiotics such as aztreonam.
본 발명의 박테리오파지 CF1은 서열번호 1로 표시되는 염기서열을 전체 유전자의 전체 또는 일부로서 포함할 수 있다. 또한, 본 발명의 박테리오파지 CF1은 서열번호 1로 표시되는 염기서열, 및 상기 염기서열의 기능적 동등물로 이루어질 수 있다. 상기 기능적 동등물이란 염기서열의 변형, 치환의 결과, 상기 서열번호 1로 표시되는 염기서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 1로 표시되는 염기서열과 실질적으로 동질의 생리활성을 나타내는 서열을 의미한다.The bacteriophage CF1 of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1 as whole or a part of the whole gene. In addition, the bacteriophage CF1 of the present invention may comprise the nucleotide sequence shown in SEQ ID NO: 1 and the functional equivalent of the nucleotide sequence. The functional equivalent means at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more of the nucleotide sequence shown in SEQ ID NO: 1 as a result of modification or substitution of the nucleotide sequence. Or more and having substantially the same physiological activity as the nucleotide sequence shown in SEQ ID NO: 1.
상기와 같은 사이트로박터 프렌디 특이적 용균활성, 내산성, 내염기성 및 내열성은 본 발명의 박테리오파지 CF1을 사이트로박터 프렌디에 의한 감염성 질병의 예방 및 치료용 조성물, 및 상기 박테리오파지 CF1를 유효성분으로 포함하는 다양한 제품에 적용함에 있어, 다양한 온도 및 pH 범위의 적용이 가능하게 한다.The bacteriophage-specific lytic activity, acid resistance, basic basicity and heat resistance of the above-mentioned site can be determined by a composition for preventing and treating infectious diseases caused by bacteriophage caused by bacteriophage CF1 of the present invention, , It is possible to apply various temperature and pH ranges.
본 발명의 박테리오파지 CF1은 도축장, 양돈 농장 부근의 하수로부터 시료를 채취하여 분리한 박테리오파지로서, 박테리오파지 CF1으로 명명하고 2016년 8월 11일에 한국미생물보존센터에 기탁번호 KFCC11682P로 기탁하였다.The bacteriophage CF1 of the present invention is a bacteriophage isolated from a sewage slaughterhouse and a pig farm near the farm and named as bacteriophage CF1 and deposited on August 11, 2016 with the deposit number KFCC11682P at the Korean Microorganism Conservation Center.
또한, 본 발명은 상기 박테리오파지 CF1을 포함하는, 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating infectious diseases caused by streptococcal bacteria comprising the bacteriophage CF1.
본 발명의 박테리오파지 CF1은 사이트로박터 프렌디에 특이적인 사멸능을 가지므로, 다양한 종류의 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료용 약학적 조성물에 사용될 수 있다. Since the bacteriophage CF1 of the present invention has a site-specific bacteriophage-specific killing ability, it can be used in a pharmaceutical composition for preventing or treating infectious diseases caused by bacteriorbide as a variety of sites.
본 발명에서, 사이트로박터 프렌디에 의한 감염성 질병은 요로감염증, 급성 방광염, 만성 방광염, 신우신염, 무증상 세균뇨, 신장결석, 수막염, 복막염, 터널 감염, 중이염, 농양 및 설사로 구성된 군으로부터 선택된 1종 이상일 수 있으나, 이에 제한되는 것은 아니다. 사이트로박터 프렌디는 사람 또는 반려동물을 포함한 동물의 요로를 통하여 감염되는 것이 일반적이나 호흡기, 혈액을 통하여 감염될 수도 있다. In the present invention, infectious diseases caused by streptococci selected from the group consisting of urinary tract infection, acute cystitis, chronic cystitis, pyelonephritis, asymptomatic bacteriuria, kidney stones, meningitis, peritonitis, tunnel infection, otitis media, abscess and diarrhea But it is not limited thereto. As a site, bacterifendies are generally transmitted through the urinary tract of an animal, including a human or companion animal, but may be transmitted through the respiratory tract or blood.
본 발명에서, 사이트로박터 프렌디에 의한 감염성 질병은 흔히 사용하는 항생제인 베타락탐계 항생제에 내성을 나타내는 사이트로박터 프렌디에 의해 발생하는 감염성 질병을 제한 없이 포함한다.In the present invention, infectious diseases caused by streptococcal bacteria include, without limitation, infectious diseases caused by bacterafendi as a site showing resistance to the commonly used antibiotic beta-lactam antibiotics.
본 발명에서 상기 감염성 질병은 에세리키아(Escherichia)속, 스태필로코커스(Staphylococcus)속, 엔테로박터 종(Enterobacter spp.) 및 프로테우스(Proteus)속으로 구성된 군으로부터 선택된 1종 이상의 균주와 사이트로박터 프렌디의 복합감염에 의한 것일 수 있다.The infectious diseases in the present invention as Escherichia (Escherichia) genus, Staphylococcus (Staphylococcus) genus, Enterobacter bakteo species (Enterobacter spp.) And Proteus (Proteus) in the at least one strain with a site selected from the group consisting bakteo It may be due to complex infections of frenched.
상기 에세리키아속의 균주는 바람직하게는 Escherichia coli이다. 상기 스태필로코커스속의 균주는 바람직하게는 Staphylococcus epidermidis , Staphylococcus aureus 등이나 이에 제한되지 않는다. 상기 프로테우스속의 균주는 바람직하게는 Proteus mirabilis , Proteus vulgaris 등이나 이에 제한되지 않는다.The strain of Escherichia is preferably Escherichia lt; / RTI > The genus Staphylococcus strain is preferably not limited to this or the like Staphylococcus epidermidis, Staphylococcus aureus. The strain in the above-mentioned Proteus is preferably Proteus mirabilis , Proteus vulgaris , and the like.
본 발명의 약학적 조성물은 1×103 내지 1×1010 PFU/mL의 박테리오파지를 포함하며, 바람직하게는 1×106 내지 1×109 PFU/mL의 박테리오파지를 포함한다. 본 발명에 사용된 용어, “PFU (plaque forming unit)”는 박테리오파지가 플라크를 형성하는 것을 수치화한 단위이다.The pharmaceutical composition of the present invention contains bacteriophage of 1 × 10 3 to 1 × 10 10 PFU / mL, preferably 1 × 10 6 to 1 × 10 9 PFU / mL of bacteriophage. As used herein, the term " PFU (plaque forming unit) " is a unit that quantifies the formation of a plaque by a bacteriophage.
본 발명의 용어 “예방”이란 조성물의 투여로 질병을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.The term " prophylactic " of the present invention means any action that inhibits the disease or delay the onset of the disease by administration of the composition.
본 발명의 용어 “치료”란 조성물의 투여로 상기 질병의 증세가 호전되거나 상기 질병의 억제 또는 경감 및 이롭게 변경되는 모든 행위를 의미한다.The term " treatment " of the present invention means all the acts of improving the symptom of the disease or inhibiting or alleviating and beneficially altering the disease upon administration of the composition.
본 발명의 약학적 조성물은 약학적으로 허용 가능한 담체를 추가로 포함할 수 있다.The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier.
본 발명에서 용어, “약학적으로 허용 가능한 담체”란 생물체를 자극하지 않고 투여 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 의미한다. 액상 용액으로 제제화되는 조성물에 있어서 허용되는 약학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로오스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 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.As used herein, the term " pharmaceutically acceptable carrier " means a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier that is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
본 발명의 약학적 조성물은 질환 부위에의 도포 또는 분무하는 방법으로 이용할 수 있으며, 그 밖에 비강 분무, 경구 투여 또는 비경구 투여를 통해 투여할 수도 있으며, 비경구 투여의 경우 정맥 내 투여, 복강 내 투여, 근육 내 투여, 피하 투여 또는 국부 투여를 이용하여 투여할 수 도 있다.The pharmaceutical composition of the present invention can be used as a method of applying or spraying on a diseased site, or may be administered through nasal spray, oral administration or parenteral administration. In the case of parenteral administration, intravenous administration, intraperitoneal administration Administration, intramuscular administration, subcutaneous administration or local administration.
상기 비강 분무의 경우 일정량의 박테리오파지 CF1을 물에 희석하여 분무기나 분무 시스템을 통해 비강으로 흡수 되도록 할 수 있다. 상기 비강 분무를 위한 비강 분무제 또는 호흡기용 제형으로는 에어로졸제 등을 들 수 있다.In the case of the nasal spray, a certain amount of bacteriophage CF1 may be diluted in water and absorbed into the nasal cavity through a sprayer or spray system. Examples of the nasal spray agent for the nasal spray or the aerosol agent for the respiratory system are mentioned.
상기 비강 분무제 또는 호흡기용 제형은, 수분산된 농축물 또는 습윤 분말을 분산시키기 위한 추진제 및/또는 기타 첨가제를 추가로 포함할 수 있다.The nasal spray or respiratory formulation may further comprise a propellant and / or other additives for dispersing the water-dispersed concentrate or wet powder.
본 발명의 약학적 조성물의 적합한 도포, 분무 및 투여량은 제제화 방법, 투여 방식, 대상이 되는 동물 및 환자의 연령, 체중, 성, 질병 증상의 정도, 음식, 투여시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사나 수의사는 목적하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다.The appropriate application, spray and dose of the pharmaceutical composition of the present invention may vary depending on factors such as formulation method, administration method, age, body weight, sex, disease symptom level, food, administration time, administration route, Response sensitivities, and the ordinarily skilled physician or veterinarian can readily determine and prescribe dosages effective for the desired treatment.
본 발명의 약학적 조성물을 유효성분으로 포함하는 경구 투여용 제형으로는, 예를 들어 정제, 트로키제, 로렌지, 수용성 또는 유성현탁액, 조제분말 또는 과립, 에멀젼, 하드 또는 소프트 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 마그네슘, 스테아르산 칼슘, 스테아릴푸마르산 나트륨 또는 폴리에틸렌글리콜 왁스와 같은 윤활유를 포함할 수 있으며, 캡슐 제형의 경우 상기 언급한 물질 외에도 지방유와 같은 액체 담체를 더 함유할 수 있다.Examples of formulations for oral administration comprising the pharmaceutical composition of the present invention as an active ingredient include tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs It can be formulated into a zero. A binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrating agent such as corn starch or sweet potato starch, a disintegrant such as magnesium stearate , Calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned materials, a liquid carrier such as a fatty oil may be further contained.
본 발명의 약학적 조성물을 유효성분으로 포함하는 비경구 투여용 제형으로는, 피하주사, 정맥주사 또는 근육 내 주사 등의 주사용 형태, 좌제 주입방식 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.Examples of the formulations for parenteral administration containing the pharmaceutical composition of the present invention as an active ingredient include injection forms such as subcutaneous injection, intravenous injection or intramuscular injection, aerosol formulations such as a suppository injection method or a respiratory apparatus It can be formulated for spraying. For formulation into injectable formulations, the compositions of the present invention may be formulated as solutions or suspensions in water with stabilizers or buffers in water, and formulated for unitary administration of ampoules or vials. When formulated for spraying, such as an aerosol formulation, a propellant or the like may be formulated with the additive such that the water-dispersed concentrate or wet powder is dispersed.
또한, 본 발명은 상기 박테리오파지 CF1을 포함하는, 항생용 조성물을 제공한다.The present invention also provides an antibiotic composition comprising the bacteriophage CF1.
본 발명에서, 용어 “항생용 조성물”은 약제 형태로 동물에게 제공되어 균을 사멸시킬 수 있는 제제를 의미하며, 방부제, 살균제, 항생제 및 항균제를 총칭하는 것이다. In the present invention, the term " antibiotic composition " means a preparation which can be given to an animal in the form of a drug to kill the bacterium, and collectively refers to an antiseptic, a bactericide, an antibiotic and an antibacterial agent.
본 발명의 박테리오파지 CF1은 기존 항생제에 비하여 사이트로박터 프렌디에 대한 특이성이 매우 높으므로, 유익균은 죽이지 않고, 특정 병원균만 사멸시킬 수 있고, 약물 내성 내지 저항성을 유도하지 않아, 기존의 항생물질에 비하여 제품수명(life cycling)이 긴 신규 항생제로서 이용될 수 있다.Since the bacteriophage CF1 of the present invention has a higher specificity for the streptavidin than the existing antibiotics, it does not kill useful bacteria, can kill only specific pathogens, does not induce drug resistance or resistance, And can be used as a new antibiotic with a long life cycling.
또한, 본 발명은 상기 박테리오파지 CF1을 포함하는, 사료 첨가용 조성물을 제공한다.The present invention also provides a composition for feed addition comprising the bacteriophage CF1.
양돈, 축산, 수산업, 반려동물 산업에서 사용되는 사료 첨가용 항생제는 질병의 예방 목적으로 사용되고 있는데, 예방 목적의 항생제 투여는 내성균 발생 가능성을 높이고 동물에 잔류하는 항생제가 사람에게 전달될 수 있어서 문제이다. 항생제가 육류를 통해 인체에 흡수되면 항생제 내성을 유발해 질병의 확산을 부를 수도 있다. 또한, 사료에 섞여 먹이는 항생제의 종류가 많고 이는 다제 내성균 발생 확률이 높아지는 문제점이 있기 때문에 좀 더 자연 친화적이면서도 기존의 항생제의 사용에서 발생한 문제를 해결할 새로운 사료 첨가용 항생물질로서 본 발명의 상기 박테리오파지 CF1을 이용할 수 있다.Feed antibiotics used in swine, livestock, fisheries and companion animal industries are used for the prevention of diseases. Antibiotic administration for prevention is problematic because it increases the possibility of resistant bacteria and antibiotics remaining in animals can be transmitted to humans . If antibiotics are absorbed into the body through meat, they may cause antibiotic resistance and spread the disease. In addition, since there are many kinds of antibiotics mixed with feed, there is a problem that the probability of occurrence of multidrug-resistant bacteria is increased. Therefore, as a new natural food additive antibiotic substance which is more environmentally friendly and solves the problems caused by the use of existing antibiotics, Can be used.
또한, 본 발명은 상기 사료 첨가용 조성물을 포함하는 사료를 제공할 수 있으며, 본 발명의 사료는 박테리오파지를 사료 첨가제 형태로 따로 제조하여 사료에 혼합시키거나, 사료 제조 시 직접 첨가시켜 제조할 수 있다. 본 발명의 사료 내 박테리오파지는 액상 또는 건조상태일 수 있으며, 바람직하게는 건조된 분말형태이다. 건조방법은 통풍건조, 자연건조, 분무건조 및 동결건조가 가능하지만, 이에 제한되는 것은 아니다. 본 발명의 박테리오파지는 분말형태로 사료 중량의 0.05 내지 10 중량%, 바람직하게는 0.1 내지 2 중량%의 성분비로 혼합될 수 있다. 또한, 상기 사료는 본 발명의 박테리오파지 외에 사료의 보존성을 높일 수 있는 통상의 첨가제들을 추가로 포함할 수 있다.In addition, the present invention can provide a feed comprising the feed composition, and the feed of the present invention can be prepared by separately preparing bacteriophage as a feed additive and mixing the feed into the feed, . The bacteriophage in the feed of the present invention may be in a liquid or dry state, preferably in the form of a dried powder. Drying methods include, but are not limited to, air drying, natural drying, spray drying and freeze drying. The bacteriophage of the present invention may be mixed in powder form in a composition ratio of 0.05 to 10% by weight, preferably 0.1 to 2% by weight of the feed weight. In addition to the bacteriophages of the present invention, the feed may further include conventional additives that can enhance the preservability of the feed.
본 발명의 사료 첨가용 조성물에는 비병원성의 다른 미생물이 추가로 첨가될 수 있다. 첨가될 수 있는 미생물로는 단백질 분해 효소, 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.), 동물의 체중을 증가시키며 우유의 산유량을 늘리고 사료의 소화 흡수율을 높이는 효과를 보여주는 아스퍼질러스 오리자에(Aspergillus oryzae)와 같은 사상균 및 사카로미세스 세레비지에 (Saccharomyces cerevisiae)와 같은 효모로 구성된 군으로부터 선택될 수 있다.In the composition for feed addition of the present invention, microorganisms other than non-pathogenic microorganisms may be further added. Examples of microorganisms that can be added include lactobacilli such as Bacillus subtilis capable of producing proteolytic enzymes, lipolytic enzymes and glycosyltransferases, lactobacilli having physiological activity and organic resolution at anaerobic conditions such as bovine, ( Lactobacillus sp.), Filamentous fungi such as Aspergillus oryzae , which increases the body weight of the animal and increases the milk yield of the animal and increases digestion and uptake of the feed, and Saccharomyces cerevisiae and yeast such as S. cerevisiae .
본 발명의 박테리오파지 CF1을 포함하는 사료에는 식물성으로는 곡물류, 근과류, 식품가공 부산물류, 조류, 섬유질류, 제약 부산물류, 유지류, 전분류, 박류, 곡물부산물류 등이 있으며, 동물성으로는 단백질류, 무기물류, 유지류, 광물성류, 유지류, 단세포 단백질, 동물성 플랑크톤류, 남은 음식물 등이 있으며 이에 제한되는 것은 아니다.The feedstuffs containing the bacteriophage CF1 of the present invention include cereals, muscle roots, food processing buses, algae, fibers, pharmaceutical buses, oils, fats and oils, But are not limited to, proteins, inorganic materials, fats, oils, fats, oils, monocellular proteins, animal plankton, and remnants.
본 발명의 사료 첨가용 조성물에는 품질 저하를 방지하기 위하여 첨가하는 결착제, 유화제, 보존제 등이 포함될 수 있고, 효용 증대를 위하여 사료에 첨가하는 아미노산제, 비타민제, 효소제, 생균제, 향미제, 비단백태질소화합물, 규산염제, 완충제, 착색제, 추출제, 올리고당 등이 포함될 수 있으며, 그 외에도 사료 혼합제 등을 추가로 포함할 수 있다.The composition for feed addition of the present invention may contain a binder, an emulsifying agent, a preservative, etc. added to prevent deterioration of quality. An amino acid, a vitamin, an enzyme, a probiotic, a flavoring agent, A nitrogen compound, a silicate, a buffer, a coloring agent, an extracting agent, an oligosaccharide, etc., and may further include a feed mixture.
또한, 본 발명은 박테리오파지 CF1을 포함하는, 음용수 첨가제를 제공한다.The present invention also provides a drinking water additive comprising bacteriophage CF1.
본 발명의 음용수 첨가제는 상기 박테리오파지 CF1 또는 이를 포함하는 조성물을 음용수 첨가제 형태로 따로 제조하여 사료 또는 음용수에 혼합하는 방식으로 사용되거나, 음용수 제조 시 직접 첨가하는 방식으로 사용할 수 있다. 상기와 같이 음용수에 혼합하여 공급함으로써 지속적으로 사이트로박터 프렌디의 숫자를 감소시킬 수 있는 효과가 있다.The additive for drinking water of the present invention can be used either by preparing the bacteriophage CF1 or a composition containing the bacteriophage CF1 separately in the form of additive for drinking water, mixing the mixture with feed or drinking water, or by directly adding it in the production of drinking water. By mixing and supplying the drinking water as described above, it is possible to continuously reduce the number of bacteria fed to the site.
본 발명에서 음용수는 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용되는 음용수를 사용할 수 있다.In the present invention, the drinking water is not particularly limited, and drinking water commonly used in the art can be used.
또한, 본 발명은 박테리오파지 CF1을 포함하는, 소독제를 제공한다.The present invention also provides a disinfectant comprising bacteriophage CF1.
사이트로박터 프렌디에 특이적 사멸능을 갖는 본 발명의 박테리오파지 CF1을 포함하는 소독제는 병원감염을 막기 위한 병원 및 보건용의 소독제로 유용하게 사용될 수 있고 일반 생활 소독제, 식품 및 조리 장소 및 설비의 소독제, 양계장, 축사 등의 건물, 축체, 음수, 깔짚, 난좌, 운반차량, 식기 등의 각종 생육 용품의 소독 등에 사용될 수 있다.The disinfectant containing the bacteriophage CF1 of the present invention having the bacteriophage-specific killing ability as a site can be usefully used as a disinfectant for hospital and public health to prevent infection of a hospital, and can be used as a general disinfectant, food, Disinfectants, poultry houses, buildings such as barns, disinfection of various growth equipments such as shaft bodies, negative water, litter, runaway, transportation vehicles and tableware.
또한, 본 발명은 상기 박테리오파지 CF1을 포함하는, 세척제를 제공한다.The present invention also provides a detergent comprising the bacteriophage CF1.
본 발명의 박테리오파지 CF1은 사이트로박터 프렌디에 특이적 사멸능을 가지므로, 상기 사이트로박터 프렌디에 노출되었거나 노출될 가능성이 있는 동물의 피부 표면 또는 신체 각 부위 등을 세척하는 용도로도 사용될 수 있다.Since the bacteriophage CF1 of the present invention has a site-specific bacteriophage-specific killing ability, it can also be used for washing skin surface or body parts of an animal which is exposed to bacteriorbide or is likely to be exposed to the site .
또한, 본 발명은 상기 박테리오파지 CF1을 개체에 투여하는 단계를 포함하는, 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료 방법을 제공한다.In addition, the present invention provides a method for preventing or treating infectious diseases caused by streptococci, comprising administering the bacteriophage CF1 to a subject.
본 발명의 방법은 사이트로박터 프렌디에 의한 감염성 질병, 예컨대 요로감염증, 급성 방광염, 만성 방광염, 신우신염, 무증상 세균뇨, 신장결석, 수막염, 복막염, 터널 감염, 중이염, 농양 또는 설사가 발생한 개체에 박테리오파지 CF1을 투여하는 단계를 포함한다. The method of the present invention can be used for the treatment of infectious diseases caused by bacteremia caused by bacteremia such as urinary tract infections, acute cystitis, chronic cystitis, pyelonephritis, asymptomatic bacteriuria, kidney stones, meningitis, peritonitis, tunnel infection, otitis media, abscess or diarrhea Lt; RTI ID = 0.0 > CF1 < / RTI >
본 발명에서, 용어 "개체"는 인간을 포함한 동물, 바람직하게는 인간, 가축류 또는 반려동물을 말하며, 가장 바람직하게는 인간, 개, 고양이, 돼지, 닭 또는 말을 뜻하나, 이에 제한되는 것은 아니다.In the present invention, the term "individual" refers to an animal including a human, preferably a human, a livestock or companion animal, most preferably a human, a dog, a cat, a pig, a chicken or a horse.
상기 용어 "가축류"는 인간에 의하여 순화, 개량되어 인간과 함께 공동생활을 하는 유용한 동물을 지칭하는 개념으로, 조류, 어류, 곤충류, 포유류를 모두 포함하는 개념이며, 상기 가축류는 돼지, 소, 닭, 말, 오리 및 개로 구성된 군에서 선택될 수 있으나, 이에 제한되는 것은 아니다.The term "livestock" is a concept that refers to useful animals that are refined and improved by humans to co-live with humans, including birds, fishes, insects, and mammals. The livestock are pigs, , Horse, duck, and dog, but is not limited thereto.
본 발명의 방법에서 상기 박테리오파지 CF1 또는 조성물은 약학적 제제의 형태로 동물에게 투여되거나, 개체의 사료 또는 음용수에 혼합하여 이를 섭식시키는 방법을 통해 투여될 수 있다.In the method of the present invention, the bacteriophage CF1 or composition may be administered to an animal in the form of a pharmaceutical preparation, or may be administered through a method of ingesting the bacteriophage CF1 or a mixture thereof into animal feed or drinking water.
본 발명의 방법에서 상기 박테리오파지 CF1 또는 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있으며, 구체적으로, 구강, 직장, 국소, 정맥 내, 복강 내, 근육 내, 동맥 내, 경피, 비측 내, 흡입 등을 통해 통상적인 방식으로 투여될 수 있다.In the method of the present invention, the administration route of the bacteriophage CF1 or the composition can be administered through various oral or parenteral route as long as it can reach the target tissue, and specifically, oral, rectal, topical, intravenous, intraperitoneal, Intravenous, intramuscular, intraarterial, transdermal, intranasal, inhalation, and the like.
본 발명의 방법에 투여되는 상기 박테리오파지 CF1의 적합한 총 1일 사용량은 올바른 의학적 판단범위 내에서 처치의에 의해 결정될 수 있다는 것은 당업자에게 자명한 일이다. 특정 개체에 대한 구체적인 치료적 유효량은 달성하고자 하는 반응의 종류와 정도, 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여시간, 투여 경로 및 조성물의 분비율, 치료기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다.It is obvious to those skilled in the art that the appropriate total daily dose of the bacteriophage CF1 administered to the method of the present invention can be determined by treatment within the scope of sound medical judgment. The specific therapeutically effective amount for a particular individual will depend upon the type and extent of the response to be achieved, the age, weight, general health, sex and diet of the patient, the time of administration, the route of administration and the fraction of the route of administration, It is preferable to apply differently depending on various factors including drugs used or co-used and similar factors well known in the medical field.
본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Terms not otherwise defined herein have meanings as commonly used in the art to which the present invention belongs.
이하, 본 발명을 실시예, 제제예에 의해 상세히 설명한다. 단, 하기 실시예, 제제예는 본 발명을 예시하는 것일 뿐 본 발명의 내용이 하기 실시예, 제제예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to Examples and Preparation Examples. However, the following examples and preparative examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and preparative examples.
실시예Example 1: One: 신규한New 박테리오파지의 분리 및 배양 Isolation and culture of bacteriophage
1.1 박테리오파지의 스크리닝 및 단일 박테리오파지의 분리1.1 Screening of bacteriophages and isolation of single bacteriophages
사이트로박터 프렌디(Citrobacter freundii)에 특이적인 박테리오파지를 스크리닝 및 분리하기 위해서 용균반 분석을 수행하였다. 시료는 박테리오파지가 존재할 것으로 예상되는 도축장이나 양돈농장 부근의 하수로부터 시료를 채취하였으며, 박테리오파지 분리에 사용된 사이트로박터 프렌디는 (주)옵티팜에서 야외 시료로부터 분리 동정된 것을 사용하였다.Site: Citrobacter To investigate and isolate the bacteriophage specific to freundii , we performed a flotation analysis. Samples were taken from the slaughterhouse where the bacteriophage is expected to exist or from the sewage near the pig farm. The site used for the bacteriophage isolation was Bactorfendi, which was isolated from outdoor samples in Opti Farm.
구체적으로, 채취한 시료를 균질화 한 후 사이트로박터 프렌디와 함께 35℃에서 하룻밤 동안 진탕배양 하였다. 상기 배양액을 4,000 rpm으로 20분간 원심분리한 후, 상등액을 공극(pore) 크기가 0.45 μm인 필터를 이용하여 여과하여 시료 여과액을 얻었다. 시료 여과액에 대해 사이트로박터 프렌디가 접종되어 있는 BHI 플레이트에 전처리한 시료를 점적하여 15시간 동안 배양하여 스크리닝하는 반점 분석법(spot test)을 수행하고 그 결과를 도 1에 나타내었다.Specifically, the collected samples were homogenized and then cultured with the ampicillin in the site at 35 DEG C overnight with shaking. The culture solution was centrifuged at 4,000 rpm for 20 minutes, and the supernatant was filtered using a filter having a pore size of 0.45 μm to obtain a sample filtrate. A spot test was performed in which a pre-treated sample was spotted on a BHI plate in which Bacteriorbia was inoculated to the sample filtrate, and the mixture was incubated for 15 hours to perform screening. The results are shown in FIG.
도 1에 나타낸 바와 같이, 사이트로박터 프렌디에 대해 용균 활성이 나타나는 시료를 확인하였으며, 용균 활성이 확인된 시료에는 사이트로박터 프렌디를 용균할 수 있는 박테리오파지가 존재하는 것으로 추정하고 용균반 형성을 확인하였다.As shown in FIG. 1, a sample showing bactericidal activity against bacteriorbione was confirmed, and it was assumed that a bacteriophage capable of lytic bacteremia was present in the sample in which lytic activity was confirmed, Respectively.
용균반 형성을 확인하기 위하여, 전배양된 사이트로박터 프렌디 150 μL를 취해서 멸균된 튜브에 넣고 3-5 mL의 BHI 탑 아가(top agar) [Brain Heart Infusion(Brain infusion solids 12.5 g/L, Beef heart infusion solids 5.0 g/L, 프로테오스 펩톤 10.0 g/L, 글루코오스 2.0 g/L, 소듐 클로라이드 5.0 g/L, 디소듐 포스페이트 2.5 g/L) + 0.6% 아가)]를 첨가하여 잘 혼합한 후 미리 만들어둔 2% BHI 아가 플레이트에 중층하였다. 실온에서 30분간 방치한 후 상기 시료 여과액을 각각 10 μL씩 점적하고, 다시 실온에서 30분간 방치한 후 35℃에서 24시간 동안 배양하여 박테리오파지의 유무를 확인하였다.To confirm the formation of the bacterial flora, 150 μL of bacteriorbide was pre-incubated in a pre-incubated site, placed in a sterile tube, and eluted with 3-5 mL of BHI top agar [Brain Heart Infusion (Brain infusion solids 12.5 g / L, (5 g / L of beef heart infusion solids, 10.0 g / L of proteospeptone, 2.0 g / L of glucose, 5.0 g / L of sodium chloride and 2.5 g / L of disodium phosphate) + 0.6% And then overlaid on 2% BHI agar plates previously prepared. After allowing to stand at room temperature for 30 minutes, the sample filtrate was dropped by 10 μL each. Each sample was left at room temperature for 30 minutes and then cultured at 35 ° C. for 24 hours to confirm presence or absence of bacteriophages.
박테리오파지의 존재를 확인한 시료로부터 박테리오파지를 순수 분리하기 위해서 소프트 아가 오버레이(soft agar overlay)법을 이용하였다. 박테리오파지가 존재하는 것으로 확인된 시료를 식염수로 적절히 희석하여 각 희석단의 시료 희석액 100 μL를 취하고, 대상 세균(사이트로박터 프렌디) 배양액 150 μL에 혼합한 후 0.6% BHI 탑 아가 3 mL을 첨가하여 잘 혼합한 뒤, 미리 만들어 둔 2% BHI 아가 플레이트에 중층하여 방치한 후 35℃에서 24시간 배양하였다. 배양한 각 플레이트에서 형성된 용균반을 확인한 후, 이를 선택하여 400 μL의 SM 완충액 (NaCl 5.8 g/L; MgSO47H2O 2 g/L; 1 M Tris-HCl (pH 7.5) 50 mL)에 현탁하고, 실온에서 4시간동안 방치하였다. 상기 박테리오파지액 100 μL를 0.6% BHI 탑 아가와 세균 배양액 500 μL와 혼합한 뒤 150 mm 지름의 BHI 플레이트에 분주하여 중층한 후 완전히 용균이 일어날 때까지 35℃에서 배양하였다. 배양이 종료된 후, BHI 플레이트에 15 mL의 SM 완충액을 가하고 실온에서 4시간 동안 천천히 교반하면서 박테리오파지 용액을 회수한 후 4,000 rpm으로 20분간 원심분리하여 상등액만 취하고 이를 0.45 μm 필터로 여과하여 최종 박테리오파지 용액을 회수하였다. 이렇게 얻어진 최종 박테리오파지의 용균반을 확인한 결과를 도 2에 나타내었다.A soft agar overlay method was used to isolate the bacteriophage from the sample which confirmed the existence of the bacteriophage. Samples that have been confirmed to contain bacteriophage are diluted appropriately with saline, 100 μL of the sample dilution at each dilution stage is taken, mixed with 150 μL of the bacterial (bacteriophage) culture, and 3 mL of 0.6% BHI topa is added , And the mixture was allowed to stand in a 2% BHI agar plate that had been previously prepared, followed by incubation at 35 ° C for 24 hours. After culturing, the plates were incubated with 400 μL of SM buffer (NaCl 5.8 g / L; MgSO 4 7H 2 O 2 g / L; 1 M Tris-HCl (pH 7.5) 50 mL) Suspended and allowed to stand at room temperature for 4 hours. 100 .mu.l of the bacteriophage solution was mixed with 500 .mu.l of 0.6% BHI top agar and bacterial culture, and the mixture was divided into 150-mm-diameter BHI plates and incubated at 35.degree. C. until lysis occurred. After incubation, 15 mL of SM buffer was added to the BHI plate, and the bacteriophage solution was recovered while slowly stirring at room temperature for 4 hours. The supernatant was centrifuged at 4,000 rpm for 20 minutes. The supernatant was collected by filtration through a 0.45 μm filter, The solution was recovered. FIG. 2 shows the result of checking the thus obtained final bacteria bacteriophage.
도 2에 나타낸 바와 같이, 2-3 mm의 크기를 가지는 용균반을 형성하는 박테리오파지를 확인하였고, 상기 박테리오파지는 사이트로박터 프렌디 특이적인 용균능을 갖는 박테리오파지임을 확인하였다.As shown in FIG. 2, a bacteriophage forming a fungus having a size of 2-3 mm was confirmed, and it was confirmed that the bacteriophage was a bacteriophage having a site-specific bacteriophage-specific fungicidal activity.
1.2 박테리오파지의 대량 배양 및 정제1.2 Bulk culture and purification of bacteriophages
상기 실시예 1.1에서 확인한 사이트로박터 프렌디 특이적 박테리오파지를 대량 배양하기 위하여, 1.1에서 회수한 최종 박테리오파지 배양액을 1.0×108 PFU/mL로 희석한 후 전배양된 사이트로박터 프렌디 1.0×109 CFU/mL과 혼합하여 MOI (Multiplicity of infection)를 0.01이 되도록 하여 35℃에서 120 rpm으로 9시간 배양하였다. 배양이 종료된 후 배양액 전량을 6,000 rpm으로 30분간 원심분리하여 상등액을 수득하였고, 이를 0.2 μm 필터로 여과하여 최종 박테리오파지 배양액을 회수하였다. 분리된 박테리오파지를 "박테리오파지 CF1(Bacteriophage CF1)"으로 명명하고, 2016년 08월 11일 한국미생물보존센터 (Korean Culture Center of Microorganisms, 서울, 한국)에 기탁하고, 기탁번호 KFCC11682P를 부여받았다.In order to massively cultivate the bacteriophage-specific bacteriophage into the site identified in Example 1.1, the final bacteriophage culture recovered at 1.1 was diluted to 1.0 x 10 8 PFU / mL, 9 CFU / mL, and the mixture was incubated at 35 ° C and 120 rpm for 9 hours to obtain MOI (multiplicity of infection) of 0.01. After the culture was completed, the whole culture was centrifuged at 6,000 rpm for 30 minutes to obtain a supernatant, which was then filtered with a 0.2 μm filter to recover the final bacteriophage culture. The isolated bacteriophage was named "Bacteriophage CF1" and deposited on August 11, 2016 at the Korean Culture Center of Microorganisms (Seoul, Korea), and received the deposit number KFCC11682P.
실시예Example 2: 박테리오파지 CF1의 전체 유전자 분석 2: Whole gene analysis of bacteriophage CF1
박테리오파지 CF1의 전체 유전자를 분석하기 위해, 박테리오파지 CF1 배양액에 20 mM EDTA, 50 μg/mL의 프로테나아제 K, 0.5% (w/v) SDS를 가한 후, 50℃에서 1시간 동안 정치하였다. 동량의 페놀-클로로포름-이소프로필 알코올 (25:24:1)과 혼합한 후 실온에서 12,000 rpm으로 10분간 원심분리하여 상등액을 회수하였다. 회수된 상등액을 동일 부피의 페놀-클로로포름-이소프로필 알코올 (25:24:1)과 혼합하여 실온에서 12,000 rpm으로 10분간 원심분리한 후 상등액을 회수하고, 상기 상등액에 3M 소듐 아세테이트를 전체 부피의 10%(v/v)가 되도록 가한 후 혼합하였다. 여기에 2배 부피의 차가운 95% 에탄올을 가하여 혼합한 뒤 -20℃에서 1시간 동안 정치하였다. 반응이 끝난 후 0℃에서 10분간 12,000 rpm으로 원심분리하여 침전물을 획득하였고, 차가운 70% 에탄올로 2회 세척한 뒤 에탄올을 완전히 제거하여 DNA 펠렛을 회수하였다. 회수한 펠렛을 최종 부피가 50 μL이 되도록 TE 완충액 (Tris-EDTA, pH8.0)에 용해시킨 후 DNA 농도를 측정하였다. 전체 유전자 분석을 위해 PacBioRSII를 이용하여 유전자 시퀀싱을 시행하였다 (마크로젠, 한국). 시퀀싱 결과로 얻은 박테리오파지 CF1 유전자의 유전자 맵을 도 3에 나타내었다.In order to analyze the entire gene of bacteriophage CF1, 20 mM EDTA, 50 μg / mL proteinase K and 0.5% (w / v) SDS were added to the bacteriophage CF1 culture and left at 50 ° C. for 1 hour. The mixture was mixed with an equal amount of phenol-chloroform-isopropyl alcohol (25: 24: 1) and centrifuged at 12,000 rpm for 10 minutes at room temperature to recover the supernatant. The recovered supernatant was mixed with an equal volume of phenol-chloroform-isopropyl alcohol (25: 24: 1) and centrifuged at 12,000 rpm for 10 minutes at room temperature. The supernatant was recovered and 3M sodium acetate was added to the supernatant in a
도 3에 나타낸 바와 같이, 박테리오파지 CF1의 전체 유전자의 크기는 50,339 bp이며, GC 함량이 43%, 전체 ORFs의 수는 85개임을 확인하였다. 분석한 박테리오파지 CF1의 전체 유전자 서열을 서열번호 1로 나타내었다. As shown in FIG. 3, it was confirmed that the size of the entire gene of bacteriophage CF1 was 50,339 bp, the GC content was 43%, and the total number of ORFs was 85. The entire gene sequence of the bacteriophage CF1 analyzed is shown in SEQ ID NO: 1.
실시예Example 3: 박테리오파지 CF1의 형태학적 특성 3: Morphological characteristics of bacteriophage CF1
박테리오파지 CF1의 형태학적 특성을 알아보기 위해, 박테리오파지 CF1을 0.01% 젤라틴 용액에 희석하여 2.5% 글루타르알데하이드(glutaraldehyde) 용액으로 고정하였다. 이를 카본 코팅된 운모판(carbon-coated mica plate, ca. 2.5 x 2.5 mm)에 적가한 후, 10분간 적응시켜 멸균 증류수로 세척하였다. 이를 구리 격자(copper grid)에 끼워 2% 우라닐 아세테이트(Uranyl acetate)에서 30-60초간 염색하고 건조시킨 후, 투과전자현미경(Tecnai G2 Spirit Twin, Bio-Transmission electron microscope, 120 kV, 120,000~400,000)으로 검경하였으며 그 결과를 도 4에 나타내었다.To examine the morphological characteristics of bacteriophage CF1, bacteriophage CF1 was diluted in 0.01% gelatin solution and fixed with 2.5% glutaraldehyde solution. This was added dropwise to a carbon-coated mica plate (ca. 2.5 x 2.5 mm), which was then adapted for 10 minutes and washed with sterile distilled water. The mixture was stained with 2% Uranyl acetate for 30-60 seconds, dried, and analyzed by transmission electron microscope (Tecnai G 2 Spirit Twin, Bio-Transmission electron microscope, 120 kV, 400,000). The results are shown in Fig.
도 4에 나타낸 바와 같이, 박테리오파지 CF1은 200-220 nm의 크기로, 정이십면체의 머리를 가지며 수축성 꼬리가 있는 형태인 마이오비리데 (Myoviridae)에 속함을 확인하였다.As shown in Fig. 4, the bacteriophage CF1 was found to belong to Myoviridae, which is 200-220 nm in size, and has a twisted-sided head and a contractile tail.
실시예Example 4: 박테리오파지 CF1의 단백질 패턴 분석 4: Protein pattern analysis of bacteriophage CF1
박테리오파지 CF1의 단백질 패턴을 분석하기 위해, 1.0x1010 PFU/mL로 준비된 박테리오파지 CF1 40 μL를 10 μL의 5X 로딩 완충액과 혼합하고, 10분간 끓인 다음 12.5% SDS-PAGE를 수행하였다. SDS-PAGE 수행 후 단백질 전기영동 결과를 도 5에 나타내었다.To analyze the protein pattern of bacteriophage CF1, 40 μL of bacteriophage CF1 prepared to 1.0 × 10 10 PFU / mL was mixed with 10 μL of 5X loading buffer, boiled for 10 minutes, and subjected to 12.5% SDS-PAGE. The results of SDS-PAGE and protein electrophoresis are shown in FIG.
도 5에 나타낸 바와 같이, 박테리오파지 CF1은 80 kDa, 65 kDa, 35 kDa, 33 kDa 및 16 kDa 크기의 주요 단백질을 가지는 것을 확인하였다.As shown in FIG. 5, it was confirmed that bacteriophage CF1 has major proteins of 80 kDa, 65 kDa, 35 kDa, 33 kDa and 16 kDa in size.
실시예Example 5: 박테리오파지 CF1의 다양한 세균에 대한 감염성 조사 5: Investigation of infectivity of bacteriophage CF1 against various bacteria
박테리오파지 CF1의 다양한 세균에 대한 감염성을 조사하였다. Infection of bacteriophage CF1 against various bacteria was investigated.
우선, 박테리오파지 CF1이 야생(양돈 농장)에서 분리한 사이트로박터 프렌디 균주(㈜옵티팜)에 대하여 감수성을 나타내는지 여부를 소프트 아가 오버레이법을 통해 확인한 결과를 표 1에 나타내었다.First, whether the bacteriophage CF1 is isolated from wild (pig farm) and whether or not the bacteriophage CF1 shows susceptibility to the bacteriophedis strain (OptiParm) is shown in Table 1 by means of soft agar overlay method.
확인된 수Number confirmed
확인되지 않은 수Unidentified number
상기 표 1에 나타낸 바와 같이, 박테리오파지 CF1은 12 균주의 사이트로박터 프렌디에 대한 용균활성을 확인한 결과, 12 균주 모두에 대해 감수성이 있는 것으로 나타났으므로 사이트로박터 프렌디에 대한 용균활성이 탁월함을 확인하였다.As shown in Table 1 above, bacteriophage CF1 was found to be susceptible to all 12 strains as a result of confirming the lectin activity against bacteriorbione by 12 strains. Thus, the lactobacillus activity against bacteriorbione was excellent Respectively.
또한, 박테리오파지 CF1이 임상 유래의 사이트로박터 프렌디에 대하여 감수성을 나타내는지 여부를 확인하기 위하여, 만성 방광염을 가진 개와 고양이로부터 분리한 사이트로박터 프렌디(㈜옵티팜)를 이용하여 소프트 아가 오버레이법을 수행하였다. 그 결과를 표 2에 나타내었다.In order to confirm whether bacteriophage CF1 is susceptible to bacteriophage as a clinical site, a site isolated from dogs and cats with chronic cystitis was treated with Bactorfendi (OptiPharm) The law was carried out. The results are shown in Table 2.
확인된 수Number confirmed
확인되지 않은 수Unidentified number
상기 표 2에 나타낸 바와 같이, 박테리오파지 CF1은 임상 균주 8종에 대하여 모두 용균활성을 나타냄을 확인하였으므로 야생 분리주 뿐만 아니라 임상 유래 사이트로박터 프렌디에 대한 용균활성 또한 탁월함을 확인하였다.As shown in Table 2, it was confirmed that bacteriophage CF1 exhibited lytic activity in all eight clinical strains. Thus, it was confirmed that lactic activity against bacteriorbide as a clinical-derived site as well as a wild isolate was excellent.
또한, 박테리오파지 CF1이 다양한 세균에 대해서 용균활성을 나타내는지 확인하기 위하여 ㈜옵티팜에서 분리하여 보유하고 있는 균주와 미생물 자원센터 등에서 분양받은 균주를 대상으로 소프트 아가 오버레이법을 통해서 감염성을 확인하였다. 그 결과를 표 3에 나타내었다.In order to confirm whether bacteriophage CF1 exhibits lytic activity against various bacteria, the strains isolated from OptiParm Co., Ltd. and purchased from the microorganism resource center were infectious through soft agar overlay method. The results are shown in Table 3.
(+: 용균활성 있음, -; 용균활성 없음)(+: Lytic activity, -; lytic activity)
상기 표 3에 나타낸 바와 같이, 사이트로박터 프렌디 외에도 다른 미생물을 대상으로 감수성 검사를 진행한 결과, 박테리오파지 CF1은 Salmonella Typhimurium, Bacillus cereus, Listeria monocytogenes, Lactobacillus plantarum 등에는 용균활성이 없는 것을 확인하였다. As shown in Table 3, the susceptibility test was carried out on other microorganisms other than the bacteriophage in the site. As a result, bacteriophage CF1 was found to be Salmonella Typhimurium, Bacillus cereus , Listeria monocytogenes , and Lactobacillus plantarum .
또한 상기 표 3에서 사용된 야생형의 사이트로박터 프렌디는 앰피실린, 페니실린, 세포탁심 등의 베타락탐계 항생제에 내성을 보이는 균주이므로, 박테리오파지 CF1은 항생제 내성이 있는 사이트로박터 프렌디에 대해서도 용균활성을 갖는 것을 확인하였다.Also, since the wild-type site used in Table 3 above is resistant to beta-lactam antibiotics such as ampicillin, penicillin, and cytotoxin, bacteriophage CF1 is an antibiotic resistant site, ≪ / RTI >
따라서, 본 발명의 박테리오파지 CF1은 사이트로박터 프렌디에 특이적으로 용균활성을 나타내는 박테리오파지임을 확인하였다.Therefore, it was confirmed that the bacteriophage CF1 of the present invention is a bacteriophage exhibiting a lytic activity specifically to the site bacteriorbione.
실시예Example 6: 박테리오파지 CF1의 pH 안정성 조사 6: Investigation of pH stability of bacteriophage CF1
박테리오파지 CF1의 pH 안정성을 확인하기 위하여, 다양한 pH 범위 (pH 2.1, 3.0, 4.0, 5.5, 7.4, 8.2, 9.0)에서 박테리오파지의 수를 측정하였다. 소듐 아세테이트 완충액, 소듐 포스페이트 완충액, Tris-HCl을 이용하여 각각의 pH에 맞게 조제한 후 1 mL로 분주하여 준비하였다. 1.0×108 PFU/mL로 준비한 박테리오파지 CF1 배양액 10 μL을 상기 용액에 첨가하여 2시간 동안 상온에서 방치하였다. 방치 후 각 반응액을 단계희석하여 소프트 아가 오버레이법에 의한 타이터(titer) 측정을 수행한 결과를 도 6에 나타내었다.The number of bacteriophages was measured in various pH ranges (pH 2.1, 3.0, 4.0, 5.5, 7.4, 8.2, 9.0) in order to confirm the pH stability of bacteriophage CF1. Sodium acetate buffer, sodium phosphate buffer, and Tris-HCl were added to each pH-adjusted solution, and the mixture was divided into 1 mL. 10 μL of the bacteriophage CF1 culture prepared at 1.0 × 10 8 PFU / mL was added to the above solution and left at room temperature for 2 hours. The reaction solution was allowed to stand, and the reaction solution was diluted stepwise, and a titer measurement was performed by soft agar overlay method. The result is shown in FIG.
도 6에 나타낸 바와 같이, pH 4-9의 범위에서는 박테리오파지 CF1이 2시간 동안 안정한 것으로 확인되었으며, pH 2에서는 박테리오파지의 수가 3 log, pH 3에서는 1 log가 감소됨을 확인하였다.As shown in FIG. 6, it was confirmed that the bacteriophage CF1 was stable for 2 hours in the range of pH 4-9, and 3 log for the number of bacteriophages and 1 log for the pH 3 at
따라서, 본 발명의 박테리오파지 CF1은 pH 4-9의 범위에서 안정함을 확인하였으므로, 박테리오파지 CF1은 pH 안정성이 우수한 박테리오파지임을 알 수 있다.Therefore, it was confirmed that the bacteriophage CF1 of the present invention is stable in the range of pH 4-9, so that bacteriophage CF1 is a bacteriophage having excellent pH stability.
실시예Example 7: 박테리오파지 CF1의 열 안정성 조사 7: Investigation of thermal stability of bacteriophage CF1
박테리오파지 CF1의 열 안정성을 확인하기 위하여, 다양한 온도 범위 (37℃, 42℃, 50℃, 60℃, 70℃, 80℃)에서 박테리오파지의 수를 측정하였다. 박테리오파지 CF1 배양액 (1.0×109 PFU/mL) 5 mL을 멸균된 튜브에 분주한 후 박테리오파지를 첨가하여 30분, 60분, 2시간 동안 각 온도에서 방치하였다. 방치 후 각 반응액을 단계희석하여 소프트 아가 오버레이법에 의한 타이터 측정을 수행한 결과를 도 7에 나타내었다.In order to confirm the thermal stability of bacteriophage CF1, the number of bacteriophages was measured in various temperature ranges (37 ° C, 42 ° C, 50 ° C, 60 ° C, 70 ° C and 80 ° C). 5 mL of the bacteriophage CF1 culture (1.0 × 10 9 PFU / mL) was dispensed into sterile tubes, and bacteriophage was added thereto and left at each temperature for 30 minutes, 60 minutes, and 2 hours. After allowing to stand, each reaction solution was diluted stepwise, and the result of performing a tester measurement by soft agar overlay method is shown in FIG.
도 7에 나타낸 바와 같이, 37-70℃의 범위에서는 30분까지는 안정한 것을 확인하였다. As shown in Fig. 7, it was confirmed that it was stable up to 30 minutes in the range of 37-70 占 폚.
따라서, 본 발명의 박테리오파지 CF1은 내열성이 우수한 박테리오파지임을 알 수 있다.Therefore, it can be seen that the bacteriophage CF1 of the present invention is a bacteriophage having excellent heat resistance.
상기 실험으로부터 박테리오파지 CF1은 사이트로박터 프렌디에 대한 특이성이 매우 높고, 사이트로박터 프렌디에 감염하여 사이트로박터 프렌디 내에서 증식함으로써 용균 활성이 우수하고 물리화학적 자극에 강하다는 장점을 가지고 있음을 확인하였다. 또한 본 발명의 박테리오파지 CF1은 사람, 동물, 식물 등 세균외의 다른 숙주에는 감염되지 않으므로, 항생제의 오남용으로 인한 항생제 내성균의 문제점, 식품 내의 항생제의 잔류 문제, 광범위한 숙주범위의 문제점을 해결할 수 있는 장점이 있다. 따라서, 본 발명의 박테리오파지 CF1은 사이트로박터 프렌디에 의한 감염성 질병의 예방 또는 치료, 항생용 조성물, 사료 첨가용 조성물, 사료, 소독제, 또는 세척제 분야에서 다양하게 사용될 수 있다.From the above experiments, bacteriophage CF1 has an extremely high specificity for bacteriorhodin to the site, has an advantage of being excellent in lytic activity and strong against physicochemical stimuli by proliferating in bacteriorpidis as a site infected with bacteriorbide Respectively. In addition, since the bacteriophage CF1 of the present invention is not infected with any host other than bacteria such as human, animal, and plant, it can solve problems of antibiotic resistant bacteria caused by misuse of antibiotics, residual antibiotics in food, have. Therefore, the bacteriophage CF1 of the present invention can be used variously in the field of prevention or treatment of infectious diseases caused by bacteremia, antibacterial composition, feed composition, feed, disinfectant, or detergent.
비록 본 발명이 상기에 언급된 바람직한 실시예로서 설명되었으나, 발명의 요지와 범위로부터 벗어남이 없이 다양한 수정이나 변형을 하는 것이 가능하다. 또한 첨부된 청구 범위는 본 발명의 요지에 속하는 이러한 수정이나 변형을 포함한다.Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.
이하 본 발명의 박테리오파지 CF1을 포함하는 감염성 질병의 예방 또는 치료용 약학적 조성물의 제제예를 설명하나, 본 발명을 한정하고자 함이 아니고 단지 이를 구체적으로 설명하고자 함이다.Hereinafter, formulation examples of pharmaceutical compositions for the prevention or treatment of infectious diseases including bacteriophage CF1 of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically explained.
제제예Formulation example 1: 의약품의 제조 1: Manufacture of medicines
1.1 주사제의 제조1.1 Manufacture of Injection
박테리오파지 CF1 1×106 PFU/mLBacteriophage CF1 1 x 10 6 PFU / mL
주사용 멸균 증류수 적량Sterile sterilized water for injection
pH 조절제 적량pH adjuster
통상의 주사제의 제조방법에 따라 1 앰플당(2 mL) 상기의 성분 함량으로 제조한다.(2 mL) per 1 ampoule in accordance with the usual injection preparation method.
1.2 1.2 액제의Liquid 제조 Produce
박테리오파지 CF1 1×106 PFU/mLBacteriophage CF1 1 x 10 6 PFU / mL
설탕 20 g20 g of sugar
이성화당 20 g20 g per isomer
레몬향 적량Lemon incense quantity
정제수를 가하여 전체 1,000 mL로 맞추었다. 통상의 액제의 제조방법에 따라 상기의 성분을 혼합한 다음, 갈색병에 충전하고 멸균시켜 액제를 제조한다.Purified water was added and the total volume was adjusted to 1,000 mL. The above components are mixed according to a usual method for producing a liquid agent, and then filled in a brown bottle and sterilized to prepare a liquid agent.
1.3 정제의 제조1.3 Manufacture of tablets
박테리오파지 CF1 1×106 PFU/mLBacteriophage CF1 1 x 10 6 PFU / mL
옥수수전분 100 mg
유당 100 mg
스테아린산 마그네슘 2 mg
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.
SEQUENCE LISTING <110> Optipharm.CO.,LTD <120> Novel Citrobacter freundii specific bacteriophage CF1 and antibacterial composition comprising the same <130> 27 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 50339 <212> DNA <213> bacteriophage <400> 1 ccagtgcttg agcgatgacc aggagcagca gctaccaacc tataaaagcg cggtgtcact 60 gacttacacc tttgcgcatg agtacgataa ccctatttat ccgctcttgc gtaaggctga 120 cgaatccggc gacgttaaag cattgcgcat gtatgtacca aaagcaaaag aaatgcgcct 180 ttgggctggt gtgctttctt tcaatgagat cccacaaacg gcggtaaacg aaatggaaac 240 cgtttctttg tcggtatccc tgaaaggtcg ctttactttc ttgccgtcta ctcaggctta 300 atttaaaggg gcgcattgcg tccctttttt atttgatgta atatcacctc aatatttaac 360 cactcaggag aaatgacaat ggctaaaatg aaattgactc ttgccccgct tccagatttc 420 aaacttccag ttaaatttgt attgccggat ggtaacgagc aaaaaattgt gtttaccgtt 480 aagcataaaa aggccagcga gattcaagag ctttaccaga aagaaggtat taaagatcct 540 gaatttatca tgaatgttgc agttggttgg gatctggagg aagaattcaa cgaagaaaac 600 gcgcaactgt tagttgatta ttatcctggc gcggcgctgg ctcttatggg atcttattta 660 agtgcgctgg cagggcaacg cgtaaaaaac taaaacgggc ggtttatctg tattaccaga 720 agccgccgac agatgcagaa ttgcaagccg taggattaac ccgcgcagac tatgaaggag 780 aagatccgcc agaggtcata tttgatgaaa gtatgatgca atcatgggat atattttgcg 840 cgatgggtac acagtggcgt agtgctggcg ctggtgctta cggttttgat tataatgttt 900 tgcctatgtt attcagaatc tataaaatag acgatgaaga aatggcctta aatgacttgc 960 gcatcatgga acaaaaagca ctggaaatga tgcaagcaaa taacaaataa agcgcctacg 1020 ggcgcttttt ttatgagggt aaaatatatg gctgaacaat ttgcaggttt aacacttggc 1080 gttaacgtcg agcagttaaa cagggctgta aagtcattgc aggactttaa aaaagcaaac 1140 gacgaagcaa agaaaggcgt tgagagcttt gttaatgagg aagaaattgc aaagcaaaga 1200 gcaaaacaat tagctgatga attggcgaaa cagagccgtg aatttaaacg cattcaggac 1260 actgttgatc cgacagcggc gaaaatgcgc aagctatctc aggccgcaag tgatcttgat 1320 aagctatggc aaaagggcat tgttcccgat gaaacgtttt tccagttggg gagtatgctg 1380 gaaactcaga tcagtaagct ggagcgtaat aaaaaggcgt taacggaaga aggccgcgca 1440 gcacttgagg aatcaaaagc caaagagaaa gcagcgttag caggaaaaac tttccttgcg 1500 gatctggaat cgcaagccgc cgcgcttgga aaaacaaaag ccgaattgct ggaaatgaaa 1560 gcggcgcaac ttggcgtaag tgctcaggcc gcgccattca ttgcacagct taaagcgcaa 1620 gaaaaacaga tgaagttaac cggaattagc gcggggcaat acaagcaagc tatggcgcaa 1680 cttccggctc agattactga cgttgtaacg tcgctggctt ccggtatgcc cgtttggtta 1740 gttgcaatcc agcagggcgg acagattaag gattctttcg gcggcgttgg taatacgttt 1800 aaagcgctgt tgtcgtttct gaatccggta aacgttggcc ttgccgcctt agcagtttca 1860 atgggtgctc ttgttaaggc tggttatgat gcgtataagt cgcaacgtga cttgcaagag 1920 gcgttggtgc tgactggtgg ctatgctggc acaagcggcg cacagttcga taagctggcg 1980 caggacatta ataacagcac tgatgcgacg ataggaaaca tccgatccat cgcaacagag 2040 cttgcaaaat ccggcaagtt cacgattgat cagattaaag caatcacgaa aactaccgct 2100 cagtggtcaa cggtaactgg tgaatcaagc gacaaaataa ccgattactt taataagatt 2160 gctggcgatc cggttaaagg tctttcagag ctaaacgagc aattcaactt tttaagcgaa 2220 gggcagttaa cttatattgc agacctggag aaaacgaaag gaaaaacgga agcagtgacg 2280 gcggcgacaa agttatttgc tgatgttatg gatcagagat tagctaaact ggcagactca 2340 gcaacgccat tagaaaagat gtggacgaac attaaaaaat gggcgtcaga tgcatggggt 2400 tgggttggag atcacacgct ggcggcgctt aacctgatta ttgacgttgt agccggaacc 2460 gttgagcagg ttcgttattt gcttaatcag ggtgatattc tcattggtga atttattgtg 2520 tcggcaacca aaacgatgca aaaaatccca gggcttgata atgttggcga ttctgttatt 2580 tcgcagcaac agaagattat caataacgct aaggagaaca acaaggagct attaaaatca 2640 attgcagaac gtaacgagcg agtcaaaaaa ggcgagcagg gttacattga catgatgaaa 2700 aatcgtgcgg cggttgaaca gcaatatgcg acaaaaacaa aagagaatat aaggaaggaa 2760 gccgaggaat tagccaaacg cgataaaaag caaaaggctg aaaaagttaa ggtatcggct 2820 ggcgataagc tggaagaaca atatcagcgt gatatcctgg cgttagaaac tcaattaaga 2880 gttttgaaag agcacaagac gatcactgat accataagcc agcaacgtaa atctttatgg 2940 gctgaacaag ctaaaattca gattttgcaa gaagcctcaa caaaacggca acttactgat 3000 gaagaaaaaa gcattttagc caacaaagat aagatcctgg cgatggctga acaaaaagct 3060 attttaggcg atcagattgt tgcgcaaacg cgccttaatc agttgcaaga tagctcaatc 3120 aagtttatcc agcagcagaa agcagcaaca gaagccctag ccaaaaccag agggttaagt 3180 gaacgtgaag cagcaagagc ggcggaacgt gcaaaggttg aggctgatta tcttgccaaa 3240 ggaggcaagg aaggagatcc gcaactcacc gcaatgatgg atgcattaga caatcgctat 3300 aaagaggaag atgctaaacg tgctgattgg ttggctggcg ctaaaaatgc ctttgcggag 3360 tacggcgagg aagcaatgaa catgtatgat aatatcggca acattgcgag ccaggcgcta 3420 aacggcctat ctcaacaaat ggctgatttt ttgactactg gacaagcaaa cttcaaagac 3480 tttgcaaaat cgatcattag ccttattgtt cagatgatca caaagatggt tatcttcaat 3540 agcatttccg gcatgatggg cggctcaacc tggacgcttg gaagtctttt aagcggtgct 3600 ggttttgctg gcggcggtta tactggcgac ggtggcaaat atgatccggc tggcgttgtt 3660 cataagggtg agtttgtttt caccaaagag gcaacgcaaa gaatcggagc taaaaacctg 3720 tatcgcatga tgcgaggtta tgcaaacggc ggtcaagtag gaagcgtgac aactggcggc 3780 gctggcatca atcgcggtgc atcgcaattt gcatttggtg atatcaacgt taacattgac 3840 aacggtcaag atccaaaagg gatggaaact ggtatcaaga tgattttcac tgaaatgatt 3900 caacggtcat gctcgcaagg tggcgaggtt tataattttg ttcatgggag ggcttaatgt 3960 tagatgaatt tacctggtgt acgcagattc agagcggcgg cggtgtaatg acaactacca 4020 ataacgacag ggaagttgtt tttggtaacg gttatcgcca aaaggcatct tctggattta 4080 acacggagcg gagggagttt tctattgttt acgctggcac tgattataaa aaagtgaaag 4140 cgttcatgac ggatcacaga ttaaagcctt tcttgtggag gatgccggat ggtgatttag 4200 ggttgtttac tgttaaagcc ggaacggtag gactaactcc aataagccca accgctcagg 4260 aagtcaaagc aacattcacg gaagaattta catccatgcg ataaatcaaa gccgccattg 4320 tgcggctttt ttattgtgtc tataatggtg ctttattgga gggcatagaa tatggctgaa 4380 aaacagatca aaaagacttt tgaaaattgt ctgcaatcgc ttttccctgg tgaaataatt 4440 acgcttgttg aggttgacgg cacaaaattt ggtgctcagg tttaccgctt tcatgctgaa 4500 aatatcgcgt acacaccaga ggaattaatg caagcgcgtg aaactggaat attgccgccg 4560 aaagatatta aattccgtgg cgaggtttac ggggcgcggc cttttgggat tactggcatc 4620 ggatttacaa gtaacggaaa ggcagagaaa ccacaattag cgctttcaaa tttagatagc 4680 cgcgtatctg cattaatcag atcatataat ggcatgatgc aagccaaagt aacaatttgg 4740 gttacatcgg ctgatttaat tgatgaagaa ggaaacgtgg aggacggcgc atatagaaag 4800 ctggtttatt atattgagcg tccgaatttt gttaatcaga cagttgcacg ttttgaatta 4860 acatcacctt atgatatgga tggaattatg atcccgccac gactcacgca aagcgtttgt 4920 tattgggcgc aaaggggatg gtatagaagc gggaaaggtt gcggatataa cggatcggca 4980 atgttcgata aagacaacaa tccggtaaca gatccgagca aggattattg cgccggaaca 5040 gtgacagcct gtaaattgcg ctttggcgat caaaatgaat tagatttcgg cggttgcgcg 5100 gttgcatcac tattgaggaa aaaccaatga ttaacgcaaa aattaaactt gaaattatgc 5160 gccatgccaa tgacatttat cctaatgaat gttgcggcct ggtaactcaa aaatcacgcg 5220 tacagaaata tcaccggatc gacaacgtaa gtaaggatcc ggaaaagcat tttgaaatgg 5280 acgctgaaca atatgcagaa gttgaggacg gcggcgcgga tatcatagcg atagttcaca 5340 gccacacagg ggaaggtgca accacaattc ctagcgcaca cgatttgtgc atgtgtgatg 5400 aaactggtgt ttcttgggtt atcgtttcga taccagaggg agacatgaga atcatagatc 5460 cacaatcacg gcctttgatt ggtcgcccct ggtcgcttgg cgcttatgat tgttgggggc 5520 ttattatggc atggcataaa gagcaaggcg tgatcctgaa tgatttccgc aagccgtatg 5580 aatggtggaa gacggaacat ggtgaaaacc tttatcaaga taattacttg aaagaaggtt 5640 ttatagaaac aggtgaaccg ccaaagccag gtgatatgat cataatgcag ttgcaagcgc 5700 cagtatggaa ccacgccgga atttatttag gcaacaatca actattgcat catgcattcg 5760 gcaagttgtc gcgagttgat ttatattctg gatggtatca ggaacacaca acaatgattt 5820 gcagacataa ggatctgaaa aatgaataag gttatcaacg taaaattatc aggctcatta 5880 ggtcgccgct ttggtgtttt ccataaatat gctgttgata gttggcctga atgcgtaagg 5940 gcattatcaa gccaggttga aggattcaaa gaatttatgc aaagtgaaat cggatcaaag 6000 atgaagtttg caattttcgt tgatggtaaa aatgttggcc tgaataatga tagtgcatgg 6060 cgttgcgctc gcgaggttcg aattgttccg gttccaacag gttcaaaaag tggcggttta 6120 ttccaagttg ttttgggtgc tgttattatg gtcactgcgt ttttcactgg tggtgcatcg 6180 ctggcggcta tgggtgcttt ttcatcggct gcatttatga tgggtggagc aatggctctt 6240 ggcggcgtaa tgcaaatgat tagcccacaa caaggaggaa tgagaatgga gagccaaagc 6300 gcagcaaata aaccatcata tgcgtttggt ggtgctgtta atactacggc ggcaggttat 6360 ccggtatgtt tgccgtatgg ttatagaacg gttggcggcg ctgtattcag tgctggctca 6420 tacgcagaag ataaacttta attatataaa cccgccgttg cgcgggtttt ttgttacatg 6480 tacaatggca gcacgtttaa atagcacaaa aggttaaaag ctatgatcaa aaatatgata 6540 actggcagta agggcggttc ttcaaagcct catacacctg ttgaaatgga agataaccta 6600 atttcaatta acaggatcag aattctttta gccgtttctg atggtgaagt tgatccagac 6660 ttttcattga aagatttata ttttgatgac gttccggtta tgaatcagga tggttcacta 6720 aactttcaga atgttaaggc agaattcagg ccagggacgc aaacgcagga ttatattcag 6780 gggtttactg atacagccag cgaaattaca gttgctcgcg atctgacagc cgcaacgcct 6840 tatattattt ctgttaccaa taaaaacctt tctgcgatcc gtattaaaat tttaatgcct 6900 cgcggggtta ctcaggaaga taacggagat ctaacaggtg ttcgcgttga atatgctgtt 6960 gatatggctg ttgatggtgc tgaatataaa gaggttttgc atgatgtaat tgaaggtaaa 7020 acaatgagcg gttacgacag aagccgccga attgatttac ctgttttcaa tgatcgtgtt 7080 ttattgcgcg tccgtcgcct gacagatagc acgtcagcaa gggtaacgga tctgattaaa 7140 atgcaaagct atgctgaagt tgtagacgct aaatttcgtt accccctgac tggtttagta 7200 tatgttgagt ttgacagcga attattccct aatgcgttgc cgaatatcag cattaagaaa 7260 aaatggaaaa tcattaatgt tccgtcaaat tatgatccta tttcccgtac ttactcaggg 7320 tcatgggatg gaacctggaa aaaggcatgg agtaataacc ctgcttttgt tctgtatgat 7380 ttgattacca atcagcgtta cggactcgat caaagagagt taggcatcgc gcttgataaa 7440 tggagcattt acgaatgtgc gcagtattgc gatcagatgg ttccagacgg taaaggcgga 7500 acagagccgc gttatctttg cgatgttgtg atccagagcc aggtagaagc atatcagctt 7560 gtgcgtgata tttgctcaat cttccgtggc atgagctttt ggaatggtga gagcctttca 7620 atcgtgatcg ataagcctcg cgatgcgtca tacatcttta ctaatgacaa cgtggttaac 7680 ggtgaattca cttacacgtt tgccagcgaa aaaagcatgt acacgcaatg caacgtgact 7740 ttcgacgacg aacaaaacat gtatcaacag gatgttgagg gagttttcga aacggaggcg 7800 gcgttacgtt ttggctataa cagcacgtca ataactgcca ttggatgcac tcgacgcagt 7860 gaggctaatc gccgtggacg ctggatttta aaaaccaacg tcaagagcac aacggtaaac 7920 tttgctacag gtctggaagg tatgatccca accgtaggcg atgtgattgt tgtatcggat 7980 aacttttggt caagcgctct aacgctgaat ctatcaggtc gcgtgatgga ggttagcggg 8040 ttgcaggtgt tcacgccgtt taaggttgac gcaagagcag gtgatcgcat tctggtaaat 8100 aagcccgatg gtaagccagt tggtcgaacc attgcgcgtg taagtgatga cggtaaaacg 8160 ctaacgctaa acacgacatt tggctttgac gttcagccag ataccatttt tgcaattgag 8220 cgaaccgata ttgcacagca acgatatgtt gtaaccggaa tcactaaggg tgacggtgat 8280 gaagaattta cctacaacat aacggctgtt gaatacgatc ctaacaaata cgatgaaatt 8340 gattatggcg taaacattga tgaccgtccg acttcaatcg tgcagcctga cattttgcca 8400 gcgccgcaaa acgtcaagat cgaatcttat tcgcgagttg tccagggcgc gagcgtggaa 8460 acaatgcacg tttcatggga taaggttgaa tatgcaagcc tttatgaaat gcagtggaga 8520 aaagacaatg gcaactggaa caacacgccg cgcacagcaa acaaggaaac ggaagttgaa 8580 ggaatttatg ccggaaacta tcacgtaagg gttagatctg ttgcagcgaa tggttcagcg 8640 tctggatggt cagccattgt aagcgccgga ttaactggca aggttggcga accggaaatg 8700 ccaattaacc ttactgcgtc tgacaatgaa gttttcggaa ttcgcgtaaa atggggtatg 8760 ccagaaggaa gcggagacac ggcatatatt gagttacatc aagcgccaaa cggttctgat 8820 ggtcatccta ttgttgatga agcaacgctc ttaacgctga taccgttccc gcaatatgag 8880 tattggcatt caatattgcc agcgggtcat gttgtctggt acaaggcaag ggctgttgac 8940 aaaatcggga atgtttctga ttggactgat tttgtgcgcg gcatggcttc cgacgatacg 9000 agcattatca cggatcatat taaggtcgat attgaaaatt ctgatggcta taagtggtta 9060 caggaaaacg caataaaggc caatgataag atccacagca cagcggaatc agtgattgaa 9120 aacgcattgg cgaatgataa agatgtgcga cgtatgcgag ttgaaaacgg caagcgtaaa 9180 gcagagttct tgcaatcgct gaaactcatt gcagatgaaa cagaagcaag ggtaacgcag 9240 gtcactcaaa tgagtgcgca atttgacgag aaattaacgg ctcaaaatag cgaattgaga 9300 gaggtaattg ctaacagcac tgaaaccatt agccaaagga ttgatcagct tacagctacg 9360 tttgagggcg aaattgacgg tgttaagcag gatatcaaag cacagataac tgatgttaac 9420 caggcaatca ccaatgaagc ggaagcgcga gcgtcagcgg atagggcgtt atcaactcaa 9480 attggtgata cccaatcagc ggttaaccag aaacttgatt cttgggttaa tgccgatagc 9540 gtaggcgcga tgtacggcgt taagttgggg ttacgttaca acgggcagga atatagcgcg 9600 ggtatggctc tttcccttgt tggtgacgga tcaggagtta aatcacaatt cctgtttgat 9660 gctggacgat ttgcgataat aaacaaccaa cagagcggag gttttacact accgtttgtt 9720 gttgagaata atcaggtgtt tatcaacagc ttacttgtga aaaatggctc tattggtaat 9780 gctcagattg cggatcagat taactctaac aactggagca gcggcgcggc tggatggatg 9840 attaataaga atggttatgc tgaatttaac cagataacgg taaggggtac agtgtacgca 9900 aacgccggat cattcactgg taacgtttac gctactgatg gttggtttag gggtactgtt 9960 tacgctgaaa aaatcgaggg cgacgttgca aaggctgttg tgcttgggtt taacggatcg 10020 gttcacattc cggcagtaaa ctacaacaga caccttgtga ttccttatgt tggtttgcat 10080 ggctatacct attcaggtgg tacgtggggc ggtggtactg tttgggttga ttcatcatat 10140 ggcggtcgct tggctaacgt tcaagctacg gctatgcatg gcggttcaag tggatacctt 10200 ctgttaccag cgggtaacgc aacaacgctt tcatatggtg gcaacctgaa ccacgctaac 10260 gcggttccag tattaacaat actgctattc aaagcataaa aaggaggtaa atcaatgccc 10320 tggcttaacg gtcggggcat tttgttttct tttgttccac gatgttcaca accgaatccg 10380 gcgtaaaatt gaaaacgtat tgaaaacaaa aagaacaaat taaatatcta tataatacat 10440 acacttagag tttaattatt attatatata tctatttgta ttcttattgt ttctattgtt 10500 ctctatggtt tggtgtggtt gtgtgttttg gattggttaa tgattatata tctcatggta 10560 tgtatttata tatggtatcc ggcgtaaatg agtgaacaat ggaacactaa aacaacaatt 10620 ggtaaaatca taaacttacg atgtgatttt gttgtcaatg ctagttgtaa acgtatgcaa 10680 aacaaaaaga acacttaaag tgtattgact acatgaaaca ttcacatata atgcagacat 10740 accaacgaga ggatgaccaa atgagcgatt ttaaagttta cacctttgac cagttaacca 10800 acgatgaata tcacgatccg aaaggctggg cggctgaata cgttagcggc tcaagcctgg 10860 cggaaatctt tgcaacttgt ccggcagcat ggaaatacag accacgcgag gaaagcaaag 10920 cgcttgtttt tggtacgcag tcacatacca actttcaaag cagtgagttg ttcgcaaaag 10980 agtaccgccg agcgccagcg ccggaagatt acgaaaactt gatcacaagc caaacggcat 11040 tagcaagcaa gttaaaatct tttggcctga ctggtacaag cgggaaaact tatccagacc 11100 tgataaaaat gatggttgat tgtggtgagg atcttaatgt tcagtggctg attgaaatga 11160 tcgcagagag ccaggcgcga gcggataacg ttgaattggt tcctgctaaa gattacgacg 11220 cttgcgttac catgcgccgt gtactggaaa gcataccgga acacaacgct tgcatgaata 11280 gcacaacagc acaacgcgag ctatcaattt tcggcactat aaaaggcgtt aaggtaaaag 11340 taaggcttga ccatgttgat atttgcaaag atgtgctggc aactgttatc tcaggttatg 11400 acggtgaagg gaatccggta tacgaaacga cgcgttttga tgaagcgatt gtgatcacag 11460 attacaaaac aacgtcaagc gcaaacccaa ccgaattttc aaggctggca ttcaatcacg 11520 gttactattt aaaaatggca ttgcagcacg acctattcaa gaaagcgtat ccagaagaaa 11580 cgcgacctgt tgttgttcga cttctggcgc aggaaaagaa agagccattt ttaccgatcg 11640 cataccgcat gacagatgaa caattaaaga tcggacgttt gcaatacatg agcgtgataa 11700 accagtttgc agaatgccag gtaaatgacg tttggccttc atatgcaaac ggagagccgg 11760 aaattgacct gcaaacgcct gattgggttc gtcgccaata caaaggtttt ttataaatag 11820 cactttttgc taaaactgcc attagatgcg tggtatacta cacgcatcaa ccaacgagag 11880 agaggaaata aaaatggaac agcaattaaa tgaacaagac attcaagatg tgcgcgatca 11940 tgtaggcgga tttgtaaatg cattagggat tatgcaacgc cagttcttgc gcgtaattga 12000 tccaagcgaa tcaccggaaa ctcttgaata tgttcgcaag gttgcatatt cgattgataa 12060 cgttgtgtta acggttttta aaatggaaaa taacgaagaa catcgcaaga tgattaatca 12120 ggcaagtgaa attatgatcc agaatttgat tgattatcac aacgagaacg aagaaaagca 12180 ttaattaaca cggcggcgca agccgccatt attaaaccgg agtcaatgaa atgaaattat 12240 cagatcaaaa cgcaaacgta attaaggcat tgtttgaggc gcgtcagtta ttcacaaaag 12300 taaaaaagga taagcaaaat acgcacctta aaaacaaata tgcaacgctt gacagtgttc 12360 ttgatgcaat tatgcctggt ctcactgata agggtttatt cctgacgcaa gatcagaaag 12420 tcagtgaaga tcttaaatca atgactgtat tcacgcgatt cattcatgtt gaatcaaatg 12480 aatggattga atatagcttt acgcttccaa tgcagaaatt agatccgcaa ggtggcggct 12540 caacaaacag ctatgcgcga cgttatgctc tttgcactgc tcttggcctg gcaacggctg 12600 acgatgacgc aaatcttgcc acaaaaaacg cgcaggattg gaaaaaagat cttgatgctt 12660 gcgataacct tacagacttg caagaaacgt ttaaaacttc ttacaagcaa tctgatgcgg 12720 ctaaccgtag aatcattaaa gagcattacg acaagctgaa agcgaaaatt gagatcggaa 12780 aggctcgagg ttttaatccg gctgaaccag cggcaaatgt tgcgaagaat gaaaaagttg 12840 aaaaccaacc gcaacaggaa gtaaaatcac agtctatcac tgactttgaa taatttatct 12900 cggggcgata acgccccgat aaggaaataa aatgcacgta atcaccggag aaattcgcaa 12960 agagccattt gtaaagcaag gcgcaaacgg cacgttatac atcgttgagc tttcggagtc 13020 atttaaagat cgtgaaggta atcgacaata caccaattac aaattcttct ttaatgctaa 13080 aagcgacggc atgaataatt ggtatcgtga agcatttcag cagggtaaag ttatttctgt 13140 atcatgcgat cagttacgca ttgatagcca ggaatacaac gggcagattt ataacacatt 13200 gacgtcggcg ggttttccta atcttttatt cagccaacgc ggagaagggc aagcgccaca 13260 acagcagcaa cctcgacagc aacaacaagc gccacgccag caagcgcctc aaaacaacga 13320 gccgcctatg gattttgatg atgatatccc attctgataa aagaaaagga gccgaaaggc 13380 tcctttttta ttacttagca atctgaataa acattgattg catttcctcg atcatctttt 13440 tcatttctgc tacttcattt tttagctcat tgatttcatc ttgttgcttc tgaatggttt 13500 cattctgctt atctgaatca tcaatcattt cattaattgc gttaaccaaa agagcagtta 13560 caccgccata gctaacacct aaatattctg aatctgaaat cttgtaaacc gcttccggta 13620 aaacagtttg cacgtcttga gctattacac cagcctcctc cgaatagaaa gaatattcat 13680 cttcatcaag attgttgcgc ttaaaataag tgtaaccttt tattgatttc actttttctt 13740 tagcgctttc aatctctttt aggtttgctt tcaggcgaat atcggaagtg ttaacccatt 13800 gcacagcaga cgcataacca gcatcgttaa acccataaac agcgcccctt acattaaaat 13860 gagtttcgca acctccagcg ccatatctgg caacatctat accaaaagcc caatcaccac 13920 cccaccttac aaccttaaca acgttaacgg ctacatcatc gcttggaagt tcaacaagca 13980 taccagcggc gcgatcacgc catgcagtaa aagcaccacc ctcaacatgt ccacctataa 14040 agcaattcgc accacccctc ctagcacctt caacataagc aaaacgtgca tcttctggag 14100 taaatgattg aatagttctg tatgcaacat tgctactctt tgtaaatgca aacatcactt 14160 gattatcagc aatagcttga agaatgccat ctccattttg cctaaatcca gtatcagtat 14220 ctccaattac tatagagtta ctaccaagag cattaccaat ccccctaccg attgttattg 14280 agttgtaatt cagcgcgttg ccactttcat caaactgata atagtttgtg tcgccacctt 14340 ctctagttac ttgaatagtt gcctttgcaa taccaccctg aatttcgttg taaacccttg 14400 aatacgaaat ctgtactcct tctgcatttt tgtttaacaa ataaagtatg ccggagtttg 14460 cagagttttt ttctgttagg cttattcctc taaaaacagg aatgtcagtt tcaccaatgc 14520 caaaattact ccttgcttgc cctatgcttt gagcgccagt gcctcctcgt tcgatgtgta 14580 atggtttgat gtttccataa tcatcttgaa caccccacac accattgtta gctacaagca 14640 tatttagttt tccatcaggt gattttatgg agttaaaatc tcctggtgtt tcgctagtaa 14700 tgcaaaaaac accttgcaca cgaaagtttt ttcttgcccc ttcaatattt tccgcaccag 14760 tgccgccagc accaacagaa agagcggatg tgctgttatc gctatttctt gcaactcgcc 14820 actcgccgtt gtttgcgatc cgtaattcat aagtccatgt agggttacga aatgagttat 14880 aatcacctgg attttgcccc ttaactgaat taacagcctc aactttcagc gcctcccttg 14940 ctaaatcttt attagcaaga tcattaagat tctgatcctt atgcagaagg ttttcagggt 15000 taacttgttg tgcccattgg cgagcttcat cacgcgcagt ctcagcgcca gcctttgcag 15060 agtcggcggc aatctttgat tgttcagcgg caagttgtga ttcctttgct gcatcgcgag 15120 cattaacaga ctcgttttta attgatccaa tttcagaaac ggcgctatct tttatttgtt 15180 gcgtctgcgt ttttattgcg tcagtgtccg ccttaatctg attggtttca ttgacagcag 15240 catccttaat ctgttgagtc tcagatacag cagaatcctt aatctgttgc gtttcatttt 15300 ttagttgctc agttgccgct ttgtcgtttg ctacctgttg agcatcagca ttaaccttgt 15360 taactaaatc ttgcaatgtt ttcagatcga agtttttaaa gaattcaatt gcttcctcaa 15420 ttaccgtttc tttactttgg taataacgca gcgtttccgc tacatcttgc gccattccgt 15480 caacagtcaa agaatcattt aacaggatca catattttcc atcaggaaca acagcgccat 15540 cagtgctaat tgctttcatt tcagtgttgc taacaatctc actaataaca gcaagtttaa 15600 taactggctg atcaagaaat acaatggttg caccagtgcg gattaatgac aaaggttcac 15660 gccattttgt gccagtacca gttataacgc cttgcgcgtc catcgcggcg gttccagtgc 15720 gatataaagc cataatttca cctcatttac ttgattaaaa tagtgtagtg attatgcgat 15780 attgctatgt tacatgcaat aaaaaacccg ccgtagcggg tttcgtcaac aacaacgaga 15840 agtcaatgaa acagattcat cgaaaagtgt tagtagcagt atgaatatta taataagtca 15900 cttgccttgt aaagcctccg ctctattcca tttttgaatt taccgttaat tggtaacatt 15960 acgtctaatt cgcgatcatc agcagcaatc aaagtatcgc gatcattagc atggcacaca 16020 acgcgcattt tgcgatctcc tttgtacctg tatgctacaa tttcaagatt tgatgcagag 16080 aaacaagccc atacttcttg tcccgtcgcc aggtggatat gttgcgcatc aatccaatca 16140 acgcaaagat agatcgtctt atcagtcttg ccagttactg caacagagcc tcgcgtataa 16200 tccttagcgt aaaaactgct ttgaccttcc tcattgacga aaagaatatt acacatttca 16260 tcatccagtt catcgctatg caccaggtgg caaggaatag cgtgaatatg ttcatcatag 16320 ccaccaggat ttatttttac gcctacttga tatgattcac cgtctagcgg gtaaaacgcc 16380 tcaaaaatgc ttagatgcgt tgcggtcatg ctttctgttt tgctcattac ttccatgcac 16440 ttttcatgag ttgcttgcga tccgaaattg taaccggagt cacgttttgc cgctttgtta 16500 gccttgttga caacctcaac cggaaccagg tttaaccatt cgcctagagt gtcgattgct 16560 tcactgtaat tttcaccacg tatgcgcatg aataatccga tccctgaatc attcccgcat 16620 tggttgcaaa tgttgccgcc gtcgcctttc tcgttgattt tgtccgtcca tctgaaacgg 16680 tctttacctc cacaataagg gcatggttga tgctttccgt taaaggttaa ttgaggaata 16740 tcacaaagtt tcatcaatgc cgcttgccac attccgcgca tctttgggat cacttcttct 16800 ttctggtaat gcatttctca acctccaaat aaaaaaacct ggcacgatga taaaccatgc 16860 caggccggaa tttttagcaa tcagtgctat cgacggaaat cacaacgcgt ttcttctgat 16920 tggcttcctt tgctttgcgc cgttcttctt tcgccttgtc gcgttcagct ttgcgccgcg 16980 ctttctcctt ctcgcgttct tctggagtca tagaccgtaa cggatcgtaa ggccgaacaa 17040 atcctcgcgc ctccgcttct tcttttgtga caaatcgcat gagctttctg ttgctgcaac 17100 gattctctaa tggcttgccg ttgttgtcaa agcgcaaatc tggacggcaa tatgaagcgc 17160 ggaaaccgtt aaaacctttg cggcggtaat ccttttggat cagttcagcg cctttagctg 17220 aaatcattcc gcgctctttc catccccaca ctgttttaag gttaacgcca atctcacgcg 17280 ccaacgcagc acagccgcca taagcgctaa ccacttcatc aagcctagct tttaatcctg 17340 cccttacttc ttcctttagt accagataac cgctaacagg tgcgcggcgc ttccggtctt 17400 taccacgcct tacgccgttg ttaccattga ttgtgcgttt gtcgatttcg cccgttgagg 17460 ccgcaatttt gattccgccc atattttcac ctatagcact ttttgctaaa actattgagt 17520 aagtttgtgt atgatatggt gcaaatggca tttagtcaaa aggataagta atgattccta 17580 acattgaaaa acagattgaa gaacttggag cggaaaagat aaaggcaata caaaaacgct 17640 tcacagttgg tgaaattgtg ccttacgaat atcaatgcgt tgcatacgtt gagatcgcaa 17700 agcgtttaag ccgttatgaa catccgtttt ttgttaaggc ggctgtatcg gctggtaaaa 17760 caatcatctt tgctatggtt gcggctcagt gccgtaaaat ggggttgaaa gcgttatttc 17820 tggcgcgtca atctgaaatt gtggatcagg attcaaaaga gattagcgcc tttggtgttc 17880 cgaattccgt ttattgcgct ggcctgaata ccaaaagcgc ttatttcccg attgttgtcg 17940 gatcggaagg cactgtaatt ggtggattga ataaagcgtt gggtgattac gtcccgcaag 18000 ttttaggtat agacgaatgt catcaagtca actggaatga tattgttgaa gccgaggaaa 18060 acggcgaaag catagagcaa atgatgacac cgaaaggtga accagttgaa gggaatccgt 18120 tgctgattgg ttcaggccgc gcacaataca ccattatcat tgcagaaatg cgccgccgtt 18180 gccttgagac ttacggtcat cagttgcgca tatttggaat gacaggatcg gaatatcgcg 18240 gaattgagcc gatacttgtc gaagataaaa gaattcctgg gttttggcgt gagcagatca 18300 caaacattga tactgattac ctggtaaaat ttggttcagt tgttcccact tattttggca 18360 atgttggtga tttgggttac gacttatcag aatttacgcc agtacatgaa tttggcgttg 18420 ctgattatga ccagaagcaa ttaaaggcaa tgagtgacaa gattcacaaa tcaggaacca 18480 tgacgcagaa aataatgcag atggttcatg aggtgatgaa aaaccgcttg tgcgctcttg 18540 ttacttgcgc tgacgaaagg cactgtaaag aggcggcggc ggcgcttcct cctggcacga 18600 aatatcacat tattaccgga aaaactggag agaaacagag gcgcttatgg cttgaggatg 18660 cttatgaagg cagggtgaaa tatatttttc aggttcaggc gctaacaact ggcgtgaacg 18720 tgccgccgtg ggatacttct gttattttgc gtaagatcgg aagtctcacg ctattaactc 18780 agcttttggg gcgtggtatg cgtcaattaa aaacatacca taaagaagta ttggggatgc 18840 acaaaacgga tcaccttgtt ttggattttt caggcacgat gtttgaaatg ggtaatttgt 18900 atttcgatcc gatgttagag caagcgcaat tccagttacg taaatcacag gacaaagatc 18960 cgaaatattg cccgatttgc ggtacagaaa attcattcta tgctcgccgt tgcattaatg 19020 aggatgaaaa cggaaatcgt tgtgaacatt tctggacgtt ccaggaatgt gaggatcaga 19080 ttgatgaccg cacaaagaaa gttattgtaa aaggttgcgg cgcaaaaaat gacgttgcgg 19140 cgcgtgtatg ccgttgctgt aatgttcagt tacgcgatcc aaatgataac ttatccggca 19200 agatgtaccg taaaaacgat tggtgcgacg tgttagattt taatgtgacg ctgacaaaaa 19260 accaggctgg cgtaatattc tgctacacgt tacgtgatag tggcggcgta gaattcaggg 19320 catacgaaaa atatttccct gaatcagaac acaagatatg taaaacgctt tggacaaagg 19380 cggttagaac acatgttata gatcgccaag ccgcaaactt tatgatcact tgccgcaatg 19440 caattaagat catgagttat gcggggcaaa tattgccgcc aaaacgtgtt acccatcgca 19500 agaacacaaa gaaagaagat ttgatcgcac agaaggagtt ttaaaaatgg ttactgataa 19560 aggcgattat ctggagtatt acgacaaatc agatccagat gacagaaaag aggaagcgca 19620 ccaggttgat tcttattcat ggttaacata tgagtttcca atgtggcttg tttggcacac 19680 taaaaacgaa gggaagaaaa caataggcac agcagtaaag gatcagcaag caggtgtaaa 19740 aaaaggagta agcgacattc taatcctgac tggttttatt ggctgcaaat atgcattcat 19800 tgcaattgag cttaaacgag ccaacaagaa aggaactaaa gtaagccagg aacaaaggga 19860 attcttgcgc cgcgttaggg aatgcggagg atttgccgca gtatgctatg gtgtagatca 19920 agtaaaattg gctatcgcag acgctacaaa atagcacttt ttgttaaaac aggcgcgacg 19980 gaatgcgcca taatgcacac atcagaacga aacggagaat taaccatgaa aaagatgtta 20040 tcagtagttg ctttatcact tgctcttgtc ggttgcatgg ataaaccaaa aacatatatt 20100 tgcggaaatg aagcgtttga tgttacaagc gaatatatga aagttgtgca aggcaaaagc 20160 acaggcgttg taatggataa tatcagcaaa aatcaatata agttgtttac gccaattgga 20220 acggctttct acacagttaa tgaaaacaca attgatatta aagttggcgt ttatcagaac 20280 actctaactt gcgaggtaaa ataaaatggc taaggatttt caggacaaag caaccattga 20340 tacttttgag ccggaaatgg attttcatga actgcaacac ggcagatatt cagatattgc 20400 gttgcgttca ggtggtcatt atcagccagt tagagaaaac ccaatgttca aggttagcgg 20460 caaccgttat gcaggaagta aaacgccgga cgttgtgcgc gacttgtggg aaactccaga 20520 tgaaattgtt gaataccttg ctgaacgtta cggcaaatat gatcttgatg cagccgcaag 20580 cgaaaacaac aaaaaatgcg ataagtttta tagcaaagag acaaactgct taaagcgttg 20640 gtggggtagc aataagcacg tttggttaaa tccaccatac agcaatccag atccatttat 20700 caaaaaagca attgagcaga tggaacacgg aaaccagatt gatattttac ttccggctga 20760 taattcaact gcatggttta gtgacgcaca aaaacacgcg gcagaaatta tctggattac 20820 tggtgaaacc tggaaggatg aagaaggtcg cgaatattcc agaactggtc gagtagcgtt 20880 tatttctggc atgacaggtg agccagttag cggaaacaat aaaggtagtg taatttttat 20940 catgcgccaa ttggaagaag gtgagcaaca gcaaacgcac tatgtgaaga ttagcgacat 21000 ttgcccttcc gtaaaaaata aacgagccaa agcgcggagt atttaaaatg aacattcgat 21060 tagccgatca catgaaatgg gatttatttg tcggtatgac aattcgctta cttgataagc 21120 atggttataa cgctgataaa atggagttga taaactgcct ggatatgaca tttgacgaaa 21180 tcaaagttgc gcctttgtac atgtgggaaa gaaaaattga gcacgacatg atacaataca 21240 aacgcagaga tggcgcacga ttttttaatg tgaaatagca ctttttgtta aaacccgcta 21300 cggcgggttt tgtattatgt tcttaacgga aaacaagagg atttcaaaaa tgaaaatcgt 21360 aaacgtcgaa tacttcaaag atgcgcaatg gctggctaaa gaatcattgt ttatgcttga 21420 tcgcaaaaat ccaaaggtta ttgcacaaat actttggaat gacgcaatgg agttacttaa 21480 aaaggcttac ggttatgaaa gtgacaaaag gtaaaaagga aacttgggag ttagcgaaaa 21540 aaggcggtct tgatgagggg atcgccaaaa tagcgcaata tttcgatatc aaagacgtgt 21600 gcgttattgt cggtaatgaa atggcatatg tggaagaaaa gccgcgcaag gttcatcgcg 21660 ttccggcaat tccgacaaca atcgactata aagaagtgat caacaaaaca aaagagcaaa 21720 gtaaaaggtt taagtgatta tgtggatcga tatattttat tacaaagatg gtgttattta 21780 ttggaaggta aggaggaacg gaagaaacgg aggcgttaag cctggggata ttgcaggatg 21840 taagaatgaa tatggatacg ttgtgattgg ttataatgga aagttgtatt atagacactc 21900 tatagtttgg gaaatgcata atggcaaaat tccagatggt tttattatag atcataaaaa 21960 tcctgttgtt gatggtcttg aatcagatga cagaatagaa aatcttagaa taataaaaca 22020 cgcagataat ttaaggaagg gatctaatag gctaagatcc agtaattcaa gtggcataac 22080 aggagtttca tatataaaac aacatgattg ctggagggct gcaatttgtg taaacggtaa 22140 aaagatgcaa aggaatttca agtcaattga tgcagcaatt aatcagcgtg tttattggga 22200 atctatttat ataaaatgag ataaatttct gttattttgt tgccgcctgt tatcgcttgg 22260 gttattatgt acgccgttgt tatgaattaa gaggatgtta aaatgagttt aaaaaagatt 22320 accgacgagc aattgatcgc tgaacgtgag gcgggtttaa aactgcgtga gattgcggaa 22380 aaatacggaa tgagcttgcg ccaagttgag caccgccaca gcaaattagc aaagcgtggc 22440 gaaatctcaa ctatcggatc acctggcttt gctgtaattg gtgagtcaaa gttagttgat 22500 aaaaacggca atactaaact aacctggatt aagacgcaca aagataaaga ccagttagag 22560 gcaataatgc aagcggctat ggctgcattt tctgacgaat tgccacgcct ggagccgcaa 22620 catgaaaaaa ttactgacta ttcgcaaacc cttgcgctat atccgatctt tgatattcat 22680 attggtgcaa tggctcataa gcatgaaagc ggcgagaatt acgacacggc aacggcagaa 22740 aagatcatga atgaattctt tgattatgcc gtcgataaag caccgaatag cgaaaaagct 22800 gtattgctga ttggtggtga tatgattcac agtgacggcc tggaggctgt aacacctgca 22860 agcggtcacg ttctggatca agatagtcgt tacgcaaagc tggtttatgt tgcaattcga 22920 gcaacgcgcc gagcagtaag cagaatgctc gacaagcaca aagaagttga gatccagatt 22980 attgagggca atcacgacca atcaggaatg atctggctac gtgctgcaat ggcggcggcg 23040 tatgaagatg aaccacgcgt taatgttgat gtttctccgc gtgtagtgca tcacacgcaa 23100 tacggaaaaa ccttccttgc ttatcatcac ggtcacacag tgcgcaagcc tgaaacgcta 23160 atgatgatgt gtgcggcaga ttggcgtgaa gatttcggaa tatcaacgag catgtatgct 23220 catgtggggc attggcatca ccaaaccgtc acagaaacga gtttaggcat tgttgaagtg 23280 catagcacta tggcagcaaa agacgcttac gccgcgcgtg gtggctggcg ttcacgtcgc 23340 agggcggcgg taattattta tgataaggaa tatggcgaaa tagggcgttt tatgttttac 23400 cctgaaatga ttgcttaatt aaattatcaa gaaaaaggct ggctaagtgc tggccttttt 23460 ttgtttttgt ggttaacata ttcacactaa atttggcatt gacattttaa tttaattgag 23520 gatcagatca tgagggactt tcttttaatg gctcaaaact ctttcggagg tgcaacggta 23580 gttggctctt ttactggtga gtttttgctt gcactggcta cgtttatttt gtttgctatt 23640 tttggctgtt ggggcgcatg gctgaaatgg cgtgatagta aagcgatccg agaggcttta 23700 gattcagggg atttaaaaac agcgcttaat ctacggcaga aagagagggt gtaatgacaa 23760 tcaagaaagg tatagccgcc accgtcacag gggcggcttt aatgttggca tcgcctttga 23820 ttgaagaaat tgagggcgta aagtataagc cttacaagga tattgcgggg atctggactg 23880 tatgccacgg cattacagga aaggatgtga ttcttggaaa ggaatatacc aggcgagaat 23940 gtgacgcgct attagcaaaa cacatgaaag tagcggctga cgctgttgat aaggcggtta 24000 atgttgaaat tcctttatca atgcgagcgg ctctatactc attcactttt aatgctggta 24060 ctggcgcgtt tcgtaagtca accatgctga aaaagatcaa caatggtgat ttatggggcg 24120 gttgtggtga gctatggaac tggacatatt accggaatcc taaaacgggt aaaaaggaaa 24180 aatcaagagg attgaagaat cgccgcgccg tcgaatataa atattgtgta agggatctga 24240 ataaatgaaa agaaaattac tggctattat gatgtgcatt gctctttctg gttgttcagc 24300 ttcaagcgtg ttacctggtc tgattggtag caagcctgat ataacggcac aggctggagc 24360 ggagaatgtg aaacagactg ttggtgtaac cgcaaagcag gatacatcaa gcaagcagga 24420 aaccacattc aaagaatcgg cagttggtca agttgacaca tccaataaaa agcaggttag 24480 cacttcaagc attaaggcag ataacatcac agcggaaaga atagagatcc gaaataatga 24540 tggtcttaat gttccggcaa tcgcaatggg tatcttgatg tttattgctg gtatgctggc 24600 ggggtggaca cttaaacgta ataaggcggc ataagccgcc ttttctttta aaaaatattg 24660 ctcattaagt cttgaaaact attgccgcta taatcctttt cttcaataat gtgagcacct 24720 ttctttttta tatcccacag cattttttca tactcgcagc ggttaatgcc aaacgtacta 24780 ccatcatcaa catatgcggt tcctttgttg ttgaaaacgt caaatgtaac attaacaacg 24840 cgaattgttt tgatagtgca ttttaacatt ttgttttcct tttgttgcta cgtttcgtta 24900 aaacaaatat acccgataca agatcgggtg ttttaacaaa aagtgctatt caaagcgcct 24960 gacgtgaatt agtagttcac cgtcatcatt tgttaactgg tgctcattat cttcaaacgc 25020 tcgcatatca ctgtaaagca acaacattag tgcatagaca aaatcatcat gcgaaatttg 25080 cattgctgaa ctttctttca aaataagatc tatcaaatgc tggcttgctg aataattctt 25140 catcgctcat tttctcaatg ttcatcatct cccacacata acgattggaa tcaccttcca 25200 ggctatgcaa ctcaaaatta aacggccttt tcatcttgtt tttgcaccag tacacgcaat 25260 gaacgccgtc aagatatcca taacccatca ttcgcgctat aaactctttg cttgagcttg 25320 aagcgaaata gcggggcgtt acccccgcat ttttggcgag cctttcacat tcacggtgac 25380 ggaatataaa tcttgctaac tcttggcggt tgaatttctc gcgagactcg caaaagcgat 25440 acagatctaa taagaacatg attaccccat taacgccgga tttacataaa caaatttacc 25500 aacgctgcaa acataattct tttcttcaag cattggcaat aattgttcct caattcgttt 25560 catcacgcct gattgacctg taaacggctt aaccttacgc gcactttcat agatagctct 25620 tgcattggta acgcctttat tttgacgtga gatcttaatg attatttcaa ttaacttgct 25680 catttctgca tcatcgccag cgtgaccgga agcattcgcg gcgttgatat atgttttgct 25740 taattcgctg aacatcacca aagcctcttg cattgtctca acttcaattt ctcgcgactt 25800 ttgcggcgta ccgtttggat taaaccaatt gcggatcgtg tgcaatactg acgcaatgcg 25860 gatcacttgc ttatccattt tacccaaagc accacgcaac attgtatgtg aatatttccc 25920 gccgtctgct aaatctggct ctaattgctg gcgagcaata ttaagaacac gcattgcaga 25980 acgtgacggc ttcaatacca cattttgttc agtcatgatg ttatgaatca actggaaata 26040 ttgcgccttt aattctttat caactggcgt gtaatcaaga tcaccatttt catcaataaa 26100 tacacgctct cctaacattg ttttttcacg tacaagcaag aaacgttcac taacaccaat 26160 accacgcgcc cctgcatcca ttatcccttt tatcgtttca tcctgggcaa ttacgctcat 26220 gcagcccaac gcaacaaaac tcatattatt ttcagagttg gcgcgagcta ttgaaacgtg 26280 tccggcgtcc catgctttta gaaccaattc gctgttagtc ttgcgatcac tgttagcgta 26340 agtcaatccg agtaagctgt tgatgcttgt tgcttcatct gaaatgacag caaagttacc 26400 ctggcgattg ttgatctttg ctaaaccttc cggcgtggtg tctgataccg gaaaaacgat 26460 atcgcacatt ttttctaact tttcctctaa ttcctccttt tcctcaaaca acgccgctaa 26520 ctcagtgccg gaacgctcag ctttcaattc cttttcaacg ccttttaatt tcgctgcgat 26580 cttctttcgc tctttcttgc gttgttcgtt gattcgctca acttcacaga ccattggcgc 26640 aatggccagg ctgttgattg ctgatttacc agttgatggc ggctgactgg ttacaacata 26700 aatagccgtt ggttgttcag tgccgtggta ctcaacttgg aagcgtccaa ccatagcggc 26760 ggaaacgcaa cctatgaaat gcatataggc cgatgattca ggaaattgaa cagattgcgc 26820 aatgctgcgt gacagtttac ccacaacatc aacatcatta cccaaagaga tcacaggata 26880 acggtcatta gcgccgttga tatccttcac aggttcccaa aagctggacg attggcggta 26940 gccgttcgac tgaatagcaa cgcgaagcgg cgatgttccc tgcgattctg caatggctat 27000 aacttgttga ggtgtcaatt tgttggtgtc aaagtcaaac atctaaaaat ctccttcttg 27060 ttgccgctaa ttataacggc aacattctac agccttttag caattcgtgc tatttgatcg 27120 gctcaaccct ggctaataac tttccgtcta taatgctatg acattcaaga gttttaggat 27180 cgaatgcaag cgaacgatgg aacgtgtgac caatcagcaa atatgaatgc ttatccctga 27240 tgcgatagac cgtttcacat tcctttaatg aggatatcgc actgaaaaca accttgtaac 27300 ggtatttttg catggcctca gaacggtgag gaatgtaatc tttttccggt aaactcatga 27360 gataaccccg tgattcttat gatagccata aattttatct gcctttactc tcgcttccct 27420 tgcttgctct actgtatcaa acaatcctaa atatcttttt ttaccgtcta ctgttaatga 27480 agcctcccat cttcccgccg ccttactcca gtaaactccc atctctccag atgaagataa 27540 aacctttcct ttatttctta aattatcaga tcttgaaacc aatctcaggt tttcaattat 27600 gttatccgtt ctatcatggt ttatgtggtc aatatcaaat cctttaggta tttcaccgta 27660 gtgcataaac catacaattc tgtgagcata tgccttaatc ccatttgcac caatagaaac 27720 ataaccattc tttgttattg ttcctgcaat aagatctggc tttactccgt ttctacgctc 27780 tcgccaatat aagacgccgt tttcatatct aagcattgac gagaggattt ttaacctctc 27840 gttttctgtc atttgtaaat agcctcaaac agataagcac cgccagcggc agagaatcca 27900 acttcttccc gatacagcgt gaaacggtcg ccgttttcgt cgaacacata tccagcaaca 27960 gcacctaaca cgcggccttg ttctatctga taacgcttat cggctttgaa agttttcttg 28020 atctgcatgg agtgatttac ccatgtgcat ttaatggttt tggtcttaac atttcggaat 28080 gttaatcctg tcaagttggc gtgaatccat ttttcatcta acttaacgaa agtatcatca 28140 ttttcgtcta cccgcgcttt gtataggcgg ttatcagatc cgataagttt gattgatttg 28200 aatttagcca tttgatttat ctccttttgt tttcgttgaa agcattatgc cggatacgtg 28260 atccggcgtt ttaacaaaaa gtgctatttc tgatttgcca ggcaaacagc aagcgcaaac 28320 ccgcgcggag ttagtgagcg gatcagtttt gttcgcttag actttcctcc gagcttttgc 28380 cagcctggat tgttgttccc tgttggcaat actgcatttg tatccggcat cacaaagccg 28440 ttacccgtcc aaaggcaagt ttttttggtg tacgcatcgc gagcgggaat gatttcagga 28500 aagtgaggat gtttatcatc ttccggcaac agacctccaa acgcgcaagg atgaaagata 28560 aaatcaggtt gccgccacat cgacgacagg acgctaacag gattctcgat catgtatggc 28620 acgttgaacc aatccgcgat ttcagccgca atcttgcatg ttgaaacggc cttatcctgg 28680 aatagcggat cacgttcacg ctttgcggca aagtgacgcg aaccgctaac agctaaatca 28740 gtgcatggag gaaatgcgat cacaaaatca ggcttgccgt aaacctcatt gcgagcctta 28800 aagctgaaat ctgaatcaat ccaaacgttc acatactcaa tgtttggatg acttacgcgc 28860 actgaatggt aatcaccatg atcggcatca tcataattga aacagattac tttatgacca 28920 cgttcagccc acggcaacgc atcataaccg gaaccgctaa aaagagaaaa aacaaacatg 28980 cttaaatcct ttgtgtgcag ccttgtaagg cgctttgttt gatagctatg caatcgcatt 29040 ggttatggcc tgaaatagcg acagaatgcg aaacaggccg agaattttag aatgggaaac 29100 agcgcttgca agatgggtcg aagttacaac cgcaatcatt gattattgcg ttaggatcag 29160 caagcattac gctactttct tgatccatat ccatagcgcc taatgcgtca tcaagcgtta 29220 tttcaagata ggcgacttta agcgcccatt ctttgttaag tccagccgcc tccgcttcat 29280 gtaggcgagc aaagaaagca tcttgaacca tatcaacccc acttctcaat aaacatgctc 29340 atttcgtcaa tcatcgcgta agcatcgctt tcagtcatgc ggcggattct accgcctgga 29400 tgttcgccaa cagcgtaacc agtttttacg cgggtaatcg ttaactgatg gaatccagaa 29460 gcgcgatcca ttttcaaaac tactttcccg tgagctttga caatgttcat gatggattca 29520 gttttcagtt tcatttttga ttctccattt tgtttgtgtg aaagtatgat accaatcaga 29580 gagatatagt ttttagcaat tcgtgctatt tacggcgata aacggatatc ctgttaaatc 29640 tgcatttcta taacgtgcag acctaaccca acagccgcct ttgctcataa ttaaagcgtt 29700 tttatggcga atgttcactc ggtaaattct tccggttttt atgtttttga aatatgtcat 29760 aatttaacct catgatattc tatgaattct tgcacatatg cgcattcgcg cggcgactcc 29820 tggcctttta gcacttcaca gccaccatta aaaagatagc aatgctggca aaggttgccg 29880 cctacgtttt cctcagcgat cttaattccg gtacacaaac cgccaccaaa cctatgagga 29940 aaatcgtaag cctggcagcg gcacgttgct tgatgccgct ttttctttgt cattaattat 30000 taccttggct gatgcgatcc atcttgcgat accaaataat catttcttcc agtacattga 30060 acagatcttc tttcttactt tctttgattg cgtcacaaat tagcttaatg tcacactcca 30120 taactccaag atattctccg tactgcttaa caccttcaat gttattcagt ttactttctt 30180 tgcggcaacg ttgcgctttg caaatcaggc taatgatgtt gctggtttct ttggaagtga 30240 tgaagtatgc gctcattgtt atttccttaa aaagtccggt tcgtttcgat gaatgcatta 30300 tgccaaaaca aaccggatca cttttaacaa aaagtgctat ttaattaaaa tacgaatacc 30360 ttttcgcacg ctttccgtcg cctccgtaat ggtcattagc aacagcaaga atccaagcgt 30420 taggaataaa aaatagcttc atcaatcact ctccattacg attaccggat caagacgttc 30480 aagcgctact ttgaaatctg caaagaaagt cgggcggcgt tcagttaatt ccgtgtaggt 30540 atcagttgcc aggcaagcgc gatcaccttc atagcacaca actcgcttac cattggcgcg 30600 gcttattgca acaacccgat aagtgttttt aatcatgaaa ccacctcaat ttcgcaaagg 30660 tgctcgacgc caggcgcata aacacgcttt ttgtcgtcag tgacgcaaag gcaaagcacc 30720 agaccgcgat catcaacgat gtgtaaccaa tcctgacggc tatacagcat ttcatattgg 30780 cggtcgctgt caaacgtcca agcgccagtg aattcaccaa acgccttaac ctttactttc 30840 ttgcctacaa aattcttgtg gctcattgtg tagcctccac agtttcaaat acggtatcaa 30900 atgccagaag tttaaatttc ttgccaggct caacaacctg aatattccag gtaatgccgc 30960 cgtgatatgc aaccatatca tcaatatcga cggtgatttg tgtacccttc ttgaacaagc 31020 ctttttcgtg gctcgcatcg cgtacagtaa ccacagtttc gaattgagtt tgaatttcca 31080 tcgttaatct cctgattggt ttcgatgggg taactatatc aagatacccc gatccttttt 31140 tagcaattcg tgctattagc caaacgcttt aatttcgaaa tcttcttctg cctgattagc 31200 aaccgccgtg aacatttgct caagagtttc aaatccgaaa tgtttagcga tctcataagc 31260 cgctttacct tccgtctgca tgtttagcgc ttcgcaatag tgcatttcca gattgttcag 31320 gtatttgttg ccagttggga ttgctgcgtc gattttgtcc cacacgatcc aaaagtttgt 31380 tttgtcagcc gctgcgagta caaagtttgt cttgtctact gcaataacta agttttccat 31440 catgtaatcc tcagttcgtt ttgttgaggt aatcatagaa attcgcgtct gattggtttt 31500 aacaaaaagt gctattcatg cagtatcagc aaaatgacca acgttcatct taacgaacaa 31560 tccgttcaac gtatcgaacg cattcacgat cacgttatac gatcacaata tgatcacgtt 31620 gatgattgat gtgtgagttg atgtgatttg atttgattag atttgattag atttgagtag 31680 caagtgagta gaaataatca caccttgccg atcagtgaaa acttttcccg caaaacataa 31740 ctctaggcta cgcatgtttc gttttgttcc ccgacgttcc catccagaca ttccgcagtt 31800 tagcgcaacg ttacacaaca cgtataacaa caaaatatct atataataca ttgaaaagta 31860 agataattat tattatatat ctatattgtt atataacttg ttttgcttgt ttctcatgat 31920 attgataccc ccttacggat ttatatagta taaaattata agataggtgc tcttatttat 31980 tataggtggc ggtgattttt agcgaaatgg tgtaacgatt tagaacatcg ttgatttgaa 32040 aggataaatt tgaaattact gttacattgc gtgatgtttt tgttgtctaa tcaaaaataa 32100 cacttgacgc acaagccagg tttgataagg tttcggcatc gagtaacaac ggaggataac 32160 caatcatggt aaaaggcaaa atcgtaaagc gtgacctatc catagttgat gcaacgccag 32220 aagatgcaac caaagatatc atgcgcctaa tgtacatagc acggatactg gaggctgcaa 32280 aaccaaacag gctatattac atcgacatta aggcatctgg tcgagatgtt aactcattgc 32340 gccaggcaat aaacactatt gcgaaagagg atcgcagaac cattaagaca ataaccactg 32400 ataacggcta tatcggcttt gttatcatcg acgttaaaga agcgtttgtg taaagaggtg 32460 aatcatggaa caaatgacaa gtaaagagtt ttgcgataaa ttgcgcgaag ctgcaagagc 32520 ggcggcaaaa ggtgaggtaa tcaagttgcc agttgcctac ataacaatga acgtccaggc 32580 agaggacgca aagaaactaa attcatcata catccgcacg atcatgaatc gtgtgccgga 32640 agtaaaagag gttggatctg taagtgtcaa gaaaaagaca agtgatgacg gagctgaata 32700 ttacgagatc gcaatcaatc gtgacacaaa gcgccgcatc gtgacaacta atgaccttcc 32760 ggcaattaaa gagcatgagc gcgaaaagct ggttgaaaag attatgcgca tatcgccgga 32820 cttctcacac ctggacgatg aaaaggcact tgttgcaatg cgagcggttg ccacgttcaa 32880 agaactgatt aaggagatcg caaaatgaaa gcattcgcct ttgtcgctgc aatgtggtgt 32940 gttgttattg atagtcctgg cgtggcgctg gttgtgttag gtctgatttt gttgggggtg 33000 tttgatgaat aagaaacttt tagatcatgc agtgctgttc tttatcgcta ctgctggatt 33060 ttcgctgttg tcaattgctt tttcaatcct tgcttttgcc gtgtcaatgc tgaaatgacg 33120 gattacatcc tgattggcaa atagcacttt ttgttaaaac tcgattgggg tagtttggca 33180 taattacccc atcgaaacga aacgaggaaa acacaatgac taccttaatc acatgggaac 33240 acgaaacaag ccagccagta acccgcgagt ttgaaactct tagccaggct tatcttttgg 33300 ctgctaacgg tggttattac aaggcgcaag tcattgatga aatgggagtg atcgtttatg 33360 aattccatgc ataatttccg cagctacacg caggaggtgg attttgaagg agtcaagttg 33420 ccacttccta atatataccg atatatcacg cgtgatgaat acggatttat caacgcatgg 33480 cgagataagc ccaaaaagat tggcaacgaa tacggcgacg gctcagagat gccgatcacg 33540 ttaggtcatc aaactggcga gaatgtagcg gctgtaatgc gcaaatatcg ccgcagcctc 33600 acaaactcat taatttctat ctttggcata caggagtaat aaaaatgttt gaacttaaac 33660 gcgtaactgt tgagatcctg aatgatggct cttacgatgg aatgaaacac ttaaccttcc 33720 cgatccgcat tgaaaattgc attgattatg ggattgaagt cggcatggtt gacgttcctc 33780 ttgatgcact tatagcggct ggatatgaca aagagaaagg ccaatgtgct gatatacctg 33840 atagctatga aagcgacaca catttcccgt tcacatatgg agaatgccgg attgttgatt 33900 gaggtgatta aatgagcagt tggcacaatg agcacgtaat gcaattctac cgacaaagaa 33960 taaagaaaat gctaaatagc actaattgct aaaactaaat caggggcatt tgctatagtg 34020 tccctagttg agttaagcaa aaggagaaac aaccatgtta aaagcaaatg aaatcaattt 34080 cccgatcaca tttaacgact gcgcaaacgg actaagcatt acattttata gtgcaacaag 34140 taacggcgtt tggagtggtg gagataaaag cagcctttca ttgtcatatt tcattaaatt 34200 gcacaatgaa cacaatccgc ataataaaat tgatgaaaga tatcgttatg ccatggcagc 34260 aaagaaagag aaaaaacgag aagataacga tttggcagat gctttgaatt atctaaacat 34320 caagcctgga caggtgtttg ttaactgcgc tgttttcacc gcctcaatta tcgcgaaaga 34380 ttatgattac atttgcgacg ttgagctaat gccagttggc gcaatcctgg ttggcattca 34440 acgtgaatgc ggggaatggg acgaactgaa attaaagccg cgccgcattg aagtacacga 34500 cgacacgatc acattacttt ccggctacgc tggcggatac aaaaccggaa acaattacag 34560 cggcgaaatc aacattaagc gcggcacgaa agtaaaaatc aaaaacagca cgaattgcta 34620 aaacagaatc ctggggttgc ggtatagtaa ccccacacca aacgaaagaa ggaaatcaaa 34680 atgaaattag tacgcaacgc aatttatggt ttacacctca aaggcggtaa aggttcaaca 34740 tatgttcacc tttttcatgt aaatgagcgc aaccaggttg atattgcgat gtatacagat 34800 cagtttctta taaaaggtga aatgggcgat aggtttaatc cgcaattcgt agaacaagat 34860 ccaacgtata gccatgacca ttgggtttgt gatgattact ataatttcat catggttgat 34920 agtcacgtta cggacacgcg gaaaataaca gttagcgact ataagcgcat gactcgaaaa 34980 gtattgaagc ataaagcgaa atagcacaaa atgttaagcg gtcatgtgtc ctgatgggta 35040 taatgaccgc atcaaccaac caaaggagaa gtaaaaatgt ctaaagagtc tatcaagcag 35100 tttctgaaat acggttctat gattgtgctc gctaccgttg tgctggctgg catcgcatat 35160 agcattattg agtttgcggg gtaattgatg tttacgatca acttatgggg aagggattat 35220 gttaatccct atgttgtgac aaaacagcgt ttcgttattt tcgacctgga cggcacattg 35280 tcaaatggtg agcaccgctt gcacctgttg ccaacggttg atttgcacct tactgaaagc 35340 tggagtgagt ttaatcgcgc cgcaaaggat gattcaccat tcatcaataa cattgccgtg 35400 tgcaacgcca tgtttgatgc cggatacctg gttatcatcc taacaggccg tagcgatgaa 35460 gttgaggcag aaacgcgagc atggttagct gaaaacggcg tttctttcga ttggctgatt 35520 atgcgccgcg cggaggacaa ccggaaagat accgtaatca aagaggaagt tttacggact 35580 ataggacttg atcgcattgt cgcatgttgg gatgactcac caaacgttat tgcacacatg 35640 cgcggactgg gcttgacggt ttaccaggtt attgactacg gcgaccaatt gcacgatcac 35700 ctaaaatctc acggcgtgga ggaattgagc gatgacagca aagctaattg aagatcggca 35760 tggtgataac gtgcttattt acatcgaagg gaaatcggtg tggattgact gcaaatatga 35820 ggatgacaca catttgaagt ttcgcggaat ctttgacaag aaaggcgcat tggagttagc 35880 ggaacacatc aagcgtcttg cggaagaatt accgaaatag aggatttatt atgtttactt 35940 ttatcttgat cgctatcgtt ttaattctgt atgtttgcgg cgctttcctg atgcgagcgc 36000 tactaaaaga ggctgacgct gatgataagg atagttggaa gccgattatt ttatggcctg 36060 gctatatcat cgtgccaatt tacgaactaa tccgcgatgg tgagtttaaa tggtaaatag 36120 cactttttgc taaaactagc taaggcacat ttgataaagt gtgccttgtc gagacaagca 36180 aggagaatga cacatgaaag tttttatgat taaacagttt gggcgcactg catattatat 36240 catcaacgac gatggatata ttttcatttc gaactatcct actcttgaag taggtcgtca 36300 tttcataagt tcagttgacg aaaagggagt tatcaatatg gttgagtgtg gaatgatggt 36360 tcccttcaat aaagaagcgg ctgaattatt ccagcaatca cagatccgaa aacgcaaccg 36420 aaatattatt tgctttactt ttgcaatggt tgtaactgtt ggctcaatct tacttggagt 36480 gtggtcgtga tgaatactga taataaagtt gaaacgcaaa agactattaa ttatctgcgt 36540 gacctggccg ataaactgga gaataaccaa attcatttaa acgagatcgt tattgaatcg 36600 cgcaacgttt gttggaacgt ggaagaagaa acattaacca tgcaagtttt aaaggcggta 36660 gacaatggct aaaattatca tccttaatgc gcctcctggt gctggtaaag acacgatagg 36720 caagatgatt tctttctatt cgcctggtga gcaatgccgt atcattagct ttaaagaacc 36780 aatgtttgat attgcattgg ctatccttgg taaagagaac tatcagcgtt tcatgttggc 36840 ttataatgac agagggaaaa aagaaaggcc acaaacattc ctgatgggta tgtcaccgcg 36900 tgagttcatg atctggatta gcgaggatgt gatcaagcct aaattcggca aggattattt 36960 cggcgtaagg tttaaccaaa aagtaaaaga gtgtgatgct cctgttgttt gcactgatgg 37020 tggttttcca gatgaaatta tcgctctaat caatgctggt aatgaggtga agctatgccg 37080 cttgcatcgc gacgggttca cgtttgcagg tgatagccgc aactacattc gaatcaatcc 37140 ctattaccat aaaaacggat atagcgagca cgattataaa ttggttgata agcaaccgaa 37200 tgtaactgta aaggaaatta ttagcgatca ccttaaatag cacgaattgc taaaacttgc 37260 gcaaggccat ttgttatagt ggccttattc aagagaagca accaatcaga ggaataagcg 37320 atgaaaatta aatgcacaaa atcaaacacc ctgaatctga cagttggcgc tatttatcac 37380 agggaagaag gtgtaaacgg gtttagccca actgtaacgc atgagtacgg cggtcaattc 37440 ttcttaaacg gtaaagacgg attatctgta aacgttggct ctttcgtact tgcagagttt 37500 gatatcgtgg aggaataaaa atgttctgga ttcattcgcc gcaagggttg atttatgtgc 37560 ctggcattga attagaagaa cgacgctacg gcgacacggt gaacatcata acaaaggatt 37620 caaaaggcaa tacgcttaaa acgtatgttt ttaacactcc ggttaaggtt gaaaaagatg 37680 attagattta ttggttttgt tgcattgctt atgtgcggaa tattgcttac tgacaatgac 37740 acttggcaaa gcgctgttgg cggcttactt atttttcttg ctggtggcat ggttggcagt 37800 gatgatgcag caaacaagaa taaactttcc gatcatttac gcagagagac aaaatgatga 37860 atagaaaaat gctggtacgc gcattcgcga agattgctaa aagatggggc gattcagcta 37920 aaacacctta cacgtttagt atgtacggtt acaagaatgc tcgcgaatgg ggtcgcatca 37980 ttgcagataa ctatgaatca gaaatgctgg aggcttacgc aagctggatt gatacgccgc 38040 taaatgaatg gggccaggct gaatatgatt gttttgctga tgaagaaatt tcttgttggt 38100 aaggagagta agcatgatcg ctaaaaatga cctttgcaac ctgttaacgg ctggtaatga 38160 ttatcaggtt ttgaatagca cagaagaccg ttattttatt gagtgtgaca atgggaatcg 38220 catgtggatt aaaaaatgct ttttcgaaag cgagcaccta agcaatgcaa agcggatcgc 38280 agatgaagca agcgcattag cagagcgtga aagaattcgc gacatagtag cggagcaggt 38340 tagaaattcg tgcttgcctg gtggcgatat cttcaaatca atgcgcggtg aaatgacggt 38400 tgaaattcaa gatcgcgcaa ttgcaaaatc agttgattct gtaattgaga atgcgcgatt 38460 caatgcgttg actgactcag atcctaacga atacgtcaaa gtgttagagc aactgcgtga 38520 agtgctgcga gtgccggaag gtgaaaatat tgtcactcat gcaaaagttg taagagcgtt 38580 ggctgatgcg ctgattggcc tgcaaaaata gcactttttg ttaaaacttc cgcgagggca 38640 tttgttaaag tgtcctcatt gaagcgaaac atagagatta cgaaatggct gtatacactg 38700 gcaagatgtt taaatgtcac aatccgattt acggcggcaa ctatgagcta atcattgatg 38760 atgatcgcat tattcttgct gatgatgacg ctgaagaata tcacgaggtt gtagcggata 38820 agtacgataa tgacgttatg atcttatcag gaaacaaccg catcaagttt tacgcagtgg 38880 aggaataata gatgatcgca atcaacctat cagaagaaca agcgaaatcg ttgctaaata 38940 gcttgggatg gcgttatggc tcgggtgaat atgagcgatt ggatgttaaa agcgatgttg 39000 caaaagaggt tttcatccag cttgagcagc aattaatgcc acgctcaaca agtgcggcag 39060 agttagcagt gtggcggcac gaaaacggat tgaacgaaat ggatattgaa aagtggaagc 39120 gtaaaaacga ggtgttagca tgatttacat tcactcttac ggcgttggca agtttggcag 39180 caagcagatc agaaacattc acgacacgtt agaagaagcg caagcacagc aacgcgtttt 39240 aggtggagtt gttcaggctt tcgaaccagt ggaagatctg gaaatgcaag aggccataaa 39300 ggaaatcgtt gttgatgaag tttcatgtat aatcgacgaa ttgagcactg atgcgccagg 39360 gttcgcaaga acatttgaac caggctcaac gcaatgggat tgctggaagg tgaaaacctt 39420 aaacaacatt cgagcaatct tggatggaaa atttaacgaa aattgatagc tcaaatagcg 39480 cctcaatcgc attctaagag gcgcattttt taggaaggtg attctatgcc tgaacacata 39540 aaaccgtcgc agtggtgcgc acagaagcaa gaagaagcat tagagcgcgg cgacactgac 39600 acagcattgc actactttga aatgtttcaa ctttggaaat caagaggact ttaaaatgtt 39660 tggacttaat gaggctgaat acaacattgt taaacgcgct gcaaaagagt gcatgaagga 39720 tctgaaagtt gaagttacga gcggcaacaa atacgatcat attgcggctg gcattatcac 39780 caaacatcac acgccaattt ctaccttaat cactcgcaca aagtttgtat ggcttgctgg 39840 ttatatagct ggtcgatggg gtaagcctgg cgagtacgaa taataagcac ttgaatcaat 39900 gaaggttgac gtcgtgttgt ctgattggta gattaaacgc cgtgaacata agaggctcaa 39960 tagagcaaag cgcggcgcgt tagccgtaaa acttaatcgg attggatcgc aaggcgaatg 40020 acaatcgcgg gaaccgtaac ccgcaaccat tcaggagaat tcacggtgca aaacaaagaa 40080 atcgcaaagg taacaattat cgttgagcgt gacggaaaga aaacgcgata caagaagcga 40140 tttaaacaat gcgaggcgat gttaggccgg atcggtgatt tcatggtgaa attgcaagag 40200 gataagccgg atgaaattca aggttaagct aaccatccgt cgcatgggtc gcaattgcgg 40260 atcatgtaaa caagatttcg aatgcgaggt tgaagcgcta aacaatgttg atgcggttca 40320 atgtgctaaa gttctttcag gtgctaaccc tgaaacacac caattttcaa ttaattatgt 40380 gaggtctatg taatgctgat gttcattgcg ggttttggtt tttgtgcgat tatctgtttc 40440 tttctctatc tttgtgtttc gtggggtgta cagcgttgcg taaaagacaa cgaattagtg 40500 atcgccattt acgaaaacaa aactgataaa tggaagatta ctaaaaactt tgaaaagatt 40560 gctgatgaaa ttgcattccg tcgcaagttt ggcaagcctg gaagcatcaa gtatattgac 40620 taatccaaag ccgaaacata acccgccttg tgcgggtttt tttgtgtctg caatttagct 40680 ataatcacgc atcaacagga ggattttatt atggctaaaa aaacagaagc gccagccgct 40740 gaaagtctaa actttaaaaa gttatggaat aagcgatatt ccgatctggt aggtagtgag 40800 attgataaac gcgcaacgct aacgccggaa caagtcatga acctgattgt tcagtatttt 40860 gagtgggcgg agctaaacgc cattaaagca ggtgaaacgg catcctttca gggtcgagtg 40920 tatcaggata cgattcacaa gccgcgtatt tttacatggg aagggttaaa gctgttttgc 40980 ggtttttccg gcacagcact agcaaagtgg aaacttaaac caggctatga tgttgttatt 41040 gagtttgcgg aatctgtaat caaagagcaa aaataccagt tagctgcaaa cggaatgatt 41100 aacagcacca tgatcgcaaa agagcttggc attgataaag gcgacacgtt caatattagc 41160 gccaacgcca aagagcaaga caatacagaa gaagcgatga aaaacgcagt cgcaagcgtc 41220 ctaagcaaga tttaaaggtg aatgctatgt tgatttggga agatttaaca aaacttgaaa 41280 agatggctat taaggaatta agcacacacg attttgatac tttcgttaaa atctggttcc 41340 ctatccagca gggcgaaaaa tggattccta actggcatca cctttatatt gcaagggcaa 41400 ttgatgaaat tattgaaggt gtgcgcaaag ataccatttt taacgtaact ccaggttcag 41460 gcaaaaccga attgctttcg atccactttc cgccttattc atatttgaaa ttgaacaagg 41520 tgagaaatct taatatttcg tttgctgata cactggttaa acgtaacagt aagcgtgtgc 41580 gcgatctggt taactcgcga gaatggcaag agctttaccc cgcaaaaact ggtacaagca 41640 aggatgatga atttcagatc cttaatgacg ctggcaaagt tcgcctggaa atgattagta 41700 aatcaatggg cggtcagatt actggtagcc gtggcggtta catcacgcct ggtgtttatt 41760 ccggttgtgt aacgcttgac gatccagaaa agccagatga tatgttttcg aaggtgaagc 41820 gtgagcgcgg tcaaatgatc gcaaagaaca ccattcgatc acgtcgcgca cattcagaaa 41880 cgccgattat cgttattcag cagcgtctac acgcgcagga tatgacctgg tttctgatga 41940 atggcggaat gggtattgaa tttgatcaga tatcaattcc ggcgatggtt acggaggaat 42000 acgggaaatc gcttcctgat tggttgcaac cgcactttga aaaggatgtg ctttcgtctg 42060 aatacatcgt tattgatggc gttaagtatt attctttttg gcctagcaag gaaagcattc 42120 atgacctgaa agcgctacgc gatgcggatt tatacacgtt cctttctcag taccagcaag 42180 agccaattgc tcttggcggc aacgcaatta acgttggatg gttccagtat tacggaaccg 42240 gagaaaaatc aacgatgcca aaaccggatc gctttgatta tacgtttatt acggctgata 42300 ccgctcagaa agaaggtgag ctaaacgact acagcgtttt atgttattgg ggcatgttca 42360 aaggccgcat ctattttatt gacggtgtgc gcggcaaatg ggaagcgcca atgttagaaa 42420 cgcagtttaa agcgtttgtt aaacagtgct ggaaccgtaa caaagaatgc ggaaacttgc 42480 gtaaaattta cgtggaagat aaagcgagcg gcacaggttt gatccagaat tgccgcaaag 42540 catttccgat agaaattacg ccagtgcaac gcgataaaga caaagtaacg cgctgcatgg 42600 acgcacagcc agtgatcaag aatggttatg ttgtattgcc tgaatcgcat cacatgcttg 42660 ctgaatttct ggcagaggcg gcggcattca cttatgatga tagccatcct catgatgata 42720 ttatggataa cctttttgat gcggttaata ttgagcttaa ccttgctgat aatgcggttg 42780 atagaatgaa gcgtttagct ggattagcga agcgttaatg tgtaaaatca aaggctggaa 42840 tattccggcc ttttttatta tgggggtatt tatgagcgac aaggtaaagg ccattactaa 42900 agaggatggt tacaatgaaa ttttcggatc taaagatgga acgtttaggc cgaatgcttt 42960 ttacatgcaa agcgcggcat ttaaaacgtt atcgcaattt tatgaagaag atggaatggc 43020 gcggcgcatt gttgatgtta ttcctgaaga aatggtaacg cctggtttta aagttgacgg 43080 cgtgaaaaat gaaaaggctt ttaaatcacg ttgggatgaa ttgcgactaa acgctaaaat 43140 tattgatgca ctttcatggt cgcgtttatt tggcggttct gcaattcttg ctgttgtggc 43200 tgataacaga atgttgaaat cacctgttaa gcctggcgca caacttgaag atattcgcgt 43260 atatgatcgc tatcagatca ccattcagga gcgtgaaact aatgcgcgga gtgttcgtta 43320 cggcgagcca aaactttata agatctcacc tggcggagac atacctgaat tctttgttca 43380 ttactcgcgt atttgcatca ttgatggtga gcgcgtatcg aatgaaaaac gccgtcaaaa 43440 tgatggttgg ggtgcaagca ttttaaacaa gcgtttgatt gaggcgattg ttgattataa 43500 ctattgccag gaattagcaa cgcaattgct acgccgaaaa caacaagcag tatggaaagc 43560 gcgtgatctg gctttgatgt gcgacgacga agaagggcgc tatgcggcgc gtttgcgttt 43620 ggctcaggtt gatgacgaaa gcggagttgg taaggcgatt ggtattgatg ccaccgatga 43680 agaatatgag gtgcttaatt ctgacgtttc aggcgttcca gagttcttac aggaaaagat 43740 tgatcgcatt gttgccctaa ctggcattca tgaaatcatc ctcaaaaata agaacactgg 43800 cggcgtttca gcgagtcaaa acacggcgct agagactttc tataagttga ttgatcgaaa 43860 gcgcggagag gattacaagc cgattcttga attccttttg ccgttcatga tcgcagaaac 43920 ggaatggtct attgaattcg aaccgttaag cgttccgagc gataaggaca aagctgaaat 43980 tatggctaag aacgttgaat ctgttgtaaa attgaaagct gaacaggcga ttgatctcaa 44040 agaaacaaga gacactttgc gatccatttg tccagatctt aaaatcaggg acaatgacaa 44100 cattgaattg ccagagccgg aagatttaga tccagagcca gggcaagagg gaggattgaa 44160 taaatgatgt ttcccgatat gacagagagc gacgttgaaa aggcgcttaa tgatctggcg 44220 gttgatatca ctgaaaacaa tgctgatgta gaggcggcaa ttgccgcctt tatgttgctg 44280 gtttacgggt tcgccgttag tgcatataaa tatcacactc gccagtttat cagaatggca 44340 aagcaaacag gcggcgcaaa taatcgatcc gttatccttc tgattggtgg tgtttacgga 44400 aatatgaatg aacgttggtt caattcaaaa tacataacct ggcgatcact tattgaggca 44460 tacgtaagga aatttgcgta taatgtgctt gatgattggg aagtacaaca gcgtttaaat 44520 gagttacgcg agcaagcaga aacaagagca cagatcagaa agcgtctaag aaaatataaa 44580 gcgtgggcta aaaatagggc gtctggtttt gttggttcgt ggtattccgt gttaatgtat 44640 caacgcgtag ttgatgccgg agttacccat tatatttggc gcactcagga agataacagg 44700 gttagacctg aacacgcgaa attggacatg aagcggatct cattggcaat aaagcacata 44760 ttcccagggc aggagtataa ttgtcgctgt tgggcacaac cggattggaa aacagagcaa 44820 gaggtatatt tatgaagaag gtgcaacgct ttgatagtgt cagagtaaag gctcattttg 44880 atgaacacgg atttttagtt gatcggccta ttgtggcgcg aattggtttg cagatttatc 44940 aaacgccata cggcgagcgt agagaattca ggccagcaag tgaagtgttt aaagctgata 45000 gcctagcgac ttacgcaggg aaaccaatca ctgttggtca cgtcactgta acgcctgaaa 45060 atgcgcgtga tgttgttgtt ggtacttgtt ccggtgctgg aattcctaac gggatcggag 45120 ttgaagcgcc tttgaacatt tacgcaaaag acgctataga aagcgccaag aaaaagcaaa 45180 cggctgaaat tagcgttggt tatacgtcga ttgatattga taagcctggt tatggctcta 45240 atgaaactgg tgaatacatc tttgaagaag atctaaaaga agatgaacag ccgccggaag 45300 gatgggtgaa atttgatgcg ctgcaaacaa gcattagcgt taaccatatt gcgttagtgt 45360 ttaagggtcg cgctggcatt gcaaaattga atcttgatag cgagcaagaa ttcccgtatg 45420 atatcgacgt taaattaaat aaagaggacg aagttatgac cgttaaaatt aaacttgatg 45480 gtgctgttga atttgacgta ccaaaagaag ttgctacttt tatcgaaacc gtgaaagcag 45540 acgctacagc ggcaaaaact aaagccgata gtctggaagc agagcgcgat gcgctacagg 45600 cgaaagttga cggcattccg gcacagattg aagaagccgt gaaaaaagca aaagctgacg 45660 ctgatgaaca tgccgcgctg gtcgctaccg cttccgaagt tggcgtaaaa tgcgacggac 45720 taaccgccaa agaaattaag atcgcatatg tgaaagaggt catgggtaat gatattagcg 45780 caaaggctga tgcatatatt gataccgctt ttgatatcgc taaagaatct gataaaatgg 45840 cggctcaacg tatcgcatta aaaggcgatg cagcaaaaaa caaacaggat gaagcggaaa 45900 cagttcttga tccttccgca cgtctgaata aagctaagta aggagaaaat attatggctc 45960 agatttcagc aagttatcat gttgcaatgg ctcgcgcctt gcctggtcaa gtttctgaca 46020 cttccgcgta taacatcgac ggcgcttgtg ttcttgatgt tgatgccgga tcaacaattt 46080 atgttggcgt tggtgttcaa catggcggag ttagcgcaag cggtgagaaa ttaattaagg 46140 ctatggcggc gtctggtaat gcttatggtg ttgctattcg atccaacttc cagaccacat 46200 ctaaagatgg tcgcatggtt tatgaggctg gtagcggaat taacgtaatg actaaagggc 46260 gcgtttggat ggtatctgta gaatcggaag cgccagcatt caaaacacct atcaagcttg 46320 ataattcagg acaggttgat ttaacgtctg gattaattga aaccacttgg atcgcaactg 46380 gagactttac ggaattccaa ggtctaaaac tcgttgaggt tcaattggta taataataag 46440 gccggattat tccggccttt tatatatggt ggttttatgt ctgattacgg ttctataata 46500 ggtatagttg aaaatgggtg cttggtagat tcaagccttt ataatataga tggctcgcca 46560 gttatggatg tttcagccgt gataggtaag gccgtgtgtg ttgatcgcat atcttgcggt 46620 tacaaaataa tcagccacaa ttttaatgaa aactcaaagc attacggaat cgttacgcgt 46680 gattatatgg atttttgcaa agaatattac gactccggag atcctgttaa cgtaataact 46740 catggtcgaa tttgggtaat cactcaggac gaacatgata ttccatcgca taaagatcct 46800 gtttttgtta aaggcgatgg ctctatatca acaagtggat ttcctactag ttggtttttt 46860 actggcgaat acgtaagata ttcacacgga tatattttac tatcagttca gttgtagtgt 46920 gctaacacaa aaaaacactt gaacgtttaa caaaaaatgc tatattaaag ccgttattat 46980 gacggctttt ctatgaggga aaatattatg tctaaagaaa tgaaatatga tgaatttgaa 47040 gcaaacgtga tcgccaacca catgcagtta cgcggcgcta aaaacgacgc atctgatatg 47100 ggtatttgga cggctcaaga gcttcataaa attaaggctc aagcctatga aaaagaatat 47160 ccggcaggtt ccgcactgcg tgtattccct gtaacgtccg agctttctga tacagataaa 47220 acttttgaat atcagacttt cgacagagta ggccacgcaa aaatcatcgc tgattacact 47280 gatgatctgc caactgttga cgcgctgatg acctctgaat tcggtaaagt gttccgcctg 47340 ggtaatgcgt tcctgatttc cattgacgaa atcaaagcag gtcaacgcac tggcaagagc 47400 ctttccactc gcaaggccaa cgccgcacag aatgcgcatg atcagcttgt aaaccacctg 47460 gttttcaaag gctctaaacc gcataaaatt atcagcgtgt ttgatcatcc aaacctgaca 47520 actatcaact ccgcaggttg gaacaacgcc gcaggtactg gtaaaaagcc ggaaaccgca 47580 caggatgaat tagagcaagc gattgaaaaa atcgaaacgc tgaccaacgg gcagcaccgc 47640 gctaatatga tcctgattcc gccgtcaatg cgtaaagttc tgatggttcg tatgccggaa 47700 acaaccatga gctatcttga ttatttcaag cagcagaatg gcggtatcac tatcgaatcc 47760 atttccgagc ttgaagatat cgacggcgca ggtactaaag cggcgctggt ttacgaaaaa 47820 gatccaatga acatgagcat tgagattcca gaagcgttta acatgctgac cgcgcaacct 47880 aaagacctgc atttcaaagt gccttgcacc tctaaatgta ctggcctgac gatttaccgt 47940 ccgttaacgc tggttctgat taaaggtctt gtagtaggct gataattcaa ttaatatagg 48000 gaaccgttaa aaggttccct ttttttatat ctggagaaaa acaaatggct aaagaaaaag 48060 aaattacact ggttaacact ggcgttgcac tgattattat tgatcgcgtt gacgttatgc 48120 caggtaagga aattaccgtt gcggaaagcg tgttagagcg gcagggtaca aaatcactgg 48180 ttgcacaagg caagctaacc gtaaaagata acagcgagct aaacgataag atcattgaag 48240 cgttcaacga aaaacgcaag ccaaacccaa ccgaaggcaa aagcaaagca cagcttgagg 48300 acggcggcga gtattaatta gaaagggcgc tcatgtagcg ccttttttat tatgggggtt 48360 attatggttg atgaaattta tggcgatatt gagatcctag agcaaattta taaaatcgcg 48420 ccagcattca agaaagttga tcctgaattg attcaggcat ggattgaact ggctaaagat 48480 ttcgtttgcg aaaagcattt caaagacaaa tatttccgcg ctgtcgctct ttacactttg 48540 cacctgatga ctcttgacgg agctatgaaa caggaaagtg agagcgtaga aagctactcg 48600 catcgtatag cgtcattttc cctgactggt gagttttcgc agacgttcag caaagttagc 48660 gacgacacaa gcggtaacac gttgcgccaa acgccgtggg gtaaaatgta tgaagttctc 48720 aacaagaaaa aaggcggcgg atttggcctt acaactgcct ttcatcggag gtgctcacga 48780 tgaattatga acaaatcaga tctatggcat cggctggaat taattttttt agcgatggaa 48840 cgggtgaatt tgattgcatt actcaacctg gaagcgttga aattgttggc ggtattgagg 48900 ttgaaaaacc tgaaataaag gtgaagatta aaggtcttgt gagagcacca cggacgcgag 48960 aagttgacgg agaggtaatt agagttaccg acaagttagg cgtttttaat gctgatgtag 49020 agcttaaaaa cggatatcag attgatatag acggcgagcg ttatgttatg gttgaaacaa 49080 gaccaatcag accaacaagc attaccgttg cttaccgtcc aataatgcgg agggttgcgg 49140 ttcatggctg attattcgat ccgagaattt catggcgacg ttaacaaatg gatcgagcaa 49200 gtagaaaacg gcttgaatga tgtgattcag atttttggtg aaaaggttca cggcgcatta 49260 gttgatattg cgccagttga tacaggacga tttaaagcca atatgcagat cacagcaaac 49320 aagccgccac tttacgcgct taaccaatat gatcgcgacg gtgaaaaaat aaaggcggaa 49380 ggtcgcagga cgctttatgc tttattgcat ggcggcggcg caattaaatc aatatacttt 49440 tccaatatgc tgatttacgc taatgctctt gaatatggtc attcaaagca agcgccagcg 49500 ggggtatttg gtatcgttgc gatccggttg cgctcttaca tggcggaagc gataagggag 49560 gcgagaaaga aaaatgcact atgaattatc tgttgcggca agaatggcgc tggctcaaga 49620 atacgaaagc gaatatatga tcgcttatga aaacgttgaa tttacgccac caaaaggcgg 49680 cggcatttgg cttaaatatg actacaaaga agcagataca ataattcatg atctgaaaag 49740 gaaatgcatt agctatattg gaatggttca gatcggaatt gaattcccgc caggttcagg 49800 aattgataag gctcgcaagc tggcaaaaaa tattgctgat ttctttgaag atggtaaaat 49860 gctttcgaat ggctatattt cagaaggtgc aaaagtgcat caagttcaga aatcagaatc 49920 aggctggttt tatccagtac gtttttatgt tcgttatgat ggttaataaa gggggctatt 49980 atgcacttac ctaacggatc acagattttt attgaaaaca cgcgcggctc agaaatcaaa 50040 gcaacagcaa tcagcaacgc aacagatccg gtgtttaccg cgtctgatac tggcggattg 50100 aaaaaaggtg attatgtgat tgtcactgca tcaacgtggg gtaaacttct tgatcgtgta 50160 ctgcgagtta aagaggttag cgataacaca agtttcactc ttgaaggtat cgacaccact 50220 gatgaaaacg tttttccgtc cggcgctact ggtagcgtta tgaaaattga atcatggaca 50280 gagatcccat gcgtacagga cttggggcag gacggcggcg agcagcagta ttacaatta 50339 SEQUENCE LISTING <110> Optipharm.CO., LTD <120> Novel Citrobacter freundii specific bacteriophage CF1 and antibacterial composition comprising the same <130> 27 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 50339 <212> DNA <213> bacteriophage <400> 1 ccagtgcttg agcgatgacc aggagcagca gctaccaacc tataaaagcg cggtgtcact 60 gacttacacc tttgcgcatg agtacgataa ccctatttat ccgctcttgc gtaaggctga 120 cgaatccggc gacgttaaag cattgcgcat gtatgtacca aaagcaaaag aaatgcgcct 180 ttgggctggt gtgctttctt tcaatgagat cccacaaacg gcggtaaacg aaatggaaac 240 cgtttctttg tcggtatccc tgaaaggtcg ctttactttc ttgccgtcta ctcaggctta 300 atttaaaggg gcgcattgcg tccctttttt atttgatgta atatcacctc aatatttaac 360 cactcaggag aaatgacaat ggctaaaatg aaattgactc ttgccccgct tccagatttc 420 gt; aagcataaaa aggccagcga gattcaagag ctttaccaga aagaaggtat taaagatcct 540 gaatttatca tgaatgttgc agttggttgg gatctggagg aagaattcaa cgaagaaaac 600 gcgcaactgt tagttgatta ttatcctggc gcggcgctgg ctcttatggg atcttattta 660 agtgcgctgg cagggcaacg cgtaaaaaac taaaacgggc ggtttatctg tattaccaga 720 agccgccgac agatgcagaa ttgcaagccg taggattaac ccgcgcagac tatgaaggag 780 aagatccgcc agaggtcata tttgatgaaa gtatgatgca atcatgggat atattttgcg 840 cgatgggtac acagtggcgt agtgctggcg ctggtgctta cggttttgat tataatgttt 900 tgcctatgtt attcagaatc tataaaatag acgatgaaga aatggcctta aatgacttgc 960 gcatcatgga acaaaaagca ctggaaatga tgcaagcaaa taacaaataa agcgcctacg 1020 ggcgcttttt ttatgagggt aaaatatatg gctgaacaat ttgcaggttt aacacttggc 1080 gttaacgtcg agcagttaaa cagggctgta aagtcattgc aggactttaa aaaagcaaac 1140 gacgaagcaa agaaaggcgt tgagagcttt gttaatgagg aagaaattgc aaagcaaaga 1200 gcaaaacaat tagctgatga attggcgaaa cagagccgtg aatttaaacg cattcaggac 1260 actgttgatc cgacagcggc gaaaatgcgc aagctatctc aggccgcaag tgatcttgat 1320 aagtatggc aaaagggcat tgttcccgat gaaacgtttt tccagttggg gagtatgctg 1380 gaaactcaga tcagtaagct ggagcgtaat aaaaaggcgt taacggaaga aggccgcgca 1440 gcacttgagg aatcaaaagc caaagagaaa gcagcgttag caggaaaaac tttccttgcg 1500 gatctggaat cgcaagccgc cgcgcttgga aaaacaaaag ccgaattgct ggaaatgaaa 1560 gcggcgcaac ttggcgtaag tgctcaggcc gcgccattca ttgcacagct taaagcgcaa 1620 gaaaaacaga tgaagttaac cggaattagc gcggggcaat acaagcaagc tatggcgcaa 1680 cttccggctc agattactga cgttgtaacg tcgctggctt ccggtatgcc cgtttggtta 1740 gttgcaatcc agcagggcgg acagattaag gattctttcg gcggcgttgg taatacgttt 1800 aaagcgctgt tgtcgtttct gaatccggta aacgttggcc ttgccgcctt agcagtttca 1860 atgggtgctc ttgttaaggc tggttatgat gcgtataagt cgcaacgtga cttgcaagag 1920 gcgttggtgc tgactggtgg ctatgctggc acaagcggcg cacagttcga taagctggcg 1980 caggacatta ataacagcac tgatgcgacg ataggaaaca tccgatccat cgcaacagag 2040 cttgcaaaat ccggcaagtt cacgattgat cagattaaag caatcacgaa aactaccgct 2100 cagtggtcaa cggtaactgg tgaatcaagc gacaaaataa ccgattactt taataagatt 2160 gctggcgatc cggttaaagg tctttcagag ctaaacgagc aattcaactt tttaagcgaa 2220 gggcagttaa cttatattgc agacctggag aaaacgaaag gaaaaacgga agcagtgacg 2280 gcggcgacaa agttatttgc tgatgttatg gatcagagat tagctaaact ggcagactca 2340 gcaacgccat tagaaaagat gtggacgaac attaaaaaat gggcgtcaga tgcatggggt 2400 tgggttggag atcacacgct ggcggcgctt aacctgatta ttgacgttgt agccggaacc 2460 gttgagcagg ttcgttattt gcttaatcag ggtgatattc tcattggtga atttattgtg 2520 tcggcaacca aaacgatgca aaaaatccca gggcttgata atgttggcga ttctgttatt 2580 tcgcagcaac agaagattat caataacgct aaggagaaca acaaggagct attaaaatca 2640 attgcagaac gtaacgagcg agtcaaaaaa ggcgagcagg gttacattga catgatgaaa 2700 aatcgtgcgg cggttgaaca gcaatatgcg acaaaaacaa aagagaatat aaggaaggaa 2760 gccgaggaat tagccaaacg cgataaaaag caaaaggctg aaaaagttaa ggtatcggct 2820 ggcgataagc tggaagaaca atatcagcgt gatatcctgg cgttagaaac tcaattaaga 2880 gtttggaaag agcacaagac gatcactgat accataagcc agcaacgtaa atctttatgg 2940 gctgaacaag ctaaaattca gattttgcaa gaagcctcaa caaaacggca acttactgat 3000 gaagaaaaaa gcattttagc caacaaagat aagatcctgg cgatggctga acaaaaagct 3060 attttaggcg atcagattgt tgcgcaaacg cgccttaatc agttgcaaga tagctcaatc 3120 aagtttatcc agcagcagaa agcagcaaca gaagccctag ccaaaaccag agggttaagt 3180 gaacgtgaag cagcaagagc ggcggaacgt gcaaaggttg aggctgatta tcttgccaaa 3240 ggaggcaagg aaggagatcc gcaactcacc gcaatgatgg atgcattaga caatcgctat 3300 aaagaggaag atgctaaacg tgctgattgg ttggctggcg ctaaaaatgc ctttgcggag 3360 tacggcgagg aagcaatgaa catgtatgat aatatcggca acattgcgag ccaggcgcta 3420 aacggcctat ctcaacaaat ggctgatttt ttgactactg gacaagcaaa cttcaaagac 3480 tttgcaaaat cgatcattag ccttattgtt cagatgatca caaagatggt tatcttcaat 3540 agcatttccg gcatgatggg cggctcaacc tggacgcttg gaagtctttt aagcggtgct 3600 ggttttgctg gcggcggtta tactggcgac ggtggcaaat atgatccggc tggcgttgtt 3660 cataagggtg agtttgtttt caccaaagag gcaacgcaaa gaatcggagc taaaaacctg 3720 tatcgcatga tgcgaggtta tgcaaacggc ggtcaagtag gaagcgtgac aactggcggc 3780 gctggcatca atcgcggtgc atcgcaattt gcatttggtg atatcaacgt taacattgac 3840 aacggtcaag atccaaaagg gatggaaact ggtatcaaga tgattttcac tgaaatgatt 3900 caacggtcat gctcgcaagg tggcgaggtt tataattttg ttcatgggag ggcttaatgt 3960 tagatgaatt tacctggtgt acgcagattc agagcggcgg cggtgtaatg acaactacca 4020 ataacgacag ggaagttgtt tttggtaacg gttatcgcca aaaggcatct tctggattta 4080 acacggagcg gagggagttt tctattgttt acgctggcac tgattataaa aaagtgaaag 4140 cgttcatgac ggatcacaga ttaaagcctt tcttgtggag gatgccggat ggtgatttag 4200 ggttgtttac tgttaaagcc ggaacggtag gactaactcc aataagccca accgctcagg 4260 aagtcaaagc aacattcacg gaagaattta catccatgcg ataaatcaaa gccgccattg 4320 tgcggctttt ttattgtgtc tataatggtg ctttattgga gggcatagaa tatggctgaa 4380 aaacagatca aaaagacttt tgaaaattgt ctgcaatcgc ttttccctgg tgaaataatt 4440 acgcttgttg aggttgacgg cacaaaattt ggtgctcagg tttaccgctt tcatgctgaa 4500 aatatcgcgt acacaccaga ggaattaatg caagcgcgtg aaactggaat attgccgccg 4560 aaagatatta aattccgtgg cgaggtttac ggggcgcggc cttttgggat tactggcatc 4620 ggatttacaa gtaacggaaa ggcagagaaa ccacaattag cgctttcaaa tttagatagc 4680 cgcgtatctg cattaatcag atcatataat ggcatgatgc aagccaaagt aacaatttgg 4740 gttacatcgg ctgatttaat tgatgaagaa ggaaacgtgg aggacggcgc atatagaaag 4800 ctggtttatt atattgagcg tccgaatttt gttaatcaga cagttgcacg ttttgaatta 4860 acatcacctt atgatatgga tggaattatg atcccgccac gactcacgca aagcgtttgt 4920 tattgggcgc aaaggggatg gtatagaagc gggaaaggtt gcggatataa cggatcggca 4980 atgttcgata aagacaacaa tccggtaaca gatccgagca aggattattg cgccggaaca 5040 gtgacagcct gtaaattgcg ctttggcgat caaaatgaat tagatttcgg cggttgcgcg 5100 gttgcatcac tattgaggaa aaaccaatga ttaacgcaaa aattaaactt gaaattatgc 5160 gccatgccaa tgacatttat cctaatgaat gttgcggcct ggtaactcaa aaatcacgcg 5220 tacagaaata tcaccggatc gacaacgtaa gtaaggatcc ggaaaagcat tttgaaatgg 5280 acgctgaaca atatgcagaa gttgaggacg gcggcgcgga tatcatagcg atagttcaca 5340 gccacacagg ggaaggtgca accacaattc ctagcgcaca cgatttgtgc atgtgtgatg 5400 aaactggtgt ttcttgggtt atcgtttcga taccagaggg agacatgaga atcatagatc 5460 cacaatcacg gcctttgatt ggtcgcccct ggtcgcttgg cgcttatgat tgttgggggc 5520 ttattatggc atggcataaa gagcaaggcg tgatcctgaa tgatttccgc aagccgtatg 5580 aatggtggaa gacggaacat ggtgaaaacc tttatcaaga taattacttg aaagaaggtt 5640 ttatagaaac aggtgaaccg ccaaagccag gtgatatgat cataatgcag ttgcaagcgc 5700 cagtatggaa ccacgccgga atttatttag gcaacaatca actattgcat catgcattcg 5760 gcaagttgtc gcgagttgat ttatattctg gatggtatca ggaacacaca acaatgattt 5820 gcagacataa ggatctgaaa aatgaataag gttatcaacg taaaattatc aggctcatta 5880 ggtcgccgct ttggtgtttt ccataaatat gctgttgata gttggcctga atgcgtaagg 5940 gcattatcaa gccaggttga aggattcaaa gaatttatgc aaagtgaaat cggatcaaag 6000 atgaagtttg caattttcgt tgatggtaaa aatgttggcc tgaataatga tagtgcatgg 6060 cgttgcgctc gcgaggttcg aattgttccg gttccaacag gttcaaaaag tggcggttta 6120 ttccaagttg ttttgggtgc tgttattatg gtcactgcgt ttttcactgg tggtgcatcg 6180 ctggcggcta tgggtgcttt ttcatcggct gcatttatga tgggtggagc aatggctctt 6240 ggcggcgtaa tgcaaatgat tagcccacaa caaggaggaa tgagaatgga gagccaaagc 6300 gcagcaaata aaccatcata tgcgtttggt ggtgctgtta atactacggc ggcaggttat 6360 ccggtatgtt tgccgtatgg ttatagaacg gttggcggcg ctgtattcag tgctggctca 6420 tacgcagaag ataaacttta attatataaa cccgccgttg cgcgggtttt ttgttacatg 6480 tacaatggca gcacgtttaa atagcacaaa aggttaaaag ctatgatcaa aaatatgata 6540 actggcagta agggcggttc ttcaaagcct catacacctg ttgaaatgga agataaccta 6600 atttcaatta acaggatcag aattctttta gccgtttctg atggtgaagt tgatccagac 6660 ttttcattga aagatttata ttttgatgac gttccggtta tgaatcagga tggttcacta 6720 aactttcaga atgttaaggc agaattcagg ccagggacgc aaacgcagga ttatattcag 6780 gggtttactg atacagccag cgaaattaca gttgctcgcg atctgacagc cgcaacgcct 6840 tatattattt ctgttaccaa taaaaacctt tctgcgatcc gtattaaaat tttaatgcct 6900 cgcggggtta ctcaggaaga taacggagat ctaacaggtg ttcgcgttga atatgctgtt 6960 gatatggctg ttgatggtgc tgaatataaa gaggttttgc atgatgtaat tgaaggtaaa 7020 acaatgagcg gttacgacag aagccgccga attgatttac ctgttttcaa tgatcgtgtt 7080 ttattgcgcg tccgtcgcct gacagatagc acgtcagcaa gggtaacgga tctgattaaa 7140 atgcaaagct atgctgaagt tgtagacgct aaatttcgtt accccctgac tggtttagta 7200 tatgttgagt ttgacagcga attattccct aatgcgttgc cgaatatcag cattaagaaa 7260 aaatggaaaa tcattaatgt tccgtcaaat tatgatccta tttcccgtac ttactcaggg 7320 tcatgggatg gaacctggaa aaaggcatgg agtaataacc ctgcttttgt tctgtatgat 7380 ttgattacca atcagcgtta cggactcgat caaagagagt taggcatcgc gcttgataaa 7440 tggagcattt acgaatgtgc gcagtattgc gatcagatgg ttccagacgg taaaggcgga 7500 acagagccgc gttatctttg cgatgttgtg atccagagcc aggtagaagc atatcagctt 7560 gtgcgtgata tttgctcaat cttccgtggc atgagctttt ggaatggtga gagcctttca 7620 atcgtgatcg ataagcctcg cgatgcgtca tacatcttta ctaatgacaa cgtggttaac 7680 ggtgaattca cttacacgtt tgccagcgaa aaaagcatgt acacgcaatg caacgtgact 7740 ttcgacgacg aacaaaacat gtatcaacag gatgttgagg gagttttcga aacggaggcg 7800 gcgttacgtt ttggctataa cagcacgtca ataactgcca ttggatgcac tcgacgcagt 7860 gaggctaatc gccgtggacg ctggatttta aaaaccaacg tcaagagcac aacggtaaac 7920 tttgctacag gtctggaagg tatgatccca accgtaggcg atgtgattgt tgtatcggat 7980 aacttttggt caagcgctct aacgctgaat ctatcaggtc gcgtgatgga ggttagcggg 8040 ttgcaggtgt tcacgccgtt taaggttgac gcaagagcag gtgatcgcat tctggtaaat 8100 aagcccgatg gtaagccagt tggtcgaacc attgcgcgtg taagtgatga cggtaaaacg 8160 ctaacgctaa acacgacatt tggctttgac gttcagccag ataccatttt tgcaattgag 8220 cgaaccgata ttgcacagca acgatatgtt gtaaccggaa tcactaaggg tgacggtgat 8280 gaagaattta cctacaacat aacggctgtt gaatacgatc ctaacaaata cgatgaaatt 8340 gattatggcg taaacattga tgaccgtccg acttcaatcg tgcagcctga cattttgcca 8400 gcgccgcaaa acgtcaagat cgaatcttat tcgcgagttg tccagggcgc gagcgtggaa 8460 acaatgcacg tttcatggga taaggttgaa tatgcaagcc tttatgaaat gcagtggaga 8520 aaagacaatg gcaactggaa caacacgccg cgcacagcaa acaaggaaac ggaagttgaa 8580 ggaatttatg ccggaaacta tcacgtaagg gttagatctg ttgcagcgaa tggttcagcg 8640 tctggatggt cagccattgt aagcgccgga ttaactggca aggttggcga accggaaatg 8700 ccaattaacc ttactgcgtc tgacaatgaa gttttcggaa ttcgcgtaaa atggggtatg 8760 ccagaaggaa gcggagacac ggcatatatt gagttacatc aagcgccaaa cggttctgat 8820 ggtcatccta ttgttgatga agcaacgctc ttaacgctga taccgttccc gcaatatgag 8880 tattggcatt caatattgcc agcgggtcat gttgtctggt acaaggcaag ggctgttgac 8940 aaaatcggga atgtttctga ttggactgat tttgtgcgcg gcatggcttc cgacgatacg 9000 agcattatca cggatcatat taaggtcgat attgaaaatt ctgatggcta taagtggtta 9060 caggaaaacg caataaaggc caatgataag atccacagca cagcggaatc agtgattgaa 9120 aacgcattgg cgaatgataa agatgtgcga cgtatgcgag ttgaaaacgg caagcgtaaa 9180 gcagagttct tgcaatcgct gaaactcatt gcagatgaaa cagaagcaag ggtaacgcag 9240 gtcactcaaa tgagtgcgca atttgacgag aaattaacgg ctcaaaatag cgaattgaga 9300 gaggtaattg ctaacagcac tgaaaccatt agccaaagga ttgatcagct tacagctacg 9360 tttgagggcg aaattgacgg tgttaagcag gatatcaaag cacagataac tgatgttaac 9420 caggcaatca ccaatgaagc ggaagcgcga gcgtcagcgg atagggcgtt atcaactcaa 9480 attggtgata cccaatcagc ggttaaccag aaacttgatt cttgggttaa tgccgatagc 9540 gtaggcgcga tgtacggcgt taagttgggg ttacgttaca acgggcagga atatagcgcg 9600 ggtatggctc tttcccttgt tggtgacgga tcaggagtta aatcacaatt cctgtttgat 9660 gctggacgat ttgcgataat aaacaaccaa cagagcggag gttttacact accgtttgtt 9720 gttgagaata atcaggtgtt tatcaacagc ttacttgtga aaaatggctc tattggtaat 9780 gctcagattg cggatcagat taactctaac aactggagca gcggcgcggc tggatggatg 9840 attaataaga atggttatgc tgaatttaac cagataacgg taaggggtac agtgtacgca 9900 aacgccggat cattcactgg taacgtttac gctactgatg gttggtttag gggtactgtt 9960 tacgctgaaa aaatcgaggg cgacgttgca aaggctgttg tgcttgggtt taacggatcg 10020 gttcacattc cggcagtaaa ctacaacaga caccttgtga ttccttatgt tggtttgcat 10080 ggctatacct attcaggtgg tacgtggggc ggtggtactg tttgggttga ttcatcatat 10140 ggcggtcgct tggctaacgt tcaagctacg gctatgcatg gcggttcaag tggatacctt 10200 ctgttaccag cgggtaacgc aacaacgctt tcatatggtg gcaacctgaa ccacgctaac 10260 gcggttccag tattaacaat actgctattc aaagcataaa aaggaggtaa atcaatgccc 10320 tggcttaacg gtcggggcat tttgttttct tttgttccac gatgttcaca accgaatccg 10380 gcgtaaaatt gaaaacgtat tgaaaacaaa aagaacaaat taaatatcta tataatacat 10440 acacttagag tttaattatt attatatata tctatttgta ttcttattgt ttctattgtt 10500 ctctatggtt tggtgtggtt gtgtgttttg gattggttaa tgattatata tctcatggta 10560 tgtatttata tatggtatcc ggcgtaaatg agtgaacaat ggaacactaa aacaacaatt 10620 ggtaaaatca taaacttacg atgtgatttt gttgtcaatg ctagttgtaa acgtatgcaa 10680 aacaaaaaga acacttaaag tgtattgact acatgaaaca ttcacatata atgcagacat 10740 accaacgaga ggatgaccaa atgagcgatt ttaaagttta cacctttgac cagttaacca 10800 acgatgaata tcacgatccg aaaggctggg cggctgaata cgttagcggc tcaagcctgg 10860 cggaaatctt tgcaacttgt ccggcagcat ggaaatacag accacgcgag gaaagcaaag 10920 cgcttgtttt tggtacgcag tcacatacca actttcaaag cagtgagttg ttcgcaaaag 10980 agtaccgccg agcgccagcg ccggaagatt acgaaaactt gatcacaagc caaacggcat 11040 tagcaagcaa gttaaaatct tttggcctga ctggtacaag cgggaaaact tatccagacc 11100 tgataaaaat gatggttgat tgtggtgagg atcttaatgt tcagtggctg attgaaatga 11160 tcgcagagag ccaggcgcga gcggataacg ttgaattggt tcctgctaaa gattacgacg 11220 cttgcgttac catgcgccgt gtactggaaa gcataccgga acacaacgct tgcatgaata 11280 gcacaacagc acaacgcgag ctatcaattt tcggcactat aaaaggcgtt aaggtaaaag 11340 taaggcttga ccatgttgat atttgcaaag atgtgctggc aactgttatc tcaggttatg 11400 acggtgaagg gaatccggta tacgaaacga cgcgttttga tgaagcgatt gtgatcacag 11460 attacaaaac aacgtcaagc gcaaacccaa ccgaattttc aaggctggca ttcaatcacg 11520 gttactattt aaaaatggca ttgcagcacg acctattcaa gaaagcgtat ccagaagaaa 11580 cgcgacctgt tgttgttcga cttctggcgc aggaaaagaa agagccattt ttaccgatcg 11640 cataccgcat gacagatgaa caattaaaga tcggacgttt gcaatacatg agcgtgataa 11700 accagtttgc agaatgccag gtaaatgacg tttggccttc atatgcaaac ggagagccgg 11760 aaattgacct gcaaacgcct gattgggttc gtcgccaata caaaggtttt ttataaatag 11820 cactttttgc taaaactgcc attagatgcg tggtatacta cacgcatcaa ccaacgagag 11880 agaggaaata aaaatggaac agcaattaaa tgaacaagac attcaagatg tgcgcgatca 11940 tgtaggcgga tttgtaaatg cattagggat tatgcaacgc cagttcttgc gcgtaattga 12000 tccaagcgaa tcaccggaaa ctcttgaata tgttcgcaag gttgcatatt cgattgataa 12060 cgttgtgtta acggttttta aaatggaaaa taacgaagaa catcgcaaga tgattaatca 12120 ggcaagtgaa attatgatcc agaatttgat tgattatcac aacgagaacg aagaaaagca 12180 ttaattaaca cggcggcgca agccgccatt attaaaccgg agtcaatgaa atgaaattat 12240 cagatcaaaa cgcaaacgta attaaggcat tgtttgaggc gcgtcagtta ttcacaaaag 12300 taaaaaagga taagcaaaat acgcacctta aaaacaaata tgcaacgctt gacagtgttc 12360 ttgatgcaat tatgcctggt ctcactgata agggtttatt cctgacgcaa gatcagaaag 12420 tcagtgaaga tcttaaatca atgactgtat tcacgcgatt cattcatgtt gaatcaaatg 12480 aatggattga atatagcttt acgcttccaa tgcagaaatt agatccgcaa ggtggcggct 12540 caacaaacag ctatgcgcga cgttatgctc tttgcactgc tcttggcctg gcaacggctg 12600 acgatgacgc aaatcttgcc acaaaaaacg cgcaggattg gaaaaaagat cttgatgctt 12660 gcgataacct tacagacttg caagaaacgt ttaaaacttc ttacaagcaa tctgatgcgg 12720 ctaaccgtag aatcattaaa gagcattacg acaagctgaa agcgaaaatt gagatcggaa 12780 aggctcgagg ttttaatccg gctgaaccag cggcaaatgt tgcgaagaat gaaaaagttg 12840 aaaaccaacc gcaacaggaa gtaaaatcac agtctatcac tgactttgaa taatttatct 12900 cggggcgata acgccccgat aaggaaataa aatgcacgta atcaccggag aaattcgcaa 12960 agagccattt gtaaagcaag gcgcaaacgg cacgttatac atcgttgagc tttcggagtc 13020 atttaaagat cgtgaaggta atcgacaata caccaattac aaattcttct ttaatgctaa 13080 aagcgacggc atgaataatt ggtatcgtga agcatttcag cagggtaaag ttatttctgt 13140 atcatgcgat cagttacgca ttgatagcca ggaatacaac gggcagattt ataacacatt 13200 gacgtcggcg ggttttccta atcttttatt cagccaacgc ggagaagggc aagcgccaca 13260 acagcagcaa cctcgacagc aacaacaagc gccacgccag caagcgcctc aaaacaacga 13320 gccgcctatg gattttgatg atgatatccc attctgataa aagaaaagga gccgaaaggc 13380 tcctttttta ttacttagca atctgaataa acattgattg catttcctcg atcatctttt 13440 tcatttctgc tacttcattt tttagctcat tgatttcatc ttgttgcttc tgaatggttt 13500 cattctgctt atctgaatca tcaatcattt cattaattgc gttaaccaaa agagcagtta 13560 caccgccata gctaacacct aaatattctg aatctgaaat cttgtaaacc gcttccggta 13620 aaacagtttg cacgtcttga gctattacac cagcctcctc cgaatagaaa gaatattcat 13680 cttcatcaag attgttgcgc ttaaaataag tgtaaccttt tattgatttc actttttctt 13740 tagcgctttc aatctctttt aggtttgctt tcaggcgaat atcggaagtg ttaacccatt 13800 gcacagcaga cgcataacca gcatcgttaa acccataaac agcgcccctt acattaaaat 13860 gagtttcgca acctccagcg ccatatctgg caacatctat accaaaagcc caatcaccac 13920 cccaccttac aaccttaaca acgttaacgg ctacatcatc gcttggaagt tcaacaagca 13980 taccagcggc gcgatcacgc catgcagtaa aagcaccacc ctcaacatgt ccacctataa 14040 agcaattcgc accacccctc ctagcacctt caacataagc aaaacgtgca tcttctggag 14100 taaatgattg aatagttctg tatgcaacat tgctactctt tgtaaatgca aacatcactt 14160 gattatcagc aatagcttga agaatgccat ctccattttg cctaaatcca gtatcagtat 14220 ctccaattac tatagagtta ctaccaagag cattaccaat ccccctaccg attgttattg 14280 agttgtaatt cagcgcgttg ccactttcat caaactgata atagtttgtg tcgccacctt 14340 ctctagttac ttgaatagtt gcctttgcaa taccaccctg aatttcgttg taaacccttg 14400 aatacgaaat ctgtactcct tctgcatttt tgtttaacaa ataaagtatg ccggagtttg 14460 cagagttttt ttctgttagg cttattcctc taaaaacagg aatgtcagtt tcaccaatgc 14520 caaaattact ccttgcttgc cctatgcttt gagcgccagt gcctcctcgt tcgatgtgta 14580 atggtttgat gtttccataa tcatcttgaa caccccacac accattgtta gctacaagca 14640 tatttagttt tccatcaggt gattttatgg agttaaaatc tcctggtgtt tcgctagtaa 14700 tgcaaaaaac accttgcaca cgaaagtttt ttcttgcccc ttcaatattt tccgcaccag 14760 tgccgccagc accaacagaa agagcggatg tgctgttatc gctatttctt gcaactcgcc 14820 actcgccgtt gtttgcgatc cgtaattcat aagtccatgt agggttacga aatgagttat 14880 aatcacctgg attttgcccc ttaactgaat taacagcctc aactttcagc gcctcccttg 14940 ctaaatcttt attagcaaga tcattaagat tctgatcctt atgcagaagg ttttcagggt 15000 taacttgttg tgcccattgg cgagcttcat cacgcgcagt ctcagcgcca gcctttgcag 15060 agtcggcggc aatctttgat tgttcagcgg caagttgtga ttcctttgct gcatcgcgag 15120 cattaacaga ctcgttttta attgatccaa tttcagaaac ggcgctatct tttatttgtt 15180 gcgtctgcgt ttttattgcg tcagtgtccg ccttaatctg attggtttca ttgacagcag 15240 catccttaat ctgttgagtc tcagatacag cagaatcctt aatctgttgc gtttcatttt 15300 ttagttgctc agttgccgct ttgtcgtttg ctacctgttg agcatcagca ttaaccttgt 15360 taactaaatc ttgcaatgtt ttcagatcga agtttttaaa gaattcaatt gcttcctcaa 15420 ttaccgtttc tttactttgg taataacgca gcgtttccgc tacatcttgc gccattccgt 15480 caacagtcaa agaatcattt aacaggatca catattttcc atcaggaaca acagcgccat 15540 cagtgctaat tgctttcatt tcagtgttgc taacaatctc actaataaca gcaagtttaa 15600 taactggctg atcaagaaat acaatggttg caccagtgcg gattaatgac aaaggttcac 15660 gccattttgt gccagtacca gttataacgc cttgcgcgtc catcgcggcg gttccagtgc 15720 gatataaagc cataatttca cctcatttac ttgattaaaa tagtgtagtg attatgcgat 15780 attgctatgt tacatgcaat aaaaaacccg ccgtagcggg tttcgtcaac aacaacgaga 15840 agtcaatgaa acagattcat cgaaaagtgt tagtagcagt atgaatatta taataagtca 15900 cttgccttgt aaagcctccg ctctattcca tttttgaatt taccgttaat tggtaacatt 15960 acgtctaatt cgcgatcatc agcagcaatc aaagtatcgc gatcattagc atggcacaca 16020 acgcgcattt tgcgatctcc tttgtacctg tatgctacaa tttcaagatt tgatgcagag 16080 aaacaagccc atacttcttg tcccgtcgcc aggtggatat gttgcgcatc aatccaatca 16140 acgcaaagat agatcgtctt atcagtcttg ccagttactg caacagagcc tcgcgtataa 16200 tccttagcgt aaaaactgct ttgaccttcc tcattgacga aaagaatatt acacatttca 16260 tcatccagtt catcgctatg caccaggtgg caaggaatag cgtgaatatg ttcatcatag 16320 ccaccaggat ttatttttac gcctacttga tatgattcac cgtctagcgg gtaaaacgcc 16380 tcaaaaatgc ttagatgcgt tgcggtcatg ctttctgttt tgctcattac ttccatgcac 16440 ttttcatgag ttgcttgcga tccgaaattg taaccggagt cacgttttgc cgctttgtta 16500 gccttgttga caacctcaac cggaaccagg tttaaccatt cgcctagagt gtcgattgct 16560 tcactgtaat tttcaccacg tatgcgcatg aataatccga tccctgaatc attcccgcat 16620 tggttgcaaa tgttgccgcc gtcgcctttc tcgttgattt tgtccgtcca tctgaaacgg 16680 tctttacctc cacaataagg gcatggttga tgctttccgt taaaggttaa ttgaggaata 16740 tcacaaagtt tcatcaatgc cgcttgccac attccgcgca tctttgggat cacttcttct 16800 ttctggtaat gcatttctca acctccaaat aaaaaaacct ggcacgatga taaaccatgc 16860 caggccggaa tttttagcaa tcagtgctat cgacggaaat cacaacgcgt ttcttctgat 16920 tggcttcctt tgctttgcgc cgttcttctt tcgccttgtc gcgttcagct ttgcgccgcg 16980 ctttctcctt ctcgcgttct tctggagtca tagaccgtaa cggatcgtaa ggccgaacaa 17040 atcctcgcgc ctccgcttct tcttttgtga caaatcgcat gagctttctg ttgctgcaac 17100 gattctctaa tggcttgccg ttgttgtcaa agcgcaaatc tggacggcaa tatgaagcgc 17160 ggaaaccgtt aaaacctttg cggcggtaat ccttttggat cagttcagcg cctttagctg 17220 aaatcattcc gcgctctttc catccccaca ctgttttaag gttaacgcca atctcacgcg 17280 ccaacgcagc acagccgcca taagcgctaa ccacttcatc aagcctagct tttaatcctg 17340 cccttacttc ttcctttagt accagataac cgctaacagg tgcgcggcgc ttccggtctt 17400 taccacgcct tacgccgttg ttaccattga ttgtgcgttt gtcgatttcg cccgttgagg 17460 ccgcaatttt gattccgccc atattttcac ctatagcact ttttgctaaa actattgagt 17520 aagtttgtgt atgatatggt gcaaatggca tttagtcaaa aggataagta atgattccta 17580 acattgaaaa acagattgaa gaacttggag cggaaaagat aaaggcaata caaaaacgct 17640 tcacagttgg tgaaattgtg ccttacgaat atcaatgcgt tgcatacgtt gagatcgcaa 17700 agcgtttaag ccgttatgaa catccgtttt ttgttaaggc ggctgtatcg gctggtaaaa 17760 caatcatctt tgctatggtt gcggctcagt gccgtaaaat ggggttgaaa gcgttatttc 17820 tggcgcgtca atctgaaatt gtggatcagg attcaaaaga gattagcgcc tttggtgttc 17880 cgaattccgt ttattgcgct ggcctgaata ccaaaagcgc ttatttcccg attgttgtcg 17940 gatcggaagg cactgtaatt ggtggattga ataaagcgtt gggtgattac gtcccgcaag 18000 ttttaggtat agacgaatgt catcaagtca actggaatga tattgttgaa gccgaggaaa 18060 acggcgaaag catagagcaa atgatgacac cgaaaggtga accagttgaa gggaatccgt 18120 tgctgattgg ttcaggccgc gcacaataca ccattatcat tgcagaaatg cgccgccgtt 18180 gccttgagac ttacggtcat cagttgcgca tatttggaat gacaggatcg gaatatcgcg 18240 gaattgagcc gatacttgtc gaagataaaa gaattcctgg gttttggcgt gagcagatca 18300 caaacattga tactgattac ctggtaaaat ttggttcagt tgttcccact tattttggca 18360 atgttggtga tttgggttac gacttatcag aatttacgcc agtacatgaa tttggcgttg 18420 ctgattatga ccagaagcaa ttaaaggcaa tgagtgacaa gattcacaaa tcaggaacca 18480 tgacgcagaa aataatgcag atggttcatg aggtgatgaa aaaccgcttg tgcgctcttg 18540 ttacttgcgc tgacgaaagg cactgtaaag aggcggcggc ggcgcttcct cctggcacga 18600 aatatcacat tattaccgga aaaactggag agaaacagag gcgcttatgg cttgaggatg 18660 cttatgaagg cagggtgaaa tatatttttc aggttcaggc gctaacaact ggcgtgaacg 18720 tgccgccgtg ggatacttct gttattttgc gtaagatcgg aagtctcacg ctattaactc 18780 agcttttggg gcgtggtatg cgtcaattaa aaacatacca taaagaagta ttggggatgc 18840 acaaaacgga tcaccttgtt ttggattttt caggcacgat gtttgaaatg ggtaatttgt 18900 atttcgatcc gatgttagag caagcgcaat tccagttacg taaatcacag gacaaagatc 18960 cgaaatattg cccgatttgc ggtacagaaa attcattcta tgctcgccgt tgcattaatg 19020 aggatgaaaa cggaaatcgt tgtgaacatt tctggacgtt ccaggaatgt gaggatcaga 19080 ttgatgaccg cacaaagaaa gttattgtaa aaggttgcgg cgcaaaaaat gacgttgcgg 19140 cgcgtgtatg ccgttgctgt aatgttcagt tacgcgatcc aaatgataac ttatccggca 19200 agatgtaccg taaaaacgat tggtgcgacg tgttagattt taatgtgacg ctgacaaaaa 19260 accaggctgg cgtaatattc tgctacacgt tacgtgatag tggcggcgta gaattcaggg 19320 catacgaaaa atatttccct gaatcagaac acaagatatg taaaacgctt tggacaaagg 19380 cggttagaac acatgttata gatcgccaag ccgcaaactt tatgatcact tgccgcaatg 19440 caattaagat catgagttat gcggggcaaa tattgccgcc aaaacgtgtt acccatcgca 19500 agaacacaaa gaaagaagat ttgatcgcac agaaggagtt ttaaaaatgg ttactgataa 19560 aggcgattat ctggagtatt acgacaaatc agatccagat gacagaaaag aggaagcgca 19620 ccaggttgat tcttattcat ggttaacata tgagtttcca atgtggcttg tttggcacac 19680 taaaaacgaa gggaagaaaa caataggcac agcagtaaag gatcagcaag caggtgtaaa 19740 aaaaggagta agcgacattc taatcctgac tggttttatt ggctgcaaat atgcattcat 19800 tgcaattgag cttaaacgag ccaacaagaa aggaactaaa gtaagccagg aacaaaggga 19860 attcttgcgc cgcgttaggg aatgcggagg atttgccgca gtatgctatg gtgtagatca 19920 agtaaaattg gctatcgcag acgctacaaa atagcacttt ttgttaaaac aggcgcgacg 19980 gaatgcgcca taatgcacac atcagaacga aacggagaat taaccatgaa aaagatgtta 20040 tcagtagttg ctttatcact tgctcttgtc ggttgcatgg ataaaccaaa aacatatatt 20100 tgcggaaatg aagcgtttga tgttacaagc gaatatatga aagttgtgca aggcaaaagc 20160 acaggcgttg taatggataa tatcagcaaa aatcaatata agttgtttac gccaattgga 20220 acggctttct acacagttaa tgaaaacaca attgatatta aagttggcgt ttatcagaac 20280 actctaactt gcgaggtaaa ataaaatggc taaggatttt caggacaaag caaccattga 20340 tacttttgag ccggaaatgg attttcatga actgcaacac ggcagatatt cagatattgc 20400 gttgcgttca ggtggtcatt atcagccagt tagagaaaac ccaatgttca aggttagcgg 20460 caaccgttat gcaggaagta aaacgccgga cgttgtgcgc gacttgtggg aaactccaga 20520 tgaaattgtt gaataccttg ctgaacgtta cggcaaatat gatcttgatg cagccgcaag 20580 cgaaaacaac aaaaaatgcg ataagtttta tagcaaagag acaaactgct taaagcgttg 20640 gtggggtagc aataagcacg tttggttaaa tccaccatac agcaatccag atccatttat 20700 caaaaaagca attgagcaga tggaacacgg aaaccagatt gatattttac ttccggctga 20760 taattcaact gcatggttta gtgacgcaca aaaacacgcg gcagaaatta tctggattac 20820 tggtgaaacc tggaaggatg aagaaggtcg cgaatattcc agaactggtc gagtagcgtt 20880 tatttctggc atgacaggtg agccagttag cggaaacaat aaaggtagtg taatttttat 20940 catgcgccaa ttggaagaag gtgagcaaca gcaaacgcac tatgtgaaga ttagcgacat 21000 ttgcccttcc gtaaaaaata aacgagccaa agcgcggagt atttaaaatg aacattcgat 21060 tagccgatca catgaaatgg gatttatttg tcggtatgac aattcgctta cttgataagc 21120 atggttataa cgctgataaa atggagttga taaactgcct ggatatgaca tttgacgaaa 21180 tcaaagttgc gcctttgtac atgtgggaaa gaaaaattga gcacgacatg atacaataca 21240 aacgcagaga tggcgcacga ttttttaatg tgaaatagca ctttttgtta aaacccgcta 21300 cggcgggttt tgtattatgt tcttaacgga aaacaagagg atttcaaaaa tgaaaatcgt 21360 aaacgtcgaa tacttcaaag atgcgcaatg gctggctaaa gaatcattgt ttatgcttga 21420 tcgcaaaaat ccaaaggtta ttgcacaaat actttggaat gacgcaatgg agttacttaa 21480 aaaggcttac ggttatgaaa gtgacaaaag gtaaaaagga aacttgggag ttagcgaaaa 21540 aaggcggtct tgatgagggg atcgccaaaa tagcgcaata tttcgatatc aaagacgtgt 21600 gcgttattgt cggtaatgaa atggcatatg tggaagaaaa gccgcgcaag gttcatcgcg 21660 ttccggcaat tccgacaaca atcgactata aagaagtgat caacaaaaca aaagagcaaa 21720 gtaaaaggtt taagtgatta tgtggatcga tatattttat tacaaagatg gtgttattta 21780 ttggaaggta aggaggaacg gaagaaacgg aggcgttaag cctggggata ttgcaggatg 21840 taagaatgaa tatggatacg ttgtgattgg ttataatgga aagttgtatt atagacactc 21900 tatagtttgg gaaatgcata atggcaaaat tccagatggt tttattatag atcataaaaa 21960 tcctgttgtt gatggtcttg aatcagatga cagaatagaa aatcttagaa taataaaaca 22020 cgcagataat ttaaggaagg gatctaatag gctaagatcc agtaattcaa gtggcataac 22080 aggagtttca tatataaaac aacatgattg ctggagggct gcaatttgtg taaacggtaa 22140 aaagatgcaa aggaatttca agtcaattga tgcagcaatt aatcagcgtg tttattggga 22200 atctatttat ataaaatgag ataaatttct gttattttgt tgccgcctgt tatcgcttgg 22260 gttattatgt acgccgttgt tatgaattaa gaggatgtta aaatgagttt aaaaaagatt 22320 accgacgagc aattgatcgc tgaacgtgag gcgggtttaa aactgcgtga gattgcggaa 22380 aaatacggaa tgagcttgcg ccaagttgag caccgccaca gcaaattagc aaagcgtggc 22440 gaaatctcaa ctatcggatc acctggcttt gctgtaattg gtgagtcaaa gttagttgat 22500 aaaaacggca atactaaact aacctggatt aagacgcaca aagataaaga ccagttagag 22560 gcaataatgc aagcggctat ggctgcattt tctgacgaat tgccacgcct ggagccgcaa 22620 catgaaaaaa ttactgacta ttcgcaaacc cttgcgctat atccgatctt tgatattcat 22680 attggtgcaa tggctcataa gcatgaaagc ggcgagaatt acgacacggc aacggcagaa 22740 aagatcatga atgaattctt tgattatgcc gtcgataaag caccgaatag cgaaaaagct 22800 gtattgctga ttggtggtga tatgattcac agtgacggcc tggaggctgt aacacctgca 22860 agcggtcacg ttctggatca agatagtcgt tacgcaaagc tggtttatgt tgcaattcga 22920 gcaacgcgcc gagcagtaag cagaatgctc gacaagcaca aagaagttga gatccagatt 22980 attgagggca atcacgacca atcaggaatg atctggctac gtgctgcaat ggcggcggcg 23040 tatgaagatg aaccacgcgt taatgttgat gtttctccgc gtgtagtgca tcacacgcaa 23100 tacggaaaaa ccttccttgc ttatcatcac ggtcacacag tgcgcaagcc tgaaacgcta 23160 atgatgatgt gtgcggcaga ttggcgtgaa gatttcggaa tatcaacgag catgtatgct 23220 catgtggggc attggcatca ccaaaccgtc acagaaacga gtttaggcat tgttgaagtg 23280 catagcacta tggcagcaaa agacgcttac gccgcgcgtg gtggctggcg ttcacgtcgc 23340 agggcggcgg taattattta tgataaggaa tatggcgaaa tagggcgttt tatgttttac 23400 cctgaaatga ttgcttaatt aaattatcaa gaaaaaggct ggctaagtgc tggccttttt 23460 ttgtttttgt ggttaacata ttcacactaa atttggcatt gacattttaa tttaattgag 23520 gatcagatca tgagggactt tcttttaatg gctcaaaact ctttcggagg tgcaacggta 23580 gttggctctt ttactggtga gtttttgctt gcactggcta cgtttatttt gtttgctatt 23640 tttggctgtt ggggcgcatg gctgaaatgg cgtgatagta aagcgatccg agaggcttta 23700 gattcagggg atttaaaaac agcgcttaat ctacggcaga aagagagggt gtaatgacaa 23760 tcaagaaagg tatagccgcc accgtcacag gggcggcttt aatgttggca tcgcctttga 23820 ttgaagaaat tgagggcgta aagtataagc cttacaagga tattgcgggg atctggactg 23880 tatgccacgg cattacagga aaggatgtga ttcttggaaa ggaatatacc aggcgagaat 23940 gtgacgcgct attagcaaaa cacatgaaag tagcggctga cgctgttgat aaggcggtta 24000 atgttgaaat tcctttatca atgcgagcgg ctctatactc attcactttt aatgctggta 24060 ctggcgcgtt tcgtaagtca accatgctga aaaagatcaa caatggtgat ttatggggcg 24120 gttgtggtga gctatggaac tggacatatt accggaatcc taaaacgggt aaaaaggaaa 24180 aatcaagagg attgaagaat cgccgcgccg tcgaatataa atattgtgta agggatctga 24240 ataaatgaaa agaaaattac tggctattat gatgtgcatt gctctttctg gttgttcagc 24300 ttcaagcgtg ttacctggtc tgattggtag caagcctgat ataacggcac aggctggagc 24360 ggagaatgtg aaacagactg ttggtgtaac cgcaaagcag gatacatcaa gcaagcagga 24420 aaccacattc aaagaatcgg cagttggtca agttgacaca tccaataaaa agcaggttag 24480 cacttcaagc attaaggcag ataacatcac agcggaaaga atagagatcc gaaataatga 24540 tggtcttaat gttccggcaa tcgcaatggg tatcttgatg tttattgctg gtatgctggc 24600 ggggtggaca cttaaacgta ataaggcggc ataagccgcc ttttctttta aaaaatattg 24660 ctcattaagt cttgaaaact attgccgcta taatcctttt cttcaataat gtgagcacct 24720 ttctttttta tatcccacag cattttttca tactcgcagc ggttaatgcc aaacgtacta 24780 ccatcatcaa catatgcggt tcctttgttg ttgaaaacgt caaatgtaac attaacaacg 24840 cgaattgttt tgatagtgca ttttaacatt ttgttttcct tttgttgcta cgtttcgtta 24900 aaacaaatat acccgataca agatcgggtg ttttaacaaa aagtgctatt caaagcgcct 24960 gacgtgaatt agtagttcac cgtcatcatt tgttaactgg tgctcattat cttcaaacgc 25020 tcgcatatca ctgtaaagca acaacattag tgcatagaca aaatcatcat gcgaaatttg 25080 cattgctgaa ctttctttca aaataagatc tatcaaatgc tggcttgctg aataattctt 25140 catcgctcat tttctcaatg ttcatcatct cccacacata acgattggaa tcaccttcca 25200 ggctatgcaa ctcaaaatta aacggccttt tcatcttgtt tttgcaccag tacacgcaat 25260 gaacgccgtc aagatatcca taacccatca ttcgcgctat aaactctttg cttgagcttg 25320 aagcgaaata gcggggcgtt acccccgcat ttttggcgag cctttcacat tcacggtgac 25380 ggaatataaa tcttgctaac tcttggcggt tgaatttctc gcgagactcg caaaagcgat 25440 acagatctaa taagaacatg attaccccat taacgccgga tttacataaa caaatttacc 25500 aacgctgcaa acataattct tttcttcaag cattggcaat aattgttcct caattcgttt 25560 catcacgcct gattgacctg taaacggctt aaccttacgc gcactttcat agatagctct 25620 tgcattggta acgcctttat tttgacgtga gatcttaatg attatttcaa ttaacttgct 25680 catttctgca tcatcgccag cgtgaccgga agcattcgcg gcgttgatat atgttttgct 25740 taattcgctg aacatcacca aagcctcttg cattgtctca acttcaattt ctcgcgactt 25800 ttgcggcgta ccgtttggat taaaccaatt gcggatcgtg tgcaatactg acgcaatgcg 25860 gatcacttgc ttatccattt tacccaaagc accacgcaac attgtatgtg aatatttccc 25920 gccgtctgct aaatctggct ctaattgctg gcgagcaata ttaagaacac gcattgcaga 25980 acgtgacggc ttcaatacca cattttgttc agtcatgatg ttatgaatca actggaaata 26040 ttgcgccttt aattctttat caactggcgt gtaatcaaga tcaccatttt catcaataaa 26100 tacacgctct cctaacattg ttttttcacg tacaagcaag aaacgttcac taacaccaat 26160 accacgcgcc cctgcatcca ttatcccttt tatcgtttca tcctgggcaa ttacgctcat 26220 gcagcccaac gcaacaaaac tcatattatt ttcagagttg gcgcgagcta ttgaaacgtg 26280 tccggcgtcc catgctttta gaaccaattc gctgttagtc ttgcgatcac tgttagcgta 26340 agtcaatccg agtaagctgt tgatgcttgt tgcttcatct gaaatgacag caaagttacc 26400 ctggcgattg ttgatctttg ctaaaccttc cggcgtggtg tctgataccg gaaaaacgat 26460 atcgcacatt ttttctaact tttcctctaa ttcctccttt tcctcaaaca acgccgctaa 26520 ctcagtgccg gaacgctcag ctttcaattc cttttcaacg ccttttaatt tcgctgcgat 26580 cttctttcgc tctttcttgc gttgttcgtt gattcgctca acttcacaga ccattggcgc 26640 aatggccagg ctgttgattg ctgatttacc agttgatggc ggctgactgg ttacaacata 26700 aatagccgtt ggttgttcag tgccgtggta ctcaacttgg aagcgtccaa ccatagcggc 26760 ggaaacgcaa cctatgaaat gcatataggc cgatgattca ggaaattgaa cagattgcgc 26820 aatgctgcgt gacagtttac ccacaacatc aacatcatta cccaaagaga tcacaggata 26880 acggtcatta gcgccgttga tatccttcac aggttcccaa aagctggacg attggcggta 26940 gccgttcgac tgaatagcaa cgcgaagcgg cgatgttccc tgcgattctg caatggctat 27000 aacttgttga ggtgtcaatt tgttggtgtc aaagtcaaac atctaaaaat ctccttcttg 27060 ttgccgctaa ttataacggc aacattctac agccttttag caattcgtgc tatttgatcg 27120 gctcaaccct ggctaataac tttccgtcta taatgctatg acattcaaga gttttaggat 27180 cgaatgcaag cgaacgatgg aacgtgtgac caatcagcaa atatgaatgc ttatccctga 27240 tgcgatagac cgtttcacat tcctttaatg aggatatcgc actgaaaaca accttgtaac 27300 ggtatttttg catggcctca gaacggtgag gaatgtaatc tttttccggt aaactcatga 27360 gataaccccg tgattcttat gatagccata aattttatct gcctttactc tcgcttccct 27420 tgcttgctct actgtatcaa acaatcctaa atatcttttt ttaccgtcta ctgttaatga 27480 agcctcccat cttcccgccg ccttactcca gtaaactccc atctctccag atgaagataa 27540 aacctttcct ttatttctta aattatcaga tcttgaaacc aatctcaggt tttcaattat 27600 gttatccgtt ctatcatggt ttatgtggtc aatatcaaat cctttaggta tttcaccgta 27660 gtgcataaac catacaattc tgtgagcata tgccttaatc ccatttgcac caatagaaac 27720 ataaccattc tttgttattg ttcctgcaat aagatctggc tttactccgt ttctacgctc 27780 tcgccaatat aagacgccgt tttcatatct aagcattgac gagaggattt ttaacctctc 27840 gttttctgtc atttgtaaat agcctcaaac agataagcac cgccagcggc agagaatcca 27900 acttcttccc gatacagcgt gaaacggtcg ccgttttcgt cgaacacata tccagcaaca 27960 gcacctaaca cgcggccttg ttctatctga taacgcttat cggctttgaa agttttcttg 28020 atctgcatgg agtgatttac ccatgtgcat ttaatggttt tggtcttaac atttcggaat 28080 gttaatcctg tcaagttggc gtgaatccat ttttcatcta acttaacgaa agtatcatca 28140 ttttcgtcta cccgcgcttt gtataggcgg ttatcagatc cgataagttt gattgatttg 28200 aatttagcca tttgatttat ctccttttgt tttcgttgaa agcattatgc cggatacgtg 28260 atccggcgtt ttaacaaaaa gtgctatttc tgatttgcca ggcaaacagc aagcgcaaac 28320 ccgcgcggag ttagtgagcg gatcagtttt gttcgcttag actttcctcc gagcttttgc 28380 cagcctggat tgttgttccc tgttggcaat actgcatttg tatccggcat cacaaagccg 28440 ttacccgtcc aaaggcaagt ttttttggtg tacgcatcgc gagcgggaat gatttcagga 28500 aagtgaggat gtttatcatc ttccggcaac agacctccaa acgcgcaagg atgaaagata 28560 aaatcaggtt gccgccacat cgacgacagg acgctaacag gattctcgat catgtatggc 28620 acgttgaacc aatccgcgat ttcagccgca atcttgcatg ttgaaacggc cttatcctgg 28680 aatagcggat cacgttcacg ctttgcggca aagtgacgcg aaccgctaac agctaaatca 28740 gtgcatggag gaaatgcgat cacaaaatca ggcttgccgt aaacctcatt gcgagcctta 28800 aagctgaaat ctgaatcaat ccaaacgttc acatactcaa tgtttggatg acttacgcgc 28860 actgaatggt aatcaccatg atcggcatca tcataattga aacagattac tttatgacca 28920 cgttcagccc acggcaacgc atcataaccg gaaccgctaa aaagagaaaa aacaaacatg 28980 cttaaatcct ttgtgtgcag ccttgtaagg cgctttgttt gatagctatg caatcgcatt 29040 ggttatggcc tgaaatagcg acagaatgcg aaacaggccg agaattttag aatgggaaac 29100 agcgcttgca agatgggtcg aagttacaac cgcaatcatt gattattgcg ttaggatcag 29160 caagcattac gctactttct tgatccatat ccatagcgcc taatgcgtca tcaagcgtta 29220 tttcaagata ggcgacttta agcgcccatt ctttgttaag tccagccgcc tccgcttcat 29280 gtaggcgagc aaagaaagca tcttgaacca tatcaacccc acttctcaat aaacatgctc 29340 atttcgtcaa tcatcgcgta agcatcgctt tcagtcatgc ggcggattct accgcctgga 29400 tgttcgccaa cagcgtaacc agtttttacg cgggtaatcg ttaactgatg gaatccagaa 29460 gcgcgatcca ttttcaaaac tactttcccg tgagctttga caatgttcat gatggattca 29520 gttttcagtt tcatttttga ttctccattt tgtttgtgtg aaagtatgat accaatcaga 29580 gagatatagt ttttagcaat tcgtgctatt tacggcgata aacggatatc ctgttaaatc 29640 tgcatttcta taacgtgcag acctaaccca acagccgcct ttgctcataa ttaaagcgtt 29700 tttatggcga atgttcactc ggtaaattct tccggttttt atgtttttga aatatgtcat 29760 aatttaacct catgatattc tatgaattct tgcacatatg cgcattcgcg cggcgactcc 29820 tggcctttta gcacttcaca gccaccatta aaaagatagc aatgctggca aaggttgccg 29880 cctacgtttt cctcagcgat cttaattccg gtacacaaac cgccaccaaa cctatgagga 29940 aaatcgtaag cctggcagcg gcacgttgct tgatgccgct ttttctttgt cattaattat 30000 taccttggct gatgcgatcc atcttgcgat accaaataat catttcttcc agtacattga 30060 acagatcttc tttcttactt tctttgattg cgtcacaaat tagcttaatg tcacactcca 30120 taactccaag atattctccg tactgcttaa caccttcaat gttattcagt ttactttctt 30180 tgcggcaacg ttgcgctttg caaatcaggc taatgatgtt gctggtttct ttggaagtga 30240 tgaagtatgc gctcattgtt atttccttaa aaagtccggt tcgtttcgat gaatgcatta 30300 tgccaaaaca aaccggatca cttttaacaa aaagtgctat ttaattaaaa tacgaatacc 30360 ttttcgcacg ctttccgtcg cctccgtaat ggtcattagc aacagcaaga atccaagcgt 30420 taggaataaa aaatagcttc atcaatcact ctccattacg attaccggat caagacgttc 30480 aagcgctact ttgaaatctg caaagaaagt cgggcggcgt tcagttaatt ccgtgtaggt 30540 atcagttgcc aggcaagcgc gatcaccttc atagcacaca actcgcttac cattggcgcg 30600 gcttattgca acaacccgat aagtgttttt aatcatgaaa ccacctcaat ttcgcaaagg 30660 tgctcgacgc caggcgcata aacacgcttt ttgtcgtcag tgacgcaaag gcaaagcacc 30720 agaccgcgat catcaacgat gtgtaaccaa tcctgacggc tatacagcat ttcatattgg 30780 cggtcgctgt caaacgtcca agcgccagtg aattcaccaa acgccttaac ctttactttc 30840 ttgcctacaa aattcttgtg gctcattgtg tagcctccac agtttcaaat acggtatcaa 30900 atgccagaag tttaaatttc ttgccaggct caacaacctg aatattccag gtaatgccgc 30960 cgtgatatgc aaccatatca tcaatatcga cggtgatttg tgtacccttc ttgaacaagc 31020 ctttttcgtg gctcgcatcg cgtacagtaa ccacagtttc gaattgagtt tgaatttcca 31080 tcgttaatct cctgattggt ttcgatgggg taactatatc aagatacccc gatccttttt 31140 tagcaattcg tgctattagc caaacgcttt aatttcgaaa tcttcttctg cctgattagc 31200 aaccgccgtg aacatttgct caagagtttc aaatccgaaa tgtttagcga tctcataagc 31260 cgctttacct tccgtctgca tgtttagcgc ttcgcaatag tgcatttcca gattgttcag 31320 gtatttgttg ccagttggga ttgctgcgtc gattttgtcc cacacgatcc aaaagtttgt 31380 tttgtcagcc gctgcgagta caaagtttgt cttgtctact gcaataacta agttttccat 31440 catgtaatcc tcagttcgtt ttgttgaggt aatcatagaa attcgcgtct gattggtttt 31500 aacaaaaagt gctattcatg cagtatcagc aaaatgacca acgttcatct taacgaacaa 31560 tccgttcaac gtatcgaacg cattcacgat cacgttatac gatcacaata tgatcacgtt 31620 gatgattgat gtgtgagttg atgtgatttg atttgattag atttgattag atttgagtag 31680 caagtgagta gaaataatca caccttgccg atcagtgaaa acttttcccg caaaacataa 31740 ctctaggcta cgcatgtttc gttttgttcc ccgacgttcc catccagaca ttccgcagtt 31800 tagcgcaacg ttacacaaca cgtataacaa caaaatatct atataataca ttgaaaagta 31860 agataattat tattatatat ctatattgtt atataacttg ttttgcttgt ttctcatgat 31920 attgataccc ccttacggat ttatatagta taaaattata agataggtgc tcttatttat 31980 tataggtggc ggtgattttt agcgaaatgg tgtaacgatt tagaacatcg ttgatttgaa 32040 aggataaatt tgaaattact gttacattgc gtgatgtttt tgttgtctaa tcaaaaataa 32100 cacttgacgc acaagccagg tttgataagg tttcggcatc gagtaacaac ggaggataac 32160 caatcatggt aaaaggcaaa atcgtaaagc gtgacctatc catagttgat gcaacgccag 32220 aagatgcaac caaagatatc atgcgcctaa tgtacatagc acggatactg gaggctgcaa 32280 aaccaaacag gctatattac atcgacatta aggcatctgg tcgagatgtt aactcattgc 32340 gccaggcaat aaacactatt gcgaaagagg atcgcagaac cattaagaca ataaccactg 32400 ataacggcta tatcggcttt gttatcatcg acgttaaaga agcgtttgtg taaagaggtg 32460 aatcatggaa caaatgacaa gtaaagagtt ttgcgataaa ttgcgcgaag ctgcaagagc 32520 ggcggcaaaa ggtgaggtaa tcaagttgcc agttgcctac ataacaatga acgtccaggc 32580 agaggacgca aagaaactaa attcatcata catccgcacg atcatgaatc gtgtgccgga 32640 agtaaaagag gttggatctg taagtgtcaa gaaaaagaca agtgatgacg gagctgaata 32700 ttacgagatc gcaatcaatc gtgacacaaa gcgccgcatc gtgacaacta atgaccttcc 32760 ggcaattaaa gagcatgagc gcgaaaagct ggttgaaaag attatgcgca tatcgccgga 32820 cttctcacac ctggacgatg aaaaggcact tgttgcaatg cgagcggttg ccacgttcaa 32880 agaactgatt aaggagatcg caaaatgaaa gcattcgcct ttgtcgctgc aatgtggtgt 32940 gttgttattg atagtcctgg cgtggcgctg gttgtgttag gtctgatttt gttgggggtg 33000 tttgatgaat aagaaacttt tagatcatgc agtgctgttc tttatcgcta ctgctggatt 33060 ttcgctgttg tcaattgctt tttcaatcct tgcttttgcc gtgtcaatgc tgaaatgacg 33120 gattacatcc tgattggcaa atagcacttt ttgttaaaac tcgattgggg tagtttggca 33180 taattacccc atcgaaacga aacgaggaaa acacaatgac taccttaatc acatgggaac 33240 acgaaacaag ccagccagta acccgcgagt ttgaaactct tagccaggct tatcttttgg 33300 ctgctaacgg tggttattac aaggcgcaag tcattgatga aatgggagtg atcgtttatg 33360 aattccatgc ataatttccg cagctacacg caggaggtgg attttgaagg agtcaagttg 33420 ccacttccta atatataccg atatatcacg cgtgatgaat acggatttat caacgcatgg 33480 cgagataagc ccaaaaagat tggcaacgaa tacggcgacg gctcagagat gccgatcacg 33540 ttaggtcatc aaactggcga gaatgtagcg gctgtaatgc gcaaatatcg ccgcagcctc 33600 acaaactcat taatttctat ctttggcata caggagtaat aaaaatgttt gaacttaaac 33660 gcgtaactgt tgagatcctg aatgatggct cttacgatgg aatgaaacac ttaaccttcc 33720 cgatccgcat tgaaaattgc attgattatg ggattgaagt cggcatggtt gacgttcctc 33780 ttgatgcact tatagcggct ggatatgaca aagagaaagg ccaatgtgct gatatacctg 33840 atagctatga aagcgacaca catttcccgt tcacatatgg agaatgccgg attgttgatt 33900 gaggtgatta aatgagcagt tggcacaatg agcacgtaat gcaattctac cgacaaagaa 33960 taaagaaaat gctaaatagc actaattgct aaaactaaat caggggcatt tgctatagtg 34020 tccctagttg agttaagcaa aaggagaaac aaccatgtta aaagcaaatg aaatcaattt 34080 cccgatcaca tttaacgact gcgcaaacgg actaagcatt acattttata gtgcaacaag 34140 taacggcgtt tggagtggtg gagataaaag cagcctttca ttgtcatatt tcattaaatt 34200 gcacaatgaa cacaatccgc ataataaaat tgatgaaaga tatcgttatg ccatggcagc 34260 aaagaaagag aaaaaacgag aagataacga tttggcagat gctttgaatt atctaaacat 34320 caagcctgga caggtgtttg ttaactgcgc tgttttcacc gcctcaatta tcgcgaaaga 34380 ttatgattac atttgcgacg ttgagctaat gccagttggc gcaatcctgg ttggcattca 34440 acgtgaatgc ggggaatggg acgaactgaa attaaagccg cgccgcattg aagtacacga 34500 cgacacgatc acattacttt ccggctacgc tggcggatac aaaaccggaa acaattacag 34560 cggcgaaatc aacattaagc gcggcacgaa agtaaaaatc aaaaacagca cgaattgcta 34620 aaacagaatc ctggggttgc ggtatagtaa ccccacacca aacgaaagaa ggaaatcaaa 34680 atgaaattag tacgcaacgc aatttatggt ttacacctca aaggcggtaa aggttcaaca 34740 tatgttcacc tttttcatgt aaatgagcgc aaccaggttg atattgcgat gtatacagat 34800 cagtttctta taaaaggtga aatgggcgat aggtttaatc cgcaattcgt agaacaagat 34860 ccaacgtata gccatgacca ttgggtttgt gatgattact ataatttcat catggttgat 34920 agtcacgtta cggacacgcg gaaaataaca gttagcgact ataagcgcat gactcgaaaa 34980 gtattgaagc ataaagcgaa atagcacaaa atgttaagcg gtcatgtgtc ctgatgggta 35040 taatgaccgc atcaaccaac caaaggagaa gtaaaaatgt ctaaagagtc tatcaagcag 35100 tttctgaaat acggttctat gattgtgctc gctaccgttg tgctggctgg catcgcatat 35160 agcattattg agtttgcggg gtaattgatg tttacgatca acttatgggg aagggattat 35220 gttaatccct atgttgtgac aaaacagcgt ttcgttattt tcgacctgga cggcacattg 35280 tcaaatggtg agcaccgctt gcacctgttg ccaacggttg atttgcacct tactgaaagc 35340 tggagtgagt ttaatcgcgc cgcaaaggat gattcaccat tcatcaataa cattgccgtg 35400 tgcaacgcca tgtttgatgc cggatacctg gttatcatcc taacaggccg tagcgatgaa 35460 gttgaggcag aaacgcgagc atggttagct gaaaacggcg tttctttcga ttggctgatt 35520 atgcgccgcg cggaggacaa ccggaaagat accgtaatca aagaggaagt tttacggact 35580 ataggacttg atcgcattgt cgcatgttgg gatgactcac caaacgttat tgcacacatg 35640 cgcggactgg gcttgacggt ttaccaggtt attgactacg gcgaccaatt gcacgatcac 35700 ctaaaatctc acggcgtgga ggaattgagc gatgacagca aagctaattg aagatcggca 35760 tggtgataac gtgcttattt acatcgaagg gaaatcggtg tggattgact gcaaatatga 35820 ggatgacaca catttgaagt ttcgcggaat ctttgacaag aaaggcgcat tggagttagc 35880 ggaacacatc aagcgtcttg cggaagaatt accgaaatag aggatttatt atgtttactt 35940 ttatcttgat cgctatcgtt ttaattctgt atgtttgcgg cgctttcctg atgcgagcgc 36000 tactaaaaga ggctgacgct gatgataagg atagttggaa gccgattatt ttatggcctg 36060 gctatatcat cgtgccaatt tacgaactaa tccgcgatgg tgagtttaaa tggtaaatag 36120 cactttttgc taaaactagc taaggcacat ttgataaagt gtgccttgtc gagacaagca 36180 aggagaatga cacatgaaag tttttatgat taaacagttt gggcgcactg catattatat 36240 catcaacgac gatggatata ttttcatttc gaactatcct actcttgaag taggtcgtca 36300 tttcataagt tcagttgacg aaaagggagt tatcaatatg gttgagtgtg gaatgatggt 36360 tcccttcaat aaagaagcgg ctgaattatt ccagcaatca cagatccgaa aacgcaaccg 36420 aaatattatt tgctttactt ttgcaatggt tgtaactgtt ggctcaatct tacttggagt 36480 gtggtcgtga tgaatactga taataaagtt gaaacgcaaa agactattaa ttatctgcgt 36540 gacctggccg ataaactgga gaataaccaa attcatttaa acgagatcgt tattgaatcg 36600 cgcaacgttt gttggaacgt ggaagaagaa acattaacca tgcaagtttt aaaggcggta 36660 gacaatggct aaaattatca tccttaatgc gcctcctggt gctggtaaag acacgatagg 36720 caagatgatt tctttctatt cgcctggtga gcaatgccgt atcattagct ttaaagaacc 36780 aatgtttgat attgcattgg ctatccttgg taaagagaac tatcagcgtt tcatgttggc 36840 ttataatgac agagggaaaa aagaaaggcc acaaacattc ctgatgggta tgtcaccgcg 36900 tgagttcatg atctggatta gcgaggatgt gatcaagcct aaattcggca aggattattt 36960 cggcgtaagg tttaaccaaa aagtaaaaga gtgtgatgct cctgttgttt gcactgatgg 37020 tggttttcca gatgaaatta tcgctctaat caatgctggt aatgaggtga agctatgccg 37080 cttgcatcgc gacgggttca cgtttgcagg tgatagccgc aactacattc gaatcaatcc 37140 ctattaccat aaaaacggat atagcgagca cgattataaa ttggttgata agcaaccgaa 37200 tgtaactgta aaggaaatta ttagcgatca ccttaaatag cacgaattgc taaaacttgc 37260 gcaaggccat ttgttatagt ggccttattc aagagaagca accaatcaga ggaataagcg 37320 atgaaaatta aatgcacaaa atcaaacacc ctgaatctga cagttggcgc tatttatcac 37380 agggaagaag gtgtaaacgg gtttagccca actgtaacgc atgagtacgg cggtcaattc 37440 ttcttaaacg gtaaagacgg attatctgta aacgttggct ctttcgtact tgcagagttt 37500 gatatcgtgg aggaataaaa atgttctgga ttcattcgcc gcaagggttg atttatgtgc 37560 ctggcattga attagaagaa cgacgctacg gcgacacggt gaacatcata acaaaggatt 37620 caaaaggcaa tacgcttaaa acgtatgttt ttaacactcc ggttaaggtt gaaaaagatg 37680 attagattta ttggttttgt tgcattgctt atgtgcggaa tattgcttac tgacaatgac 37740 acttggcaaa gcgctgttgg cggcttactt atttttcttg ctggtggcat ggttggcagt 37800 gatgatgcag caaacaagaa taaactttcc gatcatttac gcagagagac aaaatgatga 37860 atagaaaaat gctggtacgc gcattcgcga agattgctaa aagatggggc gattcagcta 37920 aaacacctta cacgtttagt atgtacggtt acaagaatgc tcgcgaatgg ggtcgcatca 37980 ttgcagataa ctatgaatca gaaatgctgg aggcttacgc aagctggatt gatacgccgc 38040 taaatgaatg gggccaggct gaatatgatt gttttgctga tgaagaaatt tcttgttggt 38100 aaggagagta agcatgatcg ctaaaaatga cctttgcaac ctgttaacgg ctggtaatga 38160 ttatcaggtt ttgaatagca cagaagaccg ttattttatt gagtgtgaca atgggaatcg 38220 catgtggatt aaaaaatgct ttttcgaaag cgagcaccta agcaatgcaa agcggatcgc 38280 agatgaagca agcgcattag cagagcgtga aagaattcgc gacatagtag cggagcaggt 38340 tagaaattcg tgcttgcctg gtggcgatat cttcaaatca atgcgcggtg aaatgacggt 38400 tgaaattcaa gatcgcgcaa ttgcaaaatc agttgattct gtaattgaga atgcgcgatt 38460 caatgcgttg actgactcag atcctaacga atacgtcaaa gtgttagagc aactgcgtga 38520 agtgctgcga gtgccggaag gtgaaaatat tgtcactcat gcaaaagttg taagagcgtt 38580 ggctgatgcg ctgattggcc tgcaaaaata gcactttttg ttaaaacttc cgcgagggca 38640 tttgttaaag tgtcctcatt gaagcgaaac atagagatta cgaaatggct gtatacactg 38700 gcaagatgtt taaatgtcac aatccgattt acggcggcaa ctatgagcta atcattgatg 38760 atgatcgcat tattcttgct gatgatgacg ctgaagaata tcacgaggtt gtagcggata 38820 agtacgataa tgacgttatg atcttatcag gaaacaaccg catcaagttt tacgcagtgg 38880 aggaataata gatgatcgca atcaacctat cagaagaaca agcgaaatcg ttgctaaata 38940 gcttgggatg gcgttatggc tcgggtgaat atgagcgatt ggatgttaaa agcgatgttg 39000 caaaagaggt tttcatccag cttgagcagc aattaatgcc acgctcaaca agtgcggcag 39060 agttagcagt gtggcggcac gaaaacggat tgaacgaaat ggatattgaa aagtggaagc 39120 gtaaaaacga ggtgttagca tgatttacat tcactcttac ggcgttggca agtttggcag 39180 caagcagatc agaaacattc acgacacgtt agaagaagcg caagcacagc aacgcgtttt 39240 aggtggagtt gttcaggctt tcgaaccagt ggaagatctg gaaatgcaag aggccataaa 39300 ggaaatcgtt gttgatgaag tttcatgtat aatcgacgaa ttgagcactg atgcgccagg 39360 gttcgcaaga acatttgaac caggctcaac gcaatgggat tgctggaagg tgaaaacctt 39420 aaacaacatt cgagcaatct tggatggaaa atttaacgaa aattgatagc tcaaatagcg 39480 cctcaatcgc attctaagag gcgcattttt taggaaggtg attctatgcc tgaacacata 39540 aaaccgtcgc agtggtgcgc acagaagcaa gaagaagcat tagagcgcgg cgacactgac 39600 acagcattgc actactttga aatgtttcaa ctttggaaat caagaggact ttaaaatgtt 39660 tggacttaat gaggctgaat acaacattgt taaacgcgct gcaaaagagt gcatgaagga 39720 tctgaaagtt gaagttacga gcggcaacaa atacgatcat attgcggctg gcattatcac 39780 caaacatcac acgccaattt ctaccttaat cactcgcaca aagtttgtat ggcttgctgg 39840 ttatatagct ggtcgatggg gtaagcctgg cgagtacgaa taataagcac ttgaatcaat 39900 gaaggttgac gtcgtgttgt ctgattggta gattaaacgc cgtgaacata agaggctcaa 39960 tagagcaaag cgcggcgcgt tagccgtaaa acttaatcgg attggatcgc aaggcgaatg 40020 acaatcgcgg gaaccgtaac ccgcaaccat tcaggagaat tcacggtgca aaacaaagaa 40080 atcgcaaagg taacaattat cgttgagcgt gacggaaaga aaacgcgata caagaagcga 40140 tttaaacaat gcgaggcgat gttaggccgg atcggtgatt tcatggtgaa attgcaagag 40200 gataagccgg atgaaattca aggttaagct aaccatccgt cgcatgggtc gcaattgcgg 40260 atcatgtaaa caagatttcg aatgcgaggt tgaagcgcta aacaatgttg atgcggttca 40320 atgtgctaaa gttctttcag gtgctaaccc tgaaacacac caattttcaa ttaattatgt 40380 gaggtctatg taatgctgat gttcattgcg ggttttggtt tttgtgcgat tatctgtttc 40440 tttctctatc tttgtgtttc gtggggtgta cagcgttgcg taaaagacaa cgaattagtg 40500 atcgccattt acgaaaacaa aactgataaa tggaagatta ctaaaaactt tgaaaagatt 40560 gctgatgaaa ttgcattccg tcgcaagttt ggcaagcctg gaagcatcaa gtatattgac 40620 taatccaaag ccgaaacata acccgccttg tgcgggtttt tttgtgtctg caatttagct 40680 ataatcacgc atcaacagga ggattttatt atggctaaaa aaacagaagc gccagccgct 40740 gaaagtctaa actttaaaaa gttatggaat aagcgatatt ccgatctggt aggtagtgag 40800 attgataaac gcgcaacgct aacgccggaa caagtcatga acctgattgt tcagtatttt 40860 ggtgggcgg agctaaacgc cattaaagca ggtgaaacgg catcctttca gggtcgagtg 40920 tatcaggata cgattcacaa gccgcgtatt tttacatggg aagggttaaa gctgttttgc 40980 ggtttttccg gcacagcact agcaaagtgg aaacttaaac caggctatga tgttgttatt 41040 gagtttgcgg aatctgtaat caaagagcaa aaataccagt tagctgcaaa cggaatgatt 41100 aacagcacca tgatcgcaaa agagcttggc attgataaag gcgacacgtt caatattagc 41160 gccaacgcca aagagcaaga caatacagaa gaagcgatga aaaacgcagt cgcaagcgtc 41220 ctaagcaaga tttaaaggtg aatgctatgt tgatttggga agatttaaca aaacttgaaa 41280 agatggctat taaggaatta agcacacacg attttgatac tttcgttaaa atctggttcc 41340 ctatccagca gggcgaaaaa tggattccta actggcatca cctttatatt gcaagggcaa 41400 ttgatgaaat tattgaaggt gtgcgcaaag ataccatttt taacgtaact ccaggttcag 41460 gcaaaaccga attgctttcg atccactttc cgccttattc atatttgaaa ttgaacaagg 41520 tgagaaatct taatatttcg tttgctgata cactggttaa acgtaacagt aagcgtgtgc 41580 gcgatctggt taactcgcga gaatggcaag agctttaccc cgcaaaaact ggtacaagca 41640 aggatgatga atttcagatc cttaatgacg ctggcaaagt tcgcctggaa atgattagta 41700 aatcaatggg cggtcagatt actggtagcc gtggcggtta catcacgcct ggtgtttatt 41760 ccggttgtgt aacgcttgac gatccagaaa agccagatga tatgttttcg aaggtgaagc 41820 gtgagcgcgg tcaaatgatc gcaaagaaca ccattcgatc acgtcgcgca cattcagaaa 41880 cgccgattat cgttattcag cagcgtctac acgcgcagga tatgacctgg tttctgatga 41940 atggcggaat gggtattgaa tttgatcaga tatcaattcc ggcgatggtt acggaggaat 42000 acgggaaatc gcttcctgat tggttgcaac cgcactttga aaaggatgtg ctttcgtctg 42060 aatacatcgt tattgatggc gttaagtatt attctttttg gcctagcaag gaaagcattc 42120 atgacctgaa agcgctacgc gatgcggatt tatacacgtt cctttctcag taccagcaag 42180 agccaattgc tcttggcggc aacgcaatta acgttggatg gttccagtat tacggaaccg 42240 gagaaaaatc aacgatgcca aaaccggatc gctttgatta tacgtttatt acggctgata 42300 ccgctcagaa agaaggtgag ctaaacgact acagcgtttt atgttattgg ggcatgttca 42360 aaggccgcat ctattttatt gacggtgtgc gcggcaaatg ggaagcgcca atgttagaaa 42420 cgcagtttaa agcgtttgtt aaacagtgct ggaaccgtaa caaagaatgc ggaaacttgc 42480 gtaaaattta cgtggaagat aaagcgagcg gcacaggttt gatccagaat tgccgcaaag 42540 catttccgat agaaattacg ccagtgcaac gcgataaaga caaagtaacg cgctgcatgg 42600 acgcacagcc agtgatcaag aatggttatg ttgtattgcc tgaatcgcat cacatgcttg 42660 ctgaatttct ggcagaggcg gcggcattca cttatgatga tagccatcct catgatgata 42720 ttatggataa cctttttgat gcggttaata ttgagcttaa ccttgctgat aatgcggttg 42780 atagaatgaa gcgtttagct ggattagcga agcgttaatg tgtaaaatca aaggctggaa 42840 tattccggcc ttttttatta tgggggtatt tatgagcgac aaggtaaagg ccattactaa 42900 agaggatggt tacaatgaaa ttttcggatc taaagatgga acgtttaggc cgaatgcttt 42960 ttacatgcaa agcgcggcat ttaaaacgtt atcgcaattt tatgaagaag atggaatggc 43020 gcggcgcatt gttgatgtta ttcctgaaga aatggtaacg cctggtttta aagttgacgg 43080 cgtgaaaaat gaaaaggctt ttaaatcacg ttgggatgaa ttgcgactaa acgctaaaat 43140 tattgatgca ctttcatggt cgcgtttatt tggcggttct gcaattcttg ctgttgtggc 43200 tgataacaga atgttgaaat cacctgttaa gcctggcgca caacttgaag atattcgcgt 43260 atatgatcgc tatcagatca ccattcagga gcgtgaaact aatgcgcgga gtgttcgtta 43320 cggcgagcca aaactttata agatctcacc tggcggagac atacctgaat tctttgttca 43380 ttactcgcgt atttgcatca ttgatggtga gcgcgtatcg aatgaaaaac gccgtcaaaa 43440 tgatggttgg ggtgcaagca ttttaaacaa gcgtttgatt gaggcgattg ttgattataa 43500 ctattgccag gaattagcaa cgcaattgct acgccgaaaa caacaagcag tatggaaagc 43560 gcgtgatctg gctttgatgt gcgacgacga agaagggcgc tatgcggcgc gtttgcgttt 43620 ggctcaggtt gatgacgaaa gcggagttgg taaggcgatt ggtattgatg ccaccgatga 43680 agaatatgag gtgcttaatt ctgacgtttc aggcgttcca gagttcttac aggaaaagat 43740 tgatcgcatt gttgccctaa ctggcattca tgaaatcatc ctcaaaaata agaacactgg 43800 cggcgtttca gcgagtcaaa acacggcgct agagactttc tataagttga ttgatcgaaa 43860 gcgcggagag gattacaagc cgattcttga attccttttg ccgttcatga tcgcagaaac 43920 ggaatggtct attgaattcg aaccgttaag cgttccgagc gataaggaca aagctgaaat 43980 tatggctaag aacgttgaat ctgttgtaaa attgaaagct gaacaggcga ttgatctcaa 44040 agaaacaaga gacactttgc gatccatttg tccagatctt aaaatcaggg acaatgacaa 44100 cattgaattg ccagagccgg aagatttaga tccagagcca gggcaagagg gaggattgaa 44160 taaatgatgt ttcccgatat gacagagagc gacgttgaaa aggcgcttaa tgatctggcg 44220 gttgatatca ctgaaaacaa tgctgatgta gaggcggcaa ttgccgcctt tatgttgctg 44280 gtttacgggt tcgccgttag tgcatataaa tatcacactc gccagtttat cagaatggca 44340 aagcaaacag gcggcgcaaa taatcgatcc gttatccttc tgattggtgg tgtttacgga 44400 aatatgaatg aacgttggtt caattcaaaa tacataacct ggcgatcact tattgaggca 44460 tacgtaagga aatttgcgta taatgtgctt gatgattggg aagtacaaca gcgtttaaat 44520 gagttacgcg agcaagcaga aacaagagca cagatcagaa agcgtctaag aaaatataaa 44580 gcgtgggcta aaaatagggc gtctggtttt gttggttcgt ggtattccgt gttaatgtat 44640 caacgcgtag ttgatgccgg agttacccat tatatttggc gcactcagga agataacagg 44700 gttagacctg aacacgcgaa attggacatg aagcggatct cattggcaat aaagcacata 44760 ttcccagggc aggagtataa ttgtcgctgt tgggcacaac cggattggaa aacagagcaa 44820 gaggtatatt tatgaagaag gtgcaacgct ttgatagtgt cagagtaaag gctcattttg 44880 atgaacacgg atttttagtt gatcggccta ttgtggcgcg aattggtttg cagatttatc 44940 aaacgccata cggcgagcgt agagaattca ggccagcaag tgaagtgttt aaagctgata 45000 gcctagcgac ttacgcaggg aaaccaatca ctgttggtca cgtcactgta acgcctgaaa 45060 atgcgcgtga tgttgttgtt ggtacttgtt ccggtgctgg aattcctaac gggatcggag 45120 ttgaagcgcc tttgaacatt tacgcaaaag acgctataga aagcgccaag aaaaagcaaa 45180 cggctgaaat tagcgttggt tatacgtcga ttgatattga taagcctggt tatggctcta 45240 atgaaactgg tgaatacatc tttgaagaag atctaaaaga agatgaacag ccgccggaag 45300 gatgggtgaa atttgatgcg ctgcaaacaa gcattagcgt taaccatatt gcgttagtgt 45360 ttaagggtcg cgctggcatt gcaaaattga atcttgatag cgagcaagaa ttcccgtatg 45420 atatcgacgt taaattaaat aaagaggacg aagttatgac cgttaaaatt aaacttgatg 45480 gtgctgttga atttgacgta ccaaaagaag ttgctacttt tatcgaaacc gtgaaagcag 45540 acgctacagc ggcaaaaact aaagccgata gtctggaagc agagcgcgat gcgctacagg 45600 cgaaagttga cggcattccg gcacagattg aagaagccgt gaaaaaagca aaagctgacg 45660 ctgatgaaca tgccgcgctg gtcgctaccg cttccgaagt tggcgtaaaa tgcgacggac 45720 taaccgccaa agaaattaag atcgcatatg tgaaagaggt catgggtaat gatattagcg 45780 caaaggctga tgcatatatt gataccgctt ttgatatcgc taaagaatct gataaaatgg 45840 cggctcaacg tatcgcatta aaaggcgatg cagcaaaaaa caaacaggat gaagcggaaa 45900 cagttcttga tccttccgca cgtctgaata aagctaagta aggagaaaat attatggctc 45960 agatttcagc aagttatcat gttgcaatgg ctcgcgcctt gcctggtcaa gtttctgaca 46020 cttccgcgta taacatcgac ggcgcttgtg ttcttgatgt tgatgccgga tcaacaattt 46080 atgttggcgt tggtgttcaa catggcggag ttagcgcaag cggtgagaaa ttaattaagg 46140 ctatggcggc gtctggtaat gcttatggtg ttgctattcg atccaacttc cagaccacat 46200 ctaaagatgg tcgcatggtt tatgaggctg gtagcggaat taacgtaatg actaaagggc 46260 gcgtttggat ggtatctgta gaatcggaag cgccagcatt caaaacacct atcaagcttg 46320 ataattcagg acaggttgat ttaacgtctg gattaattga aaccacttgg atcgcaactg 46380 gagactttac ggaattccaa ggtctaaaac tcgttgaggt tcaattggta taataataag 46440 gccggattat tccggccttt tatatatggt ggttttatgt ctgattacgg ttctataata 46500 ggtatagttg aaaatgggtg cttggtagat tcaagccttt ataatataga tggctcgcca 46560 gttatggatg tttcagccgt gataggtaag gccgtgtgtg ttgatcgcat atcttgcggt 46620 tacaaaataa tcagccacaa ttttaatgaa aactcaaagc attacggaat cgttacgcgt 46680 gattatatgg atttttgcaa agaatattac gactccggag atcctgttaa cgtaataact 46740 catggtcgaa tttgggtaat cactcaggac gaacatgata ttccatcgca taaagatcct 46800 gtttttgtta aaggcgatgg ctctatatca acaagtggat ttcctactag ttggtttttt 46860 actggcgaat acgtaagata ttcacacgga tatattttac tatcagttca gttgtagtgt 46920 gctaacacaa aaaaacactt gaacgtttaa caaaaaatgc tatattaaag ccgttattat 46980 gacggctttt ctatgaggga aaatattatg tctaaagaaa tgaaatatga tgaatttgaa 47040 gcaaacgtga tcgccaacca catgcagtta cgcggcgcta aaaacgacgc atctgatatg 47100 ggtatttgga cggctcaaga gcttcataaa attaaggctc aagcctatga aaaagaatat 47160 ccggcaggtt ccgcactgcg tgtattccct gtaacgtccg agctttctga tacagataaa 47220 acttttgaat atcagacttt cgacagagta ggccacgcaa aaatcatcgc tgattacact 47280 gatgatctgc caactgttga cgcgctgatg acctctgaat tcggtaaagt gttccgcctg 47340 ggtaatgcgt tcctgatttc cattgacgaa atcaaagcag gtcaacgcac tggcaagagc 47400 ctttccactc gcaaggccaa cgccgcacag aatgcgcatg atcagcttgt aaaccacctg 47460 gttttcaaag gctctaaacc gcataaaatt atcagcgtgt ttgatcatcc aaacctgaca 47520 actatcaact ccgcaggttg gaacaacgcc gcaggtactg gtaaaaagcc ggaaaccgca 47580 caggatgaat tagagcaagc gattgaaaaa atcgaaacgc tgaccaacgg gcagcaccgc 47640 gctaatatga tcctgattcc gccgtcaatg cgtaaagttc tgatggttcg tatgccggaa 47700 acaaccatga gctatcttga ttatttcaag cagcagaatg gcggtatcac tatcgaatcc 47760 atttccgagc ttgaagatat cgacggcgca ggtactaaag cggcgctggt ttacgaaaaa 47820 gatccaatga acatgagcat tgagattcca gaagcgttta acatgctgac cgcgcaacct 47880 aaagacctgc atttcaaagt gccttgcacc tctaaatgta ctggcctgac gatttaccgt 47940 ccgttaacgc tggttctgat taaaggtctt gtagtaggct gataattcaa ttaatatagg 48000 gaaccgttaa aaggttccct ttttttatat ctggagaaaa acaaatggct aaagaaaaag 48060 aaattacact ggttaacact ggcgttgcac tgattattat tgatcgcgtt gacgttatgc 48120 caggtaagga aattaccgtt gcggaaagcg tgttagagcg gcagggtaca aaatcactgg 48180 ttgcacaagg caagctaacc gtaaaagata acagcgagct aaacgataag atcattgaag 48240 cgttcaacga aaaacgcaag ccaaacccaa ccgaaggcaa aagcaaagca cagcttgagg 48300 acggcggcga gtattaatta gaaagggcgc tcatgtagcg ccttttttat tatgggggtt 48360 attatggttg atgaaattta tggcgatatt gagatcctag agcaaattta taaaatcgcg 48420 ccagcattca agaaagttga tcctgaattg attcaggcat ggattgaact ggctaaagat 48480 ttcgtttgcg aaaagcattt caaagacaaa tatttccgcg ctgtcgctct ttacactttg 48540 cacctgatga ctcttgacgg agctatgaaa caggaaagtg agagcgtaga aagctactcg 48600 catcgtatag cgtcattttc cctgactggt gagttttcgc agacgttcag caaagttagc 48660 gacgacacaa gcggtaacac gttgcgccaa acgccgtggg gtaaaatgta tgaagttctc 48720 aacaagaaaa aaggcggcgg atttggcctt acaactgcct ttcatcggag gtgctcacga 48780 tgaattatga acaaatcaga tctatggcat cggctggaat taattttttt agcgatggaa 48840 cgggtgaatt tgattgcatt actcaacctg gaagcgttga aattgttggc ggtattgagg 48900 ttgaaaaacc tgaaataaag gtgaagatta aaggtcttgt gagagcacca cggacgcgag 48960 aagttgacgg agaggtaatt agagttaccg acaagttagg cgtttttaat gctgatgtag 49020 agcttaaaaa cggatatcag attgatatag acggcgagcg ttatgttatg gttgaaacaa 49080 gaccaatcag accaacaagc attaccgttg cttaccgtcc aataatgcgg agggttgcgg 49140 ttcatggctg attattcgat ccgagaattt catggcgacg ttaacaaatg gatcgagcaa 49200 gtagaaaacg gcttgaatga tgtgattcag atttttggtg aaaaggttca cggcgcatta 49260 gttgatattg cgccagttga tacaggacga tttaaagcca atatgcagat cacagcaaac 49320 aagccgccac tttacgcgct taaccaatat gatcgcgacg gtgaaaaaat aaaggcggaa 49380 ggtcgcagga cgctttatgc tttattgcat ggcggcggcg caattaaatc aatatacttt 49440 tccaatatgc tgatttacgc taatgctctt gaatatggtc attcaaagca agcgccagcg 49500 ggggtatttg gtatcgttgc gatccggttg cgctcttaca tggcggaagc gataagggag 49560 gcgagaaaga aaaatgcact atgaattatc tgttgcggca agaatggcgc tggctcaaga 49620 atacgaaagc gaatatatga tcgcttatga aaacgttgaa tttacgccac caaaaggcgg 49680 cggcatttgg cttaaatatg actacaaaga agcagataca ataattcatg atctgaaaag 49740 gaaatgcatt agctatattg gaatggttca gatcggaatt gaattcccgc caggttcagg 49800 aattgataag gctcgcaagc tggcaaaaaa tattgctgat ttctttgaag atggtaaaat 49860 gctttcgaat ggctatattt cagaaggtgc aaaagtgcat caagttcaga aatcagaatc 49920 aggctggttt tatccagtac gtttttatgt tcgttatgat ggttaataaa gggggctatt 49980 atgcacttac ctaacggatc acagattttt attgaaaaca cgcgcggctc agaaatcaaa 50040 gcaacagcaa tcagcaacgc aacagatccg gtgtttaccg cgtctgatac tggcggattg 50100 aaaaaaggtg attatgtgat tgtcactgca tcaacgtggg gtaaacttct tgatcgtgta 50160 ctgcgagtta aagaggttag cgataacaca agtttcactc ttgaaggtat cgacaccact 50220 gatgaaaacg tttttccgtc cggcgctact ggtagcgtta tgaaaattga atcatggaca 50280 gagatcccat gcgtacagga cttggggcag gacggcggcg agcagcagta ttacaatta 50339
Claims (12)
Bacteriophage CF1 (KFCC11682P), consisting of the nucleotide sequence shown in SEQ ID NO: 1 and belonging to Myoviridae, having the killing activity specific for Citrobacter freundii .
The bacteriophage CF1 (KFCC11682P) according to claim 1, characterized in that the bacteriophage is stable at a pH of 4 to 9.
The bacteriophage CF1 (KFCC11682P) according to claim 1, wherein the site is bacteriorbide resistant to a beta lactam antibiotic.
The bacteriophage CF1 (KFCC11682P) according to claim 1, wherein the bacteriophage is stable at 37 to 70 占 폚.
An antibiotic composition comprising the bacteriophage CF1 (KFCC11682P) of claim 1.
A composition for feed addition comprising the bacteriophage CF1 of claim 1 (KFCC11682P).
A feed comprising the feed additive composition of claim 8.
A disinfectant comprising the bacteriophage CF1 of claim 1 (KFCC11682P).
A cleaning agent comprising the bacteriophage CF1 of claim 1 (KFCC11682P).
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Non-Patent Citations (4)
| Title |
|---|
| BMC Genomics. 2013, 14:481, pp. 1-15. |
| GenBank: KM236241.1, 2015.02.26.* |
| GenBank: KX015770.1, 2016.04.27. |
| Genome Announcements. 2015 Volume 3, Issue 1, e01434-14e, pp. 1-2.* |
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