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KR101033818B1 - 돼지의 알파에스 1 카제인 유전자, 그 프로모터 및 그의 용도 - Google Patents

돼지의 알파에스 1 카제인 유전자, 그 프로모터 및 그의 용도 Download PDF

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KR101033818B1
KR101033818B1 KR1020090058189A KR20090058189A KR101033818B1 KR 101033818 B1 KR101033818 B1 KR 101033818B1 KR 1020090058189 A KR1020090058189 A KR 1020090058189A KR 20090058189 A KR20090058189 A KR 20090058189A KR 101033818 B1 KR101033818 B1 KR 101033818B1
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여명구
강성조
안종덕
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조아제약주식회사
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Abstract

본 발명은 돼지 알파에스 1 카제인 유전자, 돼지 알파에스 1 카제인 유전자 프로모터, 및 상기 프로모터를 이용한 발현 벡터, 상기 발현 벡터를 이용한 목적단백질의 제조방법을 제공한다. 본 발명의 프로모터는 목적 단백질의 유선특이적 발현을 촉진하므로, 본 발명의 프로모터를 이용하여 형질전환된 동물은 유즙 중에 목적단백질을 고농도로 분비하게 되어 유용단백질의 생산에 유용하게 사용될 수 있다는 장점을 갖는다.
돼지 알파에스 1 카제인, 프로모터, 벡터

Description

돼지의 알파에스 1 카제인 유전자, 그 프로모터 및 그의 용도{A Gene of porcine alpha S1 casein, a Promoter of the same and Use thereof}
본 발명은 돼지 알파에스 1 카제인 유전자, 돼지 알파에스 1 카제인 유전자 프로모터, 이를 포함하는 발현벡터 및 이를 이용한 목적단백질의 제조방법에 관한 것이다.
의약 분야에서 경제적 부가가치가 높은 EPO등 유용단백질의 생산을 극대화하는 방법으로서 세포배양법에 의한 대량생산방법이 주로 사용되고 있다.
대한민국 특허출원 제94-12082호에는 변형된 재조합인간적혈구 조혈인자(rhEPO) 유전자를 포함하는 발현벡터가 개시되어 있으며 이 발현벡터로 형질전환시킨 동물세포주 COS-7(ATCC CRL 1651, African Green Monkey Kidney Cell)에서는 EPO를 대량 생산 할 수 있었으나, 이 방법에서는 계속적으로 형질전환을 시켜야 한다는 번거로움으로 인하여 대량 생산에는 부적합한 문제가 있다. 대한민국 특허 공고 제10-0232640호, 제10-0434729호 등에서도 형질전환 세포주 배양법으로 EPO를 생산하는 방법이 개시되어 있으나, 이 세포배양방법은 동물의 혈액을 배양 배지로 이용하기 때문에 생산 비용이 높아지고, 배양기술에 있어서 전문적인 지식이 요구 된다.
반면, 형질전환동물을 이용한 유용단백질 생산방법은, 동물이 분비하는 체액중에 목적단백질이 포함되므로 기존의 세포배양법에 비해 목적단백질의 생산, 분리, 정제가 용이하며, 활성 또한 우수하게 유지되므로 이 분야에 대한 관심이 급증하고 있다. 대한민국 특허 제10-0358754호에는 돼지의 유즙속에서 EPO를 생산하는 형질전환동물(새롬이)이 개시되어 있으나, 여기에 사용한 프로모터는 유즙의 구성성분 중 작은 부분을 차지하는 유청 성분의 프로모터(WAP)이었다.
본 발명자들은 유즙내에서 목적단백질을 높은 효율로 발현하는 유선특이적 프로모터를 개발하기 위하여, 연구한 결과 알파 에스 1 카제인 유전자 및 그 프로모터에 대한 염기서열을 규명하고 본 발명을 완성하였다.
본 발명에서는 돼지 알파에스 1 카제인 유전자 및 그 프로모터를 제공하고, 이를 이용하여 목적단백질을 대량 생산하는 방법을 제공하고자 한다.
본 발명은 돼지 알파에스 1 카제인 유전자를 제공한다.
본 발명의 돼지 알파에스 1 카제인 유전자는 구체적으로 프로모터, 및 3' UTR(untranslated region) 부위를 포함하는 서열번호 1의 염기서열을 포함한다.
본 발명은 또한 서열번호 1의 염기서열 중 1번째부터 9300번째까지 염기서열에 해당하는 서열번호 2의 프로모터를 제공하며, 상기 프로모터는 구조유전자의 5'쪽에 위치하여 구조유전자의 발현을 조절한다.
본 발명의 돼지 알파에스 1 카제인 유전자, 또는 프로모터는 서열번호 1 또는 서열번호 2의 염기서열에 하나 이상의 붕괴(disruption), 결실(deletion), 삽입(insertion), 점(point), 치환(substitution), 논센스(nonsense), 미스센스(misinse), 다형현상(polymorphism), 재배열 돌연변이(mutation)가 일어난 기능적 등가물 중 선택된 하나가 될 수 있다.
본 발명은 또한 서열번호 2의 프로모터 전체 또는 일부를 포함하는 발현벡터를 제공한다. 바람직하게, 서열번호 3, 또는 서열번호 4의 염기서열을 본 발명의 발현벡터는 포함한다. 상기 서열번호 2, 서열번호 3, 또는 서열번호 4의 염기서열은 벡터에 삽입되어 프로모터의 역할을 하며, 상기 서열을 프로모터 서열 또는 돼 지알파에스1 카제인 유전자 프로모터 서열이라 한다. 본 발명에서 사용하는 '돼지 알파 에스 1 카제인 유전자 프로모터'라는 용어는 돼지 알파 에스 1 카제인 유전자로부터 유래한 프로모터를 의미한다.
서열번호 3, 서열번호 4는 서열번호 1의 돼지 알파에스 1 카제인 유전자 전체 게놈 서열 중 각각 3568번째부터 9037번째까지의 염기서열, 4321번째부터 9300번째까지의 염기서열에 해당하며, 공통적으로 엑손 1 영역을 포함한다.
본 발명의 발현 벡터는 필요에 따라 적절한 위치에 조절인자들, 즉 또 다른 프로모터, 인핸서(enhancer), 선택적 표지 유전자(selective marker), 5'-UTR(untranslated region), 3'-UTR, 폴리아데닐화 신호(polyadenylation signal), 리보솜결합서열, 게놈의 특정 부위로 삽입될 수 있는 염기서열, 인트론, 또는 WPRE(woodchuck hepatitus virus posttranscriptional regulatory element) 등을 추가로 포함함으로써, 형질전환 세포주의 구축을 용이하게 하며, 목적단백질 발현량의 극대화 및 발현의 안정성을 도모할 수 있다.
선택적 표지 유전자는 네오마이신 저항성 유전자(neomycin-resistant gene) 등을 사용하며, 시판하는 벡터로부터 해당 부분을 절단하여 사용할 수 있다. 네오마이신 저항성 유전자는 세포주 구축시 사용되는 G418(2R,3S,4R,5R,6S)-5-amino-6-[(1R,2S,3S,4R,6S)-4,6-diamino-3-[(2R,3R,4R,5R)-3,5-dihydroxy-5-methyl-4-methylaminooxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-2-(1-hydroxyethyl)oxane-3,4-diol) 시약에 대해 저항성을 나타내는 유전자로, 프로모터 조절하에 단백질을 발현하는 동물세포주 구축시 효율적인 선택적 표지 유전자로 작용할 수 있다.
인슐레이터는 프로모터 부근에 존재하는 조절인자의 영향을 돕고, 위치 비의존적인(position-independent)발현을 도와주는 인자로, 프로모터의 조절하에 단백질을 안정적으로 발현할 수 있게 하며, 시판되는 벡터로부터 해당 부분을 절단하여 사용할 수 있다.
WPRE는 mRNA의 안정화에 기여하여 단백질 합성량을 증대시킬 수 있는 조절인자로, 프로모터의 조절하에 단백질을 대량으로 발현할 수 있게 한다. WPRE 역시 시판되는 벡터로부터 절단한 것일 수 있다.
본 발명의 발현벡터는 또한, 서열번호 5, 또는 서열번호 6 염기서열을 추가로 포함한다. 상기 서열번호 5, 또는 서열번호 6의 염기서열은 벡터의 3'arm을 형성하며, 형질전환 세포주의 구축을 용이하게 하며, 목적단백질 발현량의 극대화 및 발현 안정성을 도모할 수 있다.
서열번호 5, 서열번호 6은 각각 서열번호 1의 돼지 알파에스 1 카제인 유전자 전체 게놈 서열 중 26344번째부터 30599번째까지의 염기서열, 14447번째부터 19401번째까지의 염기서열에 해당한다.
돼지 알파에스 1 카제인 유전자 전체 게놈 서열 중 서열번호 3, 4, 5, 6의 염기서열 위치는 도 1에 나타낸 바와 같다.
본 발명의 벡터는 바람직하게 서열번호 3의 염기서열, 및 서열번호 5의 염기서열을 포함한다.
구체적으로 본 발명의 벡터는 도 2의 개열지도를 갖는 것으로, pBC1-pig αS1 casein벡터를 한국생명공학연구원 유전자은행(대전, 대한민국)에 기탁번호 KCTC 11324BP로 기탁하였다. 본 발명의 발현벡터 pBC1-pig αS1 casein벡터는 pBC1 벡터를 기본 골격으로 사용하며, 선택적 표지 유전자로 네오마이신 저항성 유전자가 융합되어 있다.
본 발명의 발현벡터는 프로모터 영역 염기서열의 3' 쪽으로 목적단백질을 암호화하는 염기서열을 추가로 포함함으로써, 목적단백질을 발현할 수 있다.
상기 목적단백질은 산업상 이용가능한 유용단백질로, 일 예로 의약품의 유효성분으로 사용되는 모든 단백질, 즉 EPO(erythropoietin), 알도스테론(aldosterone), 아드레노코티코트로핀(adreno-corticotropin), 혈액응고인자(blood clotting factor), 고나도트로핀(gonado-tropin), 인슐린(insulin), 프로락틴(prolactin), 또는 바소프레신(vasopressin) 등일 수 있으며, 바람직하게는 hEPO(human erythropoietin)이다.
본 발명은 상기 네오마이신 저항성 유전자, 인슐레이터, WPRE 등을 포함하는 발현벡터의 바람직한 예로 도 3의 개열지도를 갖는 벡터를 제공하며, 구체적으로 pBC1-pig αS1 casein+hEPO-WPRE 벡터를 한국생명공학연구원 유전자은행에 기탁번호 KCTC 11325BP로 기탁하였다.
본 발명의 pBC1-pig αS1 casein+hEPO-WPRE 벡터는 pBC1 벡터를 기본 골격으로 사용하며, 본 발명의 프로모터 영역의 3'쪽에 인간EPO를 암호화하는 유전자, 및 인간EPO 유전자의 3'쪽에 WPRE가 융합되어 있다.
본 발명의 발현벡터는 낙인벡터(knock-in vector)로 제조될 수 있다.
"낙인벡터"는 게놈의 특정 유전자 위치로 목적하는 유전자를 삽입할 수 있는 벡터로, 상동 재조합(homologous recombination)이 일어나도록 타겟팅하고자 하는 특정 유전자에 상동인 염기서열을 포함한다. 본 발명의 낙인벡터는 목적 단백질을 코딩하는 핵산서열이 게놈상의 알파에스 1 카제인 유전자에 삽입되는 알파에스 1 카제인 타겟팅 벡터이다.
본 발명의 낙인벡터는 바람직하게는 서열번호 4의 염기서열, 및 서열번호 6의 염기서열을 포함한다.
본 발명의 낙인벡터는 필요에 따라 포지티브 및/또는 네거티브 선별유전자를 사용하여 형질전환된 세포를 선별할 수 있다. 선별유전자란 벡터로 형질전환된 세포를 선별하기 위한 것으로, 약물내성, 영양요구성, 세포 독성제에 대한 내성 또는 표면단백질의 발현과 같은 선택가능 표현형을 부여하는 유전자들이 사용될 수 있고, 포지티브 선별유전자와 네거티브 선별유전자가 있다.
포지티브 선별유전자란 선택제가 처리된 환경에서 선택유전자를 발현하는 세포만 생존하도록 하여 포지티브 선택을 가능하게 하는 유전자로 네오마이신(Neomycin; Neo) 저항성 유전자, 하이그로마이신(Hygromycin; Hyg) 저항성 유전자 등이 있다.
네거티브 선별유전자는 무작위적 삽입이 일어난 세포를 선별하여 제거하는 네거티브 선택을 가능하게 하는 유전자로 허피스 심플렉스 바이러스-싸이미딘 키나제(Herpes simplex virus-thymidine kinase; HSV-tk) 유전자, 하이포잔틴 포스포리보실 트랜스퍼라제(hypoxanthine phosphoribosyl transferase; Hprt) 유전자, 싸이토신 디아미나제(cytosine deaminase) 유전자, 또는 디프테리아 톡신(Diphtheria toxin) 유전자 등이 있다. 네가티브 선별유전자는 프로모터 영역의 5'말단 쪽 또는 3'arm의 3'말단 쪽에 위치한다.
포지티브 선별유전자 및 네거티브 선별유전자는 별개의 프로모터, 폴리A등을 가질 수 있으며, 사용되는 프로모터는 원숭이 바이러스 40(SV40), 마우스 유방 종양 바이러스(MMTV) 프로모터, HIV의 긴 말단 반복부(LTR) 프로모터, 몰로니 바이러스, 시토메갈로바이러스(CMV) 프로모터, 엡스타인 바 바이러스(EBV) 프로모터, 로우스 사코마 바이러스(RSV) 프로모터, 또는 포스포글리세레이트키나제(phosphoglycerate kinase;PGK) 프로모터 등이 있다.
본 발명의 낙인벡터와 게놈상의 알파에스 1 카제인 유전자가 상동 재조합이 일어나면 벡터상의 목적 단백질을 코딩하는 핵산이 숙주세포의 알파에스 1 카제인 게놈 유전자로 통합(integration)되어, 숙주세포의 알파에스 1 카제인 단백질 대신 발현된다.
본 발명은 포지티브 선별유전자로 네오마이신 저항성 유전자, 및 네거티브 선별유전자로 허피스 심플렉스 바이러스-싸이미딘 키나제(Herpes simplex virus-thymidine kinase: HSV-tk)를 사용하는 낙인벡터의 바람직한 예로 도 4의 개열지도를 갖는 벡터를 제공하며, 구체적으로 Pig αS1 casein-hEPO knock in 벡터를 한국생명공학연구원 유전자은행에 기탁번호 KCTC 11326BP로 기탁하였다.
본 발명의 Pig αS1 casein-hEPO knock in 벡터는 Lox A neo벡터를 기본 골격으로 사용하며, 프로모터(도 4의 Pig aS1 casein 5' arm 부분을 의미함)의 3'쪽에 hEPO, hEPO의 3' 쪽에 포지티브 선별유전자로 네오마이신 저항성 유전자, 네오 마이신 저항성 유전자 3' 쪽에 3'arm(도 4의 Pig aS1 casein 3' arm 부분을 의미함), 및 3'arm의 3' 쪽에 허피스 심플렉스 바이러스-싸이미딘 키나제(Herpes simplex virus-thymidine kinase; HSV-tk) 유전자(TK)가 융합되어 있다.
본 발명의 벡터제작은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술분야에서 일반적으로 알려진 효소 등을 사용한다.
본 발명은 또한 본 발명의 발현 벡터를 도입시켜 형질전환시킨 동물의 체세포를 제공한다.
본 발명의 벡터가 도입되는 동물의 체세포는 돼지 등 유래로, 1차, 2차 또는 영구세포일 수 있다.
상기 세포 내로 본 발명의 벡터를 도입하는 방법은 핵산을 세포내로 도입하는 방법을 모두 사용할 수 있으며, 당 분야에서 공지된 기술 즉, 일렉트로포레이션(electroporation), 칼슘 포스페이트 공동-침전(calcium phosphate co-precipitation), 레트로바이러스 감염(retroviral infection), 미세주입법(microinjection), DEAE-덱스트란(diethylaminoethyl-dextran), 양이온 리포좀(cationic liposome) 법 등을 사용할 수 있다. 이 경우 벡터는 원형의 벡터를 적절한 제한효소로 절단하여 선형의 벡터 형태 또는 플라스미드를 제거한 선형의 벡터 형태로 도입할 수 있다.
본 발명의 프로모터 유전자는 유선조직에서만 특이적으로 단백질을 발현시킨다. 돼지의 유즙 중 단백질 구성성분은 카제인이 90%를 차지하고 있으며, 카제인은 다시 알파, 베타, 감마 카제인으로 나누어 진다. 그 중 알파 카제인이 70%로 상당부분을 차지하고, 알파카제인은 알파에스 1 카제인 및 카파카제인으로 나뉘며, 알파에스 1 카제인이 그 중 55%를 차지하므로, 돼지 알파 에스 1 카제인 프로모터를 이용한 벡터는 유즙분비하는 동물, 특히 돼지에게서 유선특이적으로 외부에서 유래한 목적 단백질을 발현하도록 할 수 있다.
본 발명은 또한 본 발명의 발현 벡터를 도입시켜 형질전환시킨 동물의 체세포의 핵을 탈핵된 난자에 핵이식하여 만든 동물 수정란을 제공한다.
"핵이식"은 핵이 제거된 난자에 세포의 핵을 넣어 이식시키는 것을 의미하며, 이런 핵이식된 수정란을 착상시켜서 태어난 개체는 핵공여 세포의 유전적 물질이 핵수여 세포질로 그대로 전달되었기 때문에 유전적으로 완전히 동일한 복제 개체가 된다.
본 발명은 또한 본 발명의 발현 벡터를 주입시킨 동물 수정란을 제공한다.
상기 주입은 구체적으로, 수정직후 전핵단계에 있는 접합체의 웅성전핵 내에 미세조작기술로서 유전자를 주입하는 미세주입법(Microinjection technique), 배아 간세포(embryonic stem cells)에 유전자를 삽입시키고 이를 배반포기의 수정란 내에 이식하는 방법(Stem cell insertion technique), 레트로바이러스 벡터(Retroviral vector)를 이용하여 유전자를 수정란 내에 주입하는 방법(Retroviral insertion technique), 유전자를 수컷의 정소 내에 주입하여 유전자를 정자에 삽입시키고 이를 난자와 수정시키는 방법(Sperm-mediated gene transfer technique) 등에 의할 수 있으며, 바람직한 예는 미세주입법이다.
본 발명은 또한 본 발명의 동물 수정란을 이식시켜 얻은 형질전환동물을 제공한다.
본 발명의 발현벡터로 형질전환될 수 있는 동물은 유즙을 분비하는 모든 동물, 즉 돼지, 생쥐, 소, 양, 또는 염소 등이다.
본 발명의 발현 벡터를 이용한 형질전환동물의 생산 방법은 통상적인 방법에 의한다.
즉, 형질전환하고자 하는 동물 중 쥐 등의 건강한 개체로부터 수정란을 채취하고, 수정란에 본 발명의 발현 벡터를 도입한 후, 정관결찰 생쥐 등을 이용하여 위임신 생쥐 등을 얻고, 이를 대리모로 하여 난관 내에 수정란을 이식한 후, 대리모로부터 얻은 자손 중 형질전환된 개체를 선별하는 과정으로 이루어질 수 있다.
또한, 돼지 등의 건강한 개체로부터 난포란을 채취하고 체외성숙용 배양액세서 배양한다. 아울러 돼지 등의 태아로부터 채취, 배양한 공여체세포로 본 발명의 발현 벡터를 도입한 후 벡터가 도입된 체세포를 선별, 배양한다. 상기 체외성숙된 난자에서 핵을 제거하고, 이 공간에 공여세포를 주입한 다음, 전기 융합을 통해 핵이식이 완료된 난자의 공여세포와 세포질을 융합시킨 후 체외배양한다. 이 복제수정란을 과배란을 유도한 수란돈에 이식한 후, 수란돈으로부터 얻은 자손 중 형질전환된 개체를 선별하는 과정을 거친다.
이후 형질전환된 것으로 확인된 개체로부터 유즙을 수거한 후, 목적단백질을 분리, 정제함으로써 목적단백질을 제조하게 된다(A. Gokana, J.J. Winchenn, A. Ben-Ghanem, A. Ahaded, J.P. Cartron, P. Lambin(1997) Chromatographic separation of recombinant human erythropoietin isoforms, Journal of chromatography, 791, 109-118).
본 발명의 목적단백질 제조방법에서 분리, 정제 방법은 통상적으로 사용되는 방법을 사용할 수 있으며, 구체적으로는 여과법 또는 크로마토그래피법 등이 될 수 있다.
이렇게 하여 제조되는 본 발명의 형질전환동물은 유즙 중에 목적단백질을 발현할 수 있다.
따라서, 본 발명의 돼지 알파에스 1 카제인 유전자, 프로모터, 이를 이용한 발현벡터 및 형질전환동물은 목적단백질의 생산분야에 유용하게 사용될 수 있다.
본 발명에서 유전공학적 기술과 관련된 사항은 샘브룩 등의 문헌(Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N. Y., 2001) 및 프레드릭 등의 문헌 (Frederick M. Ausubel et al., Current protocols in Molecular Biology volume 1, 2, 3, John Wiley & Sons, Inc., 1994)에 개시되어 있는 내용을 참조할 수 있다.
돼지 알파에스 1 카제인 유전자 프로모터는 목적 단백질의 유선특이적 발현을 촉진하므로, 본 발명의 프로모터, 및 이를 이용한 발현벡터에 의해 형질전환된 동물은 유즙 중에 목적단백질을 고농도로 분비하도록 하게 되어 의약학적으로 중요한 가치를 갖는 유용단백질의 생산분야에 유용하게 사용될 수 있다.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.
< 실시예 1> 돼지 알파 에스 1 카제인 유전자의 분리 및 클로닝
본 발명의 유선 특이적 유전자를 분리하기 위해, promega사의 pig genomic DNA library와 국립축산과학원(경기도 수원시 권선구 축산길 77 오목천동 564 국립축산과학원, 대한민국)에서 제공받은 BAC(bacterial artificial chromosome) clone을 가지고 돼지의 알파 에스 1 카제인 유전자(pig αS1 casein gene)의 염기서열(sequence)을 밝혔다.
1) pig genomic DNA library 를 이용한 돼지의 알파 에스 1 카제인 염기서열 분석
돼지의 알파 에스 1 카제인 유전자의 염기서열이 밝혀지지 않았기 때문에, 염기서열이 밝혀진 사람, 소, 말과 생쥐의 알파 에스 1 카제인 cDNA 염기서열을 비교하여 종 간에 높은 상동성과 보존된 부분을 참조하여, 돼지 알파 에스 1 카제인 PCR 증폭에 사용될 프라이머 염기서열을 제작하였다. 5'UTR 쪽 Forward primer 5'- TGACAACCATGAAACTTCTCAT-3'(서열번호 8), Reverse primer 5'- GTTCCTGATGCCTGAGAGGA-3'(서열번호 9), 3' UTR 쪽 Forward primer 5'- AACCATTTTATCTGAAGACTTTG-3'(서열번호 10), Reverse primer 5'-TCTCAGTCACTGCACACAATT-3'(서열번호 11)를 이용, 돼지의 genomic DNA 로부터 PCR 증폭(PT-200, BIO-RAD, 94℃에서 5분간 변성 과정을 거친후, 다음의 35 cycle을 수행하였다: 94℃에서 30초간 변성 후, 56℃에서 30초간 프라이머 결합, 72℃에서 5분간 신장반응)한 결과 5'UTR 쪽으로 3.3 kb의 염기서열(서열번호 12)과 3'UTR 쪽으로 303 bp의 염기서열(서열번호 13)로 이루어진 산물을 얻었다. 이를 pGEM-T vector(promega, 미국)에 클로닝(cloning) 후 염기서열 분석(sequencing)을 통해 돼지 알파 에스 1 카제인의 유전자의 일부인 것으로 확인되었으며, 5'UTR쪽 3.3 kb 와 3'UTR쪽 303 bp의 염기서열을 밝혔다.
밝혀낸 3.3 kb의 돼지 알파 에스 1 카제인 유전자의 염기서열로부터 5'UTR 쪽 탐침인자를 만들기 위해 Forward primer 5'-TGACAACCATGAAACTTCTCAT-3'(서열번호 14), Reverse primer 5'-CTAAGACTCTCATACTGAGTG-3'(서열번호 15)를 이용 PCR 증폭(PT-200, BIO-RAD, 94℃에서 5분간 변성 과정을 거친후, 다음의 35 cycle을 수행하였다: 94℃에서 30초간 변성 후, 56℃에서 30초간 프라이머 결합, 72℃에서 30초간 신장반응)으로 551 bp의 산물(서열번호 16)을 얻었다.
돼지 알파 에스 1 카제인의 염기서열을 밝히는데 사용될 탐침인자를 만들기 위해 상기에서 얻은 5'UTR 쪽 551 bp와 3'UTR쪽 303 bp 의 PCR 산물 100ng을 5분간 끓인 후, 얼음에 식혀 변성(denaturation)시켰다. 변성된 DNA를 프라이머, dNTP, [α-32P]dCTP(3000 Ci/nmol, NEN)를 함유하는 반응 완충액에 첨가한 후, 클레노우 효소(Klenow fragment; promega, 미국)를 첨가하여 37℃에서 1시간 동안 반응시켰다. 이후 반응액을 세파덱스 컬럼(Sephadex G-50 column)을 사용하여 정제함으로써, 32P로 표지된 돼지 알파 에스 1 카제인 유전자 탐침인자를 준비하였다.
돼지 알파 에스 1 카제인 유전자를 밝히기 위해, 돼지의 게놈 라이브러리를 스크리닝하였다. 본 실시예에서 돼지의 게놈 라이브러리는 promega사의 pig genomic DNA library를 사용하였다.
라이브러리를 도입할 호스트 박테리아는 다음과 같이 준비하였다.
0.2%의 말토오스(maltose)가 함유된 LB 배지(Scharlau, 스페인) 5㎖에 박테리아 콜로니(XLL blue P2, promega, 미국) 하나를 접종하여 37℃에서 하룻밤 동안 배양하였다. 상기 배양액의 1%를 다시 새로운 0.2%의 말토오스 함유 LB 배지 50㎖로 옮겨, 2.5시간 동안 배양하였다. 600㎚에서의 흡광도가 0.5 정도 되었을 때, 배양액을 2500rpm의 속도로 10분간 원심분리하였다. 그 결과 얻은 세포 침전물을 멸균한 10㎖ 황산마그네슘 용액에 현탁시켜, 최종농도가 1 ×1010세포/㎖이 되도록 하고, 실험할 때까지 4℃에 보관하였다.
라이브러리를 적정(titration)하기 위해, 라이브러리를 SM 완충용액{0.1M NaCl, 8mM MgSO4, 50mM Tris-HCl(pH 7.5), 0.01% 젤라틴}에 여러 농도로 연속 희석(serial dilution)하였다. 고체 LB배지가 담긴 플레이트(plate)를 37℃ 항온반응기(incubator)에서 데우고, 탑 아가(top agar)를 녹여 48℃로 유지되는 수조에 놓아두었다. 여러 농도로 희석된 파지 용액 10㎕ 와 상기에서 준비한 호스트 박테리아 100㎕를 혼합하여, 37℃에서 호스트 박테리아를 감염(infection)시켰다. 탑 아가에 파지로 감염된 호스트 박테리아를 첨가하고 잘 흔들어 준 후, 상기에서 준비해 둔 LB 배지 위에 부었다. 15분 후 플레이트를 거꾸로 뒤집어 37℃ 항온반응기에 서 하룻밤 동안 배양하였다. 밤새 배양한 플레이트의 배지 상에서는 파지가 호스트 박테리아 내에서 라이브러리 DNA를 복제한 후 호스트 박테리아를 용해시킨 흔적인 플라그가 형성되었으며, 이후의 실험 단계를 위해 배지를 4℃에서 1시간 이상 식혔다.
상기 플레이트에 대해, 일련 번호를 기재한 NC 필터(Amersharm Biosciences.; 영국)를 준비하고, 필터의 가운데 부분부터 닿도록 하여 상기에서 준비한 라이브러리 DNA 플레이트 위에 필터를 덮었다. 필터에 바늘을 수직으로 찔러 위치를 표시하고, 1분 후 조심스럽게 필터를 배지로부터 떼어냈다.
각 필터를 변성화 용액(denaturation solution; 0.5 M NaOH, 1.5 M NaCl; sigma, 미국), 중성화 용액(neutralization solution; 1M Tris-HCl (pH7.5), 1.5M NaCl; sigma, 미국) 및 2 ×SSC 용액(0.3M NaCl, 0.03M Sodium citrate, sigma, 미국)에 차례로 1분씩 담근 후, 80℃ 오븐에서 2시간 동안 두어, 전이된 라이브러리 DNA가 필터 상에 완전히 고정(immobilization)되도록 하였다.
고정된 필터를 밀폐용 비닐에 담아 전혼성화 용액{prehybridization solution; 50% formamide 40 ml, 20X SSPE(saline-sodium phosphate-EDTA buffer) 20 ml, 50X Dehardt's solution 8 ml, 100ng/ml salmon sperm DNA 1.2 ml, 10% SDS(Sodium Dodecyl Sulfate) 1.2 ml, 증류수 0.6 ml; sigma, 미국}을 넣어 준 후 68℃에서 1시간 동안 살살 흔들어 주면서 전혼성화 반응을 수행하였다. 전혼성화 반응 후, 준비한 탐침인자를 100 ng 첨가하고, 68℃에서 18시간 동안 살살 흔들어 혼성화 반응을 수행하였다. 혼성화 반응 후, 0.1% SDS를 함유하는 2 ×SSC 용액에 필터를 담그고, 65℃에서 10분 동안 흔들면서 세척하는 과정을 2번 반복하였다. 세척 후, 필터를 공기 중에서 건조시키고, 자기방사기록법(autoradiography)을 수행하였다. 자기방사기록과 플레이트를 대조하여, 양성 반응을 보이는 플라그를 선택하고, 선택된 플라그를 SM 완충액(0.1M NaCl, 8mM MgSO4, 50mM Tris-HCl(pH7.5), 0.01% gelatin, sigam, 미국) 500㎕에 넣고 클로로포름(chloroform) 한 방울을 첨가한 후 잘 섞어 4℃에 보관하였다. 상기와 같은 스크리닝 과정을 두번 반복하여, 양성반응을 나타내는 클론들을 최종적으로 얻었다. 최종적으로 얻은 클론을 가지고 탐침인자를 만들 때 사용했던 프라이머{정방향 프라이머(서열번호 14), 역방향 프라이머(서열번호 15)}와 T7, SP6 프라이머(Cosmo, 한국)를 서로 짝을 이뤄 PCR 증폭(PT-200, BIO-RAD, 94℃에서 5분간 변성 과정을 거친후, 다음의 35 cycle을 수행하였다: 94℃에서 30초간 변성 후, 56℃에서 30초간 프라이머 결합, 72℃에서 5분간 신장반응)을 하였다. PCR 증폭 결과 5'UTR 쪽 551 bp 탐침인자로 5' 방향으로 3.7 kb(서열번호 17)와 3'UTR 쪽 303 bp 탐침인자로 5' 방향 2.2 kb(서열번호 18)와 3' 방향 6.3kb(서열번호 19)의 산물을 얻어 pGEM-T 벡터(promega, 미국)에 클로닝 한 후 완전한 염기서열을 얻었다.
염기서열 분석은 솔젠트(solgent, 한국)에 의뢰하여 진행하였다.
2) Bac clone 을 이용한 돼지의 알파 에스 1 카제인 염기서열 분석
pig genomic DNA library를 이용한 염기서열 분석 방법에서 탐침인자를 만들기 위해 사용된 프라이머(서열번호 14 ~ 서열번호 15)를 사용하여 PCR 증폭 방 법(PT-200, BIO-RAD, 94℃에서 5분간 변성 과정을 거친 후, 다음의 35 cycle을 수행하였다: 94℃에서 30초간 변성 후, 56℃에서 30초간 프라이머 결합, 72℃에서 30초간 신장반응)으로 확인 되는 4개(155F1, 188A9, 616B6, 874E5)의 클론(clone)을 얻어 계속적인 염기서열 분석 방법을 통해 총 33kb의 완전한 염기서열(서열번호 41)을 밝혔다.
서열번호 염기서열 분석용 프라이머
20 5'-TAACGAATCCAACTAGGAACC-3'
21 5'-TCCTTCTCCAACCCTATATTC-3'
22 5'-TGAGAGGGGAATAGAAAGAAC-3'
23 5'-TATCAATAGGTCTCAGAAGATC-3'
24 5'-TAGACTTCGAGTTTGGAGGG-3'
25 5'-TATAAGGCACAAATGAGCCCTT-3'
26 5'-AAATGCTCAACATCCCTGATTA-3'
27 5'-TATTCCGTGTTCATGGATTGG-3'
28 5'-AAGTATTCTCCACTGCCTTAC-3'
29 5'-TGTGAGTATGGTAGAGAATTT-3'
30 5'-CTATTGTGAATAGAGCTGCAAT-3'
31 5'-GTGTGAGAGTGTGTACCAGTT-3'
32 5'-TGTTCCCTTGTGATATATAGC-3'
33 5'-CTTGTTCCCACAGTTCAAATG-3'
34 5'-TAGATACCTCCACCAAGAGC-3'
35 5'-TTCTCAGGTTTCCTGAGGTG-3'
36 5'-GTGCACATTTACATACTGATAG-3'
37 5'-ATCATCAATGAACTGAACAGGGT-3'
38 5'-TTGAGACCTAAGTCACAGCTA-3'
39 5'-TCCATAATAATTTATGTCAAGGG-3'
40 5'-TAAGGCAAAATGTGCATGAGTG-3'
3) 버크샤(Berkshire)종의 돼지로부터 알파 에스 1 카제인 염기서열 분석
pig genomic DNA library 분석법과 Bac clone으로부터 얻은 돼지의 알파 에스 1 카제인의 DNA 염기서열을 바탕으로 버크샤 종 돼지의 genomic DNA로부터 알파 에스 1 카제인의 염기서열을 분석하였다. genomic DNA는 첨단양돈연구소(경상남도, 대한민국)에서 분양 받은 돼지 체세포로부터 intron사(한국)의 Genomic DNA Extraction kit(cat. No. 17231)를 사용하여 분리한 것을 사용하였다. 2)에서 분석된 알파 에스 1 카제인33kb의 염기서열을 총 7부분(4.6kb, 5.7kb, 4.9kb, 5.4kb, 5.3kb, 4.7kb, 4.4kb)으로 나눈 프라이머(서열번호 42 ~ 서열번호 55)를 이용하여, PCR 증폭(PT-200, BIO-RAD, 94℃에서 5분간 변성 과정을 거친후, 다음의 35 cycle을 수행하였다: 94℃에서 30초간 변성 후, 56℃에서 30초간 프라이머 결합, 72℃에서 5분간 신장반응) 후 pGEM-T 벡터에 각각을 클로닝 한 후 염기서열을 분석하였다. 염기서열 분석은 솔젠트 (solgent, 한국)에 의뢰 하여 진행하였다.
프라이머 서열번호
정방향 4.6kb 5'-AGGATTACAAGATTGCTGTTGGA-3' 42
역방향 4.6kb 5'-AAAATCGTCAACTACCCTGATTA-3' 43
정방향 5.7kb 5'-AGCTGCAATGAACATGTGGGTG-3' 44
역방향 5.7kb 5'-CACCCACATGTTCATTGCAGCT-3' 45
정방향 4.9kb 5'-CACTCAGTATGAGAGTCTTAG-3' 46
역방향 4.9kb 5'-CTGTTCAGTTCATTGATGATTTC-3' 47
정방향 5.4kb 5'-TTTGGTTCTGCTGTGCCATAA-3' 48
역방향 5.4kb 5'-GTAGAGCTTAGAGTTCAACTC-3' 49
정방향 5.3kb 5'-CACTCAGGATGAGATTCTCTA-3' 50
역방향 5.3kb 5'-AACTGATTGATGACTACTATGTT-3' 51
정방향 4.7kb 5'-AGATCTGACACCTTCTAATTAC-3' 52
역방향 4.7kb 5'-GTGTATTCCTGCACAGCAAC-3' 53
정방향 4.4kb 5'-GTCAAACTGCCTTCTAGAGTC-3' 54
역방향 4.4kb 5'-GTAGACTTATGTGAAGCTCTG-3' 55
그 결과 버크샤 종의 돼지 알파 에스 1 카제인 genomic DNA 염기서열(서열번호 1) 및 염기서열 정보를 알아내었다.
서열번호 1의 염기서열은 돼지 알파에스 1 카제인 유전자의 전체 게놈서열이며, 크기는 33248bp이다. 서열번호 1의 염기서열 중 구조 유전자(gene) 부위는 7760번째부터 27875번째까지의 염기서열이며, 시작코돈은 9326번째부터 9328번째까 지 염기서열이고, 종결코돈은 25982번째부터 25983번째까지 염기서열과 26593번째 염기 부분이다. 또한 5'UTR부위는 7760번째부터 7804번째까지의 염기서열, 및 9314번째부터 9325번째까지 염기서열이고, 3'UTR부위는 26594번째부터 26636번째까지 염기서열, 및 27482번째부터 27875번째까지 염기서열, 폴리에이시그널부위는 27855번째부터 27860번째까지 염기서열이다. 엑손부위는 7760번째부터 7804번째까지 염기서열, 9314번째부터 9376번째까지 염기서열, 12653번째부터 12685번째까지 염기서열, 13117번째부터 13137번째까지 염기서열, 13537번째부터 13578번째까지 염기서열, 14049번째부터 14072번째까지 염기서열, 14780번째부터 14803번째까지 염기서열, 16568번째부터 16591번째까지 염기서열, 17617번째부터 17646번째까지 염기서열, 18423번째부터 18446번째까지 염기서열, 18533번째부터 18580번째까지 염기서열, 20563번째부터 20604번째까지 염기서열, 22369번째부터 22386번째까지 염기서열, 22868번째부터 22906번째까지 염기서열, 24004번째부터 24030번째까지 염기서열, 25018번째부터 25041번째까지 염기서열, 25835번째부터 25983번째까지 염기서열, 26593번째부터 26636번째까지 염기서열, 및 27482번째부터 27875번째까지 염기서열 부분이고, 인트론 부위는 7805번째부터 9313번째까지 염기서열, 9377번째부터 12652번째까지 염기서열, 12686번째부터 13116번째까지 염기서열, 13138번째부터 13536번째까지 염기서열, 13579번째부터 14018번째까지 염기서열, 14073번째부터 14779번째까지 염기서열, 14804번째부터 16567번째까지 염기서열, 16592번째부터 17616번째까지 염기서열, 17647번째부터 18422번째까지 염기서열, 18447번째부터 18532번째까지 염기서열, 18581번째부터 20562번째까지 염기서열, 20605번째부 터 22368번째까지 염기서열, 22387번째부터 22867번째까지 염기서열, 22907번째부터 24003번째까지 염기서열, 24031번째부터 25017번째까지 염기서열, 25042번째부터 25834번째까지 염기서열, 25984번째부터 26592번째까지 염기서열, 및 26637번째부터 27481번째까지 염기서열이며, CDS(coding sequence)는 9326번째부터 9376번째까지 염기서열, 12653번째부터 12685번째까지 염기서열, 13117번째부터 13137번째까지 염기서열, 13537번째부터 13578번째까지 염기서열, 14049번째부터 14072번째까지 염기서열, 14780번째부터 14803번째까지 염기서열, 16568번째부터 16591번째까지 염기서열, 17617번째부터 17646번째까지 염기서열, 18423번째부터 18446번째까지 염기서열, 18533번째부터 18580번째까지 염기서열, 20563번째부터 20604번째까지 염기서열, 22369번째부터 22386번째까지 염기서열, 22868번째부터 22906번째까지 염기서열, 24004번째부터 24030번째까지 염기서열, 25018번째부터 25041번째까지 염기서열, 25835번째부터 25983번째까지 염기서열, 및 26593번째 염기서열이다.
또한, 알파 에스 1 카제인 아미노산 염기서열(서열번호 7)를 분석하였다.
상기 탐침인자의 위치와 탐침인자로 찾아낸 염기서열의 위치 구조는 도 1에 도시된 바와 같다.
도 1의 63/12-ATG-48은 돼지 알파 에스 1 카제인 2번째 엑손의 염기서열 63개중 12개의 염기서열 다음 ATG (시작코돈)가 존재하며 이후로 48개의 엑손 2번째 염기서열을 갖는 것을 의미한다. 149/147-TG는 돼지 알파 에스 1 카제인 17번째 엑손의 염기서열 149개중 147개의 염기서열 다음 TGA (종결코돈) 중 TG 가 존재 하 며, 44/A-43은 돼지 알파 에스 1 카제인 18번째 엑손의 염기서열 44개중 종결코돈의 A가 존재하고 이후로 43개의 엑손 염기서열을 갖는 것을 의미한다.
분석한 돼지 알파 에스 1 카제인 염기서열과 정보는 미국 국립생물공학정보센터에 등록 (EU025875)하였다.
< 실시예 2> pBC1 - pig α S1 casein 클로닝 벡터 구축
pBC1 벡터(인비트로젠, 미국)의 앰피실린(Ampicillin) 저항성을 가진 유전자를 네오마이신(Neomycin) 저항성 유전자로 치환한 벡터[G418 drug에 저항할 수 있는 neo' 유전자는 pEGFP-N1 벡터(Clontech, 미국)로부터 정방향 프라이머 5'-GCGGCCGCGCGCGTCAGGTGGCAC-3'(서열번호 81)와 역방향 프라이머인 5'-CGATCGGACGCTCAGTGGAACGAAAACTC-3'(서열번호 82)을 이용하여 1.9 kb PCR 산물(서열번호 83)을 증폭하여 수득하였으며, 이 후 pGEM T-easy 벡터에 클로닝하였다. T-벡터에 클로닝된 1.9 kb neo 유전자는 NotI과 PvuI 제한효소{New England Biolabs (NEB), 미국}로 절단하여 삽입 부위를 준비하였다. 또한, pBC1 벡터의 amp 유전자 (ampicillin-resistance gene) 부위를 NotI과 PvuI 제한효소로 절단하여 제거한 후 벡터 부위를 준비하였다. 이렇게 준비된 두 삽입조각 및 벡터 부위를 라이게이션을 통해, neo 유전자(neomycin-resistance gene)가 삽입된 pBC1 벡터를 구축하였다.]에 유산양(Goat) 베타카제인 (beta casein) 프로모터 (promoter) 부위와 3'genomic DNA 부위를 각각 돼지 알파 에스 1 카제인 프로모터 염기서열과 3'arm 염기서열로 치환시킨 클로닝 벡터를 구축하였다.
돼지 알파 에스 1 카제인 프로모터 염기서열 5.5kb(서열번호 3)와 3'arm 염기서열 4.3kb(서열번호 5)를 프라이머 염기서열(서열번호 56 ~ 서열번호 59)을 이용한 PCR증폭(PT-200, BIO-RAD, 94℃에서 5분간 변성 과정을 거친후, 다음의 35 cycle을 수행하였다: 94℃에서 30초간 변성 후, 56℃에서 30초간 프라이머 결합, 72℃에서 5분간 신장반응)으로 산물을 얻은 뒤 각각 pGEM-T벡터(Promega, 미국)에 클로닝하였다.
프라이머 서열번호
프로모터 증폭용 정방향 프라이머 5'-GGATCCGGCTGTCGTTTTGTTATGATT-3' 56
프로모터 증폭용 역방향 프라이머 5'-CTCGAGAACTAAAAGGCACAGGGAACT-3' 57
3'arm 증폭용 정방향 프라이머 5'-CTCGAGTTACAATTCAGTGTGGGGAAT-3' 58
3'arm 증폭용 역방향 프라이머 5'-GCGGCCGCCAGCTTTATTACAGGCAGAGG-3' 59
돼지의 알파 에스 1 카제인 프로모터 염기서열에 존재하는 BamHI site(GGATCC) 2곳을 하기와 같이 반복적인 point mutation을 통해 BamHI 제한 효소로 잘리지 않게 하였다. point mutation을 위해 2곳 중 먼저 한 곳을 정해 프라이머를 제작하였다. 돼지 알파 에스 1 카제인 5' 프로모터 부위가 포함된 pGEM-T 벡터 DNA를 정제 후 주형으로 사용될 DNA 20 ng 과 한 쌍의 point mutation 프라이머를 사용하여 95℃ 30 초 (1반복), 95℃ 30초, 55℃ 1분, 72℃ 8분 30초 (15 반복)의 조건으로 PCR 증폭하였다. 주형으로 사용된(point mutation 이 안된) DNA를 제거하기 위해 MutazymeTM 효소(iNtRON Biotechnology, 대한민국)를 1 ul 첨가 후 37 ℃ 에서 1시간 반응시켰다. 상기 반응물 10 ul를 DH10B competent cell(Invitrogen, 미국) 에 형질전환(Transfomation) 시키고 LB+Ampicillin 고체 배지(sigma, 미국)에 plating 후 37℃에서 20시간 배양시켰다. LB+Ampicillin 고체 배지에서 자란 콜로니를 LB+Ampicillin 액체 배지에 키운 후 DNA를 정제하여 염기서열 분석 방법으로 BamHI site가 point mutation(GGATCC -> GGACCC) 되었는지 확인하였다. 한 곳에 Point mutation된 콜로니의 DNA를 사용하여 남은 한 곳의 BamHI site도 상기와 같은 방법으로 point mutation 시켰다. 여기서 사용된 point mutation 방법은 인트론사의 제품 Site Direct Mutagenesis kit를 사용하였다.
point mutation에 사용된 프라이머 염기서열은 이하의 표와 같다.
프라이머 서열번호
1차 포인트 뮤테이션용 정방향 프라이머 5'-TATATACTACATCTTCCGGGTCCAATCATCTGTTGATGG-3' 60
1차 포인트 뮤테이션용 역방향 프라이머 5'-CCATCAACAGATGATTGGACCCGGAAGATGTAGTATATA-3' 61
2차 포인트 뮤테이션용 정방향 프라이머 5'-AAGACGTGGCTTGGGTCCCACGTTGCTGT-3' 62
2차 포인트 뮤테이션용 역방향 프라이머 5'-ACAGCAACGTGGGACCCAAGCCACGTCTT-3' 63
pGEM-T 벡터에 들어 있는 돼지 알파 에스 1 카제인 프로모터를 BamHI과 XhoI으로 잘라 8.5kb의 벡터를 준비하였다. 또한, 3'arm을 포함하는 염기서열 부분을 XhoI과 NotI으로 잘라 4.3kb의 삽입물(insert)(서열번호 5)을 준비하였다. 각각의 조각을 라이게이션(ligation)하여 pGEM-T-pig αS1 casein 5'+3'을 클로닝하였다.
pBC1 벡터를 BamHI과 NotI으로 잘라 10kb의 벡터를 준비하였고 pGEM-T-pig αS1 casein 5'+3'벡터를 BamHI과 NotI으로 잘라 9.8kb의 삽입물을 준비하였다. 각각의 조각을 라이게이션하여 pBC1-pig αS1 casein 클로닝 벡터를 구축하였다.
완성된 pBC1-pig αS1 casein 클로닝 벡터의 구조는 도 2와 같다.
도 2의 P αS1 casein은 엑손 1(E1)을 포함하는 돼지 aS1카제인 프로모터 염기서열(서열번호 2)이다. 엑손 1은 서열번호 1의 염기서열 중 5'-3'방향으로 가장 먼저 배열되는 엑손을 의미한다.
도 2의 αS1-casein 3'genomic DNA는 엑손 18(E18), 엑손 19(E19), 및 인트론 18(IVS 18)을 포함하는 3'arm의 염기서열(서열번호 5)이다. 엑손 18, 엑손 19는 각각 서열번호 1의 염기서열 중 5'-3'방향으로 18번째와 19번째 엑손을 의미하며, 인트론 18은 18번째 인트론을 의미한다.
XhoI 제한효소 자리를 갖게 되므로, 목적단백질의 유전자를 벡터에 삽입할 수 있다.
2Xβ-globin insulator, 및 pBR322는 pBC1벡터로 부터 기원하는 인슐레이터와 벡터구성요소이며, Neomycin은 네오마이신저항성유전자로 pEGFP-N1 벡터(Clontech, 미국)로부터 기원한다.
상기 방법으로 제조된 pBC1-pig aS1 casein 벡터를 한국생명공학연구원 유전자은행(대전, 대한민국)에 기탁번호 KCTC 11324BP로 기탁하였다.
< 실시예 3> pBC1 - pig aS1 casein + hEPO - WPRE 벡터 구축
pBC1 벡터(인비트로젠, 미국)의 엠피실린(Ampicillin) 저항성을 가진 유전자를 네오마이신(Neomycin) 저항성 유전자로 치환한 벡터{G418 drug에 저항할 수 있는 neo' 유전자는 pEGFP-N1 벡터(Clontech, 미국)로부터 정방향 프라이머 5'-GCGGCCGCGCGCGTCAGGTGGCAC-3'(서열번호 81)와 역방향 프라이머인 5'- CGATCGGACGCTCAGTGGAACGAAAACTC-3'(서열번호 82)을 이용하여 1.9 kb PCR 산물(서열번호 83)을 증폭하여 수득하였으며, 이 후 pGEM T-easy 벡터에 클로닝하였다. T-벡터에 클로닝된 1.9 kb neo 유전자는 NotI과 PvuI 제한효소(New England Biolabs (NEB), 미국)로 절단하여 삽입 부위를 준비하였다. 또한, pBC1 벡터의 amp 유전자 (ampicillin-resistance gene) 부위를 NotI과 PvuI 제한효소로 절단하여 제거한 후 벡터 부위를 준비하였다. 이렇게 준비된 두 삽입조각 및 벡터 부위를 라이게이션을 통해, neo 유전자(neomycin-resistance gene)가 삽입된 pBC1 벡터를 구축하였다.}에 에리트로포이에틴(hEPO)을 클로닝 하고 벡터에 존재하는 유산양(Goat) 베타카제인 (beta casein) 프로모터 (promoter) 부위와 3' genomic DNA 부위를 돼지 알파 에스 1 카제인 프로모터 염기서열(서열번호 3) 및 3'arm 염기서열(서열번호 5)로 치환시켰다. 또한 mRNA를 안정화하여 단백질 발현을 증대시킨다고 알려진 WPRE(woodchuck hepatitus virus post-transcriptional regulatory element)를 hEPO 3' 쪽에 추가함으로써 hEPO 단백질의 발현을 극대화시키도록 하였다.
hEPO와 WPRE 각각을 PCR 증폭(PT-200, BIO-RAD, 94에서 5분간 변성 과정을 거친후, 다음의 35 cycle을 수행하였다: 94℃에서 30초간 변성 후, 56℃에서 30초간 프라이머 결합, 72℃에서 hEPO는 2분 30초, WPRE는 30초간 신장반응)으로 2.3kb(서열번호 69)와 0.6kb(서열번호 70)의 산물을 얻어 pGEM-T 벡터에 클로닝 후 염기서열을 확인하였다. hEPO가 들어있는 pGEM-T벡터를 EcoRV와 NotI(New England Biolabs (NEB), 미국)으로 자르고, WPRE가 들어있는 pGEM-T 벡터는 EcoRV와 NotI으로 잘라 각각의 조각을 라이게이션 하였다.
hEPO와 WPRE PCR 증폭을 위해 사용한 프라이머 염기서열은 이하의 표와 같다.
프라이머 서열번호
hEPO증폭용 정방향 프라이머 5'-GGATCCTGTGGTCACCCGGCGCGC-3' 64
hEPO증폭용 역방향 프라이머 5'-GATATCCCATGGGACAGGCTGGCGCT-3' 65
WPRE증폭용 정방향 프라이머 5'-GATATCTCTGTTCCTGTTAATCAACCTC -3' 66
WPRE증폭용 역방향 프라이머 5'-GCGGCCGCGAGCCCGAGGCGAAACAG-3' 67
pBC1 벡터를 BamHI과 NotI으로 잘라 유산양의 베타 카제인 프로모터와 3'genomic DAN부분을 각각 잘라내어 벡터를 준비하였고, pGEM-T 벡터에 클로닝된 hEPO+WPRE를 BamHI과 NotI으로 잘라 2.9kb의 삽입물을 준비하였다. 준비된 벡터와 삽입물을 라이게이션하여 pBC1+hEPO-WPRE를 구축하였다. pBC1+hEPO-WPRE에 돼지 알파 에스 1 카제인의 프로모터와 3'arm을 클로닝 하기 위해 PCR 증폭으로 돼지 알파 에스 1 프로모터 염기서열 5.4kb(서열번호 3)과 3'arm 염기서열 4.3kb(서열번호 5)를 각각의 pGEM-T 벡터(Promega, 미국)에 클로닝하였다.
돼지 알파 에스 1 카제인 프로모터 염기서열과 3'arm 염기서열을 PCR증폭 시키기 위해 사용한 프라이머 염기서열은 하기 표와 같다.
프라이머 서열번호
프로모터 증폭용 정방향 프라이머 5'-GGATCCGGCTGTCGTTTTGTTATGATT-3' 70
프로모터 증폭용 역방향 프라이머 5'-GGATCCAACTAAAAGGCACAGGGAACT-3' 71
3'arm 증폭용 정방향 프라이머 5'-GCGGCCGCTTACAATTCAGTGTGGGGAAT-3' 72
3'arm 증폭용 역방향 프라이머 5'-GCGGCCGCCAGCTTTATTACAGGCAGAGG-3' 73
돼지의 알파 에스 1 카제인 프로모터 염기서열에 존재하는 BamHI site(GGATCC) 2곳을 프라이머(서열번호 60 ~ 서열번호 63)를 이용하여 인트론 제품 Site Direct Mutagenesis로 point mutation을 시켰다. pBC1 + hEPO-WPRE벡터를 BamHI으로 자르고 Alkaline Phosphatase(CIP)를 30분간 처리하여 벡터를 준비하고 point mutatation된 돼지 알파 에스 1 카제인 5' 프로모터 DNA가 들어 있는 pGEM-T 벡터를 BamHI으로 잘라 5.5kb의 삽입물(서열번호 3)을 준비하였다. 각각의 조각을 라이게이션 하여 pBC1 pig αS1 casein 5'+hEPO-WPRE벡터를 클로닝하였다. pBC pig αS1 casein 5'+hEPO-WPRE벡터를 NotI으로 잘라 CIP를 30분간 처리하여 벡터를 준비하고 3'arm DNA가 들어 있는 pGEM-T 벡터를 NotI으로 잘라 4.3kb의 삽입물(서열번호 5)을 준비하였다. 각각의 조각을 라이게이션하여 pBC1-pig αS1 casein +hEPO-WPRE 벡터를 구축하였다.
완성된 pBC1-pig αS1 casein+hEPO-WPRE 벡터의 구조는 도 3과 같다.
도 3의 P αS1 casein은 돼지 aS1 카제인 프로모터 염기서열(서열번호 3)을 의미하며, αS1-casein 3'genomic DNA는 3'arm 염기서열(서열번호 6)을 의미한다.
hEPO는 인간EPO유전자이며, WPRE는 woodchuck hepatitus virus post-transcriptional regulatory element 유전자이다.
2Xβ-globin insulator, 및 pBR322는 pBC1벡터로 부터 기원하는 인슐레이터와 벡터구성요소이며, Neomycin은 네오마이신저항성유전자로 pEGFP-N1벡터(Clontech, 미국)로부터 기원한다.
상기 방법으로 제조된 pBC1-pig aS1 casein+hEPO-WPRE 벡터를 한국생명공학연구원 유전자은행(대전, 대한민국)에 기탁번호 KCTC 11325BP로 기탁하였다.
< 실시예 4> 돼지 알파 에스 1 카제인 유전자를 이용한 낙인벡터( Knock -invector) Pig α S1 casein - hEPO knock - in 벡터의 구축
1) pGEM -T- hEPO 벡터의 클로닝
TK유전자 선별 방법을 이용하여 특정 부위에 올바른 유전자도입 여부를 확인 할 수 있는 돼지 알파 에스 1 카제인hEPO knock-in 벡터의 구축을 위해, 먼저, 돼지 알파 에스 1 카제인 유전자에 엑손 2번 영역의 시작부터 시작 코돈까지를 포함하고 시작 코돈 이후 부터는 hEPO 유전자의 염기서열이 증폭 될 수 있도록 하는 특이적인 프라이머 2쌍(서열번호 74 ~ 76)을 준비하였다. 상기에서 준비한 돼지 알파 에스 1 카제인 엑손 2번 영역을 포함하는 프라이머를 가지고 인간의 genomic DNA(조아제약, 대한민국 특허 제10-0769291호의 pBC1-hEPO벡터)로부터 일차 PCR증폭(PT-200, BIO-RAD, 94℃에서 5분간 변성 과정을 거친후, 다음의 30 cycle을 수행하였다: 94℃에서 30초간 변성 후, 56℃에서 30초간 프라이머 결합, 72℃에서 2분 30초간 신장반응)한 후, 여기서 나온 PCR산물을 다시 주형으로 하여 이차 PCR 증폭(PT-200, BIO-RAD, 94℃에서 5분간 변성 과정을 거친후, 다음의 30 cycle을 수행하였다: 94℃에서 30초간 변성 후, 56℃에서 30초간 프라이머 결합, 72℃에서 2분 30초간 신장반응)을 하였다. 돼지 알파 에스1 카제인 엑손 2번 영역부터 시작 코돈까지의 염기서열을 포함하는 PCR증폭 산물 2.3kb hEPO 유전자(서열번호 68)를 pGEM-T 벡터(Promega, 미국)에 클로닝(pGEM-T-hEPO) 하였다.
hEPO PCR 증폭에 사용된 프라이머 염기서열은 다음의 표와 같다.
프라이머 서열번호
hEPO 증폭용 일차 정방향 프라이머 5'-GTGTTGACAACCATGGGGGTGCACGGTGAGTACTC-3' 74
hEPO 증폭용 이차 정방향 프라이머 5'-GATATCTTTTCTTATATAGGTGTTGACAACCATGGGGG-3' 75
hEPO 증폭용 역방향 프라이머 5'-GAATTCATGGGACAGGCTGGCGCTGA-3' 76
2) pGEM -T- pig α S1 casein 5' arm , pGEM -T- pig α S1 casein 3' arm 의 구축
돼지 알파 에스 1 카제인 유전자의 프로모터 염기서열(5' arm)과 3' arm 염기서열을 클로닝하기 위해, 서열번호 77 ~ 서열번호 80의 프라이머를 제작하여 돼지 genomic DNA로부터 PCR 증폭으로 산물 5.0kb(서열번호 4)와 4.9kb(서열번호 6)를 얻어 pGEM-T 벡터에 클로닝(pGEM-T-pig αS1 casein 5'arm, pGEM-T-pig αS1 casein 3'arm)하였다.
프라이머 서열번호
프로모터 증폭용 정방향 프라이머 5'-GTCGACAGCTGCAATGAACATGTGGGTG-3' 77
프로모터 증폭용 역방향 프라이머 5'-GATATCCAAAATAAAAATTTAGGTCTGACAG-3' 78
3'arm 증폭용 정방향 프라이머 5'-GCGGCCGCATGGCATATGGAAGTTCCCAGG-3' 79
3'arm 증폭용 역방향 프라이머 5'-CCGCGGTGGGAACTTCCATATGCCAT-3' 80
3) Lox A neo - hEPO 벡터 구축
Lox A neo 벡터(Gerard Karsenty's, Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032)를 EcoR V, EcoR I 제한 효소(New England Biolabs (NEB), 미국)로 잘라 벡터를 준비하였고 클로닝된 pGEM-T-hEPO 벡터를 EcoR V, EcoR I 제한 효소로 잘라 2.3kb의 삽입물(서열번호 68)을 준비하였다. 각각의 조각을 ligation 하여 Lox A neo-hEPO 벡터를 구축하였다.
4) Lox A neo - hEPO - poly A 벡터 구축
Lox A neo-hEPO 벡터 3'쪽으로 RNA 안정화를 위한 poly A 염기 서열을 삽입 하기 위해 Lox A neo-hEPO 벡터를 EcoR I 제한 효소로 자르고 Alkaline phosphatase(New England Biolabs (NEB), 미국)를 30분간 처리하여 벡터를 준비하 였다. 또한, pcDNA3 벡터(Invitrogen, 미국)에서 유래된 BGH(Bovine Growth Hormone) poly A를 EcoR I 제한 효소로 잘라 0.3kb의 삽입물을 준비하였다. 각각의 조각을 ligation 하여 Lox A neo-hEPO-poly A 벡터를 구축하였다.
5) Lox A neo - hEPO - poly A-5' arm 벡터 구축
Lox A neo-hEPO-poly A 벡터 5'쪽으로 돼지 알파 에스 1 카제인 5' arm을 삽입 하기위해 Lox A neo-hEPO-poly A 벡터를 Sal I, EcoR V 제한 효소(New England Biolabs (NEB), 미국)로 잘라 벡터를 준비하고, 클로닝된 pGEM-T-pig αS1 5'arm 벡터를 Sal I, EcoR V 제한 효소로 잘라 5.0kb의 삽입물(서열번호 4)을 준비하였다. 각각의 조각을 ligation 하여 Lox A neo-hEPO-poly A-5'arm 벡터를 구축하였다.
6) Lox A neo - hEPO - poly A-5' arm -3' arm 벡터 구축
Lox A neo-hEPO-poly A-5'arm 벡터 3'쪽으로 3'arm을 삽입하기 위해 Lox A neo-hEPO-poly A-5'arm 벡터를 Not I 제한효소(New England Biolabs (NEB), 미국)로 자르고 alkaline phosphatase를 30분간 처리하여 벡터를 준비하였다. 클로닝된 pGEM-T-pig αS1 3'arm 벡터를 Not I 제한 효소로 잘라 4.9kb의 삽입물(서열번호 6)을 준비하였다. 각각의 조각을 ligation 하여 Lox A neo-hEPO-poly A-5'arm-3'arm 벡터를 구축하였다.
7) Lox A neo - hEPO - poly A-5' arm -3' arm - TK 벡터 구축
Lox A neo-hEPO-poly A-5'arm-3'arm 벡터 3' 쪽으로 세포 사멸 유전자인 허피스 심플렉스 바이러스-싸이미딘 키나제(Herpes simplex virus-thymidine kinase: HSV-tk) 유전자를 삽입 하기 위해 Lox A neo-hEPO-poly A-5'arm-3'arm 벡터를 Sac II제한 효소(New England Biolabs (NEB), 미국)로 자르고 alkaline phosphatase를 30분간 처리하여 벡터를 준비하였다. pBS-TK 벡터(Gerard Karsenty's, Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032)를 Not I 제한 효소로 잘라 2.3kb의 삽입물(허피스 심플렉스 바이러스-싸이미딘 키나제 유전자 암호화 부분)을 준비하였다. 각각의 조각을 ligation 하여 Lox A neo-hEPO-poly A-5'arm-3'arm-TK 벡터(Pig aS1 casein-hEPO knock-in 벡터)를 구축하였다.
완성된 Pig αS1 casein-hEPO knock-in 벡터의 구조는 도 4와 같다.
도 4의 Pig αS1 casein 5' arm은 돼지 αS1 카제인 프로모터(서열번호 4)를 의미하며, Pig αS1 casein 3' arm은 3'arm(서열번호 6)을 의미한다.
hEPO는 인간EPO유전자이며, poly A는 폴리 A 시그널을 코딩하는 유전자이며, Neo cassette는 네오마이신 저항성 유전자로 포지티브 선별 유전자이며, PGK promoter는 포스포글리세레이트키나제(phosphoglycerate kinase;PGK) 프로모터이며, TK는 허피스 심플렉스 바이러스-싸이미딘 키나제(Herpes simplex virus-thymidine kinase: HSV-tk) 유전자로 네거티브 선별 유전자이며, pBS-TK벡터로부터 기원한다.
상기 방법으로 제조된 Pig aS1 casein-hEPO knock-in벡터를 한국생명공학연구원 유전자은행(대전, 대한민국)에 기탁번호 KCTC 11326BP로 기탁하였다.
< 실시예 5> 형질전환 세포주 제조 및 hEPO 발현 확인
1) 외래 유전자 도입 (Transfection)
생쥐 유선 세포 (HC11, 국립축산과학원, 대한민국), 생쥐 근육 세포 (C2C12, ATCC, 미국), 인간 간세포 (HepG2, ATCC, 미국), 인간 신장 세포 (Caki, ATCC, 미국), 인간 혈액 세포 (U937, ATCC, 미국), 랫트 뇌 세포 (C6, ATCC, 미국) 등을 아래와 같은 배양 조건으로 배양기에서 배양하였다.
HC11 - RPMI 1640 (Gibco, 미국), 10% Fetal Bovine Serum(FBS, HyClone, 미국), 1 % penicillin streptomycin (HyClone, 미국), insulin (sigma, 미국)5 ug/ml, 39℃, CO2 5%
C2C12, HepG2 - DMEM (Gibco, 미국), 10 % FBS, 1 % penicillin streptomycin, 37℃, CO2 5%
Caki - McCoy's 5A (Gibco, 미국), 10 % FBS, 1% penicillin streptomycin, 37℃, CO2 5%
U937 - RPMI 1640 (Gibco, 미국), 10 % FBS, 1 % penicillin streptomycin, 37℃, CO2 5%
80~90 % 정도 자란 세포주를 trypsin (HyClone) 을 이용하여 세포배양접시에서 떼어낸 후 1500 rpm에서 5분간 원심 분리 후 상층액을 제거하였다. Hemocytometer(Reichert, 미국)를 이용하여 세포수를 계수한 후 60 mm 세포배양 접 시에 5 x 105 cells/dish로 세포를 16 시간~20 시간 배양기에서 배양하였다. 외래 유전자 도입은 lipofectamine (invitrogen, 미국)을 사용하였으며 60 mm 세포배양 접시당 실시예 2 와 3에서 제조한 벡터 4 ug을 도입함으로써, 외래유전자를 도입하였다. HC 11 세포주는 외래 유전자 도입 4 시간 후 insulin (sigma, 미국) 5 ug/ml, prolactin (sigma, 미국) 5 ug/ml, hydrocortisone (sigma, 미국) 5 ug/ml을 첨가하였다.
2) 역전사 (Reverse Transcription, RT)
외래유전자 도입 후 24 시간 후에 easy-BLUE total RNA Extraction Solution(iNtRON Biotechnology, 대한민국)을 이용하여 RNA를 정제 하였다. 정제된 RNA 4 ug 과 역전사 효소인 superscript III reverse transcriptase (invitrogen, 미국)을 가지고 역전사 (reverse transcription)를 수행하였다. 이때 외래유전자로 넣어준 DNA의 오염을 막기 위해 DNase I (invitrogen)을 처리하였다. 역전사는 10 pM oligo dT 1 ul와 10 mM dNTPs 1 ul를 넣은 후 65℃에서 5분간 반응 후 얼음 안에서 1분간 반응하였다. 다음으로 5X buffer 4 ul 와 0.1 M DTT 1 ul, reverse transcriptase 1 ul 를 넣은 후 50℃에서 60분 반응 후 70℃에서 15분간 반응 시켜 cDNA를 합성하였다.
3) 실시간 PCR (Real Time - PCR)
민감도가 우수한 실시간 PCR 방법을 이용하여 각 세포주에 도입 시킨 EPO 발현을 확인하였다. 실시간 PCR은 역전사 효소를 이용하여 만든 cDNA를 주형으로 SYBR Green qPCR kit (FINNZYMES, 핀란드)와 DNA engine Opticon 2 (BIO-RAD, 미국)를 이용하여 실시간 PCR (94℃에서 5분간 변성 과정을 거친 후, 다음의 50 cycle을 수행하였다: 94℃에서 30초간 변성 후, 56℃에서 30초간 프라이머 결합, 72℃에서 60초간 신장반응 후 형광 값 측정)을 하였다. 형광 값 보정을 위하여 벡터내의 Neo 유전자와 세포내의 beta actin 유전자를 기준으로 하였다. 결과 분석은 GeneExMacro 3.0 (BIO-RAD) 프로그램을 사용하였다. PCR에 사용한 프라이머(서열번호 84 ~ 91)는 하기 표와 같다.
프라이머 서열번호
EPO 증폭용 정방향 5'-CAAGGAGGCCGAGAATATCA-3' 84
EPO 증폭용 역방향 5'-AAGTGTCAGCAGTGATTGTTCG-3' 85
Neo 증폭용 정방향 5'-GCTACCCGTGATATTGCTGAA-3' 86
Neo 증폭용 역방향 5'-CAACACCGTGCGTTTTATTCT-3' 87
인간 beta actin 증폭용 정방향 5'-CGTGGGCCGCCCTAGGCACCA-3' 88
인간 beta actin 증폭용 역방향 5'-TTGGCCTTAGGGTTCAGGGGGG-3' 89
생쥐 beta actin 증폭용 정방향 5'-TGTGATGGTGGGAATGGGTCAG-3' 90
생쥐 beta actin 증폭용 역방향 5'-TTTGATGTCACGCACGATTTTCC-3' 91
실시간 PCR 결과를 도 5에 나타내었다.
도 5는 pBC1-pig αS1 casein 벡터와 pBC1-pig αS1 casein+hEPO-WPRE 벡터를 도입 시킨 유선세포 및 유선세포 이외의 조직세포에서의 hEPO 발현결과를 나타내며, 같은 세포주에 대조군으로 pBC1 벡터 (invitrogen) 와 pBC1-hEPO-WPRE (조아제약, 대한민국 특허 제 10-0769291 호) 벡터를 도입시켰다. x 축은 각각의 세포주를 나타내고, y 축은 pBC1-pig αS1 casein 벡터와 pBC1 벡터의 결과 값을 1로 하였을 경우 각 세포주에서 발현되는 hEPO의 상대적 비율을 나타낸다. 도 5에서 pPAC는 실시예 2의 pBC1-pig αS1 casein 벡터를 의미하고 pPAC-hEPO는 실시예 3의 pBC1-pig αS1 casein+hEPO-WPRE를 의미한다. pBC1은 염소 프로모터를 갖고 유선특이적으로 발현하도록 만들어 판매되는 invitrogen 사의 벡터이고, pBC1-hEPO는 pBC1-hEPO-WPRE를 의미한다.
도 5에서 보는 바와 같이 hEPO는 생쥐 유선세포 HC11 세포에서 가장 많이 발현하였고 인간 간세포 HepG2와 생쥐 근육세포 C2C12 세포에서는 약하게 발현 하였으며 다른 조직의 세포에서는 발현되지 않음을 확인하였다.
따라서, 본 발명의 벡터로 형질전환된 유선세포에서 목적하는 단백질을 생산할 수 있음을 알 수 있다.
< 실시예 6> 미세주입법에 의한 동물수정란과 그를 이용한 형질전환동물 구축 및 EPO 제조
1) 유전자의 정제
실시예 3에서 제조한 벡터를 Sal 1 (NEB, R0138)을 이용하여 선형으로 만든 후 Qiagen사의 QIAquick Gel Extraction kit(Q-28706)를 이용하여 정제하여 주입용 버퍼(10mM Tris-HCl, 0.1mM EDTA, pH 7.4)에 용리시켜 최종 농도를 2ng/ul로 5ul씩 분주하여 -20℃에 보관하였다.
2) 과배란 암컷 쥐로부터 수정란의 회수
8주령의 C57BL/6 암컷 쥐(오리엔트 바이오, 대한민국)를 5 IU의 pregnant mare serum gonadotropin (PMSG; Intervet, Netherlands)의 주사 후 46시간 뒤에 5 IU의 human chorionic gonadotropin (hCG, Intervet)을 주사하여 과배란을 유도하 였다. 12시간마다 빛 조절(7 a.m. to 7 p.m.)이 되며, PMSG는 11 a.m에 hCG는 9 a.m에 복강투여 방법으로 주입되었다. hCG 주입 후 같은 종의 수컷 쥐와 교미를 시켜 다음날 암컷 쥐의 질입구(vaginal plugs)를 확인하여 정상적으로 교미가 진행되었음을 확인한 후 난관을 적출하였다. 난자-난구세포 복합체를 분리시키기 위해 적출된 난관을 0.1% hyaluronidase (Sigma, H3884)가 포함된 M2 (Sigma, M7167) 배양액으로 옮긴 후 팽대부 부분을 찢었다. 잠시 후 난구세포가 분리된 수정란만을 회수하여 깨끗한 M2 배양액에 씻어서 M16 (Sigma, M7292) 배양액으로 옮겨 미세주입 실험 전까지 37℃, 5% CO 2 인큐베이터에서 배양하였다.
3) 유전자의 미세주입
챔버 슬라이드(Nunclon, Denmark)에 M2 배양액을 소량 떨어뜨린 후 증발을 막기 위해 오일로 덮어 미세주입용 디쉬를 준비한 후 회수된 수정란을 올려놓았다. 수정란의 미세주입은 수정란의 미세조작을 위한 장비인 OLYMPUS 1X71 TH4-200 inverted microscope (Narishige, Tokyo, Japan)에서 수행하였다. Microloader (Eppendorf, Hamburg, Germany)를 이용하여 femtojet automatic injector (Eppendorf)에 연결된 femtotip injection pipette (Eppendorf)에 미리 준비된 1)의 정제 유전자를 로딩한다. holding pipette으로 난자를 잡고 수정란의 전핵에 현미경의 초점을 맞춘 후 femtotip injection pipette을 이용하여 난자의 투명대를 뚫고 전핵까지 들어간 후 femtojet automatic injector를 이용하여 상기 유전자를 전핵에 주입하여 전핵의 팽창을 확인하면 난자로부터 injection pipette을 빼내였 다. 미세주입 후 살아남은 난자는 M16 배양액으로 옮겨 37℃, 5% CO 2 인큐베이터에 넣어두었다.
4) 수정란 이식
미세주입 실험 하루 전날 6주령의 BDF-1 암컷 쥐(오리엔트 바이오, 대한민국)를 같은 종의 거세된 수컷과 교미를 시켜 가임신을 유도하였다. 실험전 암컷 쥐의 질입구(vaginal plugs)를 확인하여 정상적으로 교미가 진행되어 가임신이 유도된 암컷쥐에 avertin (Sigma)을 복강 주입하여 마취시킨 후 쥐 등의 옆구리와 흉각 사이의 중간부분을 절개하여 난소 지방을 당겨 난소 및 난관을 꺼냈다. 난소 지방을 수술용 집게로 고정시키고 난소와 난관 사이를 둘러싸고 있는 포낭을 찢어 난관 입구를 확인 한 후 미리 준비된 이식용 파이펫을 난관 입구에 삽입하여 난자를 이식하였다. 수정란 이식은 양쪽의 난관 모두에 수행되었다. 이식 후 확인을 위한 마커 버블 4개와 15개의 미세주입 수정란 및 최종 마커 버블 한 개로 이루어진 이식용 파이펫을 이용하였다.
5) 유전자 도입 확인
수정란 이식 3주 후 새끼가 태어나면 새끼의 꼬리를 잘라 Dneasy Blood&Tissue kit (Qiagen, Q-69506)를 이용하여 genomic DNA를 추출하였다. 돼지 알파 에스 1 카제인 유전자가 도입된 형질전환 쥐의 확인을 위하여 추출된 DNA에서 EPO-WPRE 유전자 부위와 WPRE-3‘arm 유전자 부위를 증폭 시키는 프라이머를 이용하여 PCR 증폭실험(PT-200, BIO-RAD, 94℃에서 5분간 변성 과정을 거친후, 다음의 35 cycle을 수행하였다: 94℃에서 30초간 변성 후, 55℃에서 30초간 프라이머 결합, 72℃에서 30초간 신장반응)을 실시하여 돼지 알파 에스 1 카제인 유전자의 도입을 확인하였다.
EPO-WPRE PCR 증폭에 사용된 프라이머 염기서열은 다음의 표와 같다.
프라이머 서열번호
EPO-WPRE 증폭용 정방향 프라이머 5'-AACTCTTCCGAGTCTACTCCA-3' 92
EPO-WPRE 증폭용 역방향 프라이머 5'-CTCCTCATAAAGAGACAGCAAC-3' 93
WPRE-3'arm 증폭용 정방향 프라이머 5'-TTCCTGTTAATCAACCTCTGG-3' 94
WPRE-3'arm 증폭용 역방향 프라이머 5'-TACCAAAGGCCATAATTGTGG-3' 95
그 결과를 도 6에 나타내었다. 도 6은 실시예 3의 발현벡터 pBC1-pig alpha S1 casein+hEPO-WPRE 벡터로 형질전환된 생쥐를 선별하기 위한 PCR 결과를 나타낸다. 도 6의 EPO-WPRE는 EPO-WPRE 유전자 부위에 대한 결과이고, WPRE-3'arm는 EPO-alpha S1 3'arm 유전자 부위에 대한 결과이다. M은 크기마커(size marker), V는 pBC1-pig-alpha S1 casein-EPO-WPRE 벡터, N은 음성대조를 위한 정상 마우스 genomic DNA, 숫자는 각각의 개체를 의미한다.
그 결과로부터 유전자 도입 여부를 확인하여 형질전환된 쥐를 선발하였다.
6)형질전환 실험동물의 번식 및 유전자 전이 확인
돼지 알파 에스 1 카제인 유전자 도입이 확인된 형질전환 쥐 중 암컷은 성성숙 시기인 6주령이 지난 후 정상 수컷과 교배하여 새끼를 생산한 후, 새끼에 대하여 상기 5)와 동일한 방법으로 외래 유전자의 도입을 확인하였다.
도 7은 실시예 3의 발현벡터 pBC1-pig alpha S1 casein+hEPO-WPRE 벡터로 형질전환된 생쥐를 선별하기 위한 PCR 결과를 나타낸다. 도 7의 EPO-WPRE는 EPO-WPRE 유전자 부위에 대한 결과이고, WPRE-alpha S1은 EPO-alpha S1 3'arm 유전자 부위에 대한 결과이다. M은 크기마커(size marker), V는 pBC1-pig-alpha S1 casein-EPO-WPRE 벡터, N은 음성대조를 위한 정상 마우스 genomic DNA, 1-1 ~ 1-6은 형질전환 쥐의 새끼를 의미한다.
7) 형질전환 어미쥐의 유즙에서 EPO 의 존재여부 및 함량확인
어미쥐를 수유 7일 후 새끼쥐를 격리시키고, 2시간후 옥시토신 10 IU를 복강 주사함과 동시에 유선을 맛사지 하면서 유즙을 채취하였다. 유즙을 이용하여 Western blot assay를 수행하였다. 12% SDS-PAGE한 젤을 PVDF막(millipore, 미국)에 transfer 한 후 5% Skim milk blocking solution에서 항온처리 하였다. 막에 항-인간 EPO 항체(1:1,000, hEPO anti-rabbit antibody, R&D systems Cat. No. AB-286-NA, Lot No. HX01, 미국)를 제조사의 프로토콜에 따라 첨가하여 1시간 동안 상온에서 항온처리 하였다. 30분 동안 TBST buffer(tris buffered saline buffer, 0.01% tween-20)로 막을 씻어준 다음 퍼옥시다제로 레이블 된 항 토끼 항체(1: 3,000, peroxidase labeled anti-rabbit antibody, GE healthcare, Cat. No. NA9340V, Lot No. 348424, 영국)를 첨가하고 1시간 동안 상온에서 항온처리한 후 TBST buffer로 세정한 후 필름에 노출하였다.
그 결과를 도 8에 나타내었다. 도 8은 형질전환된 어미쥐의 유즙을 이용하여 웨스텃블럿 어세이한 결과를 나타낸다. 도 8의 1~4는 EPO 스탠다드(calbiochem, 미국), 5 ~ 6은 시료를 나타낸다.
그 결과 유즙에서 32 KDa 분자량을 가진 단백질이 발현됨을 확인하였다.
또한, 어미쥐의 유즙내 hEPO의 농도를 확인하기 위하여 ELISA kit(Stem Cell Technology)를 이용하여 사용설명서에 따라 ELISA(Enzyme-linked immunosorbent assay)를 수행하였다. 그 결과 유즙내 hEPO가 50,000-200,000 IU/ml이 발현됨을 확인하였다.
도 1은 본 발명의 돼지 알파 에스 1 카제인의 염기서열을 밝히는데 사용된 탐침인자의 위치와 탐침인자에 의해 찾아낸 염기서열의 위치를 나타낸다.
도 2는 본 발명의 일 실시예인 pBC1-pig αS1 casein벡터의 구조를 나타낸다.
도 3은 본 발명의 일 실시예인 pBC1-pig αS1 casein+hEPO-WPRE벡터의 구조를 나타낸다.
도 4는 본 발명의 일 실시예인 Pig αS1 casein-hEPO knock-in 벡터의 구조를 나타낸다.
도 5는 본 발명의 일 실시예인 Pig αS1 casein+hEPO-WPRE 벡터를 도입시킨 세포주에서 hEPO의 발현 결과를 나타낸 그래프이다.
도 6은 본 발명의 일 실시예인 pBC1-pig αS1 casein+hEPO-WPRE 벡터로 형질전환 된 생쥐를 선별하기 위한 PCR 결과를 나타낸다.
도 7는 본 발명의 일 실시예인 발현벡터 pBC1-pig αS1 casein+hEPO-WPRE 벡터로 형질전환된 생쥐의 새끼에게서 형질전환 여부를 확인한 PCR 결과를 나타낸다.
도 8은 본 발명의 일 실시예인 형질전환 된 어미쥐의 유즙을 이용하여 Western blot assay 한 결과를 나타낸다.
<110> CHO-A PHARM Co., LTD. <120> A Gene of porcine alpha S1 casein, a promoter of the same and use thereof <130> P09-053-CHOA <150> KR10-2008-0062765 <151> 2008-06-30 <160> 95 <170> KopatentIn 1.71 <210> 1 <211> 33247 <212> DNA <213> pig <220> <221> gene <222> (1)..(33247) <223> gene of porcine alpah S1 casein <400> 1 aggattacaa gattgctgtt ggataatgct ggatataaga aattgcatat ctttattctg 60 taacctcagc agaagtattg tgaagtttgt ctttagcaat gctgagatgt tctttactag 120 ttctttttac tctatttctc ggggcttaaa tctttcatct tatatgcatg ttaggcattc 180 tccaccagct aggatgttat ttcctccatc acctatccga agattcgaca ccttgaatac 240 attgcttttg ttgagtaaaa gaataagcag tatgatatat tagaaagagc acaggctctg 300 atgttgggag atctgagtca tcatttactc attagctggt gcaagcattt tgtatggaaa 360 ttaatttact tattaatttt attactttta tttttacttt tattttttgc catttctagg 420 gccgctcccg cagcatatgg aagttcccag gctacgggtc taattggagc tgtagctgcc 480 agcctatgcc agatccttag caacgcagga tccaagtcgc gtctgcaaac tacaccacag 540 ctcacggcaa caccagatcc ttaacccatt gaggaaggtc agggattgaa cctgcaacct 600 catggttcct agttggattc gttaagcact gtgccacaat gggaactcct taatttactt 660 aatttataaa agtttagttt ccttatctac aaaagggaaa ataatgactt gtttctctac 720 attacatgat tcttataagg agtagttgtg aaaataatta taagaagact ttagaaatta 780 taaagaagat tataaattga caaagaaatg aatctatttc attctgctga tggatgactt 840 attaatctat ctggtaaact gtggttctga tggccaataa tgcctgttaa atacattgta 900 tcctcataat aatattgtta aacagggact gccaatgatt ggaccccagg aaagtttatc 960 agtcatgaaa accacatttt gtcaagtcag aagaactatt actgataaga aaaaaatctc 1020 tttacccacc aaattacagt ctttcagaaa gaaatcacta ggtgagattt agttaaccaa 1080 gttaaatttt agcccagttt tcccagatgt cttgagtagg tgaaagcttc cttcatggca 1140 agatgaatga atttttggtt ggaggtaaga ggcaggtagt ggtcttggca cgacatagac 1200 agataaactg ttacttgtga gatatcatac ctacaaaaag aatatagggt tggataagga 1260 gatgctttat tgtagacatt gctataaagg taaacacttt catggaaata caattcagca 1320 gttttagcat ctttgttttt gccatatgtg ttacatattt ttacaatttt catatgcttt 1380 taatttttta aaaggtattt tacatacaaa ctttcattat tgggtatttt aaaagagaag 1440 actgtgtatt acattttcaa atctttcagt ttctagctac tcaaagtgtg ctgcatgggt 1500 caccagcagt gacatcacct tagagcttat taaaaattca ttagctcctg gagttcccat 1560 cgtgatgcag cagaaacaaa tttgactagg aaccatgagg ttgcaggttc aatccctgcc 1620 tccctcagtg ggttaaaaat ccgacattac tgtgagctgt ggtataggtc acagacccgg 1680 ctgggatctg gtgttgctgt gactctagca tagtccagca gaaacagctc tgattagacc 1740 tctagcctgg aaaccttcat atgccgctgg tgccctaaaa ggacaaaaga gaaaaaaaaa 1800 aatacgttag ttcaggacac acctcagatt tactaaatct gaatctgaat tttaacataa 1860 tatcctggtg attcataagc acattaaagc attgttgtag ttcattgttc tgtgagaaca 1920 tcaaatagta taggatttca tcaataaaat tcagaagttc tttctattcc cctctcatat 1980 tccatcccaa gctttcttca gttgtggtca ctaataaaaa ttttgccttc acacacacac 2040 acacacacac acacacacac acataaggtc tttttagggc tgcacccatg gcatatggag 2100 tttcccaggc taagggtcaa atcagagctg tggccgctgg cctatgtcac tgccacaaca 2160 acaccagatc tgaattgcat ctgtaactta caccacagct catggcaata ccgatcctta 2220 acccactgag caaggccagg gatcaatcct gcatcttcat gtttactagt cagatttgtt 2280 tctgctgagc catgacagga accctgcctt taccctttta tacattttta atgcacatac 2340 aatatacaag tatgattcaa gacttctcga aagttaacaa atccacaaga taggaaaatg 2400 atttgggcac aaaataatta gttgaggttt tttttaaaaa aaaaattcta ataatgatcg 2460 ttttaaatga gcacttactt tgtctcaggc tctcttctaa gtgtgtgatc taattttttc 2520 ctctcgatca cacaggaagt ggatgtcctt aaacagatgt ggacactgag gcacagtgat 2580 tttgcaagat taccctgctg gtcattgaga gccagaaatt aaatttacaa tttgacctca 2640 aaatcttgtc ttgcaacaac tacatcatgt gtttcattta agatcttctg agacctattg 2700 atacagcact aaaaaggata ttgttctaaa cacaaacata aaatcaagca taagttccat 2760 atttccaggg atatcacata ccacacctta ccgaaagaac tctattttag ttctagatgc 2820 caagattcca cttgtgcaat acttaagagt aatgattcgg ggggggggga tgcactgggg 2880 gtttgggatg gaaatgctat aaaactgggt tgtgatgata attgtacaac tataaatgta 2940 ataaattcat tgagaaatat agaaaaaaat aaaataaaat cccccctccc aaaaaaagaa 3000 ttcactgtgg atcctctctt agattccaaa cagtatgtgg gtttctagag agaaacaaag 3060 atgtctttca tattctcagt aaatttcctt ctggaagttt cactgttaaa agacattttc 3120 ctagaagact caccagtcat aaacccaaat attcctggga gaatgctgac aaactagctt 3180 tgaaattcat gttctgaatt attgactgtc tcatacagaa tgagttctcc aatgaggttt 3240 aatgtggacc agaatatgta gcaacagtac acaaaacttt gcagagtaaa gtttacctcc 3300 tgactcacag ttcccttccc tccaaactcg aagtctattg ctaatgtttc ctctcctttt 3360 tgtgctagtt tttctaagaa gtatgaatct aggaaagaat gtttccaata ttgaaacttg 3420 aaacacaaga aagctttcag cttggttgtc ccttatctga cctatttcca ctttcactga 3480 cttagggatt ctgttggatt ttcaaaaccg tcgtgatagt actagagtag ctttgccttt 3540 gtgtgccaaa gatagttttg gtttatggct gtcgttttgt tatgattatt acaaagactc 3600 tcccatactc ataagtgttc catgttgaat gatcagttat atgttcatcc tacatatgac 3660 tattacatca gaaaatcgct attcaggtaa taatttcatt ctttctttac tcacaggaca 3720 aaggcctgtt taactacaaa tcatctaaat atgtgtctca aatgtgaact gtgatttttc 3780 ttttttagtg gtttgaactg aacaattttt ttttaattta tttttttccc actgtaaagc 3840 aaggggatca agttatcctt acatgtatac atttttattc cccacccttt gttctgttgc 3900 aatatgagta tctagacata gttctcaatg ctactcagta ggatctcctt gtaaatctac 3960 tctaagttgt gtctgatcag cccaagctcc caatccctcc cactccctcc ctctcccatc 4020 aggcagccac aagtctattt tccaagtcca tgattttctt ttctgtggaa gggctcattt 4080 gtgccttata taagattcca ggtatcagtg atatatcatt tggtatttgt ctttctcttt 4140 ctgacttact tcactcagta tgagagtctc tagttccatc catgttgctg caaatggcat 4200 tatgccattc tttttatggc tgagtagtat tccattgtgt gtatatacta catcttccgg 4260 atccaatcat ctgttgatgg acatttgggt tgtttccatg tcctggctat tgtgaataga 4320 gctgcaatga acatgtgggt gcatgtgtct ctttcaaggt aagttttgtc tggatatatg 4380 cccaagagtg ggattgcagg gtcacatggt agttctatgt gtagatttct aaggtatctc 4440 caaactgttc tctatagtgg ctgtaccagc ttacattcac accaacagtg caggagggtt 4500 cccttttctc cacccccccc ccagcatttg ttatttgtgg acttatcaat gatggccatt 4560 ctgactggtg tgaggtggta tctcatggta gttttgtttg catttctcta ataatcaggg 4620 atgttgagca ttttttcatg tgcttgttgg ccatctgtac atcttccttt gagaaatgtc 4680 tattcaggtc ttttgcccat ttttccattg ggttggttgg cttttttgct gttgagttgt 4740 ataagttgct tgtatattct agagattaag cccttgtcca ttgcatcatt tgaagctatt 4800 ttctcccatt ctgtaagttg tctttttgtt ttcttttggg tttcctttgc tgtgcaaaag 4860 cttgtcagtt tgatgaggtc ccattggttt atttttgctc ttatttctgt tgctttggga 4920 gattgacctg agaaaatatt catgatgttg atgtcagaga gtattttgcc aatgttctct 4980 tccaggagtt tgatggtgtc ttgtcttata tttaagtctt tcagccattt tgagtttatt 5040 tttgtgcatg gtgtgagagt gtgtaccagt ttcgttaatt ttcatgcagc tgtccaggtt 5100 tcccagcaat gcttgctgaa tagactttct ttttcccatt ttatgttctt gcctcctttg 5160 tcaaagatta attgaccata gttgtcaaag tttatttctg ggttctctat tctgttccat 5220 tggtctgtct gttttgatag cagtggcatg ttgttttgat gactgtggct ttgtaatatt 5280 ttttgaagtc tgggaaagtt atgcctcctg cttgattttt gtttctcagg attgctttgg 5340 caattctgag tcttttgtgg ttccatataa aattttggat tgtttgttct agttctgtga 5400 aaaacgtcat cggtaatttg atagggattg cattgaatct gtatattact ttgggtagta 5460 tggccatttt tacattattg atttttccaa tccatgaaca cggaatatct ttccatttct 5520 ttacatcttc tttgatttct ttgattatag ttttatatga actgaacaac tttaagtgat 5580 aaaagcaaaa ggaaaaatac gaatataaag caaattgaca taagctaaaa ttttgcagga 5640 tttgacgttg tataaatcta caatgaatat gtttgctcga attacagtga acagtcatat 5700 tttacaagta taagaatgat ttatttcaaa atacaaactt aattaactat attctatatc 5760 taataagcaa agtgaagatt gatccttatc cataggtact agaaaaatct gtgttttgag 5820 tttatgagaa ttcctatggt ggatacatgt gttcactaag agttggcttg tctttaaaag 5880 ttctgattgt tcttctttga tggcaagcct tattattatc aaatctaaga ctccagtctc 5940 agttttttga gtctctattt ttggactatc aggaatatta aaattatcag ctttttgtat 6000 gtgaattacc cactatagta tgagactaca gattttgtct tacccaactt ttatttcccc 6060 tgtgtcttgt ctcatttctt cacctctgta aataaataat gtaagaatga gtaaacaaat 6120 gaggatacag tagctttaca caacttacag tatgatcctg aattggaaat aaaataagtc 6180 agttatcctg gatgcattct caggaaaaga caagagccgg gtattgtaag gcagtggaga 6240 atacttgttc tcagcccctt ggataaatca gagtaaatag aaaatactag tgcttttttg 6300 acattgatgt aatgcagtca gcaaggacga tactatccaa agagaagttt aacatggaaa 6360 actatagcct tgtctatccc cagtatggaa agcctgagcc actgcgaaat ttcttttaac 6420 cccaaataat gttcctacca tatgactgta aattggctgt gaatatcact atggatttat 6480 tattttattt ttaaattttt ttggtctttt ttctttttag ggctgcaccc atggcatatg 6540 gaggttccca ggctaggggt cgaattggag ttgtagctgc cagcctacac cacagccaca 6600 gcaatgccag atctgagctg tgtctgcaac ttacaccaca gctcatggca acaccagatc 6660 attgacccct tgagtgaggc cagggatcga acctgcaacc tcatggttcc tagtcagatt 6720 catttcccct gcgccacaat gagaattcca gaattttttt aaatatatag tgtgatacct 6780 ttctgtaaac aagcagtcac aatcaacaat tttttaaatc cagctctatg tatagatatt 6840 ttattcagca tgcaattttt ttcctaaaat taacaatgcc agttaattct aggattatat 6900 ttcaggactg gaaagaaagt ttttttttcc ttttatttac ttactttaaa aggtggaaaa 6960 ttggagttat ggttgatttt ttgggggggg ggagtattta aaaattgtat tcttaaataa 7020 aaattattct tgaataatta tttttaatta agaaatctaa caattaaatt aatgaatact 7080 atcacaacac atatacccaa aataaagcaa gcagaaaatt atttggtgta gttaaaatac 7140 taccaaagtt tataaggcaa ttgtattttc tttttggtta aaaaaaagat cagatcacat 7200 ataaggtaac ttactccaca aggtaactta cttagaatac ttagaataaa tacttagaag 7260 acttagaata aataataggg aataaataga gttttaaaag gtgaaataga tgatgaaatc 7320 ttctcatggt ctagtacaat tataaaaatt aaaaattttt gatgatttta ttttgtctca 7380 agaatttccc ttacaggtat tgactttttc aaaagctgta aaggaaattt tattgctata 7440 ttaatctttc caattatcca tttaacttaa aaagcatgtt cttataataa ccataaatat 7500 ggaattttta tgtatcttaa ttttgaataa tgtcattcca tttcctgtat aatttggtat 7560 catagcatga atcactcctt tgttgaaaac tctcctcaga atttcttggg agaaaaattg 7620 gacagaaaat taatttcctc tttgagagaa ttcttagaat ttaaatgaca ctattggttg 7680 aactgaaacc acaaaattag cattttacta atcactaggt ttaaatattt gtgaaacaaa 7740 gagatctgcc accatcttga tcatcagctc agcttgcttc ttctttccgg tcttgggttc 7800 aaggtatttc atttacatat agcaaaatgt gatatattat gatttcaatc tgtctaattt 7860 ttcactcctc actaaaaaat atgcactggt aacttttctg tgtgattcca aatattgata 7920 ccttttaatg atatactggt ggcttaaaaa tgcatttgca aatgtcgatg ccatctatct 7980 cagagcttta gttgaaaaat aatagtttta taaagaccaa attttttttg ccaaatttta 8040 tgaaaactta ttatgtgaaa taatttataa tctttttaaa gatcatagtg aggatcattt 8100 ctggtagaat atttcaagac catttttatt ccatgtcatt aggttaataa aattaattct 8160 ataaaggata tgtcaatgat atacacagat ataaatgact actttttaaa agatggttag 8220 atttggatat ttggaaaaat gcaaatgaat aaaaccagta aactcatttt ggatttataa 8280 atatgtcttc cttacaaatg cagttagatt ctacaatatg tagactgaaa cagtatgtat 8340 aaaataagct gattagtttg ttggctaatg tataaacaaa ttgcatgtat attatgactt 8400 tcctttccta atttctctgg aaaccagttt cccaggacat aagttctaag tatctctggg 8460 ttcttgtaat ttgatggaac tctagaagtc acacatgata agacatcaga atcttatgat 8520 tctgctcaat gaagtcgtct ttatgcagtc atgtcatgga tatagcaacg tagaaaaaca 8580 taacataata gctagacttt aaaaaaaaat tgatggaggt taaatgtttc tacataatat 8640 gcaccaacag tgtttttccc aaagacgctg aaaaagcagg attctctaac atagacctag 8700 aaaaacacct tcaaaaaatt gcagataggg agttcccgtt gtggctcagt ggttaacaaa 8760 tccgactagg aaccatgagg ttgcaggttc aatccctggc cttgctcagt gggttaaagg 8820 atctggcatt gatgtgagct gtggtgtagg tcgaagacgt ggcttggatc ccacgttgct 8880 gtggctctgg tgtaggctgg tggctacagc tctgattcaa cccctagcct gggaacctcc 8940 acatgccgtg ggagcggccc aaaaaaaggc aaaaagacca aaaaaaaatt tttttttcag 9000 ataaaattaa atgccagttc cctgtgcctt ttagtttatt atcaattttt agcaaatctg 9060 atggtctaag aggaaatatt taaaataatt aattgtagta ttcttaaatt tagtagtatt 9120 taaatattaa tgtttatgta ttcctctgac aaaaccctat taccacttca aggatcaaat 9180 gttttgtttt agagggtgat actggtgttt cttatctcat ataagcacta agcaagataa 9240 tttgaatgat aaatttttct tgtgagtaaa ttttctgtca gacctaaatt tttattttgt 9300 tttcttatat aggtgttgac aaccatgaaa cttctcatct ttatctgtct tgcagctgtt 9360 gcccttgcca ggcctgtgag tatggtagag aatttagagg cttctagatt cttgattgaa 9420 attacctgat atcaaacaca agaaactgag gataataatc ttaaaagtat tgaatgatct 9480 ctaattacct tttgaagcct tgatattaaa actgtagaaa tccttcacat cttgatcatt 9540 attacatagt tcattcaaag tcatcactcc aaataaaatc tgagttgaaa tataaatgcc 9600 tcacagtaaa aaaataaaaa caaaaaatga aaagaaaaga aaaaggaata atgtatttaa 9660 caacatagta aatagaatca atgagtgtta ttacgctctt tgcctgggtc caataaagaa 9720 ttagcatata tttaaacata caagtccatg attttttctg tggaagggct catttgtgcc 9780 ttatataaga ttccaggtat cagtgatata tcatttggta tttgtctttc tctttctgac 9840 ttacttcact cagtatgaga gtcttagttc catccatgtt gctgcaaatg gcattatgcc 9900 attcttttta tggctgagta gtattccatt gtgtgtatat actacatctt ccgaatccaa 9960 tcatctgttg atggacattt gggttgtttc catgtcctgg ctattgtgaa tagagctgca 10020 atgaacatgt gggtgcatgt gtctctttca aggtaagttt tgtctggata tatgcccaag 10080 agtgggattg cagggtcaca tggtagttct atgtgtagat ttctaaggta tctccaaact 10140 gttctctata gtggctgtac cagcttacat tcacaccaac agtgcaggag ggttcccttt 10200 tctccacccc cccccagcat ttgttatttg tggacttatc aatgatggcc attctgactg 10260 gtgtgaggtg gtatcttgtg gtagttttga tttgcatttc tctaataatc agggatgttg 10320 agcatttttt catgtgcttg ttggccatct gtacatcttc ctttgagaaa gtctattcag 10380 gtcttttgcc catttttcca ttggggtgtt ggcttttttg ctgttgagtt gtataagttg 10440 tttgtatatt ttagagatta agcccttgtc agttgcatcg tttgaaacta ttttctccca 10500 ttctgtaagt tgtctttttg ttttcttttg ggtttccttt gctgtgcaaa agcttgtcag 10560 tttgatgagg tcccattggt ttatttttgc tcttatttct gttgctttgg gagattgacc 10620 tgagaaaata ttcatgatgt tgatgtcaga gagtattttg ccaatgttct cttccaggag 10680 tttgatggtg tcttgtctta tatttaagtc tttcagccat tttgagttta tttttgtgca 10740 tggtgtgaga gtgtgtacca gtttcgttaa ttttcatgca gctgtccagg tttcccagca 10800 atgcttgctg aatagacttt ctttttccca ttttatgttc ttgcctcctt tgtcaaagat 10860 taattgaacc tttccataga aaagaaaatc atgaatttgg agaataaact tgtggttgcc 10920 aaaggggagg gagagggagt ggtgtggttg aggagcttgg ggttaataga tataaactat 10980 tgcctttgga atggattagc aatgagaccc tgctgtgtag cactgggaac tatgtccagt 11040 cacttatgat ggagcatgat aatgtgcgaa aatagaatgt gtacatgtat gtgtaactgg 11100 gtccccatgc tgtacagtag aaaaaaaatg tgttgagaaa ataactatta caaaaatgag 11160 aattatgaaa taaataaata aataaacaaa catacatgtc caatattttc ttaccaaata 11220 taacagaatg ccgcctattg cagtgtatcc tacttttgga acctaagtca aaccctcaca 11280 tgagatgact catatcaacc aatatttccc aaggtgtaga aaactgagtt attctgctga 11340 ttcagagatt taattattat ggtccttgta gacaaggaaa ctaggtaata taaattaaaa 11400 tcacttttct tctcaaaaaa aaaaaagatt cagttactta tgtagagcaa aaattattct 11460 gttcccttgt gatatatagc tagcaaagat ggaagggata aactggaatt gcttgacaaa 11520 gaaaccatcc agataaaata ataatcttgg atatttgggc attgtaatta ttaaatatag 11580 gtgtaaaatt ttttggaatt ctgagatatt ctatgatgag actcctattt tctaaatttt 11640 tgtttggata atgcaggatc actaataacc taactttcct tttttttttt tttttttttt 11700 ttgcttttta gggccatacc cacagcatat ggaggttctc aggctaggag tcaaatcaga 11760 gctgcagctg gcctaaccac agccacagca acattggaat cagatccgag ctgtgtctgt 11820 gatgtacacc acagctcaca gcaatgctgg atccttaacc cactgagtga gaccagggat 11880 caaacccaca tccccataaa cactagttag ctttgttact ccagagccac tatgggaact 11940 ccaaaccaac ctgttagaca ctagagcttt ttctatgcta caccttaagc accaatagga 12000 agaaaatcta tgatagcaag ttacaagaaa aaggatccaa ctttttcaga aaagtgtcac 12060 tgttagtctt tttacaaaag aaatgggtat agtttcccca gcaaacagat tctataattc 12120 tattccaaga aaacatcatt tttaatgcta ccatttaaca aacataaatc ttgttcccac 12180 agttcaaatg tagattgagt taaattttta tataattaac tgattataaa aataaaatcc 12240 agaaaatgtt taatgaagaa aagtatatat tttgtttcaa aaattcataa tagttatttt 12300 tagaactacc atgtaatata aatagttcag aaaaattaaa tttatcattc tggatcaaga 12360 caaattaatt ttttttttca gtctttttac acatttttct cagttctcca tcatacccca 12420 atgacaggaa atcatttact tttctgtgat ttttatcaag aaaataacca ttttttttct 12480 tcccagggaa acttggggtc agatgaattt agctattaaa atacacatca ttcttaaata 12540 gcaaatgtta catttacagt attctatttg ttttgtgata ttattatcat attttgattg 12600 cattttgttt tctttttgtt tgtttttaaa ttcttgcatt gtttttcaca gaaacctcct 12660 ctcaggcatc aggaacacct tcaggtgagt gctattctct gtgttctaag aactcactaa 12720 ttgtgggact gagcaatgat atattggtca tacatagact ctggtcctac tccacccttc 12780 tctataagac attcagtttc acattcaccg aggaataagg gcttcagttc aagtattaaa 12840 ctggttattg atctagtctc acaaacctta gagagaaggg tatagggact tacataaata 12900 tttaattaat atttaacatg gtttatttaa tataatgttg cttctttcca tctcttttgc 12960 atgtacccaa atatatgatt gacaatttgc tctcatttta cacactgaat cttaaattag 13020 atacctccac caagagcctt tacaagagtg attaatcctc cactctgtcc actaattgaa 13080 ttaataccat tttatttatt tttcattctt tacagaacga gccagacagc agagaggtaa 13140 agttctttct ttccctcgac aaatctctcc ttcagttctc catgatgaat gttaaatatc 13200 ttctttgttt caatttttat ttgtctttgg tactcaggta cttggagtac tttctcaatt 13260 ctttgctttc attacactcc ttttcagttc ctcctttgac acctgacgag tattaagagt 13320 atgaacttac taaacctcta ggcttggtaa agaaggaacc aaaaataatg actcctttag 13380 attttaaaat tagattataa aactaaaact aattatctcc taacttacct aagaaatatt 13440 ttggtttgcc taagtaaatg gggaagttgt gttcaaatgg aaaaatattc tccttttctg 13500 aatcatgttt ataattcaca attgaatttc tacaggaact cttcaaagaa agaaagtttc 13560 tcaggtttcc tgaggtggta agcattatcc acttcttaaa tgacaaatat atttttccgg 13620 aaaaaatcaa tttaattttt tgtttataaa tgtgtttttc acttgatttg atcaaacttt 13680 ttcttcattt tccaaagctc ccacaaataa tattgaaatc agatatgcaa atattaggag 13740 ttgctttaaa tattaaagaa ctgctttaaa tattaacttg tgcctctata tctgactctt 13800 tgcaaaacac aaacgctttt ttttaatatc ctatgtagag ttttaaaatg tccatgatta 13860 tgatgatgga atgttctcta tctctaaata atacctcata tgtgttgtgt tttctataat 13920 tttgtgactg aattgtcaca taggaaaaaa aatagaaatt ttacttcaag accactgttt 13980 agaagatttg atatgaaccc atctttgtct aaatgatttt taacataacc tttctttttt 14040 tttgtagcct ttattaagtc agttcagaca ggtaagaaat tctccaccag atatacagta 14100 gagttaacaa gggaagcaat cttggtttct gtctagtaag tgctgtggtg tgggtgttgt 14160 gccaattctt tattgccttt ttatggaaat aaacactaga cttaactgaa tcaggcagat 14220 gaaatcaaga gaactcagaa ctacgtcata aataaggtga aagataatag caacaacatt 14280 tagtggaaca aaattttaaa tgattttaag tgcacattta catactgata gcttaaacaa 14340 agaaagctca gtgaggcgtc tcagtgcaag aaatgataca gcaataaata ttagtgcatt 14400 cataaaaagc atatttgttt atttattgct ttttagggct gtacccatgg catatggaag 14460 ttcccaggct aggggtcgat cagagctgta gaccccgacc tacaccacag ccacagcaac 14520 tcaggatcca agccacatct gcgacctaca ccacagctca cggcaatgct ggatccccaa 14580 cccactgagc aaggccaggg attgaaccgg caacctcatg gatactagtc ggatttggtt 14640 ctgctgtgcc ataatgggaa ctccaggcct gtttattttc ttaatttgca gcagcacaat 14700 gtaaagtgtt ttctcatagc tatccattca tttatgtccc tctttactaa tgtttatctc 14760 ttctttttta tccttaagga aatcatcaat gaactgaaca gggtaaggaa cattaatgat 14820 atttaaatta ttttaaaatt cattctttca aaaatatatt agctacactt tttagtttta 14880 acaaagagaa ctcctgagaa aaaaacagta ataaactcaa ggtatcaaaa tctttcttat 14940 gatagtgtgt caaaaagtat attcttgcag ttcaaaagtg ttttgattta gaaaaacagt 15000 atttttcctg tgatatttat accttcactg aactttcaaa atgactaatg aattctactc 15060 ataattctaa atatttattt tattgattta attttgtatc tatgaataga caaggcatta 15120 atatgaatga atgaatgggt gcaattttgg actaaccaat tttttgcact tatcaccaaa 15180 actgaaagaa ttctttatta aaggttttat aaaaaaacaa tattatctgc acctagaatg 15240 ttttacataa tcacagttgg ttattctctt tctttacaca tgagtttctg ggccggggat 15300 cagatccaag ctgcacttga gacctaagtc acagctactt caatggagga cccttaaccc 15360 actttgctgg gctgggtatg aacccagcgc ttcctagtgc cacagatccc attgcaccac 15420 aggggaacct ctaacgcata tttttttaaa atctggtctg tcagattttt agtagttttg 15480 gtattgagac aacacaggtg ccactgaaga taaaaaaata tatcttccta actatccttc 15540 atacttgaat gatcatcctt tcttggcagg atgctaggag tgaatcaact gaagtaagat 15600 tctttattgt aaaactatta aatataatgt aaggaaaaga aagaaataaa atcatttcct 15660 ttaaatatcc taatgaaaat gaatgactaa ttcttttagg tcaaaactaa gacagatatc 15720 tctaattcaa agagagaaaa aaaaaaacat acaatgttat catctgtgag ccataattag 15780 ctggataact aaatcaatgg tattatattg agcttaaatt ctatggatca tgtcactact 15840 ccctgtactg ctatggtcat gaaaactgaa cacaacattc tagatggaga aaatccattt 15900 tgcttaaata tttatacacc attgaaccaa tgcttcatag acttttattt attacataat 15960 ctttaatcca atacttctgc ttaatagcca aaagccaagt aaaggaaaaa aagtaagggt 16020 agttaaagga aaatgtagga ttatttacac tgatttacca caacaaataa gaatccataa 16080 taatttatgt caagggagtt cccgtcatgg cgcagtggtt aacgaatctg actaggaacc 16140 atgaggttga gggttcgatc cctgctcttg ctcagtgggt taaggatccg gtgttgccct 16200 gagctgtggt gtaggtcgca gacgcggctc ggatcctgca ttactgtgac cctggtgtag 16260 gctggcagct acagctctga ttcgacccct agcctgggaa ctttcatatg ccgtgggagc 16320 ggcccaagaa atggcaaaaa gacaaaaaaa acaaacaaac aaacaaacaa aaattttatg 16380 ttaaactcag aaatgcagat tagggaggta aattctttgg ttagcctggt aggtaggctt 16440 ttttctttcc ttatcactgg ctcttaccac atatttctat ttcttttggc atctatttta 16500 tttgataatt attattttac atttgaatat ttgtcataaa aaataaattc tcttttcttt 16560 tctaagaatc atggcatgga aggccatgag gtaagaccct tattgtaata aactctacac 16620 ttacataaca tccatagtat atactctatg ctctatttta agaaaactct cttctcaaat 16680 tgagcaagat tgaacttccc aaacaaggtt attataccca agaatgtaca atgttgtgcc 16740 agataatgtt aaaattagaa gaggaaatgt gtcttttaat tgataatcag ggaaaatgtt 16800 atttattaaa ataaagggtg taaggcaaaa tgtgcatgag tgtttcaaat gaaatatgag 16860 cctccaaaaa aaaagaaaaa aaaacctgtc tctaccacct cagggtataa actcactcct 16920 gtttgtgagg gtagtcttgg ggagaaagaa tctggttgca gtaattactt aggatcaatg 16980 gcctatgcta cttaatccaa aagcatggaa aaaatccact agcccacaat atttggttaa 17040 tcaactttcc caagagatga atcatctgtt gactattaaa atcaatcttt taggtatgta 17100 cttgagaaca aaaattatat ccatatttaa atataaacgt ccgtgagtta gaaaaaaatc 17160 taaatagttc aaagtgtaga tgttttaaaa gctaatgtaa ttttagttcc ataccaaaat 17220 ttgttgttgt attttcatgt ttcaatttat tcccttcaaa gaagccccaa ggatacatga 17280 gaataaatat cccacccggg gttctgggtg cccaagctca gtagggcact tccttccaaa 17340 tttcatatat attttgcatt ctactcaacc acatatctat gtttgattta ataaaatgtt 17400 gttttttagt gattggttat tacattccca catccaacat attttaaata aaattgacaa 17460 ccccaaaaag gtacgttata ttggccctga atgtttcatt accaattgct ttcattctaa 17520 acaaggagtt agcaacccag tatgaaagtg tggaacaaat ttccttctaa ttctaaaagt 17580 cacagtgttg ggaacactga ttttctctct tttagcaaag gggatctagc agttcatcaa 17640 gtgaggtaaa tcattttgat gttaattcag tatctcagtt agaaaatgtt tatgaaaact 17700 tgttgtgcta tgaatgttac acatcccata aggtctcatg gtacaggctc tatgtctaca 17760 gctctaccct aattttaaca tacaggctat gagcccaaaa gatataataa cgcaaatact 17820 tgtcagatga aattacagaa ttgtttctgc gctaacaatt ctatctggct atccatggtg 17880 ccccattttt tttctctaat ttttttgcct ttcctaggtt ttggtagtac taaatatttt 17940 atttaaaatt actataagag ctatgcttct aaatcattaa tagaaacttg gtatttcctg 18000 tgcagacatc tactgattct aggttaactg ggtactggaa tcctttactc cttgttaacc 18060 taaaagagaa aaagagcagg catagaaggt cctttcgtaa actttgggag aagaaaattt 18120 caaaataaac ccaacccagt tatgcggagt ttctgggaga tcagtaaaag tgcatcgaat 18180 atttcacatc tacctccaac attctgaata acttccctgt tgaaatcagt gagaagagga 18240 aagggaatct tgagtacaac ctgtaccttg aattattcat cttatctcag ttaccaagga 18300 atgggtgcct aagatcaatt tatcacagtt aagcaacatg gtaactggct aattagtatt 18360 cataccttga gtataaatta ataagtcata aaactaacac tgcatgtttt tattttttaa 18420 ggaagttgtt ggcaatagtg ctgaggtgag atatacttac taaatttaaa atacattcac 18480 gttatccagg atgtgttaaa atttacttgt actttttttt ttctttttta gcagaagcac 18540 gttcaaaaag aagaagatgt gccctcccaa agctatctgg taaaatttta ctaaaagttt 18600 atcaaaggca aatgtaccaa ggaatgagta tgaatgttgt actgatacat tatttctcct 18660 tctcaacctc tgctacacct taatacatag taagccgtct aacagactct agatgtttac 18720 tgatcccctg caaaaataaa gctaacaact tttttatccc agggtttttg tttgtttgtt 18780 tgtttgtttt gtcttttgtc tttttagggc cgcacctgca gcacatggag gttcccaggc 18840 taggggtcta atcagagctg tagctgctgg cctgcatcac agccacagca atgccacatc 18900 caaaccgcat ctgcaaccta caccacagct cccagcaacg ctgggtccct aacccactga 18960 gcaaggccag ggatcaaacc ggaacctcat ggttcctagt cagattcgtt tccactgcgc 19020 catgatggga actcctatcc caggttatta attcatgctt tcatgagatt tgtgttttat 19080 agtctatttt tggatctgaa taacatagta ttttttttat ttttttaatt ttcccactgt 19140 acagcaaggg ggtcaggtta tccttacatg tatacattgc agttacagtt ttttccccca 19200 ccctttcttc tgttgcaaca tgagtatcta gacatagttc tcaatgctat tcagcaggat 19260 ctccttgtaa atctattcta ggtgtgtctg ataagcccaa gctcccgatc cctcccactc 19320 cctccccctc ccatcaggca accacaagtc tcttctccaa gtccatgatt ttcttttctg 19380 aggagatgtt catttgtgct ggatattaga ttccagttat aagtgatatc atatggtatt 19440 tgcctttgtc tttctggctc atttcactca ggatgagatt ctctagttcc atccatgttg 19500 ctgcaaatgg cattatgtca tcctttttta tggctgagta gtattccatt gtgtatatat 19560 actacatctt ccgaatccaa tcatctgttg atggacattt ggattgtttc catatcctgg 19620 ctattgtgaa tacggctgca atgaacatgc gggtgcatgt gtctctttta agtagagctt 19680 tgtccggata gatgcccaag agtgggattg cggggtcata tggaagttct atgtatagat 19740 ttctaaggta tgtccaaact gttctccata gtggctgtac cagtttacat tcccaccagc 19800 agtgcaggag ggttcccttt tctccacagc ccctccagca cttgttattt gtggatttat 19860 taatgatggc cattctgact ggtgtgaggt ggtatctcat ggtagttttg atttgcattt 19920 ctctactaat tagggatgtt gagcattttt tcatgtgttt gttggccatc tgaataacat 19980 agtatttaaa tggcacccta cagatttggc cagatttgag ttgaactcta agctctacct 20040 tttactagca tgtgatgtta gttatttatc tgctataagt ctgcaaccta aactctaaaa 20100 tgataatata atgatagcaa tacacagcac aaagcaagca ctcggtaaac gtggcaagtc 20160 tcacgaaact taacatgttt ttgcccactt catctactat tctgatttaa tttggccaaa 20220 gttaaatcct ttgtcagaaa gcaaaaacaa ttttcaattt tcttagcaat tctgacataa 20280 gacacactgc aaataaaaat gaatattcat tcatatttga cagcatggta atttatttca 20340 cattaaaagt aagttatgaa agtagttttt ctagtattaa aatataattg gattagtcca 20400 aattatctgt ggttgaagta acattaaaag aggattacta gttcttcctt ctttgcacct 20460 aggaaaggtc atgttatgat tctagttata ttacttgaaa atgataggat gagactggat 20520 tgtctccaag caatggacaa tatttttttt ttcttgaata gggacatctt caaggactga 20580 acaaatacaa actgcgccag ctggtaatat ctttattata ataacacaaa actaaactgt 20640 gcaaaatcaa aataattaag ttgtaaattg ggtccacctc tgaatatttt tttaagaaat 20700 tatttattct agacttgacc attaataata actgacaaat aatccaaatg attgcctatg 20760 tatttactgt cacagctatt tgtttctatt tatgccgaga gtagtattgt aaatcaggag 20820 gtgttcctgt cgtggcacag tggaaacgaa tctgactagg aaccatgagg ttgcaggttc 20880 aatccctggc ctcgctcaga gggttaagga tctggcattg ctatgagcag tggtgtaggc 20940 cggcagctat agctctgatt agaccactag cctgggaacc tccacaggcc acaggtgcag 21000 gcctgaaagg acaaaagaca aaataatcat atccataatt gctaagcagg gagaaatttc 21060 ctttttttac taaattcttt atcaaaccct cttagaccct cccacatgat gaattatatt 21120 cagaagttta ttctttttat tgttgttgca aattcagtct tgaaaggtgt tccataaata 21180 tgcctgcttc ttaaagcaca gatatgccaa ataaaccttc catttaagga aagagtgtag 21240 actcaggaga aggaaccagg agtcctaaat attcaacttg cttttgtcaa aattctctaa 21300 gaaagaggag cttttttgct gcaccttggg ttaaggggcc atcattgtca cagctgtggc 21360 tctggttgct gctgtggcac aggtttgatc cctgaactgg gaattttcac atgccatatg 21420 tgtggccaaa aaaaaaaaaa aaaaccaaat aaacataaac aaaaagctct aagaggaaag 21480 aattggcaga ttgattgatc taatggtcta tgaatccatt tccttaggtg tgaaatggaa 21540 atacagttat tctcactccc tggtgttatt ttgaatatta aatgagcaat cgcctactca 21600 ggtctcccta agtccagctt gtagcaagtc cagtaaaagc ttcattcctc cttctcctgc 21660 ccccactcca cagcctctag aaaatttgct gcgctcatga gacttctgat aacattatga 21720 aagtctttca tcaggatatg tgtttgaact ataatgtcat aaactcttta tcagcagagg 21780 tatggataga taaaaaacat atcaattagc aagccctttc aattagcaag cccttcttcc 21840 actatttaat tttgatcctt ttgagtatca caatctttca tttggaaggt caaaaaaaat 21900 ttcttagcta tagcaaataa acaacctaat taggacttta gggaaaaaag cttgtcagtt 21960 taggtaaatt accttttcac attttcttct gttatatatg taagatgaag agagttaata 22020 atgaatttaa tttgcacctc agtttttact cttatttcaa tacctgagtt acctgtgttt 22080 ataaagtcca aataaattta aatttatcag tacttcactg gaattcatca ttactgatct 22140 aagaaattac ctgaataatc agttttagga cttagctctg atatgacaag gtcataaccc 22200 ccagaagtgt taggaccagt aaggcattgc tcattatgtt cattgtattt catagatggt 22260 catcagtttt tagcatagtc ctcaagtgca tgaaagtact tagtaaatct ctgttattca 22320 tatgaataaa atcaatatac taaaagaaga tgttctttct ttcttaggaa gctattcatg 22380 accaggtaaa gttatttatt tattaagtgt aaaatatttt agtatttcct tcatgtgtta 22440 tatttttata atgtgcattc cttttttttt tataacagaa atatatccca atagagaaat 22500 ttatctccta cccatatctg gtaatatgtt atttaaaatt caccaaagat aatatttaaa 22560 taattgatta atagtcttat atggaaaata tgtattctta gagaaatgat aggtttttct 22620 ttttcccagg gtatgagttt tataaattct ttattttcca aactatacta agtctagcct 22680 ttagttgaat atttttttct aaagttagaa aataaaaagt gtattgctat tttttttcat 22740 aattttgacc tagaatactg tctatcctaa atttcatgga tgatgatata ctcttggtac 22800 attgtaaaat gaatgtgttg tatcagaaga tatctaagta atttaaaatg tctttccttt 22860 aaataggaac ttcacagaac aaatgaggac aagcataccc aacaggtaat attttgttta 22920 ataaattaca cagttatatt gtaaagttta aatatgtttg tttttaaata ccctcatact 22980 tttgaggggg tttctttctt aggcagctct ttttattttt ttgcattttg gggccacacc 23040 caaggcatat ggaagttccc atactagggg ccgaattgaa actgtagctg ctgacctaca 23100 atgacaacgc cggatcctta acccactgag tgaggccacg gatggaacct acatcctcat 23160 ggttgctagt tgggttcgtt actgctgagc cacaaggggg actctgcagg cagttatttc 23220 ttattcccag ctttctgaaa taatttatat tgatcaatca gagatgagaa ccatgtgtca 23280 ctcttctttg gaataagatt tgttctatcc ggtctgtctc acctgacgtg gtcagcctat 23340 ttataattct gagcactatc aagaattcat ctccatggtt acattaattt cccctttaag 23400 atattcttta attttgcatt tttgtccata atttgattct taaattcaat ttacatttag 23460 gaaactcaaa ctcaccatta tgctctttta agcaaagaga gtggagattt actaactgac 23520 actttctcaa gcaaccatct ggagccatag taaatgtctg tccaaaaaaa atcttcctga 23580 tcctctgttt tctcctctgt gaagtgagat tatatataat acctaacaga aactgactct 23640 tctcctgccc tcccttccac tgatgcccta gtgctcctgt ggcttctgct ttccttttta 23700 aggcaatgat gagttaccat gaaacatatg ttactctacc agctctgatt cctgtgaata 23760 gctacaccaa ttccaatcat ggaggctccc aatcactatt tgctgtttag ggaatcttat 23820 aaggatggta gagtatgttt catatgtcta agaagaaact tcctttagag aaggcaattg 23880 ggaaaatttt agtatgagtt ctcagatctc taacataaaa agcatttcaa gtaggttgct 23940 tcttacagct ttggttttat ttagccttaa aaaagtaact ttaattctct ttttctttcc 24000 agggagagcc tatgaaagga gtgaatcagg taagagtgag tgtgtgagtg tgtgtgtgtg 24060 tgtgtgtatt aatactgccc cataagctag tgctatgcta gtatttcttg gctattgact 24120 ggtgttggac tctctagagc tggttctaac attgctgtgg aagatctgat agatctgatt 24180 gccaaaggaa atgaatgaat gattctatgg ccatctgtga gtgatgatag cttcaggtca 24240 actttaagtc aggacaatct caaccagcta tttacattgt taaaatttga cctgttatta 24300 atctatagta tcatgtcatg aaaataattt gagttttcaa tcttagatct gacaccttct 24360 aattacagga ccttgagcaa attgtgctat tctctttgag cactgctttt tcatttgtga 24420 aattaatata tggacctatc catttatttc ctccccaaac ttccatatcc tgatttttgg 24480 agtgtctttc tatatcttca cttttagaag tagtagctct gcattcaatt cagtttcttc 24540 agtctctctc tctctctctc tctctctcta tatatatata tatatatata tatatatata 24600 tatatatata tatatataca cacacacaga cacacacaca cggcatgcta attaaaaaga 24660 atgcaaagta aaccacggag ttttctcaat atgaatgtta ttaggaagaa aaaaatttcc 24720 tcaagattca aaacatagta gtcatcaatc agttttatgt gcatctttac tgtgcatcag 24780 atttactctc aatttcttct gaaattctct aatcacttct gagctaaagt aagagaaaag 24840 gcaattactc acttcatttg agatttaaag ggtacactaa cctcaacttt tcatgtaatt 24900 ccaaaaaaaa agagggtgat gattataatc tcaataagaa tatttgatag caataattta 24960 atcatggtat ttaattggat ttaaattaca aaactatttt tcccctctct gttaaggaac 25020 aggcctactt ctattttgag gtaaatttat tttattttat tcttttatcc aaatgattta 25080 taaaggaaaa gtattggtaa acatttataa tatagtaatc tttatgtagg taaccacagc 25140 aaaactggaa atgtttttat tttttattcc atcaaaaagc acatattttc acctaaatat 25200 atagagaatt atgttatgca taaaaacaag taaaacatca tgatagatga acagagtcac 25260 ctatcctgaa atttcagaaa ggaaaatggt ttggcaccaa aactttataa ttaggaaagg 25320 ataatgtgtt aggaattgga gaattctttt ccctttattc ctggcatttc tgagagcaga 25380 ggtatggacc ccgagtggga ggcccttcct tttgtttgag ctcagtgtct tcatggaaaa 25440 ttaagctaaa gaacaaaatg gccaaaaaag tcctttccag tccacaagtc catgaggtta 25500 taaattttat aataattaaa ttcacacccc tacattcctc tgggctctgg taactggact 25560 ccgattagta atgcagattc gtggagttca atgctgaata ttgaccttga agaagttatt 25620 tcttcattac tcacaagtct cccaaaaagc cctttccaaa agttcctttg ccatgatcca 25680 ctgcatggaa gaatgtgatt tctccgtttt ccttgcagag tagatatctc ttgtgatgct 25740 aatagccatg tcagaagtga ataaaatgct tctttttttc agagattcta aagagcaatt 25800 tcccatatcc tgttgctatt tcattctctc tagcctctcc accagttcta ccagcttgat 25860 gcctatccct atgctacctg gtattatcct ccacaatata ttgctcaccc attattcacc 25920 aacatccctc aacccactgc ccctgagaag ggtggaaaaa ctgagattat gcctcagtgg 25980 tggtaagttc attttaatga ctgtatattg atgttctacc aaaggaaata aaagaaaact 26040 tcttaaagaa cataccataa aaacagattt agaataaaca tgacaaaatc aatatctaga 26100 gcgtcatagt agaattttcc aaaatgggaa attggcagga cgttctgata tctgcagcta 26160 atgttaatcc actactcagg aacttgtgga gcagcgctct ctgttctttg agattcattc 26220 tgatgaagtc aggaaaaagt tttctatcca aagcaaaaac acagtaattt cattttatcc 26280 tccttacaat tttactaatc tctaaaggct tttcttttgg ttatatatac ccatgatata 26340 cattacaatt cagtgtgggg aataaagcac agattttgac atccaaaagt cccaaatcca 26400 aatcctgacc ttttttgctt acttaaaata atgtattaat gcttattttt ataattctga 26460 aggtgattaa agacaataat ctgttaagca tagtgctggg aagatacata gcagtcagtt 26520 tttattgatt tagtaaaatt gtactgctga ctatcttcat cacatgattt taagaatttt 26580 tgttttttca gaagaattaa gtgaattctc aggaactcca caattatggc ctttggtaag 26640 ttggaaatca tttgtggaac catcgatcct cttttcgttt aaagactcat tacaaagata 26700 ggactgtaga ctataaagat tttttttcct gtagttgagc tccttgtgga cacattagca 26760 cttagataat aattaaattg gcttggacat ttgcaaatgt ttgtttcata attatactat 26820 atgtaaatag caatcaaatt agataatttt aatgaatata atttattata ttgaacccct 26880 atacaagtat aggagcatga atgctactaa ttttccatca agatgtgacc ttgagatgct 26940 ggtaaactca acagtgggat tctatttttt atgatcacta caataaaaat ccttagcaag 27000 tcatgtgata aaaccaagta tttgtttctc aacaagaaaa cagactttaa cgtctacaga 27060 cttgttttaa ttaacttcat ccattgtact ggtgtttccg attgtatgtc agtagagggg 27120 tgtgtgtgtg tgtgtgtgtg tgtgtgtgta tgttttccct ttctagtggt ggaaatttcc 27180 cttccaactg attgaattaa ggaaaatgac aaaacatatg ggaaaggttt ttctttgaat 27240 tccttggcca tattttctct gttactgcaa aagaaaatac tacctagcaa taaattgtct 27300 atataactta aaattatcag gataaatggg tacatatgtc agtcacagga aagaacaaat 27360 aactttgtga gtttcatctt aaaatgaaga gaaaatgatt attcaaatgc atcataacag 27420 tgtctcttcc attcaaaaca tgtaatataa ccaaccacat atttcttttt ctatttacag 27480 atgtgactga aaataccatg cttgaaattt ctcctctcca tctaccatgt agaaccattt 27540 tatctgaaga ctttgactgt tcttttagaa cagggaaatc gcaaatcgaa gtcaatcttc 27600 cttcttgaat tctttactct atattagata gcatataatc cttttccttt ggcaaagttg 27660 tcctaacagt ttagtgtcta aatttcagtt gtatcatgcc agtaggaaga ccactgaatc 27720 agagggaatt aaaagtcttt actaaatttc aatatggaaa ttttgtttaa aaagcctttg 27780 aattgcttct cctgtaagtg ccatcatttc aaataattgt gtgcagtgac tgagattttt 27840 cttccttctt ttcaataaat tacattttaa ggcacaactc ctattttttg tcattattcc 27900 attcagcaga atttgcacaa tcctgttaac agtctttatg cctgtaacat tttattttca 27960 ctaagttttt attacacttt caaccacaat tcaatgaaca aaatggtaaa tcttcatgcc 28020 tagctgatgc tgacaggtta taagctgggt ctaggatctt tcatttgaag tcacctgtct 28080 ataggatatt ctccatgaga acataggtgt ggctgcagag aagaacagtg gtatgacagt 28140 tgcgggtgca ctgggaattt gaaaaacatg gctaagctgt ttatttatgt caccagaacc 28200 tgtttgagcc aaactcattt gattatttga ttattataca cttactttta taattaggta 28260 gattagataa taaacattta ataggttaga ccatggcata cagtaacaca atactcagtg 28320 tcctcccgaa ctccagtgaa gagttatttc tcaaaatgac aatagtcgct tgcattaata 28380 ccctcaaaac cctgctggaa tttactttcc agagtttatt ccagggtccc caacaaaccc 28440 tatctgctgt gactctttaa gaaacttctt tccaccaggt cataaaatac aggtggcaag 28500 gtctcttgta ccacaaccta aacgacctat ttagttagca gttctgtctc ttatcgatta 28560 tatatataat tgatatgcac atatatgcat aacacctaaa cacgtatttt ttgcctataa 28620 cgctttttta ccagttgtca cttggtttgg tgacactagt ctccttccat tttccctgag 28680 gttggaaatg caatttcaat tactttgcac ctctcaccag agttctcagt caattgttta 28740 gcacaaaaga atttcataaa agtttgccct tcaaggaaaa gtttaaagga gaataattta 28800 tcctttgtag acacagggaa gtgcagggcc attagaacta agcatatcta tttatggatg 28860 tttaaatatt tctctttcaa ttatgattgt caaactgcct tctagagtct cacaaataac 28920 agaagcagaa aaaaatacag ttgtgaaaaa acagtgctga cagtgaactg aagaatatgt 28980 gcattcctca ttggctgccc cttccaatgc attgttgctg tgcaggaata cacaggtcaa 29040 atgtaaccaa atttggggac ttctaggtga aactagaaat gtggattggt gtgtaagatc 29100 tcctattttt aaatattggt ctagtgtttt ttaaatataa aatacacagt ctccttctct 29160 ttattggcca gacccaaccc ctctaacact agtctatgta ctatagtgta ctttaagtca 29220 ttaagtaaag gactttctac ttccagtcca atcaaggttg aatcccagtt acagtgaagt 29280 gaaagtgaga agtaggacat tagggtaata attagctaca gtaaacaaag aaatcatgat 29340 tccatcatga caaatacaca gtaagtgtta ggggctacat ggggctcatt gttaaaatgg 29400 ctcatcatgc tgacccatga gactgaccat cttgttcaag cgacatcctg tttttgccac 29460 tggtgcctat tttcccaaga ctacaagacc aggggggacc acacacctcc agccttctca 29520 agattatgag tgcaccctac cgtgagatac ctctgacttt ctcatgacga tgtgaccacc 29580 agagtccacc tgcaggtgaa agataaacta taaactaacc ttcccccgct tcaggaatca 29640 atttcccctc tgcagagtat aagaaggccc tgctagaagg gcgggggggc tgactcttct 29700 caaaggtcag tcagcctctc ctttttcctt ctaataaatt ttcttctctt tgcctgaaca 29760 cccaattcct tctctttttc tccatgctcg ccttacagta agaccttcag gagcacttgg 29820 gagcaggcgg ttctgtaaat tttccatacc ttcccatata ttccactttc aatagttggg 29880 ttcctattgt cctaactttc acaagaacta tccattttat ggattcatgt catattaatc 29940 actatagatt agattttgca tgccctcccg aattcatatg ttgaagccct aactccccca 30000 tgtgatgcac ttggaggtgg catctttgga ggcaaatagg tcatgtgagt gggatatcta 30060 tgagtgggat tagtgcccct ctaagaaaag ctaggagaga gcttgtctat ctttctgcct 30120 tgtgaggata cagcagaaag gcaggcacct gcaggccaag agggagcctt caccggacac 30180 aggatctgct ggcacctgga tcttggtaaa tgttatttgt aactgtatgt ctgtgagttg 30240 ctatgggcag gatgtactgg ctggtaaaca ttgtttagta ataagtggat gtggtttaca 30300 ccagatttag gagaatctag gagtggtgag gtaaaatggg gtttggcatt caattgttga 30360 ctcattatat agctaaaata tgcctgcttg atgagcttat tgtaagagat ctcacaagtc 30420 attttgagct tcctagtatt gaggtgcttt gcacaccctc tgttagtggc ctaagaactg 30480 aagataacac atgtcttgtg ctaccaagca gtaggaggat aaatgaagtt tgctcttgag 30540 atctcagtac ctctttcatc tttcaatgca tacccttctc ctgttgttgt actctgtcct 30600 ctgcctgtaa taaagctgtt ctgttagtgt aaactgtatg ggtctccttt aactatcaaa 30660 aatgttaggt cttgtgtgat taccacctcg ctgtaatata cagcaaaaaa aaaaaaaaaa 30720 aaaagggaga attcatctgc aagttctctc tcatctcctg cttctcattc tgaaattttc 30780 acctcagtgg gggctaaaac tatgcatata acagtagcat taaccaacta ttacagtttc 30840 tgaggaaacc cggtcctgtg ctatagtaca caatcccatt caagtccaac agcaaagagg 30900 tgagtctgca ttgatgaggt cccaaccaca gggaaaagga agaagacagg caatatactg 30960 aatgcacaca tcgcaatgaa tcaccacaga tacttacttc caggctaagg gaaagaatat 31020 cgctagttcc ccagcacttt ctcacatttt ctgtcaagtt gtaccttccc tcagataacc 31080 aatagtctag cttatcacac aagaatttaa tcgatcatta gattctaata aattccactt 31140 attggattac atattccatt atattttggc ctgattactg tcattcaatg ttatttatat 31200 aaaattcacc cttgtttttt ttgtggcagt aactcattca tttcaactgc agtatgatat 31260 cccattccac tttagaccat gacttattta tacaaaatac atttatacaa aatacaaatt 31320 tatacaaaat aacgtaaatg ttaattcaca tttatgttga ttttagtttg gggtattagc 31380 attgttagga gtattgggta aatgttttta gaatacatat gaaagcatat tatggtttcc 31440 ttttggttgt atacctaaga gaagaaatgt tagttataat ataagcagat gtttattttt 31500 gacaaagact cataatactt tccgagttgc ttgaaccatt ttatatttat tttaacatta 31560 cctagcattt attcagtttg cttatttttg taaatacttg gttttaccaa ttcttctcat 31620 ttcagctatt ctggaggttg tgtcatgata tctggtattt ttaacttgca ttttccagag 31680 aaataatggt attgaacatc ttttcatata tttattgaca attaaaataa cctctttggt 31740 gaagtgccca ttcaaagata ttgcttgtgc actattatat tagttgcctt tgttattgaa 31800 atatgcccaa ttaaattttg gacaatttgg ggactggatt cagacaaaaa gttctgctgg 31860 ccatgtcagg tttgagatat taattagata ctccgatgtg gagaagaatg cagttgagca 31920 cttgaatttg gagttcagga aaaaataagt ttgaggacat acatgtggaa gcttcaagcc 31980 cacgaatgga cttaaagtca tggaatcaaa caacatcaat catagtaagg ctaatcccta 32040 gggtattcca cacattgcat gtttggaaaa ggagaaaatg ccagaagaga cgatgcaaag 32100 aaataactgg tgagttaaga gggaaatcaa gggcttatca ggcttaggat ttcgaataaa 32160 atttgcgata tttcaaaaaa aaaaaaaaaa aaaaaggaat gaccaactgg accaaatagc 32220 actgatagtt tgggtaaaat aaacatagat tggtaaaatg aatgttgttg gtaatcttga 32280 aaaaagtggt taccataggg gtaaagctca atgggaacag gttgatgaga gaataccatg 32340 tgaaacaata gacacggtag tttggacact tcctttctga tggttaccat agagcagtct 32400 aaagtactgc caaggatgga attttttctt taaagatggg agacattata gcctaagtat 32460 ggcttgcact tgatctgagg taaggaaaac actgaggatg caggaaagat gaaattactg 32520 ctagaaacaa gtccttgtgt aaacacaagg ggagggaatc aagtttacaa atgtaggagt 32580 tgtgtaacca agcaggaccc tgtgaagcct tcccacagtg gatccacact cttgtcctct 32640 gcctgcattt ttgtctatag aaaaacttta gtcaaagaat cagtttgatc agagaagtga 32700 gaaaatacag agaaaaagga atacagtcaa gcaagacaga gtaataatag tttagccact 32760 cagcaatgtc aaggacttat ggttcttcct catggactat agataatatg ctgagccatg 32820 tccttggaac ggttttgcag gtgctcaaac ccctaccagg cggaagaagt caactgcatg 32880 ctgcccacaa acatgtagac cccagaccag ttggaaacag aaggttgatg atgctgactc 32940 ccaattacct catcaccaaa cagttaggaa aatatccaag ggctgatcat gcccggctcc 33000 ttgaagagta agagtaagag tcctcaccac tcctccaaga ggcacacagt ccccgaagca 33060 ctagcctgct gtgtaccctc tgcctggcaa ttaaagctac ttttgccggt ttccacattt 33120 ctatttggcg tcagtgtata gaggcagccg atacttcggt gatagttggt agacagataa 33180 ttcattgcaa tgaaaaaatt actccacaga cagagacaag ggatatcaga gcttcacata 33240 agtctac 33247 <210> 2 <211> 9300 <212> DNA <213> pig <220> <221> promoter <222> (1)..(9300) <223> promoter of porcine alpah S1 casein <400> 2 aggattacaa gattgctgtt ggataatgct ggatataaga aattgcatat ctttattctg 60 tagacctcag cagaagtatt gtgaagtttg tctttagcaa tgctgagatg ttctttacta 120 gttcttttta ctctatttct cggggcttaa atctttcatc ttatatgcat gttaggcatt 180 ctccaccagc taggatgtta tttcctccat cacctatccg aagattcgac accttgaata 240 cattgctttt gttgagtaaa agaataagca gtatgatata ttagaaagag cacaggctct 300 gatgttggga gatctgagtc atcatttact cattagctgg tgcaagcatt ttgtatggaa 360 attaatttac ttattaattt tattactttt atttttactt ttattttttg ccatttctag 420 ggccgctccc gcagcatatg gaagttccca ggctacgggt ctaattggag ctgtagctgc 480 cagcctatgc cagatcctta gcaacgcagg atccaagtcg cgtctgcaaa ctacaccaca 540 gctcacggca acaccagatc cttaacccat tgaggaaggt cagggattga acctgcaacc 600 tcatggttcc tagttggatt cgttaagcac tgtgccacaa tgggaactcc ttaatttact 660 taatttataa aagtttagtt tccttatcta caaaagggaa aataatgact tgtttctcta 720 cattacatga ttcttataag gagtagttgt gaaaataatt ataagaagac tttagaaatt 780 ataaagaaga ttataaattg acaaagaaat gaatctattt cattctgctg atggatgact 840 tattaatcta tctggtaaac tgtggttctg atggccaata atgcctgtta aatacattgt 900 atcctcataa taatattgtt aaacagggac tgccaatgat tggaccccag gaaagtttat 960 cagtcatgaa aaccacattt tgtcaagtca gaagaactat tactgataag aaaaaaatct 1020 ctttacccac caaattacag tctttcagaa agaaatcact aggtgagatt tagttaacca 1080 agttaaattt tagcccagtt ttcccagatg tcttgagtag gtgaaagctt ccttcatggc 1140 aagatgaatg aatttttggt tggaggtaag aggcaggtag tggtcttggc acgacataga 1200 cagataaact gttacttgtg agatatcata cctacaaaaa gaatataggg ttggataagg 1260 agatgcttta ttgtagacat tgctataaag gtaaacactt tcatggaaat acaattcagc 1320 agttttagca tctttgtttt tgccatatgt gttacatatt tttacaattt tcatatgctt 1380 ttaatttttt aaaaggtatt ttacatacaa actttcatta ttgggtattt taaaagagaa 1440 gactgtgtat tacattttca aatctttcag tttctagcta ctcaaagtgt gctgcatggg 1500 tcaccagcag tgacatcacc ttagagctta ttaaaaattc attagctcct ggagttccca 1560 tcgtgatgca gcagaaacaa atttgactag gaaccatgag gttgcaggtt caatccctgc 1620 ctccctcagt gggttaaaaa tccgacatta ctgtgagctg tggtataggt cacagacccg 1680 gctgggatct ggtgttgctg tgactctagc atagtccagc agaaacagct ctgattagac 1740 ctctagcctg gaaaccttca tatgccgctg gtgccctaaa aggacaaaag agaaaaaaaa 1800 aaatacgtta gttcaggaca cacctcagat ttactaaatc tgaatctgaa ttttaacata 1860 atatcctggt gattcataag cacattaaag cattgttgta gttcattgtt ctgtgagaac 1920 atcaaatagt ataggatttc atcaataaaa ttcagaagtt ctttctattc ccctctcata 1980 ttccatccca agctttcttc agttgtggtc actaataaaa attttgcctt cacacacaca 2040 cacacacaca cacacacaca cacataaggt ctttttaggg ctgcacccat ggcatatgga 2100 gtttcccagg ctaagggtca aatcagagct gtggccgctg gcctatgtca ctgccacaac 2160 aacaccagat ctgaattgca tctgtaactt acaccacagc tcatggcaat accgatcctt 2220 aacccactga gcaaggccag ggatcaatcc tgcatcttca tgtttactag tcagatttgt 2280 ttctgctgag ccatgacagg aaccctgcct ttaccctttt atacattttt aatgcacata 2340 caatatacaa gtatgattca agacttctcg aaagttaaca aatccacaag ataggaaaat 2400 gatttgggca caaaataatt agttgaggtt ttttttaaaa aaaaaattct aataatgatc 2460 gttttaaatg agcacttact ttgtctcagg ctctcttcta agtgtgtgat ctaatttttt 2520 cctctcgatc acacaggaag tggatgtcct taaacagatg tggacactga ggcacagtga 2580 ttttgcaaga ttaccctgct ggtcattgag agccagaaat taaatttaca atttgacctc 2640 aaaatcttgt cttgcaacaa ctacatcatg tgtttcattt aagatcttct gagacctatt 2700 gatacagcac taaaaaggat attgttctaa acacaaacat aaaatcaagc ataagttcca 2760 tatttccagg gatatcacat accacacctt accgaaagaa ctctatttta gttctagatg 2820 ccaagattcc acttgtgcaa tacttaagag taatgattcg gggggggggg atgcactggg 2880 ggtttgggat ggaaatgcta taaaactggg ttgtgatgat aattgtacaa ctataaatgt 2940 aataaattca ttgagaaata tagaaaaaaa taaaataaaa tcccccctcc caaaaaaaga 3000 attcactgtg gatcctctct tagattccaa acagtatgtg ggtttctaga gagaaacaaa 3060 gatgtctttc atattctcag taaatttcct tctggaagtt tcactgttaa aagacatttt 3120 cctagaagac tcaccagtca taaacccaaa tattcctggg agaatgctga caaactagct 3180 ttgaaattca tgttctgaat tattgactgt ctcatacaga atgagttctc caatgaggtt 3240 taatgtggac cagaatatgt agcaacagta cacaaaactt tgcagagtaa agtttacctc 3300 ctgactcaca gttcccttcc ctccaaactc gaagtctatt gctaatgttt cctctccttt 3360 ttgtgctagt ttttctaaga agtatgaatc taggaaagaa tgtttccaat attgaaactt 3420 gaaacacaag aaagctttca gcttggttgt cccttatctg acctatttcc actttcactg 3480 acttagggat tctgttggat tttcaaaacc gtcgtgatag tactagagta gctttgcctt 3540 tgtgtgccaa agatagtttt ggtttatggc tgtcgttttg ttatgattat tacaaagact 3600 ctcccatact cataagtgtt ccatgttgaa tgatcagtta tatgttcatc ctacatatga 3660 ctattacatc agaaaatcgc tattcaggta ataatttcat tctttcttta ctcacaggac 3720 aaaggcctgt ttaactacaa atcatctaaa tatgtgtctc aaatgtgaac tgtgattttt 3780 cttttttagt ggtttgaact gaacaatttt tttttaattt atttttttcc cactgtaaag 3840 caaggggatc aagttatcct tacatgtata catttttatt ccccaccctt tgttctgttg 3900 caatatgagt atctagacat agttctcaat gctactcagt aggatctcct tgtaaatcta 3960 ctctaagttg tgtctgatca gcccaagctc ccaatccctc ccactccctc cctctcccat 4020 caggcagcca caagtctatt ttccaagtcc atgattttct tttctgtgga agggctcatt 4080 tgtgccttat ataagattcc aggtatcagt gatatatcat ttggtatttg tctttctctt 4140 tctgacttac ttcactcagt atgagagtct ctagttccat ccatgttgct gcaaatggca 4200 ttatgccatt ctttttatgg ctgagtagta ttccattgtg tgtatatact acatcttccg 4260 gatccaatca tctgttgatg gacatttggg ttgtttccat gtcctggcta ttgtgaatag 4320 agctgcaatg aacatgtggg tgcatgtgtc tctttcaagg taagttttgt ctggatatat 4380 gcccaagagt gggattgcag ggtcacatgg tagttctatg tgtagatttc taaggtatct 4440 ccaaactgtt ctctatagtg gctgtaccag cttacattca caccaacagt gcaggagggt 4500 tcccttttct ccaccccccc cccagcattt gttatttgtg gacttatcaa tgatggccat 4560 tctgactggt gtgaggtggt atctcatggt agttttgttt gcatttctct aataatcagg 4620 gatgttgagc attttttcat gtgcttgttg gccatctgta catcttcctt tgagaaatgt 4680 ctattcaggt cttttgccca tttttccatt gggttggttg gcttttttgc tgttgagttg 4740 tataagttgc ttgtatattc tagagattaa gcccttgtcc attgcatcat ttgaagctat 4800 tttctcccat tctgtaagtt gtctttttgt tttcttttgg gtttcctttg ctgtgcaaaa 4860 gcttgtcagt ttgatgaggt cccattggtt tatttttgct cttatttctg ttgctttggg 4920 agattgacct gagaaaatat tcatgatgtt gatgtcagag agtattttgc caatgttctc 4980 ttccaggagt ttgatggtgt cttgtcttat atttaagtct ttcagccatt ttgagtttat 5040 ttttgtgcat ggtgtgagag tgtgtaccag tttcgttaat tttcatgcag ctgtccaggt 5100 ttcccagcaa tgcttgctga atagactttc tttttcccat tttatgttct tgcctccttt 5160 gtcaaagatt aattgaccat agttgtcaaa gtttatttct gggttctcta ttctgttcca 5220 ttggtctgtc tgttttgata gcagtggcat gttgttttga tgactgtggc tttgtaatat 5280 tttttgaagt ctgggaaagt tatgcctcct gcttgatttt tgtttctcag gattgctttg 5340 gcaattctga gtcttttgtg gttccatata aaattttgga ttgtttgttc tagttctgtg 5400 aaaaacgtca tcggtaattt gatagggatt gcattgaatc tgtatattac tttgggtagt 5460 atggccattt ttacattatt gatttttcca atccatgaac acggaatatc tttccatttc 5520 tttacatctt ctttgatttc tttgattata gttttatatg aactgaacaa ctttaagtga 5580 taaaagcaaa aggaaaaata cgaatataaa gcaaattgac ataagctaaa attttgcagg 5640 atttgacgtt gtataaatct acaatgaata tgtttgctcg aattacagtg aacagtcata 5700 ttttacaagt ataagaatga tttatttcaa aatacaaact taattaacta tattctatat 5760 ctaataagca aagtgaagat tgatccttat ccataggtac tagaaaaatc tgtgttttga 5820 gtttatgaga attcctatgg tggatacatg tgttcactaa gagttggctt gtctttaaaa 5880 gttctgattg ttcttctttg atggcaagcc ttattattat caaatctaag actccagtct 5940 cagttttttg agtctctatt tttggactat caggaatatt aaaattatca gctttttgta 6000 tgtgaattac ccactatagt atgagactac agattttgtc ttacccaact tttatttccc 6060 ctgtgtcttg tctcatttct tcacctctgt aaataaataa tgtaagaatg agtaaacaaa 6120 tgaggataca gtagctttac acaacttaca gtatgatcct gaattggaaa taaaataagt 6180 cagttatcct ggatgcattc tcaggaaaag acaagagccg ggtattgtaa ggcagtggag 6240 aatacttgtt ctcagcccct tggataaatc agagtaaata gaaaatacta gtgctttttt 6300 gacattgatg taatgcagtc agcaaggacg atactatcca aagagaagtt taacatggaa 6360 aactatagcc ttgtctatcc ccagtatgga aagcctgagc cactgcgaaa tttcttttaa 6420 ccccaaataa tgttcctacc atatgactgt aaattggctg tgaatatcac tatggattta 6480 ttattttatt tttaaatttt tttggtcttt tttcttttta gggctgcacc catggcatat 6540 ggaggttccc aggctagggg tcgaattgga gttgtagctg ccagcctaca ccacagccac 6600 agcaatgcca gatctgagct gtgtctgcaa cttacaccac agctcatggc aacaccagat 6660 cattgacccc ttgagtgagg ccagggatcg aacctgcaac ctcatggttc ctagtcagat 6720 tcatttcccc tgcgccacaa tgagaattcc agaatttttt taaatatata gtgtgatacc 6780 tttctgtaaa caagcagtca caatcaacaa ttttttaaat ccagctctat gtatagatat 6840 tttattcagc atgcaatttt tttcctaaaa ttaacaatgc cagttaattc taggattata 6900 tttcaggact ggaaagaaag tttttttttc cttttattta cttactttaa aaggtggaaa 6960 attggagtta tggttgattt tttggggggg gggagtattt aaaaattgta ttcttaaata 7020 aaaattattc ttgaataatt atttttaatt aagaaatcta acaattaaat taatgaatac 7080 tatcacaaca catataccca aaataaagca agcagaaaat tatttggtgt agttaaaata 7140 ctaccaaagt ttataaggca attgtatttt ctttttggtt aaaaaaaaga tcagatcaca 7200 tataaggtaa cttactccac aaggtaactt acttagaata cttagaataa atacttagaa 7260 gacttagaat aaataatagg gaataaatag agttttaaaa ggtgaaatag atgatgaaat 7320 cttctcatgg tctagtacaa ttataaaaat taaaaatttt tgatgatttt attttgtctc 7380 aagaatttcc cttacaggta ttgacttttt caaaagctgt aaaggaaatt ttattgctat 7440 attaatcttt ccaattatcc atttaactta aaaagcatgt tcttataata accataaata 7500 tggaattttt atgtatctta attttgaata atgtcattcc atttcctgta taatttggta 7560 tcatagcatg aatcactcct ttgttgaaaa ctctcctcag aatttcttgg gagaaaaatt 7620 ggacagaaaa ttaatttcct ctttgagaga attcttagaa tttaaatgac actattggtt 7680 gaactgaaac cacaaaatta gcattttact aatcactagg tttaaatatt tgtgaaacaa 7740 agagatctgc caccatcttg atcatcagct cagcttgctt cttctttccg gtcttgggtt 7800 caaggtattt catttacata tagcaaaatg tgatatatta tgatttcaat ctgtctaatt 7860 tttcactcct cactaaaaaa tatgcactgg taacttttct gtgtgattcc aaatattgat 7920 accttttaat gatatactgg tggcttaaaa atgcatttgc aaatgtcgat gccatctatc 7980 tcagagcttt agttgaaaaa taatagtttt ataaagacca aatttttttt gccaaatttt 8040 atgaaaactt attatgtgaa ataatttata atctttttaa agatcatagt gaggatcatt 8100 tctggtagaa tatttcaaga ccatttttat tccatgtcat taggttaata aaattaattc 8160 tataaaggat atgtcaatga tatacacaga tataaatgac tactttttaa aagatggtta 8220 gatttggata tttggaaaaa tgcaaatgaa taaaaccagt aaactcattt tggatttata 8280 aatatgtctt ccttacaaat gcagttagat tctacaatat gtagactgaa acagtatgta 8340 taaaataagc tgattagttt gttggctaat gtataaacaa attgcatgta tattatgact 8400 ttcctttcct aatttctctg gaaaccagtt tcccaggaca taagttctaa gtatctctgg 8460 gttcttgtaa tttgatggaa ctctagaagt cacacatgat aagacatcag aatcttatga 8520 ttctgctcaa tgaagtcgtc tttatgcagt catgtcatgg atatagcaac gtagaaaaac 8580 ataacataat agctagactt taaaaaaaaa ttgatggagg ttaaatgttt ctacataata 8640 tgcaccaaca gtgtttttcc caaagacgct gaaaaagcag gattctctaa catagaccta 8700 gaaaaacacc ttcaaaaaat tgcagatagg gagttcccgt tgtggctcag tggttaacaa 8760 atccgactag gaaccatgag gttgcaggtt caatccctgg ccttgctcag tgggttaaag 8820 gatctggcat tgatgtgagc tgtggtgtag gtcgaagacg tggcttggat cccacgttgc 8880 tgtggctctg gtgtaggctg gtggctacag ctctgattca acccctagcc tgggaacctc 8940 cacatgccgt gggagcggcc caaaaaaagg caaaaagacc aaaaaaaaat ttttttttca 9000 gataaaatta aatgccagtt ccctgtgcct tttagtttat tatcaatttt tagcaaatct 9060 gatggtctaa gaggaaatat ttaaaataat taattgtagt attcttaaat ttagtagtat 9120 ttaaatatta atgtttatgt attcctctga caaaacccta ttaccacttc aaggatcaaa 9180 tgttttgttt tagagggtga tactggtgtt tcttatctca tataagcact aagcaagata 9240 atttgaatga taaatttttc ttgtgagtaa attttctgtc agacctaaat ttttattttg 9300 9300 <210> 3 <211> 5470 <212> DNA <213> pig <220> <221> promoter <222> (1)..(5470) <223> Pig aS1 casein promoter 5.5kb <400> 3 ggctgtcgtt ttgttatgat tattacaaag actctcccat actcataagt gttccatgtt 60 gaatgatcag ttatatgttc atcctacata tgactattac atcagaaaat cgctattcag 120 gtaataattt cattctttct ttactcacag gacaaaggcc tgtttaacta caaatcatct 180 aaatatgtgt ctcaaatgtg aactgtgatt tttctttttt agtggtttga actgaacaat 240 ttttttttaa tttatttttt tcccactgta aagcaagggg atcaagttat ccttacatgt 300 atacattttt attccccacc ctttgttctg ttgcaatatg agtatctaga catagttctc 360 aatgctactc agtaggatct ccttgtaaat ctactctaag ttgtgtctga tcagcccaag 420 ctcccaatcc ctcccactcc ctccctctcc catcaggcag ccacaagtct attttccaag 480 tccatgattt tcttttctgt ggaagggctc atttgtgcct tatataagat tccaggtatc 540 agtgatatat catttggtat ttgtctttct ctttctgact tacttcactc agtatgagag 600 tctctagttc catccatgtt gctgcaaatg gcattatgcc attcttttta tggctgagta 660 gtattccatt gtgtgtatat actacatctt ccggatccaa tcatctgttg atggacattt 720 gggttgtttc catgtcctgg ctattgtgaa tagagctgca atgaacatgt gggtgcatgt 780 gtctctttca aggtaagttt tgtctggata tatgcccaag agtgggattg cagggtcaca 840 tggtagttct atgtgtagat ttctaaggta tctccaaact gttctctata gtggctgtac 900 cagcttacat tcacaccaac agtgcaggag ggttcccttt tctccacccc ccccccagca 960 tttgttattt gtggacttat caatgatggc cattctgact ggtgtgaggt ggtatctcat 1020 ggtagttttg tttgcatttc tctaataatc agggatgttg agcatttttt catgtgcttg 1080 ttggccatct gtacatcttc ctttgagaaa tgtctattca ggtcttttgc ccatttttcc 1140 attgggttgg ttggcttttt tgctgttgag ttgtataagt tgcttgtata ttctagagat 1200 taagcccttg tccattgcat catttgaagc tattttctcc cattctgtaa gttgtctttt 1260 tgttttcttt tgggtttcct ttgctgtgca aaagcttgtc agtttgatga ggtcccattg 1320 gtttattttt gctcttattt ctgttgcttt gggagattga cctgagaaaa tattcatgat 1380 gttgatgtca gagagtattt tgccaatgtt ctcttccagg agtttgatgg tgtcttgtct 1440 tatatttaag tctttcagcc attttgagtt tatttttgtg catggtgtga gagtgtgtac 1500 cagtttcgtt aattttcatg cagctgtcca ggtttcccag caatgcttgc tgaatagact 1560 ttctttttcc cattttatgt tcttgcctcc tttgtcaaag attaattgac catagttgtc 1620 aaagtttatt tctgggttct ctattctgtt ccattggtct gtctgttttg atagcagtgg 1680 catgttgttt tgatgactgt ggctttgtaa tattttttga agtctgggaa agttatgcct 1740 cctgcttgat ttttgtttct caggattgct ttggcaattc tgagtctttt gtggttccat 1800 ataaaatttt ggattgtttg ttctagttct gtgaaaaacg tcatcggtaa tttgataggg 1860 attgcattga atctgtatat tactttgggt agtatggcca tttttacatt attgattttt 1920 ccaatccatg aacacggaat atctttccat ttctttacat cttctttgat ttctttgatt 1980 atagttttat atgaactgaa caactttaag tgataaaagc aaaaggaaaa atacgaatat 2040 aaagcaaatt gacataagct aaaattttgc aggatttgac gttgtataaa tctacaatga 2100 atatgtttgc tcgaattaca gtgaacagtc atattttaca agtataagaa tgatttattt 2160 caaaatacaa acttaattaa ctatattcta tatctaataa gcaaagtgaa gattgatcct 2220 tatccatagg tactagaaaa atctgtgttt tgagtttatg agaattccta tggtggatac 2280 atgtgttcac taagagttgg cttgtcttta aaagttctga ttgttcttct ttgatggcaa 2340 gccttattat tatcaaatct aagactccag tctcagtttt ttgagtctct atttttggac 2400 tatcaggaat attaaaatta tcagcttttt gtatgtgaat tacccactat agtatgagac 2460 tacagatttt gtcttaccca acttttattt cccctgtgtc ttgtctcatt tcttcacctc 2520 tgtaaataaa taatgtaaga atgagtaaac aaatgaggat acagtagctt tacacaactt 2580 acagtatgat cctgaattgg aaataaaata agtcagttat cctggatgca ttctcaggaa 2640 aagacaagag ccgggtattg taaggcagtg gagaatactt gttctcagcc ccttggataa 2700 atcagagtaa atagaaaata ctagtgcttt tttgacattg atgtaatgca gtcagcaagg 2760 acgatactat ccaaagagaa gtttaacatg gaaaactata gccttgtcta tccccagtat 2820 ggaaagcctg agccactgcg aaatttcttt taaccccaaa taatgttcct accatatgac 2880 tgtaaattgg ctgtgaatat cactatggat ttattatttt atttttaaat ttttttggtc 2940 ttttttcttt ttagggctgc acccatggca tatggaggtt cccaggctag gggtcgaatt 3000 ggagttgtag ctgccagcct acaccacagc cacagcaatg ccagatctga gctgtgtctg 3060 caacttacac cacagctcat ggcaacacca gatcattgac cccttgagtg aggccaggga 3120 tcgaacctgc aacctcatgg ttcctagtca gattcatttc ccctgcgcca caatgagaat 3180 tccagaattt ttttaaatat atagtgtgat acctttctgt aaacaagcag tcacaatcaa 3240 caatttttta aatccagctc tatgtataga tattttattc agcatgcaat ttttttccta 3300 aaattaacaa tgccagttaa ttctaggatt atatttcagg actggaaaga aagttttttt 3360 ttccttttat ttacttactt taaaaggtgg aaaattggag ttatggttga ttttttgggg 3420 ggggggagta tttaaaaatt gtattcttaa ataaaaatta ttcttgaata attattttta 3480 attaagaaat ctaacaatta aattaatgaa tactatcaca acacatatac ccaaaataaa 3540 gcaagcagaa aattatttgg tgtagttaaa atactaccaa agtttataag gcaattgtat 3600 tttctttttg gttaaaaaaa agatcagatc acatataagg taacttactc cacaaggtaa 3660 cttacttaga atacttagaa taaatactta gaagacttag aataaataat agggaataaa 3720 tagagtttta aaaggtgaaa tagatgatga aatcttctca tggtctagta caattataaa 3780 aattaaaaat ttttgatgat tttattttgt ctcaagaatt tcccttacag gtattgactt 3840 tttcaaaagc tgtaaaggaa attttattgc tatattaatc tttccaatta tccatttaac 3900 ttaaaaagca tgttcttata ataaccataa atatggaatt tttatgtatc ttaattttga 3960 ataatgtcat tccatttcct gtataatttg gtatcatagc atgaatcact cctttgttga 4020 aaactctcct cagaatttct tgggagaaaa attggacaga aaattaattt cctctttgag 4080 agaattctta gaatttaaat gacactattg gttgaactga aaccacaaaa ttagcatttt 4140 actaatcact aggtttaaat atttgtgaaa caaagagatc tgccaccatc ttgatcatca 4200 gctcagcttg cttcttcttt ccggtcttgg gttcaaggta tttcatttac atatagcaaa 4260 atgtgatata ttatgatttc aatctgtcta atttttcact cctcactaaa aaatatgcac 4320 tggtaacttt tctgtgtgat tccaaatatt gatacctttt aatgatatac tggtggctta 4380 aaaatgcatt tgcaaatgtc gatgccatct atctcagagc tttagttgaa aaataatagt 4440 tttataaaga ccaaattttt tttgccaaat tttatgaaaa cttattatgt gaaataattt 4500 ataatctttt taaagatcat agtgaggatc atttctggta gaatatttca agaccatttt 4560 tattccatgt cattaggtta ataaaattaa ttctataaag gatatgtcaa tgatatacac 4620 agatataaat gactactttt taaaagatgg ttagatttgg atatttggaa aaatgcaaat 4680 gaataaaacc agtaaactca ttttggattt ataaatatgt cttccttaca aatgcagtta 4740 gattctacaa tatgtagact gaaacagtat gtataaaata agctgattag tttgttggct 4800 aatgtataaa caaattgcat gtatattatg actttccttt cctaatttct ctggaaacca 4860 gtttcccagg acataagttc taagtatctc tgggttcttg taatttgatg gaactctaga 4920 agtcacacat gataagacat cagaatctta tgattctgct caatgaagtc gtctttatgc 4980 agtcatgtca tggatatagc aacgtagaaa aacataacat aatagctaga ctttaaaaaa 5040 aaattgatgg aggttaaatg tttctacata atatgcacca acagtgtttt tcccaaagac 5100 gctgaaaaag caggattctc taacatagac ctagaaaaac accttcaaaa aattgcagat 5160 agggagttcc cgttgtggct cagtggttaa caaatccgac taggaaccat gaggttgcag 5220 gttcaatccc tggccttgct cagtgggtta aaggatctgg cattgatgtg agctgtggtg 5280 taggtcgaag acgtggcttg gatcccacgt tgctgtggct ctggtgtagg ctggtggcta 5340 cagctctgat tcaaccccta gcctgggaac ctccacatgc cgtgggagcg gcccaaaaaa 5400 aggcaaaaag accaaaaaaa aatttttttt tcagataaaa ttaaatgcca gttccctgtg 5460 ccttttagtt 5470 <210> 4 <211> 4980 <212> DNA <213> pig <220> <221> promoter <222> (1)..(4980) <223> Pig aS1 casein promoter 5.0kb <400> 4 agctgcaatg aacatgtggg tgcatgtgtc tctttcaagg taagttttgt ctggatatat 60 gcccaagagt gggattgcag ggtcacatgg tagttctatg tgtagatttc taaggtatct 120 ccaaactgtt ctctatagtg gctgtaccag cttacattca caccaacagt gcaggagggt 180 tcccttttct ccaccccccc cccagcattt gttatttgtg gacttatcaa tgatggccat 240 tctgactggt gtgaggtggt atctcatggt agttttgttt gcatttctct aataatcagg 300 gatgttgagc attttttcat gtgcttgttg gccatctgta catcttcctt tgagaaatgt 360 ctattcaggt cttttgccca tttttccatt gggttggttg gcttttttgc tgttgagttg 420 tataagttgc ttgtatattc tagagattaa gcccttgtcc attgcatcat ttgaagctat 480 tttctcccat tctgtaagtt gtctttttgt tttcttttgg gtttcctttg ctgtgcaaaa 540 gcttgtcagt ttgatgaggt cccattggtt tatttttgct cttatttctg ttgctttggg 600 agattgacct gagaaaatat tcatgatgtt gatgtcagag agtattttgc caatgttctc 660 ttccaggagt ttgatggtgt cttgtcttat atttaagtct ttcagccatt ttgagtttat 720 ttttgtgcat ggtgtgagag tgtgtaccag tttcgttaat tttcatgcag ctgtccaggt 780 ttcccagcaa tgcttgctga atagactttc tttttcccat tttatgttct tgcctccttt 840 gtcaaagatt aattgaccat agttgtcaaa gtttatttct gggttctcta ttctgttcca 900 ttggtctgtc tgttttgata gcagtggcat gttgttttga tgactgtggc tttgtaatat 960 tttttgaagt ctgggaaagt tatgcctcct gcttgatttt tgtttctcag gattgctttg 1020 gcaattctga gtcttttgtg gttccatata aaattttgga ttgtttgttc tagttctgtg 1080 aaaaacgtca tcggtaattt gatagggatt gcattgaatc tgtatattac tttgggtagt 1140 atggccattt ttacattatt gatttttcca atccatgaac acggaatatc tttccatttc 1200 tttacatctt ctttgatttc tttgattata gttttatatg aactgaacaa ctttaagtga 1260 taaaagcaaa aggaaaaata cgaatataaa gcaaattgac ataagctaaa attttgcagg 1320 atttgacgtt gtataaatct acaatgaata tgtttgctcg aattacagtg aacagtcata 1380 ttttacaagt ataagaatga tttatttcaa aatacaaact taattaacta tattctatat 1440 ctaataagca aagtgaagat tgatccttat ccataggtac tagaaaaatc tgtgttttga 1500 gtttatgaga attcctatgg tggatacatg tgttcactaa gagttggctt gtctttaaaa 1560 gttctgattg ttcttctttg atggcaagcc ttattattat caaatctaag actccagtct 1620 cagttttttg agtctctatt tttggactat caggaatatt aaaattatca gctttttgta 1680 tgtgaattac ccactatagt atgagactac agattttgtc ttacccaact tttatttccc 1740 ctgtgtcttg tctcatttct tcacctctgt aaataaataa tgtaagaatg agtaaacaaa 1800 tgaggataca gtagctttac acaacttaca gtatgatcct gaattggaaa taaaataagt 1860 cagttatcct ggatgcattc tcaggaaaag acaagagccg ggtattgtaa ggcagtggag 1920 aatacttgtt ctcagcccct tggataaatc agagtaaata gaaaatacta gtgctttttt 1980 gacattgatg taatgcagtc agcaaggacg atactatcca aagagaagtt taacatggaa 2040 aactatagcc ttgtctatcc ccagtatgga aagcctgagc cactgcgaaa tttcttttaa 2100 ccccaaataa tgttcctacc atatgactgt aaattggctg tgaatatcac tatggattta 2160 ttattttatt tttaaatttt tttggtcttt tttcttttta gggctgcacc catggcatat 2220 ggaggttccc aggctagggg tcgaattgga gttgtagctg ccagcctaca ccacagccac 2280 agcaatgcca gatctgagct gtgtctgcaa cttacaccac agctcatggc aacaccagat 2340 cattgacccc ttgagtgagg ccagggatcg aacctgcaac ctcatggttc ctagtcagat 2400 tcatttcccc tgcgccacaa tgagaattcc agaatttttt taaatatata gtgtgatacc 2460 tttctgtaaa caagcagtca caatcaacaa ttttttaaat ccagctctat gtatagatat 2520 tttattcagc atgcaatttt tttcctaaaa ttaacaatgc cagttaattc taggattata 2580 tttcaggact ggaaagaaag tttttttttc cttttattta cttactttaa aaggtggaaa 2640 attggagtta tggttgattt tttggggggg gggagtattt aaaaattgta ttcttaaata 2700 aaaattattc ttgaataatt atttttaatt aagaaatcta acaattaaat taatgaatac 2760 tatcacaaca catataccca aaataaagca agcagaaaat tatttggtgt agttaaaata 2820 ctaccaaagt ttataaggca attgtatttt ctttttggtt aaaaaaaaga tcagatcaca 2880 tataaggtaa cttactccac aaggtaactt acttagaata cttagaataa atacttagaa 2940 gacttagaat aaataatagg gaataaatag agttttaaaa ggtgaaatag atgatgaaat 3000 cttctcatgg tctagtacaa ttataaaaat taaaaatttt tgatgatttt attttgtctc 3060 aagaatttcc cttacaggta ttgacttttt caaaagctgt aaaggaaatt ttattgctat 3120 attaatcttt ccaattatcc atttaactta aaaagcatgt tcttataata accataaata 3180 tggaattttt atgtatctta attttgaata atgtcattcc atttcctgta taatttggta 3240 tcatagcatg aatcactcct ttgttgaaaa ctctcctcag aatttcttgg gagaaaaatt 3300 ggacagaaaa ttaatttcct ctttgagaga attcttagaa tttaaatgac actattggtt 3360 gaactgaaac cacaaaatta gcattttact aatcactagg tttaaatatt tgtgaaacaa 3420 agagatctgc caccatcttg atcatcagct cagcttgctt cttctttccg gtcttgggtt 3480 caaggtattt catttacata tagcaaaatg tgatatatta tgatttcaat ctgtctaatt 3540 tttcactcct cactaaaaaa tatgcactgg taacttttct gtgtgattcc aaatattgat 3600 accttttaat gatatactgg tggcttaaaa atgcatttgc aaatgtcgat gccatctatc 3660 tcagagcttt agttgaaaaa taatagtttt ataaagacca aatttttttt gccaaatttt 3720 atgaaaactt attatgtgaa ataatttata atctttttaa agatcatagt gaggatcatt 3780 tctggtagaa tatttcaaga ccatttttat tccatgtcat taggttaata aaattaattc 3840 tataaaggat atgtcaatga tatacacaga tataaatgac tactttttaa aagatggtta 3900 gatttggata tttggaaaaa tgcaaatgaa taaaaccagt aaactcattt tggatttata 3960 aatatgtctt ccttacaaat gcagttagat tctacaatat gtagactgaa acagtatgta 4020 taaaataagc tgattagttt gttggctaat gtataaacaa attgcatgta tattatgact 4080 ttcctttcct aatttctctg gaaaccagtt tcccaggaca taagttctaa gtatctctgg 4140 gttcttgtaa tttgatggaa ctctagaagt cacacatgat aagacatcag aatcttatga 4200 ttctgctcaa tgaagtcgtc tttatgcagt catgtcatgg atatagcaac gtagaaaaac 4260 ataacataat agctagactt taaaaaaaaa ttgatggagg ttaaatgttt ctacataata 4320 tgcaccaaca gtgtttttcc caaagacgct gaaaaagcag gattctctaa catagaccta 4380 gaaaaacacc ttcaaaaaat tgcagatagg gagttcccgt tgtggctcag tggttaacaa 4440 atccgactag gaaccatgag gttgcaggtt caatccctgg ccttgctcag tgggttaaag 4500 gatctggcat tgatgtgagc tgtggtgtag gtcgaagacg tggcttggat cccacgttgc 4560 tgtggctctg gtgtaggctg gtggctacag ctctgattca acccctagcc tgggaacctc 4620 cacatgccgt gggagcggcc caaaaaaagg caaaaagacc aaaaaaaaat ttttttttca 4680 gataaaatta aatgccagtt ccctgtgcct tttagtttat tatcaatttt tagcaaatct 4740 gatggtctaa gaggaaatat ttaaaataat taattgtagt attcttaaat ttagtagtat 4800 ttaaatatta atgtttatgt attcctctga caaaacccta ttaccacttc aaggatcaaa 4860 tgttttgttt tagagggtga tactggtgtt tcttatctca tataagcact aagcaagata 4920 atttgaatga taaatttttc ttgtgagtaa attttctgtc agacctaaat ttttattttg 4980 4980 <210> 5 <211> 4276 <212> DNA <213> Artificial Sequence <220> <223> 3'arm 4.3kb <400> 5 ttacaattca gtgtggggaa taaagcacag attttgacat ccaaaagtcc caaatccaaa 60 tcctgacctt ttttgcttac ttaaaataat gtattaatgc ttatttttat aattctgaag 120 gtgattaaag acaataatct gttaagcata gtgctgggaa gatacatagc agtcagtttt 180 tattgattta gtaaaattgt actgctgact atcttcatca catgatttta agaatttttg 240 ttttttcaga agaattaagt gaattctcag gaactccaca attatggcct ttggtaagtt 300 ggaaatcatt tgtggaacca tcgatcctct tttcgtttaa agactcatta caaagatagg 360 actgtagact ataaagattt tttttcctgt agttgagctc cttgtggaca cattagcact 420 tagataataa ttaaattggc ttggacattt gcaaatgttt gtttcataat tatactatat 480 gtaaatagca atcaaattag ataattttaa tgaatataat ttattatatt gaacccctat 540 acaagtatag gagcatgaat gctactaatt ttccatcaag atgtgacctt gagatgctgg 600 taaactcaac agtgggattc tattttttat gatcactaca ataaaaatcc ttagcaagtc 660 atgtgataaa accaagtatt tgtttctcaa caagaaaaca gactttaacg tctacagact 720 tgttttaatt aacttcatcc attgtactgg tgtttccgat tgtatgtcag tagaggggtg 780 tgtgtgtgtg tgtgtgtgtg tgtgtgtatg ttttcccttt ctagtggtgg aaatttccct 840 tccaactgat tgaattaagg aaaatgacaa aacatatggg aaaggttttt ctttgaattc 900 cttggccata ttttctctgt tactgcaaaa gaaaatacta cctagcaata aattgtctat 960 ataacttaaa attatcagga taaatgggta catatgtcag tcacaggaaa gaacaaataa 1020 ctttgtgagt ttcatcttaa aatgaagaga aaatgattat tcaaatgcat cataacagtg 1080 tctcttccat tcaaaacatg taatataacc aaccacatat ttctttttct atttacagat 1140 gtgactgaaa ataccatgct tgaaatttct cctctccatc taccatgtag aaccatttta 1200 tctgaagact ttgactgttc ttttagaaca gggaaatcgc aaatcgaagt caatcttcct 1260 tcttgaattc tttactctat attagatagc atataatcct tttcctttgg caaagttgtc 1320 ctaacagttt agtgtctaaa tttcagttgt atcatgccag taggaagacc actgaatcag 1380 agggaattaa aagtctttac taaatttcaa tatggaaatt ttgtttaaaa agcctttgaa 1440 ttgcttctcc tgtaagtgcc atcatttcaa ataattgtgt gcagtgactg agatttttct 1500 tccttctttt caataaatta cattttaagg cacaactcct attttttgtc attattccat 1560 tcagcagaat ttgcacaatc ctgttaacag tctttatgcc tgtaacattt tattttcact 1620 aagtttttat tacactttca accacaattc aatgaacaaa atggtaaatc ttcatgccta 1680 gctgatgctg acaggttata agctgggtct aggatctttc atttgaagtc acctgtctat 1740 aggatattct ccatgagaac ataggtgtgg ctgcagagaa gaacagtggt atgacagttg 1800 cgggtgcact gggaatttga aaaacatggc taagctgttt atttatgtca ccagaacctg 1860 tttgagccaa actcatttga ttatttgatt attatacact tacttttata attaggtaga 1920 ttagataata aacatttaat aggttagacc atggcataca gtaacacaat actcagtgtc 1980 ctcccgaact ccagtgaaga gttatttctc aaaatgacaa tagtcgcttg cattaatacc 2040 ctcaaaaccc tgctggaatt tactttccag agtttattcc agggtcccca acaaacccta 2100 tctgctgtga ctctttaaga aacttctttc caccaggtca taaaatacag gtggcaaggt 2160 ctcttgtacc acaacctaaa cgacctattt agttagcagt tctgtctctt atcgattata 2220 tatataattg atatgcacat atatgcataa cacctaaaca cgtatttttt gcctataacg 2280 cttttttacc agttgtcact tggtttggtg acactagtct ccttccattt tccctgaggt 2340 tggaaatgca atttcaatta ctttgcacct ctcaccagag ttctcagtca attgtttagc 2400 acaaaagaat ttcataaaag tttgcccttc aaggaaaagt ttaaaggaga ataatttatc 2460 ctttgtagac acagggaagt gcagggccat tagaactaag catatctatt tatggatgtt 2520 taaatatttc tctttcaatt atgattgtca aactgccttc tagagtctca caaataacag 2580 aagcagaaaa aaatacagtt gtgaaaaaac agtgctgaca gtgaactgaa gaatatgtgc 2640 attcctcatt ggctgcccct tccaatgcat tgttgctgtg caggaataca caggtcaaat 2700 gtaaccaaat ttggggactt ctaggtgaaa ctagaaatgt ggattggtgt gtaagatctc 2760 ctatttttaa atattggtct agtgtttttt aaatataaaa tacacagtct ccttctcttt 2820 attggccaga cccaacccct ctaacactag tctatgtact atagtgtact ttaagtcatt 2880 aagtaaagga ctttctactt ccagtccaat caaggttgaa tcccagttac agtgaagtga 2940 aagtgagaag taggacatta gggtaataat tagctacagt aaacaaagaa atcatgattc 3000 catcatgaca aatacacagt aagtgttagg ggctacatgg ggctcattgt taaaatggct 3060 catcatgctg acccatgaga ctgaccatct tgttcaagcg acatcctgtt tttgccactg 3120 gtgcctattt tcccaagact acaagaccag gggggaccac acacctccag ccttctcaag 3180 attatgagtg caccctaccg tgagatacct ctgactttct catgacgatg tgaccaccag 3240 agtccacctg caggtgaaag ataaactata aactaacctt cccccgcttc aggaatcaat 3300 ttcccctctg cagagtataa gaaggccctg ctagaagggc gggggggctg actcttctca 3360 aaggtcagtc agcctctcct ttttccttct aataaatttt cttctctttg cctgaacacc 3420 caattccttc tctttttctc catgctcgcc ttacagtaag accttcagga gcacttggga 3480 gcaggcggtt ctgtaaattt tccatacctt cccatatatt ccactttcaa tagttgggtt 3540 cctattgtcc taactttcac aagaactatc cattttatgg attcatgtca tattaatcac 3600 tatagattag attttgcatg ccctcccgaa ttcatatgtt gaagccctaa ctcccccatg 3660 tgatgcactt ggaggtggca tctttggagg caaataggtc atgtgagtgg gatatctatg 3720 agtgggatta gtgcccctct aagaaaagct aggagagagc ttgtctatct ttctgccttg 3780 tgaggataca gcagaaaggc aggcacctgc aggccaagag ggagccttca ccggacacag 3840 gatctgctgg cacctggatc ttggtaaatg ttatttgtaa ctgtatgtct gtgagttgct 3900 atgggcagga tgtactggct ggtaaacatt gtttagtaat aagtggatgt ggtttacacc 3960 agatttagga gaatctagga gtggtgaggt aaaatggggt ttggcattca attgttgact 4020 cattatatag ctaaaatatg cctgcttgat gagcttattg taagagatct cacaagtcat 4080 tttgagcttc ctagtattga ggtgctttgc acaccctctg ttagtggcct aagaactgaa 4140 gataacacat gtcttgtgct accaagcagt aggaggataa atgaagtttg ctcttgagat 4200 ctcagtacct ctttcatctt tcaatgcata cccttctcct gttgttgtac tctgtcctct 4260 gcctgtaata aagctg 4276 <210> 6 <211> 4955 <212> DNA <213> Artificial Sequence <220> <223> 3'arm 4.9kb <400> 6 catggcatat ggaagttccc aggctagggg tcgatcagag ctgtagaccc cgacctacac 60 cacagccaca gcaactcagg atccaagcca catctgcgac ctacaccaca gctcacggca 120 atgctggatc cccaacccac tgagcaaggc cagggattga accggcaacc tcatggatac 180 tagtcggatt tggttctgct gtgccataat gggaactcca ggcctgttta ttttcttaat 240 ttgcagcagc acaatgtaaa gtgttttctc atagctatcc attcatttat gtccctcttt 300 actaatgttt atctcttctt ttttatcctt aaggaaatca tcaatgaact gaacagggta 360 aggaacatta atgatattta aattatttta aaattcattc tttcaaaaat atattagcta 420 cactttttag ttttaacaaa gagaactcct gagaaaaaaa cagtaataaa ctcaaggtat 480 caaaatcttt cttatgatag tgtgtcaaaa agtatattct tgcagttcaa aagtgttttg 540 atttagaaaa acagtatttt tcctgtgata tttatacctt cactgaactt tcaaaatgac 600 taatgaattc tactcataat tctaaatatt tattttattg atttaatttt gtatctatga 660 atagacaagg cattaatatg aatgaatgaa tgggtgcaat tttggactaa ccaatttttt 720 gcacttatca ccaaaactga aagaattctt tattaaaggt tttataaaaa aacaatatta 780 tctgcaccta gaatgtttta cataatcaca gttggttatt ctctttcttt acacatgagt 840 ttctgggccg gggatcagat ccaagctgca cttgagacct aagtcacagc tacttcaatg 900 gaggaccctt aacccacttt gctgggctgg gtatgaaccc agcgcttcct agtgccacag 960 atcccattgc accacagggg aacctctaac gcatattttt ttaaaatctg gtctgtcaga 1020 tttttagtag ttttggtatt gagacaacac aggtgccact gaagataaaa aaatatatct 1080 tcctaactat ccttcatact tgaatgatca tcctttcttg gcaggatgct aggagtgaat 1140 caactgaagt aagattcttt attgtaaaac tattaaatat aatgtaagga aaagaaagaa 1200 ataaaatcat ttcctttaaa tatcctaatg aaaatgaatg actaattctt ttaggtcaaa 1260 actaagacag atatctctaa ttcaaagaga gaaaaaaaaa aacatacaat gttatcatct 1320 gtgagccata attagctgga taactaaatc aatggtatta tattgagctt aaattctatg 1380 gatcatgtca ctactccctg tactgctatg gtcatgaaaa ctgaacacaa cattctagat 1440 ggagaaaatc cattttgctt aaatatttat acaccattga accaatgctt catagacttt 1500 tatttattac ataatcttta atccaatact tctgcttaat agccaaaagc caagtaaagg 1560 aaaaaaagta agggtagtta aaggaaaatg taggattatt tacactgatt taccacaaca 1620 aataagaatc cataataatt tatgtcaagg gagttcccgt catggcgcag tggttaacga 1680 atctgactag gaaccatgag gttgagggtt cgatccctgc tcttgctcag tgggttaagg 1740 atccggtgtt gccctgagct gtggtgtagg tcgcagacgc ggctcggatc ctgcattact 1800 gtgaccctgg tgtaggctgg cagctacagc tctgattcga cccctagcct gggaactttc 1860 atatgccgtg ggagcggccc aagaaatggc aaaaagacaa aaaaaacaaa caaacaaaca 1920 aacaaaaatt ttatgttaaa ctcagaaatg cagattaggg aggtaaattc tttggttagc 1980 ctggtaggta ggcttttttc tttccttatc actggctctt accacatatt tctatttctt 2040 ttggcatcta ttttatttga taattattat tttacatttg aatatttgtc ataaaaaata 2100 aattctcttt tcttttctaa gaatcatggc atggaaggcc atgaggtaag acccttattg 2160 taataaactc tacacttaca taacatccat agtatatact ctatgctcta ttttaagaaa 2220 actctcttct caaattgagc aagattgaac ttcccaaaca aggttattat acccaagaat 2280 gtacaatgtt gtgccagata atgttaaaat tagaagagga aatgtgtctt ttaattgata 2340 atcagggaaa atgttattta ttaaaataaa gggtgtaagg caaaatgtgc atgagtgttt 2400 caaatgaaat atgagcctcc aaaaaaaaag aaaaaaaaac ctgtctctac cacctcaggg 2460 tataaactca ctcctgtttg tgagggtagt cttggggaga aagaatctgg ttgcagtaat 2520 tacttaggat caatggccta tgctacttaa tccaaaagca tggaaaaaat ccactagccc 2580 acaatatttg gttaatcaac tttcccaaga gatgaatcat ctgttgacta ttaaaatcaa 2640 tcttttaggt atgtacttga gaacaaaaat tatatccata tttaaatata aacgtccgtg 2700 agttagaaaa aaatctaaat agttcaaagt gtagatgttt taaaagctaa tgtaatttta 2760 gttccatacc aaaatttgtt gttgtatttt catgtttcaa tttattccct tcaaagaagc 2820 cccaaggata catgagaata aatatcccac ccggggttct gggtgcccaa gctcagtagg 2880 gcacttcctt ccaaatttca tatatatttt gcattctact caaccacata tctatgtttg 2940 atttaataaa atgttgtttt ttagtgattg gttattacat tcccacatcc aacatatttt 3000 aaataaaatt gacaacccca aaaaggtacg ttatattggc cctgaatgtt tcattaccaa 3060 ttgctttcat tctaaacaag gagttagcaa cccagtatga aagtgtggaa caaatttcct 3120 tctaattcta aaagtcacag tgttgggaac actgattttc tctcttttag caaaggggat 3180 ctagcagttc atcaagtgag gtaaatcatt ttgatgttaa ttcagtatct cagttagaaa 3240 atgtttatga aaacttgttg tgctatgaat gttacacatc ccataaggtc tcatggtaca 3300 ggctctatgt ctacagctct accctaattt taacatacag gctatgagcc caaaagatat 3360 aataacgcaa atacttgtca gatgaaatta cagaattgtt tctgcgctaa caattctatc 3420 tggctatcca tggtgcccca ttttttttct ctaatttttt tgcctttcct aggttttggt 3480 agtactaaat attttattta aaattactat aagagctatg cttctaaatc attaatagaa 3540 acttggtatt tcctgtgcag acatctactg attctaggtt aactgggtac tggaatcctt 3600 tactccttgt taacctaaaa gagaaaaaga gcaggcatag aaggtccttt cgtaaacttt 3660 gggagaagaa aatttcaaaa taaacccaac ccagttatgc ggagtttctg ggagatcagt 3720 aaaagtgcat cgaatatttc acatctacct ccaacattct gaataacttc cctgttgaaa 3780 tcagtgagaa gaggaaaggg aatcttgagt acaacctgta ccttgaatta ttcatcttat 3840 ctcagttacc aaggaatggg tgcctaagat caatttatca cagttaagca acatggtaac 3900 tggctaatta gtattcatac cttgagtata aattaataag tcataaaact aacactgcat 3960 gtttttattt tttaaggaag ttgttggcaa tagtgctgag gtgagatata cttactaaat 4020 ttaaaataca ttcacgttat ccaggatgtg ttaaaattta cttgtacttt tttttttctt 4080 ttttagcaga agcacgttca aaaagaagaa gatgtgccct cccaaagcta tctggtaaaa 4140 ttttactaaa agtttatcaa aggcaaatgt accaaggaat gagtatgaat gttgtactga 4200 tacattattt ctccttctca acctctgcta caccttaata catagtaagc cgtctaacag 4260 actctagatg tttactgatc ccctgcaaaa ataaagctaa caactttttt atcccagggt 4320 ttttgtttgt ttgtttgttt gttttgtctt ttgtcttttt agggccgcac ctgcagcaca 4380 tggaggttcc caggctaggg gtctaatcag agctgtagct gctggcctgc atcacagcca 4440 cagcaatgcc acatccaaac cgcatctgca acctacacca cagctcccag caacgctggg 4500 tccctaaccc actgagcaag gccagggatc aaaccggaac ctcatggttc ctagtcagat 4560 tcgtttccac tgcgccatga tgggaactcc tatcccaggt tattaattca tgctttcatg 4620 agatttgtgt tttatagtct atttttggat ctgaataaca tagtattttt tttatttttt 4680 taattttccc actgtacagc aagggggtca ggttatcctt acatgtatac attgcagtta 4740 cagttttttc ccccaccctt tcttctgttg caacatgagt atctagacat agttctcaat 4800 gctattcagc aggatctcct tgtaaatcta ttctaggtgt gtctgataag cccaagctcc 4860 cgatccctcc cactccctcc ccctcccatc aggcaaccac aagtctcttc tccaagtcca 4920 tgattttctt ttctgaggag atgttcattt gtgct 4955 <210> 7 <211> 206 <212> PRT <213> pig <220> <221> PEPTIDE <222> (1)..(206) <223> amino acid of porcine alpah S1 casein <400> 7 Met Lys Leu Leu Ile Phe Ile Cys Leu Ala Ala Val Ala Leu Ala Arg 1 5 10 15 Pro Lys Pro Pro Leu Arg His Gln Glu His Leu Gln Asn Glu Pro Asp 20 25 30 Ser Arg Glu Glu Leu Phe Lys Glu Arg Lys Phe Leu Arg Phe Pro Glu 35 40 45 Val Pro Leu Leu Ser Gln Phe Arg Gln Glu Ile Ile Asn Glu Leu Asn 50 55 60 Arg Asn His Gly Met Glu Gly His Glu Gln Arg Gly Ser Ser Ser Ser 65 70 75 80 Ser Ser Glu Glu Val Val Gly Asn Ser Ala Glu Gln Lys His Val Gln 85 90 95 Lys Glu Glu Asp Val Pro Ser Gln Ser Tyr Leu Gly His Leu Gln Gly 100 105 110 Leu Asn Lys Tyr Lys Leu Arg Gln Leu Glu Ala Ile His Asp Gln Glu 115 120 125 Leu His Arg Thr Asn Glu Asp Lys His Thr Gln Gln Gly Glu Pro Met 130 135 140 Lys Gly Val Asn Gln Glu Gln Ala Tyr Phe Tyr Phe Glu Pro Leu His 145 150 155 160 Gln Phe Tyr Gln Leu Asp Ala Tyr Pro Tyr Ala Thr Trp Tyr Tyr Pro 165 170 175 Pro Gln Tyr Ile Ala His Pro Leu Phe Thr Asn Ile Pro Gln Pro Thr 180 185 190 Ala Pro Glu Lys Gly Gly Lys Thr Glu Ile Met Pro Gln Trp 195 200 205 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR forward primer <400> 8 tgacaaccat gaaacttctc at 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR reverse primer <400> 9 gttcctgatg cctgagagga 20 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 3' UTR forward primer <400> 10 aaccatttta tctgaagact ttg 23 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 3' UTR reverse primer <400> 11 tctcagtcac tgcacacaat t 21 <210> 12 <211> 3360 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR sequence <400> 12 tgacaaccat gaaacttctc atctttatct gtcttgcagc tgttgccctt gccaggcctg 60 tgagtatggt agagaattta gaagcttcta gattcttgat tgaaattacc tgatatcaaa 120 cacaagaaac tgaggataat aatcttaaaa gtattgaatg atctctaatt accttttgaa 180 gccttgatat taaaactgta gaaatccttc acatcttgat cattattaca tagttcattc 240 aaagtcatca ctccaaataa aatctgagtt gaaatataaa tgcctcacag taaaaaaata 300 aaaacaaaaa atgaaaagaa aagaaaaagg aataatgtat ttaacaacat agtaaataga 360 atcaatgagt gttattacgc tctttgcctg ggtccaataa agaattagca tatatttaaa 420 catacaagtc catgattttt tctgtggaag ggctcatttg tgccttatat aagattccag 480 gtatcagtga tatatcattt ggtatttgtc tttctctttc tgacttactt cactcagtat 540 gagagtctta gttccatcca tgttgctgca aatggcatta tgccattctt tttatggctg 600 agtagtattc cattgtgtgt atatactaca tcttccgaat ccaatcatct gttgatggac 660 atttgggttg tttccatgtc ctggctattg tgaatagagc tgcaatgaac atgtgggtgc 720 atgtgtctct ttcaaggtaa gttttgtctg gatatatgcc caagagtggg attgcagggt 780 cacatggtag ttctatgtgt agatttctaa ggtatctcca aactgttctc tatagtggct 840 gtaccagctt acattcacac caacagtgca ggagggttcc cttttctcca ccccccccca 900 gcatttgtta tttgtggact tatcaatgat ggccattctg actggtgtga ggtggtatct 960 tgtggtagtt ttgatttgca tttctctaat aatcagggat gttgagcatt ttttcatgtg 1020 cttgttggcc atctgtacat cttcctttga gaaagtctat tcaggtcttt tgcccatttt 1080 tccattgggg tgttggcttt tttgctgttg agttgtataa gttgtttgta tattttagag 1140 attaagccct tgtcagttgc atcgtttgaa actattttct cccattctgt aagttgtctt 1200 tttgttttct tttgggtttc ctttgctgtg caaaagcttg tcagtttgat gaggtcccat 1260 tggtttattt ctgctcttat ttctgttgct ttgggagatt gacctgagaa aatattcatg 1320 atgttgatgt cagagagtat tttgccaatg ttctcttcca ggagtttgat ggtgtcttgt 1380 cttatattta agtctttcag ccattttgag tttatttttg tgcatggtgt gagagtgtgt 1440 accagtttcg ttaattttca tgcagctgtc caggtttccc agcaatgctt gctgaataga 1500 ctttcttttt cccactttat gttcttgcct cctttgtcaa agattaattg aacctttcca 1560 tagaaaagaa aatcatgaat ttggagaata aacttgtggt tgccaaaggg gagggagagg 1620 gagtggtgtg gttgaggagc ttggggttaa tagatataaa ctattgcctt tggaatggat 1680 tagcaatgag accctgctgt gtagcactgg gaactatgtc cagtcactta tgatggagca 1740 tgataatgtg cgaaaataga atgtgtacat gtatgtgtaa ctgggtcccc atgctgtaca 1800 gtagaaaaaa aatgtgttga gaaaataact attacaaaaa tgagaattat gaaataaata 1860 aataaataaa caaacataca tgtccaatat tttcttacca aatataacag aatgccgcct 1920 attgcagtgt atcctacttt tggaacctaa gtcaaaccct cacatgagat gactcatatc 1980 aaccaatatt tcccaaggtg tagaaaactg agttattctg ctgattcaga gatttaatta 2040 ttatggtcct tgtagacaag gaaactaggt aatataaatt aaaatcactt ttcttctcaa 2100 aaaaaaaaaa gattcagtta cttatgtaga gcaaaaatta ttctgttccc ttgtgatata 2160 tagctagcaa agatggaagg gataaactgg aattgcttga caaagaaacc atccagataa 2220 aataataatc ttggatattt gggcattgta attattaaat ataggtgtaa aattttttgg 2280 aattctgaga tattctatga tgagactcct atttcctaaa tttttgtttg gataatgcag 2340 gatcactaat aacctaactt tccttttttt tttttttttt ttttttgctt tttagggcca 2400 tacccacagc atatggaggt tctcaggcta ggagtcaaat cagagctgcc ggtggcctaa 2460 ccacagccac agcaactttg gaatcaaatc tgagctgtgt ctgggatgta ccccacagct 2520 cacagcagtg gtggatcctt aacccactga gtgaggccag ggatcaaacc cacctcccca 2580 taaacactat ttagctttgt taccccagac cccctatggg aactccaaac caacctgtta 2640 gacactagag ctttttctat gctacacctt aagcaccaat aggaagaaaa tctatgatag 2700 caagttacaa gaaaaaggat ccaacttttt cagaaaagtg tcactgttag tctttttaca 2760 aaagaaatgg gtatagtttc cccagcaaac agattctata attctattcc aagaaaacat 2820 catttttaat gctaccattt aacaaacata aatcttgttc ccacagttca aatgtagatt 2880 gagttaaatt tttatataat taactgatta taaaaataaa atccagaaaa tgtttaatga 2940 agaaaagtat atattttgtt tcaaaaattc ataatagtta tttttagaac taccatgtaa 3000 tataaatagt tcagaaaaat taaatttatc attttggatc aagacaaatt aatttttttt 3060 ttcagtcttt ttacacattt ttctcagttc tccatcatac cccaatgaca ggaaatcatt 3120 tacttttctg tgatttttat caagaaaata accatttttt ttcttcccag ggaaacttgg 3180 ggtcagatga atttagctat taaaatacac atcattttta aatagcaaat gttacattta 3240 cagtattcta tttgttttgt gatattatta tcatattttg attgcatttt gttttctttt 3300 tgtttgtttt taaattcttg cattgttttt cacagaaacc tcctctcagg catcaggaac 3360 3360 <210> 13 <211> 303 <212> DNA <213> Artificial Sequence <220> <223> 3' UTR sequence <400> 13 aaccatttta tctgaagact ttgactgttc ttttagaaca gggaaatcgc aaatcgaagt 60 caatcttcct tcttgaattc tttactctat attagatagc atataatcct tttcctttgg 120 caaagttgtc ctaacagttt agtgtctaaa tttcagttgt atcatgccag taggaagacc 180 actgaatcag agggaattaa aagtctttac taaatttcaa tatggaaatt ttgtttaaaa 240 agcctttgaa ttgcttctcc tgtaagtgcc atcatttcaa ataattgtgt gcagtgactg 300 aga 303 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR probe forward primer <400> 14 tgacaaccat gaaacttctc at 22 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR probe reverse primer <400> 15 ctaagactct catactgagt g 21 <210> 16 <211> 551 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR probe sequence <400> 16 tgacaaccat gaaacttctc atctttatct gtcttgcagc tgttgccctt gccaggcctg 60 tgagtatggt agagaattta gaggcttcta gattcttgat tgaaattacc tgatatcaaa 120 cacaagaaac tgaggataat aatcttaaaa gtattgaatg atctctaatt accttttgaa 180 gccttgatat taaaactgta gaaatccttc acatcttgat cattattaca tagttcattc 240 aaagtcatca ctccaaataa aatctgagtt gaaatataaa tgcctcacag taaaaaaata 300 aaaacaaaaa atgaaaagaa aagaaaaagg aataatgtat ttaacaacat agtaaataga 360 atcaatgagt gttattacgc tctttgcctg ggtccaataa agaattagca tatatttaaa 420 catacaagtc catgattttt tctgtggaag ggctcatttg tgccttatat aagattccag 480 gtatcagtga tatatcattt ggtatttgtc tttctctttc tgacttactt cactcagtat 540 gagagtctta g 551 <210> 17 <211> 3735 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR sequence <400> 17 aggtgacact atagaagagc tcgaggatcc tgaattggaa ataaaataag tcagttatcc 60 tggatgcatt ctcaggaaaa gacaagagcc gggtattgta aggcagtgga gaatacttgt 120 tctcagcccc ttggataaat cagagtaaat agaaaatact agtacttttt tgacattgat 180 gtaatgcagt cagcaaggac gatactatcc aaagaggagt ttaacatgga aaactatagc 240 cttgtctatc cccagtatgg aaagcctgag ccactgcgaa atttctttta accccaaata 300 atgttcctac catatgactg taaattggct gtgaatatca ctatggattt attattttat 360 ttttaaattt ttttggtctt ttttcttttt agggctgcac ccatggcata tggaggttcc 420 caggctaggg gtcgaattgg agttgtagct gccagcctac accacagcca cagcaatgcc 480 agatctgagc tgtgtctgca acttacacca cagctcatgg caacaccaga tcattgaccc 540 cttgagtgag gccagggatc gaacctgcaa cctcatggtt cctagtcaga ttcatttccc 600 ctgcgccaca atgagaattc cagaattttt ttaaatatat agtgtgatac ctttctgtaa 660 acaagcagtc acaatcaaca attttttaaa tccagctcta tgtatagata ttttattcag 720 catgcaattt ttttcctaaa attaacaatg ccagttaatt ctaggattat atttcagact 780 ggaaagaaag tttttttttc cttttattta cttactttaa aaggtggaaa attggagtta 840 tggttgattt tttggggggg ggagtattta aaaattgtat tcttaaataa aaattattct 900 tgaataatta tttttaatta agaaatctaa caattaaatt aatgaatact atcacaacac 960 atatacccaa aataaagcaa gcagaaaatt atttggtgta gttaaaatac taccaaagtt 1020 tataaggcaa ttgtattttc tttttggtta aaaaaaagat cagatcacat ataaggtaac 1080 ttactccaca aggtaactta cttagaatac ttagaataaa tacttagaag acttagaata 1140 aataataggg aataaataga gttttaaaag gtgaaataga tgatgaaatc ttctcatggt 1200 ctagtacaat tataaaaatt aaaaattttt gatgatttta ttttgtctca agaatttccc 1260 ttacaggtat tgactttttc aaaagctgta aaggaaattt tattgctata ttaatctttc 1320 caattatcca tttaacttaa aaagcatgtt cttataataa ccataaatat ggaattttta 1380 tgtatcttaa ttttgaataa tgtcattcca tttcctgtat aatttggtgt catagcatga 1440 atcactcctt tgttgaaaac tctcctcaga atttcttggg agaaaattgg acagaaaatt 1500 aatttcctct ttgagagaat tcttagaatt taaatgacac tattggttga actgaaacca 1560 caaaattagc attttactaa tcactaggtt taaatatttg tgaaacaaag agatctgcca 1620 ccatcttgat catcagctca gcttgcttct tctttccggt cttgggttca aggtatttca 1680 tttacatata gcaaaatgtg atatattatg atttcaatct gtctaatttt tcactcctca 1740 ctaaaaaata tgcactggta acttttctgt gtgattccaa atattgatac cttttaatga 1800 tatactggtg gcttaaaaat gcatttgcaa atgtcgatgc catctatctc agagctttag 1860 ttgaaaaata atagttttat aaagaccaaa tttttttgcc aaattttatg aaaacttatt 1920 atgtgaaata atttataatc tttttaaaga tcatagtgag gatcatttct ggtagaatat 1980 ttcaagacca tttttattcc atgtcattag gttaataaaa ttaattctat aaaggatatg 2040 ccaatgatat acacagatat aaatgactac tttttaaaag atggttagat ttggatattt 2100 ggaaaaatgc aaatgaataa aaccagtaaa ctcattttgg atttataaat atgtcttcct 2160 tacaaatgca gttagattct acaatatgta gactgaaaca gtatgtataa aataagctga 2220 ttagtttgtt ggctaatgta taaacaaatt gcatgtatat tatgactttc ttttcctaat 2280 ttctctggaa accagtttcc ccaggacata agttctaagt atctctgggt tcttgtaatt 2340 tgatggaact ctagaagtca cacatgataa gacatcagaa tcttatgatt ctgctcaatg 2400 aagtcgtctt tatgcagtca tgtcatggat atagcaacgt agaaaaacat aacataatag 2460 ctagacttta aaaaaaaatt gatggaggtt aaatgtttct acataatatg caccaacagt 2520 gtttttccca aagacgctga aaaagcagga ttctctaaca tagacctaga aaaacacctt 2580 caaaaaattg cagataggga gttcccgttg tggctcagtg gttaacaaat ccgactagga 2640 accatgaggt tgcaggttca atccctggcc ttgctcagtg ggttaaagga tctggcattg 2700 atgtgagctg tggtgtaggt cgaagacgtg gcttggatcc cacgttgctg tggctctggt 2760 gtaggctggt ggctacagct ctgattcaac ccctagcctg ggaacctcca catgccgtgg 2820 gagcggccca aaaaaaggca aaaagaccaa aaaaaaattt tttttcagat aaaattaaat 2880 gccagttccc tgtgcctttt agtttattat caatttttag caaatctgat ggtctaagag 2940 gaaatattta aaataattaa ttgtagtatt cttaaattta gtagtattta aatattaatg 3000 tttatgtatt cctctgacaa aaccctatta ccacttcaag gatcaaatgt tttgttttag 3060 agggtgatac tggtgtttct tatctcatat aagcactaag caagataatt tgaatgataa 3120 atttttcttg tgagtaaatt ttctgtcaga cctaaatttt tattttgttt tcttatatag 3180 gtgttgacaa ccatgaaact tctcatcttt atctgtcttg cagctgttgc ccttgccagg 3240 cctgtgagta tggtagagaa tttagaagct tctagattct tgattgaaat tacctgatat 3300 caaacacaag aaactgagga taataatctt aaaagtattg aatgatctct aattaccttt 3360 tgaagccttg atattaaaac tgtagaaatc cttcacatct tgatcattat tacatagttc 3420 attcaaagtc atcactccaa ataaaatctg agttgaaata taaatgcctc acagtaaaaa 3480 aataaaaaca aaaaatgaaa agaaaagaaa aaggaataat gtatttaaca acatagtaaa 3540 tagaatcaat gagtgttatt acgctctttg cctgggtcca ataaagaatt agcatatatt 3600 taaacataca agtccatgat tttttctgtg gaagggctca tttgtgcctt atataagatt 3660 ccaggtatca gtgatatatc atttggtatt tgtctttctc tttctgactt acttcactca 3720 gtatgagagt cttag 3735 <210> 18 <211> 2179 <212> DNA <213> Artificial Sequence <220> <223> 3' UTR sequence <400> 18 aatacgactc actatagggc gtcgactcga tccactgcat ggaagaatgt gatttctcca 60 ttttccttgc agagtagata tctcttgtga tgctaatagc catgtcagaa gtgaatagaa 120 tgcttctttt tttcagagat tctaaagagc aatttcccat atcctgttgc tatttcattc 180 tctctagcct ctccaccagt tctaccagct tgatgcctat ccctatgcta cctggtatta 240 tcctccacaa tatattgctc acccattatt caccaacatc cctcaaccca ctgcccctga 300 gaagggtgga aaaactgaga ttatgcctca gtggtggtaa gttcatttta atgactgtat 360 attgatgttc taccaaagga aataaaagaa aacttcttaa agaacatacc ataaaaacag 420 atttagaata aacatgacaa aatcaatatc tagagcgtcg tagtagaatt ttccaaaatg 480 ggaaattggc aggacgttct gatatctgca gctaatgtta atccactact caggaacttg 540 tggagcagcg ctctctgttc tttgagattc attctgatga agtcaggaaa aagttttcta 600 tccaaagcaa aaacacagta atttcacttt atcctcctta caattttact aatctctaaa 660 ggcttttctt ttggttatat atacccatga tatacattac aattcagtgt ggggaataaa 720 gcacagattt tggcatccaa aagtcccaaa tccaaatcct gacctttttt gcttacttaa 780 aataatgcat taatgcttat ttttataatt ctgaaggtga ttaaagacaa taatctgtta 840 agcatagtgc tgggaagata catagcagtc agtttttatt gatttagtaa aattgtactg 900 ctgactacct tcatcacatg attttaagaa tttttgtttt ttcagaagaa ttaagtgaat 960 tctcaggaac tccacaatta tggcctttgg taagttggaa atcatttgtg gaaccattga 1020 tcctcttttc gtttaaagac tcattacaaa gataggactg tagactataa agattttttt 1080 tcctgtagtt gagctccttg tggacacatt agcacttaga taataattaa attggcttgg 1140 acatttgcaa atgtttgttt cataattata ctatatgtaa atagcaatca aattagataa 1200 ttttaatgaa tataatttat tatattgaac ccctatacaa gtataggagc atgaatgcta 1260 ctaattttcc atcaagatgt gaccttgaga tgctggtaaa ctcaacagtg ggattctatt 1320 ttttatgatc actacaataa aaatccttag caagtcatgt gataaaacca agtatttgtt 1380 tctcaacaag aaaacagact ttaacgtcta cagacttgtt ttaattaact tcatccattg 1440 tactggtgtt tccgattgta tgtcagtaga ggtgtgtgtg tgtgtgtgtg tgtgtgtttt 1500 ccctttctag tggtggaaat ttcccttcca actgattgaa ttaaggaaaa tgacaaaaca 1560 tatgggaaag gtttttcttt gaattccttg gccatatttt ctctgttact gcaaaagaaa 1620 atactaccta gcaataaatt gtctatataa cttaaaatta tcaggataaa tgggtacata 1680 tgtcagtcac aggaaagaac aaataacttt gtgagtttca tcttaaaatg aagagaaaat 1740 gattattcaa atgcatcata acagtgtctc ttccattcaa aacatgtaat ataaccaacc 1800 acatatttct ttttctattt acagatgtga ctgaaaatac catgcttgaa atttctcctc 1860 tccatctacc atgtagaacc attttatctg aagactttga ctgttctttt agaacaggga 1920 aatcgcaaat cgaagtcaat cttccttctt gaattcttta ctctatatta gatagcatat 1980 aatccttttc ccttggcaaa gttgtcctaa cagtttagtg tctaaatttc agttgtatca 2040 tgccagtagg aagaccactg aatcagaggg aattaaaagt ctttactaaa tttcaatatg 2100 gaaattttgt ttaaaaagcc tttgaattgc ttctcctgta agtgccatca tttcaaataa 2160 ttgtgtgcag tgactgaga 2179 <210> 19 <211> 6357 <212> DNA <213> Artificial Sequence <220> <223> 3' UTR sequence <400> 19 aaccatttta tctgaagact ttgactgttc ttttagaaca gggaaatcgc aaatcgaagt 60 caatcttcct tcttgaattc tttactctat attagatagc atataatcct tttcccttgg 120 caaagttgtc ctaacagttt agtgtctaaa tttcagttgt atcatgccag taggaagacc 180 actgaatcag agggaattaa aagtctttac taaatttcaa tatggaaatt ttgtttaaaa 240 agcctttgaa ttgcttctcc tgtaagtgcc atcatttcaa ataattgtgt gcagtgactg 300 agatttttct tccttctttt caataaatta cattttaagg cacaactcct attttttgtc 360 attattccat tcagcagaat ttgcacaatc ctgttaacag tctttatgcc tgtaacattt 420 tattttcact aaatttttat tacactttca accacaattc aatgaacaaa atggtaaatc 480 ttcatgccta gctgatgctg acaggttata agctgggtct aggatctttc atttgaagtc 540 acctgtctat aggatattct ccatgagaac ataggtgtgg ctgcagagaa gaacagtggt 600 atgacagttg cgggtgcact gggaatttga aaaacatggc taagctgttt atttatgtca 660 ccagaacctg tttgagccaa actcatttga ttatttgatt attatacact tacttttata 720 attaggtaga ttagataata aacatttaat aggttagacc atggcataca gtaacacaat 780 actcagtgtc ctcccgaact ccaatgaaga gttatttctc aaaatgacaa tagtcgcttg 840 cattaatacc ctcaaaaccc tgctggaatt tactttccag agtttattcc agggtcccca 900 acaaacccta tctgctgtga ctctttaaga aacttctttc caccaggtca taaaatacag 960 gtggcaaggt ctcttgtacc acaacctaaa cgacctattt agttagcagt tctgtctctt 1020 atcgattata tatataattg atatgcacat atatgcataa cacctaaaca tgtatttttt 1080 gcctataacg cttttttacc agttgtcact tggtttggtg acactagtct ccttccattt 1140 tccctgaggt tggaaatgca atttcaatta ctttgcacct ctcaccagag ttctcagtca 1200 attgtttagc acaaaagaat ttcataaaag tttacccttc aaggaaaagt ttaaaggaga 1260 ataatttatc ctttgtagac acagggaagt gcagggccat tagaactaag catatctatt 1320 tatggatgtt taaatatttc tctttcaatt atgattgtca aactgccttc tagagtctta 1380 caaataacag aagcagaaaa aaatacagtt gtgaaaaaac agtgctgaca gtgaactgaa 1440 gaatatgtgc attcctcatt ggctgcccct tccaatgcat tgttgctgtg caggaataca 1500 caggtcaaat gtaaccaaat ttggggactt ctaggtgaac tagaaatgtg gattgtgtgt 1560 aagatctcct atttttaaat attggtctag tgttttttaa atataaaata cacagtctcc 1620 ttctctttat tggccagacc caacccctct aacactagtc tatgtactat agtgtacttt 1680 aagtcattaa gtaaaggact ttctacttcc agtccaatca aggttgaatc ccagttacag 1740 tgaagtgaaa gtgagaagta ggacattagg gtaataatta gctacagtaa acaaagaaat 1800 catgattcca tcatgacaaa tacacagtaa gtgttagggg ttacatgggg ctcattgtta 1860 aaatggctca tcatgctgac ccatgagact gaccatcttg ttcaagcgac atcctgtttt 1920 tgccactggt gcctattttc ccaagactac aagaccaggg gggaccacac acctccagct 1980 ttctcaagat tatgagtgca ccctaccgtg agatacctct gactttctca tgacgatgtg 2040 accaccagag tccacctgca ggtgaaagat aaactataaa ctaaccttcc cccgcttcag 2100 gaatcaattt cccctctgca gagtataaga aggccctgct agaagggcgg gggggctgac 2160 tcttctcaaa ggtcagtcag cctctccttt ttccttctaa taaattttct tctctttgcc 2220 tgaacaccca attccttctc tttttctcca tgctcccctt acagtaagac cttcaggagc 2280 acttgggagc aggcggttct gtaaattttc cataccttcc catatattcc actttcaata 2340 gttgggttcc tattgtccta actttcacaa gaactatcca ttttatggat tcatgtcata 2400 ttaatcacta tagattagat tttgcatgcc ctcccgaatt catatgttga agccctaact 2460 cccccatgtg atgcacttgg aggtggcatc tttggaggca aataggtcat gtgagtggga 2520 tatctatgag tgggattagt gcccctctaa gaaaagctag gagagagctt gtctatcttt 2580 ctgccttgtg aggatacagc agaaaggcag gcacctgcag gccaagaggg agccttcacc 2640 ggacacagga tctgctggca cctggatctt ggtaaatgtt atttgtaact gtatgtctgt 2700 gagttgctat gggcaggatg tactggctgg taaacattgt ttagtaataa gtggatgtgg 2760 tttacaccag atttaggaga atctaggagt ggtgaagtaa aatggggttt ggcattcaat 2820 tgttgactca ttatatagct aaaatatgcc tgcttgatga gcttattgta agagatctca 2880 caagtcattt tgagcttcct agtattgagg tgctttgcac accctctgtt agtggcctaa 2940 gaactgaaga taacacatgt cttgtgctac caagcagtag gaggataaat gaagtttgct 3000 cttgagatct cagtacctct ttcatctttc aatgcatacc cttctcctgt tgttgtactc 3060 tgtcctctgc ctgtaataaa gctgttctgt tagtgtaaac tgtatgggtc tcctttaact 3120 atcaaaaatg ttaggtcttg tgtgattacc acctcgctgt aatatacagc aaaaaaaaaa 3180 aaaaaaaaaa aaaggagaat tcatctgcaa gttctctctc atctcctgct tctcattctg 3240 aaattttcac ctcagtgggg gctaaaacta tgcatataac agtagcatta accaactatt 3300 acagtttctg aggaaacccg gtcctgtgct atagtacaca atcccattca agtccaacag 3360 caaagaggtg agtctgcatt gatgaggtcc caaccacagg gaaaaggaag aagacaggca 3420 atatactgaa tgcacacatc acaatgaatc accacagata cttacttcca ggctaaggga 3480 aagaatatcg ctagttcccc agcactttct cacattttct gtcaagttgt accttccctc 3540 agataaccaa tagtctagct tatcacacaa gaatttaatc gatcattaga ttctaataaa 3600 ttccacttat tggattacat attccattat attttggcct gattactgtc attcaatgtt 3660 atttatataa aattcaccct tgtttttttg tggcagtaac tcattcattt caactgcagt 3720 atgatatccc attccacttt agaccatgac ttatttatac aaaatacatt tatacaaaat 3780 acaaatttat acaaaataac gtaaatgtta attcacattt atgttgattt tagtttaggg 3840 tattagcatt gttaggagta ttgggtaaat gtttttagaa tacatatgaa agcatattat 3900 ggtttccttt tggttgtata cctaagagaa gaaatgttag ttataatata agcagatgtt 3960 tatttttgac aaagactcat aatactttcc gagttgcttg aaccatttta tatttatttt 4020 aacattacct agcatttatt cagtttgctt atttttgtaa atacttggtt ttaccaattc 4080 ttctcattca gctattctgg aggttgtgtc atgatatctg gtatttttaa cttgcatttt 4140 ccagagaaat aatggtattg aacatctttt catatattta ttgacaatta aaataacctc 4200 tttggtgaag tgcccattca aagatattgc ttgtgcacta ttatattagt tgcctttgtt 4260 attgaaatat gcccaattaa attttggaca atttggggac tggattcaga caaaaagttc 4320 tgctggccat gtcaggtttg agatattaat tagatactcc gatgtggaga agaatgcagt 4380 tgagcacttg aatttggagt tcaggaaaaa ataagtttga ggacatacat gtggaagctt 4440 caagcccatg aatggactta aagtcatgga atcaaacaac atcaatcata gtaaggctaa 4500 tccctagggt attccacaca ttgcatgttt ggaaaaggag aaaatgccag aagagacgat 4560 gcaaagaaat aactggtgag ttaagaggga aatcaagggc ttatcaggct taggatttca 4620 aataaaattt gcgatatttc aaaaaaaaaa aaaaaaaaaa ggaatgatca actggaccaa 4680 atagcactga tagtttgggt aaaataaaca tagattggta aaatgaatgt tgttggtaat 4740 cttgaaaaaa gtggttacca taggggtaaa gctcaatggg aacaggttga tgagagaata 4800 ccatgtgaaa caatagacac agtagtttgg acacttcctt tctgatggtt accatagagc 4860 agtctaaagt actgccaagg atggaatttt ttctttaaag atgggagata ttatagccta 4920 agtatggctt gcacttgatc ctgaggtaag gaaaacactg aggatgcagg aaagatgaaa 4980 ttactgctag aaacaagtcc ttgtgtaaac acaaggggag ggaatcaagt ttacaaatgt 5040 aggagttgtg taaccaagca ggaccctgtg aagccttccc acagtggatc cacactcttg 5100 tcctctgcct gcatttttgt ctatagaaaa actttagtca aagaatcagt ttgatcagag 5160 aagtgagaaa atacagagaa aaaggaatac agtcaagcaa gacagagtaa taatagttta 5220 gccactcagc aatgtcaagg acttatggtt cttcctcatg gactatagat aatatgctga 5280 gccatgtcct tggaacggtt ttgcaggtgc tcaaacccct accaggcgga agaagtcaac 5340 tgcatgctgc ccacaaacat gtagacccca gaccagttgg aaacagaagg ttgatgatgc 5400 tgactcccaa ttacctcatc accaaacagt taggaaaata tccaagggct gatcatgccc 5460 ggctccttga agagtaagag taagagtcct caccactcct ccaagaggca cacagtcctc 5520 gaagcactag cctgctgtgt accctctgcc tggcaattaa agctactttt gccggtttcc 5580 acatttctat ttggcgtcag tgtatagagg cagccgatac ttcggtgata gttggtagac 5640 agataattca ttgcaatgaa aaaattactc cacagacaga gacaagggat atcagagctt 5700 cacataagtc tactctcttg ggcattagtg gctaaaacaa catccttcag ctcattctca 5760 ttgtgacact ggcactggct gctctagtgt aagtgaggaa tgtggagcca cacggacacc 5820 catcctgcac tgtgtggggt gctattcaca ttaaaacttc aataaaaccc tttcaactga 5880 ctgtgttata gatgcaaaaa ctgaatcttt gaaagcctaa cttatacagc accatttagc 5940 caccatgtta cagagccgtg attcagattt tacattggaa gccttcttat ctaggaggat 6000 cataaaattt aacatccaat gcagatactt ttgagagtga aaggcacaag tataatagtt 6060 cagctgagat aagaggtata aaccaagact ttctagagta tattcagcat tagatcaccc 6120 tgccttttcc tccatttttg atataattta gtactcaagt caaagcattc tactcaattt 6180 caatttttcc gtggctattt ttaacattaa taataacttt aagttaacta ataaagaggc 6240 cagttaggtt tggaaaatta taataaaaac aggaaaactt aaatatgcct aaggattaat 6300 ttattgaaat gactggttag gaaattataa ttccataaat tatttttata catattt 6357 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 20 taacgaatcc aactaggaac c 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 21 tccttctcca accctatatt c 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 22 tgagagggga atagaaagaa c 21 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 23 tatcaatagg tctcagaaga tc 22 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 24 tagacttcga gtttggaggg 20 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 25 tataaggcac aaatgagccc tt 22 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 26 aaatgctcaa catccctgat ta 22 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 27 tattccgtgt tcatggattg g 21 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 28 aagtattctc cactgcctta c 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 29 tgtgagtatg gtagagaatt t 21 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 30 ctattgtgaa tagagctgca at 22 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 31 gtgtgagagt gtgtaccagt t 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 32 tgttcccttg tgatatatag c 21 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 33 cttgttccca cagttcaaat g 21 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 34 tagatacctc caccaagagc 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 35 ttctcaggtt tcctgaggtg 20 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 36 gtgcacattt acatactgat ag 22 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 37 atcatcaatg aactgaacag ggt 23 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 38 ttgagaccta agtcacagct a 21 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 39 tccataataa tttatgtcaa ggg 23 <210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sequencing primer <400> 40 taaggcaaaa tgtgcatgag tg 22 <210> 41 <211> 34071 <212> DNA <213> pig <220> <221> gene <222> (1)..(34071) <223> pig aS1 casein DNA sequence <400> 41 ttactgaaag agcttcnctt acttcttagt tancctctat gtcatagaaa gaaattntat 60 tttaaacttt accaatgtaa aatttccctc tctgctctca tattgcttta aggcacttca 120 gactttctaa gagtagcaaa cattcttttg agctattaaa agttagttaa tcaatgaaga 180 cattattatt attattattg atattatagt agttacttta aggaaaaagg attacaagat 240 tgctgttgga taatgctgga tataagaaat tgcatatctt tattctgtag acctcagcag 300 aagtattgtg aagtttgtct ttagcaatgc tgagatgttc tttactagtt ctttttactc 360 tatttctcgg ggcttaaatc tttcatctta tatgcatgtt aggcattctc caccagctag 420 gatgttattt cctccatcac ctatccgaag attcgacacc ttgaatacat tgcttttgtt 480 gagtaaaaga ataagcagta tgatatatta gaaagagcac aggctctgat gttgggagat 540 ctgagtcatc atttactcat tagctggtgc aagcattttg tatggaaatt aatttactta 600 ttaattttat tacttttatt tttactttta ttttttgcca tttctagggc cgctcccgca 660 gcatatggaa gttcccaggc tacgggtcta attggagctg tagctgccag cctatgccag 720 atccttagca acgcaggatc caagtcgcgt ctgcaaacta caccacagct cacggcaaca 780 ccagatcctt aacccattga ggaaggtcag ggattgaacc tgcaacctca tggttcctag 840 ttggattcgt taagcactgt gccacaatgg gaactcctta atttacttaa tttataaaag 900 tttagtttcc ttatctacaa aagggaaaat aatgacttgt ttctctacat tacatgattc 960 ttataaggag tagttgtgaa aataattata agaagacttt agaaattata aagaagatta 1020 taaattgaca aagaaatgaa tctatttcat tctgctgatg gatgacttat taatctatct 1080 ggtaaactgt ggttctgatg gccaataatg cctgttaaat acattgtatc ctcataataa 1140 tattgttaaa cagggactgc caatgattgg accccaggaa agtttatcag tcatgaaaac 1200 cacattttgt caagtcagaa gaactattac tgataagaaa aaaatctctt tacccaccaa 1260 attacagtct ttcagaaaga aatcactagg tgagatttag ttaaccaagt taaattttag 1320 cccagttttc ccagatgtct tgagtaggtg aaagcttcct tcatggcaag atgaatgaat 1380 ttttggttgg aggtaagagg caggtagtgg tcttggcacg acatagacag ataaactgtt 1440 acttgtgaga tatcatacct acaaaaagaa tatagggttg gataaggaga tgctttattg 1500 tagacattgc tataaaggta aacactttca tggaaataca attcagcagt tttagcatct 1560 ttgtttttgc catatgtgtt acatattttt acaattttca tatgctttta attttttaaa 1620 aggtatttta catacaaact ttcattattg ggtattttaa aagagaagac tgtgtattac 1680 attttcaaat ctttcagttt ctagctactc aaagtgtgct gcatgggtca ccagcagtga 1740 catcacttag agcttattaa aaattcatta gctcctggag ttcccatcgt gatgcagcag 1800 aaacaaattt gactaggaac catgaggtgc aggttcaatc cctgcctccc tcagtgggtt 1860 aaaaatccga cattactgtg agctgtggta taggtcgcag acccggctgg gatctggtgt 1920 tgctgtgact ctagcatagt ccagcagaaa cagctctgat tagacctcta gcctggaaac 1980 cttcatatgc cgctggtgcc ctaaaaggac aaaagagaaa aaaaaaaata cgttagttca 2040 ggacacacct cagatttact aaatctgaat ctgaatttta acataatatc ctggtgattc 2100 ataagcacat taaagcattg ttgtagttca ttgttctgtg agaacatcaa atagtatagg 2160 atttcatcaa taaaattcag aagttctttc tattcccctc tcatattcca tcccaagctt 2220 tcttcagttg tggtcactaa taaaaatttt gccttcacac acacacacac acacacacac 2280 acacacacat aaggtctttt tagggctgca cccatggcat atggagtttc ccaggctaag 2340 ggtcaaatca gagctgtggc cgctggccta tgtcactgcc acaacaacac cagatctgaa 2400 ttgcatctgt aacttacacc acagctcatg gcaataccga tccttaaccc actgagcaag 2460 gccagggatc aatcctgcat cttcatgttt actagtcaga ttcgtttctg ctgagccatg 2520 acaggaaacc ctgcctttac cttttataca tttttaatgc acatacaata tacaagtatg 2580 attcaagact tctcgaaagt taacaaatcc acaagatagg aaaatgattt gggcacaaaa 2640 taattagttg aggttttttt taaaaaaaaa aattctaata atgatcgttt taaatgagca 2700 cttactttgt ctcaggctct cttctaagtg tgtgatctaa ttttttcctc tcgatcacac 2760 aggaagtgga tgttcttaaa cagatgtgga cactgaggca cagtgatttt gcaagattac 2820 cctgctggtc attgagagcc agaaattaaa tttacaattt gacctcaaaa tcttgtcttg 2880 caacaactac atcatgtgtt tcatttaaga tcttctgaga cctattgata cagcactaaa 2940 aaggatattg ttctaaacac aaacataaaa tcaagcataa gttccatatt tccagggata 3000 tcacatacca caccttaccg aaagaactct attttagttc tagatgccaa gattccactt 3060 gtgcaatact taagagtaat gattcggggg gggggggatg cactgggggt ttgggatgga 3120 aatgctataa aactgggttg tgatgataat tgtacaacta taaatgtaat aaattcattg 3180 agaaatatag aaaaaaataa aataaaatcc cccctcccaa aaaaagaatt cactgtggat 3240 cctctcttag attccaaaca gtatgtgggt ttctagagaa aaacaaagat gtctttcata 3300 ttctcagtaa atttccttct ggaagtttca ctgttaaaag acattttcct agaagactca 3360 ccagtcataa acccaaatat tcctgggaga atgctgacag actagctttg aaattcatgt 3420 tgtgaattat tgactgtctc atacagaatg agttctccaa tgaggtttaa tgtggaccag 3480 aatatgtagc aacagtacac aaaacttgca gagtaaagtt tacctcctga ctcacagttc 3540 ccttccctcc aaactcgaag tctattgcta atgtttcctc tccttattgt gctagttttt 3600 ctaagaagta tgaatctagg aaagaatgtt tccaatattg aaacttgaaa cacaagaaag 3660 ctttcagctt ggttgtccct tatctgacct atttccactt tcactgactt agggattctg 3720 ttggattttc aaaaccgtcg tgatagtact agagtagctt tgcctttgtg tgccaaagat 3780 agttttggtt tatggctgtc gttttgttat gattattaca aagactctcc cattctcata 3840 agtgttccat gttgaatgat cagttatatg ttcatcctac atatgactat tacatcagaa 3900 aatcgctatt caggtaataa tttcattctt tctttactca caggacaaag gcctgttaac 3960 tacaaatcat ctaaatagtg tctcaaatgt gaactgtgat tttctttttt agtgttgaac 4020 tgaacaattt tttttaattt attttttcca ctgtaaagca agggatcaag ttatccttac 4080 atgtatacat tttattcccc accctttgtc tgttgcaata tgagtatcta gacatagttc 4140 tcaatgctac tcagtaggat ctccttgtaa atctactcta agttgtgtct gatcagccca 4200 agctcccaat ccctcccact ccctccctct cccatcaggc agccacaagt ctattttcca 4260 agtccatgat tttcttttct gtggaagggc tcatttgtgc cttatataag attccaggta 4320 tcagtgatat atcatttggt atttgtcttt ctctttctga cttacttcac tcagtatgag 4380 agtctctagt tccatccatg ttgctgcaaa tggcattatg ccattctttt tatggctgag 4440 tagtattcca ttgtgtgtat atactacatc ttccgaatcc aatcatctgt tgatggacat 4500 ttgggttgtt tccatgtcct ggctattgtg aatagagctg caatgaacat gtgggtgcat 4560 gtgtctcttt caaggtaagt tttgtctgga tatatgccca agagtgggat tgcagggtca 4620 catggtagtt ctatgtgtag atttctaagg tatctccaaa ctgttctcta tagtggctgt 4680 accagcttac attcacacca acagtgcagg agggttccct tttctccacc ccccccccag 4740 catttgttat ttgtggactt atcaatgatg gccattctga ctggtgtgag gtggtatctc 4800 atggtagttt tgtttgcatt tctctaataa tcagggatgt tgagcatttt ttcatgtgct 4860 tgttggccat ctgtacatct ccttgagaaa tgtctattca ggtctttgcc cattttccat 4920 tgggttgatt ggcttttttg ctgttgagct gtataagtgc ttgtatattc tagagattaa 4980 gcccttgtcc attgcatcat ttgaagctat tttctcccat tctgtaagtt gtctttttgt 5040 tttcttttgg gtttcctttg ctgtgcaaaa gcttgtcagt ttgatgaggt cccattggtt 5100 tatttttgct cttatttctg ttgctttggg agattgacct gagaaaatat tcatgatgtt 5160 gatgtcagag agtattttgc caatgttctc ttccaggagt ttgatggtgt cttgtcttat 5220 atttaagtct ttcagccatt ttgagtttat ttttgtgcat ggtgtgagag tgtgtaccag 5280 tttcgttaat tttcatgcag ctgtccaggt ttcccagcaa tgcttgctga atagactttc 5340 tttttcccat tttatgttct tgcctccttt gtcaaagatt aattgaccat agttgtcaaa 5400 gtttatttct gggttctcta ttctgttcca ttggtctgtc tgttttgata gcagtggcat 5460 gttgttttga tgactgtggc tttgtaatat tttttgaagt ctgggaaagt tatgcctcct 5520 gcttgatttt tgtttctcag gattgctttg gcaattctga gtcttttgtg gttccatata 5580 aaattttgga ttgtttgttc tagttctgtg aaaaacgtca tcggtaattt gatagggatt 5640 gcattgaatc tgtatattac tttgggtagt atggccattt ttacattatt gatttttcca 5700 atccatgaac acggaatatc tttccatttc tttacatctt ctttgatttc tttgattata 5760 gttttatatg aactgaacaa ctttaagtga taaaagcaaa aggaaaaata ctaatataaa 5820 gcaaattgac ataagctaaa attttgcagg atttgacgtt gtataaatct acaatgaata 5880 tgtttgctcg aattacagtg aacagtcata ttttacaagt ataagaatga tttatttcaa 5940 aatacaaact taattaacta tattctatat ctaataagca aagtgaagat tgatccttat 6000 ccataggtac tagaaaaatc tgtgttttga gtttatgaga attcctatgg tggatacatg 6060 tgttcactaa gagttggctt gtctttaaaa gttctgattg ttcttctttg atggcaagcc 6120 ttattttatc aaatctaaga ctccagtctc agttttttga gtctctaatt ttggactatc 6180 aggaatatta aaattatcag ctttttgtat gtgaattacc cactatagta tgagactaca 6240 gatttgtctt actcaacttt tatttcccct gtgtcttgtc tcatttcttc acctctgtaa 6300 ataaataatg taagaatgag taaacaaatg aggatacagt agctttacac aacttacagt 6360 atgatcctga attggaaata aaataagtca gttatcctgg atgcattctc aggaaaagac 6420 aagagccggg tattgtaagg cagtggagaa tacttgttct cagccccttg gataaatcag 6480 agtaaataga aaatactagt acttttttga cattgatgta atgcagtcag caaggacgat 6540 actatccaaa gaggagttta acatggaaaa ctatagcctt gtctatcccc agtatggaaa 6600 gcctgagcca ctgcgaaatt tcttttaacc ccaaataatg ttcctaccat atgactgtaa 6660 attggctgtg aatatcacta tggatttatt attttatttt taaatttttt tggtcttttt 6720 tctttttagg gctgcaccca tggcatatgg aggttcccag gctaggggtc gaattggagt 6780 tgtagctgcc agcctacacc acagccacag caatgccaga tctgagctgt gtctgcaact 6840 tacaccacag ctcatggcaa caccagatca ttgacccctt gagtgaggcc agggatcgaa 6900 cctgcaacct catggttcct agtcagattc atttcccctg cgccacaatg agaattccag 6960 aattttttta aatatatagt gtgatacctt tctgtaaaca agcagtcaca atcaacaatt 7020 ttttaaatcc agctctatgt atagatattt tattcagcat gcaatttttt tcctaaaatt 7080 aacaatgcca gttaattcta ggattatatt tcagactgga aagaaagttt ttttttcctt 7140 ttatttactt actttaaaag gtggaaaatt ggagttatgg ttgatttttt ggggggggga 7200 gtatttaaaa attgtattct taaataaaaa ttattcttga ataattattt ttaattaaga 7260 aatctaacaa ttaaattaat gaatactatc acaacacata tacccaaaat aaagcaagca 7320 gaaaattatt tggtgtagtt aaaatactac caaagtttat aaggcaattg tattttcttt 7380 ttggttaaaa aaaagatcag atcacatata aggtaactta ctccacaagg taacttactt 7440 agaatactta gaataaatac ttagaagact tagaataaat aatagggaat aaatagagtt 7500 ttaaaaggtg aaatagatga tgaaatcttc tcatggtcta gtacaattat aaaaattaaa 7560 aatttttgat gattttattt tgtctcaaga atttccctta caggtattga ctttttcaaa 7620 agctgtaaag gaaattttat tgctatatta atctttccaa ttatccattt aacttaaaaa 7680 gcatgttctt ataataacca taaatatgga atttttatgt atcttaattt tgaataatgt 7740 cattccattt cctgtataat ttggtgtcat agcatgaatc actcctttgt tgaaaactct 7800 cctcagaatt tcttgggaga aaattggaca gaaaattaat ttcctctttg agagaattct 7860 tagaatttaa atgacactat tggttgaact gaaaccacaa aattagcatt ttactaatca 7920 ctaggtttaa atatttgtga aacaaagaga tctgccacca tcttgatcat cagctcagct 7980 tgcttcttct ttccggtctt gggttcaagg tatttcattt acatatagca aaatgtgata 8040 tattatgatt tcaatctgtc taatttttca ctcctcacta aaaaatatgc actggtaact 8100 tttctgtgtg attccaaata ttgatacctt ttaatgatat actggtggct taaaaatgca 8160 tttgcaaatg tcgatgccat ctatctcaga gctttagttg aaaaataata gttttataaa 8220 gaccaaattt ttttgccaaa ttttatgaaa acttattatg tgaaataatt tataatcttt 8280 ttaaagatca tagtgaggat catttctggt agaatatttc aagaccattt ttattccatg 8340 tcattaggtt aataaaatta attctataaa ggatatgcca atgatataca cagatataaa 8400 tgactacttt ttaaaagatg gttagatttg gatatttgga aaaatgcaaa tgaataaaac 8460 cagtaaactc attttggatt tataaatatg tcttccttac aaatgcagtt agattctaca 8520 atatgtagac tgaaacagta tgtataaaat aagctgatta gtttgttggc taatgtataa 8580 acaaattgca tgtatattat gactttcttt tcctaatttc tctggaaacc agtttcccca 8640 ggacataagt tctaagtatc tctgggttct tgtaatttga tggaactcta gaagtcacac 8700 atgataagac atcagaatct tatgattctg ctcaatgaag tcgtctttat gcagtcatgt 8760 catggatata gcaacgtaga aaaacataac ataatagcta gactttaaaa aaaaattgat 8820 ggaggttaaa tgtttctaca taatatgcac caacagtgtt tttcccaaag acgctgaaaa 8880 agcaggattc tctaacatag acctagaaaa acaccttcaa aaaattgcag atagggagtt 8940 cccgttgtgg ctcagtggtt aacaaatccg actaggaacc atgaggttgc aggttcaatc 9000 cctggccttg ctcagtgggt taaaggatct ggcattgatg tgagctgtgg tgtaggtcga 9060 agacgtggct tggatcccac gttgctgtgg ctctggtgta ggctggtggc tacagctctg 9120 attcaacccc tagcctggga acctccacat gccgtgggag cggcccaaaa aaaggcaaaa 9180 agaccaaaaa aaaatttttt ttcagataaa attaaatgcc agttccctgt gccttttagt 9240 ttattatcaa tttttagcaa atctgatggt ctaagaggaa atatttaaaa taattaattg 9300 tagtattctt aaatttagta gtatttaaat attaatgttt atgtattcct ctgacaaaac 9360 cctattacca cttcaaggat caaatgtttt gttttagagg gtgatactgg tgtttcttat 9420 ctcatataag cactaagcaa gataatttga atgataaatt tttcttgtga gtaaattttc 9480 tgtcagacct aaatttttat tttgttttct tatataggtg ttgacaacca tgaaacttct 9540 catctttatc tgtcttgcag ctgttgccct tgccaggcct gtgagtatgg tagagaattt 9600 agaagcttct agattcttga ttgaaattac ctgatatcaa acacaagaaa ctgaggataa 9660 taatcttaaa agtattgaat gatctctaat taccttttga agccttgata ttaaaactgt 9720 agaaatcctt cacatcttga tcattattac atagttcatt caaagtcatc actccaaata 9780 aaatctgagt tgaaatataa atgcctcaca gtaaaaaaat aaaaacaaaa aatgaaaaga 9840 aaagaaaaag gaataatgta tttaacaaca tagtaaatag aatcaatgag tgttattacg 9900 ctctttgcct gggtccaata aagaattagc atatatttaa acatacaagt ccatgatttt 9960 ttctgtggaa gggctcattt gtgccttata taagattcca ggtatcagtg atatatcatt 10020 tggtatttgt ctttctcttt ctgacttact tcactcagta tgagagtctt agttccatcc 10080 atgttgctgc aaatggcatt atgccattct ttttatggct gagtagtatt ccattgtgtg 10140 tatatactac atcttccgaa tccaatcatc tgttgatgga catttgggtt gtttccatgt 10200 cctggctatt gtgaatagag ctgcaatgaa catgtgggtg catgtgtctc tttcaaggta 10260 agttttgtct ggatatatgc ccaagagtgg gattgcaggg tcacatggta gttctatgtg 10320 tagatttcta aggtatctcc aaactgttct ctatagtggc tgtaccagct tacattcaca 10380 ccaacagtgc aggagggttc ccttttctcc accccccccc agcatttgtt atttgtggac 10440 ttatcaatga tggccattct gactggtgtg aggtggtatc ttgtggtagt tttgatttgc 10500 atttctctaa taatcaggga tgttgagcat tttttcatgt gcttgttggc catctgtaca 10560 tcttcctttg agaaagtcta ttcaggtctt ttgcccattt ttccattggg gtgttggctt 10620 ttttgctgtt gagttgtata agttgtttgt atattttaga gattaagccc ttgtcagttg 10680 catcgtttga aactattttc tcccattctg taagttgtct ttttgttttc ttttgggttt 10740 cctttgctgt gcaaaagctt gtcagtttga tgaggtccca ttggtttatt tctgctctta 10800 tttctgttgc tttgggagat tgacctgaga aaatattcat gatgttgatg tcagagagta 10860 ttttgccaat gttctcttcc aggagtttga tggtgtcttg tcttatattt aagtctttca 10920 gccattttga gtttattttt gtgcatggtg tgagagtgtg taccagtttc gttaattttc 10980 atgcagctgt ccaggtttcc cagcaatgct tgctgaatag actttctttt tcccacttta 11040 tgttcttgcc tcctttgtca aagattaatt gaacctttcc atagaaaaga aaatcatgaa 11100 tttggagaat aaacttgtgg ttgccaaagg ggagggagag ggagtggtgt ggttgaggag 11160 cttggggtta atagatataa actattgcct ttggaatgga ttagcaatga gaccctgctg 11220 tgtagcactg ggaactatgt ccagtcactt atgatggagc atgataatgt gcgaaaatag 11280 aatgtgtaca tgtatgtgta actgggtccc catgctgtac agtagaaaaa aaatgtgttg 11340 agaaaataac tattacaaaa atgagaatta tgaaataaat aaataaataa acaaacatac 11400 atgtccaata ttttcttacc aaatataaca gaatgccgcc tattgcagtg tatcctactt 11460 ttggaaccta agtcaaaccc tcacatgaga tgactcatat caaccaatat ttcccaaggt 11520 gtagaaaact gagttattct gctgattcag agatttaatt attatggtcc ttgtagacaa 11580 ggaaactagg taatataaat taaaatcact tttcttctca aaaaaaaaaa agattcagtt 11640 acttatgtag agcaaaaatt attctgttcc cttgtgatat atagctagca aagatggaag 11700 ggataaactg gaattgcttg acaaagaaac catccagata aaataataat cttggatatt 11760 tgggcattgt aattattaaa tataggtgta aaattttttg gaattctgag atattctatg 11820 atgagactcc tatttcctaa atttttgttt ggataatgca ggatcactaa taacctaact 11880 ttcctttttt tttttttttt tttttttgct ttttagggcc atacccacag catatggagg 11940 ttctcaggct aggagtcaaa tcagagctgc cggtggccta accacagcca cagcaacttt 12000 ggaatcaaat ctgagctgtg tctgggatgt accccacagc tcacagcagt ggtggatcct 12060 taacccactg agtgaggcca gggatcaaac ccacctcccc ataaacacta tttagctttg 12120 ttaccccaga ccccctatgg gaactccaaa ccaacctgtt agacactaga gctttttcta 12180 tgctacacct taagcaccaa taggaagaaa atctatgata gcaagttaca agaaaaagga 12240 tccaactttt tcagaaaagt gtcactgtta gtctttttac aaaagaaatg ggtatagttt 12300 ccccagcaaa cagattctat aattctattc caagaaaaca tcatttttaa tgctaccatt 12360 taacaaacat aaatcttgtt cccacagttc aaatgtagat tgagttaaat ttttatataa 12420 ttaactgatt ataaaaataa aatccagaaa atgtttaatg aagaaaagta tatattttgt 12480 ttcaaaaatt cataatagtt atttttagaa ctaccatgta atataaatag ttcagaaaaa 12540 ttaaatttat cattttggat caagacaaat taattttttt tttcagtctt tttacacatt 12600 tttctcagtt ctccatcata ccccaatgac aggaaatcat ttacttttct gtgattttta 12660 tcaagaaaat aaccattttt tttcttccca gggaaacttg gggtcagatg aatttagcta 12720 ttaaaataca catcattttt aaatagcaaa tgttacattt acagtattct atttgttttg 12780 tgatattatt atcatatttt gattgcattt tgttttcttt ttgtttgttt ttaaattctt 12840 gcattgtttt tcacagaaac ctcctctcag gcatcaggaa caccttcaag gtgagtgcta 12900 ttttctgtgt tctaagaact cactaattgt gggactgagc aatgatatat tggtcataca 12960 tagactctgg tcctactcca cccttctcta taagacattc agtttcacat tcaccgagga 13020 ataagggctt cagttcaagt attaaactgg ttattgatct agtctcacaa accttagaga 13080 gaagggtata gggacttaca taaatattta attaatattt aacatggttt atttaatata 13140 atgttgcttc tttccatctc ttttgcatgt acccaaatat atgattgaca atttgctctc 13200 attttacaca ctgaatctta aattagatac ctccaccaag agcctttaca agagtgatta 13260 atcctccact ctgtccacta attgaattaa taccatttta tttatttttc attctttaca 13320 gaatcgagcc agacagcaga gaggtaaagt tctttctttc cctcgacaaa tctctccttc 13380 agttctccat gatgaatgtt aaatatcttc tttgtttcaa tttttatttg tctttggtac 13440 tcagtacttg gagtactttc tcattctttg ctttcattac actccttttc agttcctcct 13500 ttgacacctg acgagtatta agagtatgaa cttactaaac ctctaggctt ggtaaagaag 13560 gaaccaaaaa taatgactcc tttagatttt aaaattagat tataaaacta aaactaatta 13620 tctcctaact tacctaagaa atattttggt ttgcctaagt aaatggggaa gttgtgttca 13680 aatggaaaaa tattctcctt ttctgaatca tgtttataat tcacaattga atttctacag 13740 gaactcttca aagaaagaaa gtttctcagg tttcctgagg tggtaagcat tatccacttc 13800 ttaaatgaca aatatatttt tccggaaaaa atcaatttaa ttttttgttt ataaatgtgt 13860 ttttcacttg atttgatcaa actttttctt cattttccaa agctcccaca aataatattg 13920 aaatcagata tgcaaatatt aggagttgct ttaaatatta aagaactgct ttaaatatta 13980 acttgtgcct ctatatctga ctctttgcaa aacacaaacg ctttttttta atatcctatg 14040 tagagtttta aaatgtccat gattatgatg atggaatgtt ctctatctct aaataatacc 14100 tcatatgtgt tgtgttttct ataattttgt gactgaattg tcacatagga aaaaaaatag 14160 aaattttact tcaagaccac tgtttagaag atttgatatg aacccatctt tgtctaaatg 14220 atttttaaca taacctttct ttttttttgt agcctttatt aagtcagttc agacaggtaa 14280 gaaattctcc accagatata cagtagagtt aacaagggaa gcaatcttgg tttctgtcta 14340 gtaagtgctg tggtgtgggt gttgtgccaa ttctttattg cctttttatg gaaataaaca 14400 ctagacttaa ctgaatcagg cagatgaaat caagagaact cagaactacg tcataaataa 14460 ggtgaaagat aatagcaaca acatttagtg gaacaaaatt ttaaatgatt ttaagtgcac 14520 atttacatac tgatagctta aacaaagaaa gctcagtgag gcgtctcagt gcaagaaatg 14580 atacagcaat aaatattagt gcattcataa aaagcatatt tgtttattta ttgcttttta 14640 gggctgtacc catggcatat ggaagttccc aggctagggg tcgatcagag ctgtagaccc 14700 cgacctacac cacagccaca gcaactcagg atccaagcca catctgcgac ctacaccaca 14760 gctcacggca atgctggatc cccaacccac tgagcaaggc cagggattga accggcaacc 14820 tcatggatac tagtcggatt tggttctgct gtgccataat gggaactcca ggcctgttta 14880 tttttttaat ttgcagcagc acaatgtaaa gtgttttctc atagctatcc attcatttat 14940 gtccctcttt actaatgttt atctcttctt ttttatcctt aaggaaatca tcaatgaact 15000 gaacagggta aggaacatta atgatattta aattatttta aaattcattc tttcaaaaat 15060 atattagcta cactttttag ttttaacaaa gagaactcct gagaaaaaaa cagtaataaa 15120 ctcaaggtat caaaatcttt cttatgatag tgtgtcaaaa agtatattct tgcagttcaa 15180 aagtgttttg atttagaaaa acagtatttt tcctgtgata tttatacctt cactgaactt 15240 tcaaaatgac taatgaattc tactcataat tctaaatatt tattttattg atttaatttt 15300 gtatctatga atagacaagg cattaatatg aatgaatgaa tgggtgcaat tttggactaa 15360 ccaatttttt gcacttatca ccaaaactga aagaattctt tattaaaggt tttataaaaa 15420 aacaatatta tctgcaccta gaatgtttta cataatcaca gttggttatt ctctttcttt 15480 acacatgagt ttctgggccg gggatcagat ccaagctgca cttgagacct aagtcacagc 15540 tacttcaatg gaggaccctt aacccacttt gctgggctgg gtatgaaccc agcgcttcct 15600 agtgccacag atcccattgc accacagggg aacctctaac gcatattttt ttaaaatctg 15660 gtctgtcaga tttttagtag ttttggtatt gagacaacac aggtgccact gaagataaaa 15720 aaatatatct tcctaactat ccttcatact tgaatgatca tcctttcttg gcaggatgct 15780 aggagtgaat caactgaagt aagattcttt attgtaaaac tattaaatat aatgtaagga 15840 aaagaaagaa ataaaatcat ttcctttaaa tatcctaatg aaaatgaatg actaattctt 15900 ttaggtcaaa actaagacag atatctctaa ttcaaagaga gaaaaaaaaa aacatacaat 15960 gttatcatct gtgagccata attagctgga taactaaatc aatggtatta tattgagctt 16020 aaattctatg gatcatgtca ctactccctg tactgctatg gtcatgaaaa ctgaacacaa 16080 cattctagat ggagaaaatc cattttgctt aaatatttat acaccattga accaatgctt 16140 catagacttt tatttattac ataatcttta atccaatact tctgcttaat agccaaaagc 16200 caagtaaagg aaaaaaagta agggtagtta aaggaaaatg taggattatt tacactgatt 16260 taccacaaca aataagaatc cataataatt tatgtcaagg gagttcccgt catggcgcag 16320 tggttaacga atctgactag gaaccatgag gttgagggtt cgatccctgc tcttgctcag 16380 tgggttaagg atccggtgtt gccctgagct gtggtgtagg tcgcagacgc ggctcggatc 16440 ctgcattact gtgaccctgg tgtaggctgg cagctacagc tctgattcga cccctagcct 16500 gggaactttc atatgccgtg ggagcggccc aagaaatggc aaaaagacaa aaaaaacaaa 16560 caaacaaaca aacaaaaatt ttatgttaaa ctcagaaatg cagattaggg aggtaaattc 16620 tttggttagc ctggtaggta ggcttttttc tttccttatc actggctctt accacatatt 16680 tctatttctt ttggcatcta ttttatttga taattattat tttacatttg aatatttgtc 16740 ataaaaaata aattctcttt tcttttctaa gaatcatggc atggaaggcc atgaggtaag 16800 acccttattg taataaactc tacacttaca taacatccat agtatatact ctatgctcta 16860 ttttaagaaa actctcttct caaattgagc aagattgaac ttcccaaaca aggttattat 16920 acccaagaat gtacaatgtt gtgccagata atgttaaaat tagaagagga aatgtgtctt 16980 ttaattgata atcagggaaa atgttattta ttaaaataaa gggtgtaagg caaaatgtgc 17040 atgagtgttt caaatgaaat atgagcctcc aaaaaaaaag aaaaaaaaac ctgtctctac 17100 cacctcaggg tataaactca ctcctgtttg tgagggtagt cttggggaga aagaatctgg 17160 ttgcagtaat tacttaggat caatggccta tgctacttaa tccaaaagca tggaaaaaat 17220 ccactagccc acaatatttg gttaatcaac tttcccaaga gatgaatcat ctgttgacta 17280 ttaaaatcaa tcttttaggt atgtacttga gaacaaaaat tatatccata tttaaatata 17340 aacgtccgtg agttagaaaa aaatctaaat agttcaaagt gtagatgttt taaaagctaa 17400 tgtaatttta gttccatacc aaaatttgtt gttgtatttt catgtttcaa tttattccct 17460 tcaaagaagc cccaaggata catgagaata aatatcccac ccggggttct gggtgcccaa 17520 gctcagtagg gcacttcctt ccaaatttca tatatatttt gcattctact caaccacata 17580 tctatgtttg atttaataaa atgttgtttt ttagtgattg gttattacat tcccacatcc 17640 aacatatttt aaataaaatt gacaacccca aaaaggtacg ttatattggc cctgaatgtt 17700 tcattaccaa ttgctttcat tctaaacaag gagttagcaa cccagtatga aagtgtggaa 17760 caaatttcct tctaattcta aaagtcacag tgttgggaac actgattttc tctcttttag 17820 caaaggggat ctagcagttc atcaagtgag gtaaatcatt ttgatgttaa ttcagtatct 17880 cagttagaaa atgtttatga aaacttgttg tgctatgaat gttacacatc ccataaggtc 17940 tcatggtaca ggctctatgt ctacagctct accctaattt taacatacag gctatgagcc 18000 caaaagatat aataacgcaa atacttgtca gatgaaatta cagaattgtt tctgcgctaa 18060 caattctatc tggctatcca tggtgcccca ttttttttct ctaatttttt tgcctttcct 18120 aggttttggt agtactaaat attttattta aaattactat aagagctatg cttctaaatc 18180 attaatagaa acttggtatt tcctgtgcag acatctactg attctaggtt aactgggtac 18240 tggaatcctt tactccttgt taacctaaaa gagaaaaaga gcaggcatag aaggtccttt 18300 cgtaaacttt gggagaagaa aatttcaaaa taaacccaac ccagttatgc ggagtttctg 18360 ggagatcagt aaaagtgcat cgaatatttc acatctacct ccaacattct gaataacttc 18420 cctgttgaaa tcagtgagaa gaggaaaggg aatcttgagt acaacctgta ccttgaatta 18480 ttcatcttat ctcagttacc aaggaatggg tgcctaagat caatttatca cagttaagca 18540 acatggtaac tggctaatta gtattcatac cttgagtata aattaataag tcataaaact 18600 aacactgcat gtttttattt tttaaggaag ttgttggcaa tagtgctgag gtgagatata 18660 cttactaaat ttaaaataca ttcacgttat ccaggatgtg ttaaaattta cttgtacttt 18720 tttttttctt ttttagcaga agcacgttca aaaagaagaa gatgtgccct cccaaagcta 18780 tctggtaaaa ttttactaaa agtttatcaa aggcaaatgt accaaggaat gagtatgaat 18840 gttgtactga tacattattt ctccttctca acctctgcta caccttaata catagtaagc 18900 cgtctaacag actctagatg tttactgatc ccctgcaaaa ataaagctaa caactttttt 18960 atcccagggt ttttgtttgt ttgtttgttt gttttgtctt ttgtcttttt agggccgcac 19020 ctgcagcaca tggaggttcc caggctaggg gtctaatcag agctgtagct gctggcctgc 19080 atcacagcca cagcaatgcc acatccaaac cgcatctgca acctacacca cagctcccag 19140 caacgctggg tccctaaccc actgagcaag gccagggatc aaaccggaac ctcatggttc 19200 ctagtcagat tcgtttccac tgcgccatga tgggaactcc tatcccaggt tattaattca 19260 tgctttcatg agatttgtgt tttatagtct atttttggat ctgaataaca tagtattttt 19320 tttatttttt taattttccc actgtacagc aagggggtca ggttatcctt acatgtatac 19380 attgcagtta cagttttttc ccccaccctt tcttctgttg caacatgagt atctagacat 19440 agttctcaat gctattcagc aggatctcct tgtaaatcta ttctaggtgt gtctgataag 19500 cccaagctcc cgatccctcc cactccctcc ccctcccatc aggcaaccac aagtctcttc 19560 tccaagtcca tgattttctt ttctgaggag atgttcattt gtgctggata ttagattcca 19620 gttataagtg atatcatatg gtatttgcct ttgtctttct ggctcatttc actcaggatg 19680 agattctcta gttccatcca tgttgctgca aatggcatta tgtcatcctt ttttatggct 19740 gagtagtatt ccattgtgta tatatactac atcttccgaa tccaatcatc tgttgatgga 19800 catttggatt gtttccatat cctggctatt gtgaatacgg ctgcaatgaa catgcgggtg 19860 catgtgtctc ttttaagtag agctttgtcc ggatagatgc ccaagagtgg gattgcgggg 19920 tcatatggaa gttctatgta tagatttcta aggtatgtcc aaactgttct ccatagtggc 19980 tgtaccagtt tacattccca ccagcagtgc aggagggttc ccttttctcc acagcccctc 20040 cagcacttgt tatttgtgga tttattaatg atggccattc tgactggtgt gaggtggtat 20100 ctcatggtag ttttgatttg catttctcta ctaattaggg atgttgagca ttttttcatg 20160 tgtttgttgg ccatctgaat aacatagtat ttaaatggca ccctacagat ttggccagat 20220 ttgagttgaa ctctaagctc taccttttac tagcatgtga tgttagttat ttatctgcta 20280 taagtctgca acctaaactc taaaatgata atataatgat agcaatacac agcacaaagc 20340 aagcactcgg taaacgtggc aagtctcacg aaacttaaca tgtttttgcc cacttcatct 20400 actattctga tttaatttgg ccaaagttaa atcctttgtc agaaagcaaa aacaattttc 20460 aattttctta gcaattctga cataagacac actgcaaata aaaatgaata ttcattcata 20520 tttgacagca tggtaattta tttcacatta aaagtaagtt atgaaagtag tttttctagt 20580 attaaaatat aattggatta gtccaaatta tctgtggttg aagtaacatt aaaagaggat 20640 tactagttct tccttctttg cacctaggaa aggtcatgtt atgattctag ttatattact 20700 tgaaaatgat aggatgagac tggattgtct ccaagcaatg gacaatattt tttttttctt 20760 gaatagggac atcttcaagg actgaacaaa tacaaactgc gccagctggt aatatcttta 20820 ttataataac acaaaactaa actgtgcaaa atcaaaataa ttaagttgta aattgggtcc 20880 acctctgaat atttttttaa gaaattattt attctagact tgaccattaa taataactga 20940 caaataatcc aaatgattgc ctatgtattt actgtcacag ctatttgttt ctatttatgc 21000 cgagagtagt attgtaaatc aggaggtgtt cctgtcgtgg cacagtggaa acgaatctga 21060 ctaggaacca tgaggttgca ggttcaatcc ctggcctcgc tcagagggtt aaggatctgg 21120 cattgctatg agcagtggtg taggccggca gctatagctc tgattagacc actagcctgg 21180 gaacctccac aggccacagg tgcaggcctg aaaggacaaa agacaaaata atcatatcca 21240 taattgctaa gcagggagaa atttcctttt tttactaaat tctttatcaa accctcttag 21300 accctcccac atgatgaatt atattcagaa gtttattctt tttattgttg ttgcaaattc 21360 agtcttgaaa ggtgttccat aaatatgcct gcttcttaaa gcacagatat gccaaataaa 21420 ccttccattt aaggaaagag tgtagactca ggagaaggaa ccaggagtcc taaatattca 21480 acttgctttt gtcaaaattc tctaagaaag aggagctttt ttgctgcacc ttgggttaag 21540 gggccatcat tgtcacagct gtggctctgg ttgctgctgt ggcacaggtt tgatccctga 21600 actgggaatt ttcacatgcc atatgtgtgg ccaaaaaaaa aaaaaaaaaa ccaaataaac 21660 ataaacaaaa agctctaaga ggaaagaatt ggcagattga ttgatctaat ggtctatgaa 21720 tccatttcct taggtgtgaa atggaaatac agttattctc actccctggt gttattttga 21780 atattaaatg agcaatcgcc tactcaggtc tccctaagtc cagcttgtag caagtccagt 21840 aaaagcttca ttcctccttc tcctgccccc actccacagc ctctagaaaa tttgctgcgc 21900 tcatgagact tctgataaca ttatgaaagt ctttcatcag gatatgtgtt tgaactataa 21960 tgtcataaac tctttatcag cagaggtatg gatagataaa aaacatatca attagcaagc 22020 ccttcttcca ctatttaatt ttgatccttt tgagtatcac aatctttcat ttggaaggtc 22080 aaaaaaaatt tcttagctat agcaaataaa caacctaatt aggactttag ggaaaaaagc 22140 ttgtcagttt aggtaaatta ccttttcaca ttttcttctg ttatatatgt aagatgaaga 22200 gagttaataa tgaatttaat ttgcacctca gtttttactc ttatttcaat acctgagtta 22260 cctgtgttta taaagtccaa ataaatttaa atttatcagt acttcactgg aattcatcat 22320 tactgatcta agaaattacc tgaataatca gttttaggac ttagctctga tatgacaagg 22380 tcataacccc cagaagtgtt aggaccagta aggcattgct cattatgttc attgtatttc 22440 atagatggtc atcagttttt agcatagtcc tcaagtgcat gaaagtactt agtaaatctc 22500 tgttattcac atatgaataa aatcaatata ctaaaagaag atgttctttc tttcttagga 22560 agctattcat gaccaggtaa agttatttat ttattaagtg taaaatattt tagtatttcc 22620 ttcatgtgtt atatttttat aatgtgcatt cctttttttt ttataacaga aatatatccc 22680 aatagagaaa tttatctcct acccatatct ggtaatatgt tatttaaaat tcaccaaaga 22740 taatatttaa ataattgatt aatagtctta tatggaaaat atgtattctt agagaaatga 22800 taggtttttc tttttcccag ggtatgagtt ttataaattc tttattttcc aaactatact 22860 aagtctagcc tttagttgaa tatttttttc taaagttaga aaataaaaag tgtattgcta 22920 ttttttttca taattttgac ctagaatact gtctatccta aatttcatgg atgatgatat 22980 actcttggta cattgtaaaa tgaatgtgtt gtatcagaag atatctaagt aatttaaaat 23040 gtctttcctt taaataggaa cttcacagaa caaatgagga caagcatacc caacaggtaa 23100 tattttgttt aataaattac acagttatat tgtaaagttt aaatatgttt gtttttaaat 23160 accctcatac ttttgagggg gtttctttct taggcagctc tttttatttt tttgcatttt 23220 ggggccacac ccaaggcata tggaagttcc catactaggg gccgaattga aactgtagct 23280 gctgacctac aatgacaacg ccggatcctt aacccactga gtgaggccac ggatggaacc 23340 tacatcctca tggttgctag ttgggttcgt tactgctgag ccacaagggg gactctgcag 23400 gcagttattt cttattccca gctttctgaa ataatttata ttgatcaatc agagatgaga 23460 accatgtgtc actcttcttt ggaataagat ttgttctatc cggtctgtct cacctgacgt 23520 ggtcagccta tttataattc tgagcactat caagaattca tctccatggt tacattaatt 23580 tcccctttaa gatattcttt aattttgcat ttttgtccat aatttgattc ttaaattcaa 23640 tttacattta ggaaactcaa actcaccatt atgctctttt aagcaaagag agtggagatt 23700 tactaactga cactttctca agcaaccatc tggagccata gtaaatgtct gtccaaaaaa 23760 aatcttcctg atcctctgtt ttctcctctg tgaagtgaga ttatatataa tacctaacag 23820 aaactgactc ttctcctgcc ctcccttcca ctgatgccct agtgctcctg tggcttctgc 23880 tttccttttt aaggcaatga tgagttacca tgaaacatat gttactctac cagctctgat 23940 tcctgtgaat agctacacca attccaatca tggaggctcc caatcactat ttgctgttta 24000 gggaatctta taaggatggt agagtatgtt tcatatgtct aagaagaaac ttcctttaga 24060 gaaggcaatt gggaaaattt tagtatgagt tctcagatct ctaacataaa aagcatttca 24120 agtaggttgc ttcttacagc tttggtttta tttagcctta aaaaagtaac tttaattctc 24180 tttttctttc cagggagagc ctatgaaagg agtgaatcag gtaagagtga gtgtgtgagt 24240 gtgtgtgtgt gtgtgtgtat taatactgcc ccataagcta gtgctatgct agtatttctt 24300 ggctattgac tggtgttgga ctctctagag ctggttctaa cattgctgtg gaagatctga 24360 tagatctgat tgccaaagga aatgaatgaa tgattctatg gccatctgtg agtgatgata 24420 gcttcaggtc aactttaagt caggacaatc tcaaccagct atttacattg ttaaaatttg 24480 acctgttatt aatctatagt atcatgtcat gaaaataatt tgagttttca atcttagatc 24540 tgacaccttc taattacagg accttgagca aattgtgcta ttctctttga gcactgcttt 24600 ttcatttgtg aaattaatat atggacctat ccatttattt cctccccaaa cttccatatc 24660 ctgatttttg gagtgtcttt ctatatcttc acttttagaa gtagtagctc tgcattcaat 24720 tcagtttctt cagtctctct ctctctctct ctctctctct atatatatat atatatatat 24780 atatatatat atatatatat atatatatac acacacacag acacacacac acggcatgct 24840 aattaaaaag aatgcaaagt aaaccacgga gttttctcaa tatgaatgtt attaggaaga 24900 aaaaaatttc ctcaagattc aaaacatagt agtcatcaat cagttttatg tgcatcttta 24960 ctgtgcatca gatttactct caatttcttc tgaaattctc taatcacttc tgagctaaag 25020 taagagaaaa ggcaattact cacttcattt gagatttaaa gggtacacta acctcaactt 25080 ttcatgtaat tccaaaaaaa aaagagggtg atgattataa tctcaataag aatatttgat 25140 agcaataatt taatcatggt atttaattgg atttaaatta caaaactatt tttcccctct 25200 ctgttaagga acaggcctac ttctattttg aggtaaattt attttatttt attcttttat 25260 ccaaatgatt tataaaggaa aagtattggt aaacatttat aatatagtaa tctttatgta 25320 ggtaaccaca gcaaaactgg aaatgttttt attttttatt ccatcaaaaa gcacatattt 25380 tcacctaaat atatagagaa ttatgttatg cataaaaaca agtaaaacat catgatagat 25440 gaacagagtc acctatcctg aaatttcaga aaggaaaatg gtttggcacc aaaactttat 25500 aattaggaaa ggataatgtg ttaggaattg gagaattctt ttccctttat tcctggcatt 25560 tctgagagca gaggtatgga ccccgagtgg gaggcccttc cttttgtttg agctcagtgt 25620 cttcatggaa aattaagcta aagaacaaaa tggccaaaaa agtcctttcc agtccacaag 25680 tccatgaggt tataaatttt ataataatta aattcacacc cctacattcc tctgggctct 25740 ggtaactgga ctccgattag taatgcagat tcgtggggtt caatgctgaa tattgacctt 25800 gaagaagtta tttcttcatt actcacaagt ctcccaaaaa gccctttcca aaagttcctt 25860 tgccatgatc cactgcatgg aagaatgtga tttctccatt ttccttgcag agtagatatc 25920 tcttgtgatg ctaatagcca tgtcagaagt gaatagaatg cttctttttt tcagagattc 25980 taaagagcaa tttcccatat cctgttgcta tttcattctc tctagcctct ccaccagttc 26040 taccagcttg atgcctatcc ctatgctacc tggtattatc ctccacaata tattgctcac 26100 ccattattca ccaacatccc tcaacccact gcccctgaga agggtggaaa aactgagatt 26160 atgcctcagt ggtggtaagt tcattttaat gactgtatat tgatgttcta ccaaaggaaa 26220 taaaagaaaa cttcttaaag aacataccat aaaaacagat ttagaataaa catgacaaaa 26280 tcaatatcta gagcgtcgta gtagaatttt ccaaaatggg aaattggcag gacgttctga 26340 tatctgcagc taatgttaat ccactactca ggaacttgtg gagcagcgct ctctgttctt 26400 tgagattcat tctgatgaag tcaggaaaaa gttttctatc caaagcaaaa acacagtaat 26460 ttcactttat cctccttaca attttactaa tctctaaagg cttttctttt ggttatatat 26520 acccatgata tacattacaa ttcagtgtgg ggaataaagc acagattttg gcatccaaaa 26580 gtcccaaatc caaatcctga ccttttttgc ttacttaaaa taatgcatta atgcttattt 26640 ttataattct gaaggtgatt aaagacaata atctgttaag catagtgctg ggaagataca 26700 tagcagtcag tttttattga tttagtaaaa ttgtactgct gactaccttc atcacatgat 26760 tttaagaatt tttgtttttt cagaagaatt aagtgaattc tcaggaactc cacaattatg 26820 gcctttggta agttggaaat catttgtgga accattgatc ctcttttcgt ttaaagactc 26880 attacaaaga taggactgta gactataaag attttttttc ctgtagttga gctccttgtg 26940 gacacattag cacttagata ataattaaat tggcttggac atttgcaaat gtttgtttca 27000 taattatact atatgtaaat agcaatcaaa ttagataatt ttaatgaata taatttatta 27060 tattgaaccc ctatacaagt ataggagcat gaatgctact aattttccat caagatgtga 27120 ccttgagatg ctggtaaact caacagtggg attctatttt ttatgatcac tacaataaaa 27180 atccttagca agtcatgtga taaaaccaag tatttgtttc tcaacaagaa aacagacttt 27240 aacgtctaca gacttgtttt aattaacttc atccattgta ctggtgtttc cgattgtatg 27300 tcagtagagg tgtgtgtgtg tgtgtgtgtg tgtgttttcc ctttctagtg gtggaaattt 27360 cccttccaac tgattgaatt aaggaaaatg acaaaacata tgggaaaggt ttttctttga 27420 attccttggc catattttct ctgttactgc aaaagaaaat actacctagc aataaattgt 27480 ctatataact taaaattatc aggataaatg ggtacatatg tcagtcacag gaaagaacaa 27540 ataactttgt gagtttcatc ttaaaatgaa gagaaaatga ttattcaaat gcatcataac 27600 agtgtctctt ccattcaaaa catgtaatat aaccaaccac atatttcttt ttctatttac 27660 agatgtgact gaaaatacca tgcttgaaat ttctcctctc catctaccat gtagaaccat 27720 tttatctgaa gactttgact gttcttttag aacagggaaa tcgcaaatcg aagtcaatct 27780 tccttcttga attctttact ctatattaga tagcatataa tccttttcct ttggcaaagt 27840 tgtcctaaca gtttagtgtc taaatttcag ttgtatcatg ccagtaggaa gaccactgaa 27900 tcagagggaa ttaaaagtct ttactaaatt tcaatatgga aattttgttt aaaaagcctt 27960 tgaattgctt ctcctgtaag tgccatcatt tcaaataatt gtgtgcagtg actgagattt 28020 ttcttccttc ttttcaataa attacatttt aaggcacaac tcctattttt tgtcattatt 28080 ccattcagca gaatttgcac aatcctgtta acagtcttta tgcctgtaac attttatttt 28140 cactaaattt ttattacact ttcaaccaca attcaatgaa caaaatggta aatcttcatg 28200 cctagctgat gctgacaggt tataagctgg gtctaggatc tttcatttga agtcacctgt 28260 ctataggata ttctccatga gaacataggt gtggctgcag agaagaacag tggtatgaca 28320 gttgcgggtg cactgggaat ttgaaaaaca tggctaagct gtttatttat gtcaccagaa 28380 cctgtttgag ccaaactcat ttgattattt gattattata cacttacttt tataattagg 28440 tagattagat aataaacatt taataggtta gaccatggca tacagtaaca caatactcag 28500 tgtcctcccg aactccaatg aagagttatt tctcaaaatg acaatagtcg cttgcattaa 28560 taccctcaaa accctgctgg aatttacttt ccagagttta ttccagggtc cccaacaaac 28620 cctatctgct gtgactcttt aagaaacttc tttccaccag gtcataaaat acaggtggca 28680 aggtctcttg taccacaacc taaacgacct atttagttag cagttctgtc tcttatcgat 28740 tatatatata attgatatgc acatatatgc ataacaccta aacatgtatt ttttgcctat 28800 aacgcttttt taccagttgt cacttggttt ggtgacacta gtctccttcc attttccctg 28860 aggttggaaa tgcaatttca attactttgc acctctcacc agagttctca gtcaattgtt 28920 tagcacaaaa gaatttcata aaagtttacc cttcaaggaa aagtttaaag gagaataatt 28980 tatcctttgt agacacaggg aagtgcaggg ccattagaac taagcatatc tatttatgga 29040 tgtttaaata tttctctttc aattatgatt gtcaaactgc cttctagagt cttacaaata 29100 acagaagcag aaaaaaatac agttgtgaaa aaacagtgct gacagtgaac tgaagaatat 29160 gtgcattcct cattggctgc cccttccaat gcattgttgc tgtgcaggaa tacacaggtc 29220 aaatgtaacc aaatttgggg acttctaggt gaactagaaa tgtggattgt gtgtaagatc 29280 tcctattttt aaatattggt ctagtgtttt ttaaatataa aatacacagt ctccttctct 29340 ttattggcca gacccaaccc ctctaacact agtctatgta ctatagtgta ctttaagtca 29400 ttaagtaaag gactttctac ttccagtcca atcaaggttg aatcccagtt acagtgaagt 29460 gaaagtgaga agtaggacat tagggtaata attagctaca gtaaacaaag aaatcatgat 29520 tccatcatga caaatacaca gtaagtgtta ggggttacat ggggctcatt gttaaaatgg 29580 ctcatcatgc tgacccatga gactgaccat cttgttcaag cgacatcctg tttttgccac 29640 tggtgcctat tttcccaaga ctacaagacc aggggggacc acacacctcc agctttctca 29700 agattatgag tgcaccctac cgtgagatac ctctgacttt ctcatgacga tgtgaccacc 29760 agagtccacc tgcaggtgaa agataaacta taaactaacc ttcccccgct tcaggaatca 29820 atttcccctc tgcagagtat aagaaggccc tgctagaagg gcgggggggc tgactcttct 29880 caaaggtcag tcagcctctc ctttttcctt ctaataaatt ttcttctctt tgcctgaaca 29940 cccaattcct tctctttttc tccatgctcc ccttacagta agaccttcag gagcacttgg 30000 gagcaggcgg ttctgtaaat tttccatacc ttcccatata ttccactttc aatagttggg 30060 ttcctattgt cctaactttc acaagaacta tccattttat ggattcatgt catattaatc 30120 actatagatt agattttgca tgccctcccg aattcatatg ttgaagccct aactccccca 30180 tgtgatgcac ttggaggtgg catctttgga ggcaaatagg tcatgtgagt gggatatcta 30240 tgagtgggat tagtgcccct ctaagaaaag ctaggagaga gcttgtctat ctttctgcct 30300 tgtgaggata cagcagaaag gcaggcacct gcaggccaag agggagcctt caccggacac 30360 aggatctgct ggcacctgga tcttggtaaa tgttatttgt aactgtatgt ctgtgagttg 30420 ctatgggcag gatgtactgg ctggtaaaca ttgtttagta ataagtggat gtggtttaca 30480 ccagatttag gagaatctag gagtggtgaa gtaaaatggg gtttggcatt caattgttga 30540 ctcattatat agctaaaata tgcctgcttg atgagcttat tgtaagagat ctcacaagtc 30600 attttgagct tcctagtatt gaggtgcttt gcacaccctc tgttagtggc ctaagaactg 30660 aagataacac atgtcttgtg ctaccaagca gtaggaggat aaatgaagtt tgctcttgag 30720 atctcagtac ctctttcatc tttcaatgca tacccttctc ctgttgttgt actctgtcct 30780 ctgcctgtaa taaagctgtt ctgttagtgt aaactgtatg ggtctccttt aactatcaaa 30840 aatgttaggt cttgtgtgat taccacctcg ctgtaatata cagcaaaaaa aaaaaaaaaa 30900 aaaaaaagga gaattcatct gcaagttctc tctcatctcc tgcttctcat tctgaaattt 30960 tcacctcagt gggggctaaa actatgcata taacagtagc attaaccaac tattacagtt 31020 tctgaggaaa cccggtcctg tgctatagta cacaatccca ttcaagtcca acagcaaaga 31080 ggtgagtctg cattgatgag gtcccaacca cagggaaaag gaagaagaca ggcaatatac 31140 tgaatgcaca catcacaatg aatcaccaca gatacttact tccaggctaa gggaaagaat 31200 atcgctagtt ccccagcact ttctcacatt ttctgtcaag ttgtaccttc cctcagataa 31260 ccaatagtct agcttatcac acaagaattt aatcgatcat tagattctaa taaattccac 31320 ttattggatt acatattcca ttatattttg gcctgattac tgtcattcaa tgttatttat 31380 ataaaattca cccttgtttt tttgtggcag taactcattc atttcaactg cagtatgata 31440 tcccattcca ctttagacca tgacttattt atacaaaata catttataca aaatacaaat 31500 ttatacaaaa taacgtaaat gttaattcac atttatgttg attttagttt agggtattag 31560 cattgttagg agtattgggt aaatgttttt agaatacata tgaaagcata ttatggtttc 31620 cttttggttg tatacctaag agaagaaatg ttagttataa tataagcaga tgtttatttt 31680 tgacaaagac tcataatact ttccgagttg cttgaaccat tttatattta ttttaacatt 31740 acctagcatt tattcagttt gcttattttt gtaaatactt ggttttacca attcttctca 31800 ttcagctatt ctggaggttg tgtcatgata tctggtattt ttaacttgca ttttccagag 31860 aaataatggt attgaacatc ttttcatata tttattgaca attaaaataa cctctttggt 31920 gaagtgccca ttcaaagata ttgcttgtgc actattatat tagttgcctt tgttattgaa 31980 atatgcccaa ttaaattttg gacaatttgg ggactggatt cagacaaaaa gttctgctgg 32040 ccatgtcagg tttgagatat taattagata ctccgatgtg gagaagaatg cagttgagca 32100 cttgaatttg gagttcagga aaaaataagt ttgaggacat acatgtggaa gcttcaagcc 32160 catgaatgga cttaaagtca tggaatcaaa caacatcaat catagtaagg ctaatcccta 32220 gggtattcca cacattgcat gtttggaaaa ggagaaaatg ccagaagaga cgatgcaaag 32280 aaataactgg tgagttaaga gggaaatcaa gggcttatca ggcttaggat ttcaaataaa 32340 atttgcgata tttcaaaaaa aaaaaaaaaa aaaaggaatg atcaactgga ccaaatagca 32400 ctgatagttt gggtaaaata aacatagatt ggtaaaatga atgttgttgg taatcttgaa 32460 aaaagtggtt accatagggg taaagctcaa tgggaacagg ttgatgagag aataccatgt 32520 gaaacaatag acacagtagt ttggacactt cctttctgat ggttaccata gagcagtcta 32580 aagtactgcc aaggatggaa ttttttcttt aaagatggga gatattatag cctaagtatg 32640 gcttgcactt gatcctgagg taaggaaaac actgaggatg caggaaagat gaaattactg 32700 ctagaaacaa gtccttgtgt aaacacaagg ggagggaatc aagtttacaa atgtaggagt 32760 tgtgtaacca agcaggaccc tgtgaagcct tcccacagtg gatccacact cttgtcctct 32820 gcctgcattt ttgtctatag aaaaacttta gtcaaagaat cagtttgatc agagaagtga 32880 gaaaatacag agaaaaagga atacagtcaa gcaagacaga gtaataatag tttagccact 32940 cagcaatgtc aaggacttat ggttcttcct catggactat agataatatg ctgagccatg 33000 tccttggaac ggttttgcag gtgctcaaac ccctaccagg cggaagaagt caactgcatg 33060 ctgcccacaa acatgtagac cccagaccag ttggaaacag aaggttgatg atgctgactc 33120 ccaattacct catcaccaaa cagttaggaa aatatccaag ggctgatcat gcccggctcc 33180 ttgaagagta agagtaagag tcctcaccac tcctccaaga ggcacacagt cctcgaagca 33240 ctagcctgct gtgtaccctc tgcctggcaa ttaaagctac ttttgccggt ttccacattt 33300 ctatttggcg tcagtgtata gaggcagccg atacttcggt gatagttggt agacagataa 33360 ttcattgcaa tgaaaaaatt actccacaga cagagacaag ggatatcaga gcttcacata 33420 agtctactct cttgggcatt agtggctaaa acaacatcct tcagctcatt ctcattgtga 33480 cactggcact ggctgctcta gtgtaagtga ggaatgtgga gccacacgga cacccatcct 33540 gcactgtgtg gggtgctatt cacattaaaa cttcaataaa accctttcaa ctgactgtgt 33600 tatagatgca aaaactgaat ctttgaaagc ctaacttata cagcaccatt tagccaccat 33660 gttacagagc cgtgattcag attttacatt ggaagccttc ttatctagga ggatcataaa 33720 atttaacatc caatgcagat acttttgaga gtgaaaggca caagtataat agttcagctg 33780 agataagagg tataaaccaa gactttctag agtatattca gcattagatc accctgcctt 33840 ttcctccatt tttgatataa tttagtactc aagtcaaagc attctactca atttcaattt 33900 ttccgtggct atttttaaca ttaataataa ctttaagtta actaataaag aggccagtta 33960 ggtttggaaa attataataa aaacaggaaa acttaaatat gcctaaggat taatttattg 34020 aaatgactgg ttaggaaatt ataattccat aaattatttt tatacatatt t 34071 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 4.6kb forward primer <400> 42 aggattacaa gattgctgtt gga 23 <210> 43 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 4.6kb reverse primer <400> 43 aaaatcgtca actaccctga tta 23 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 5.7kb forward primer <400> 44 agctgcaatg aacatgtggg tg 22 <210> 45 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 5.7kb reverse primer <400> 45 cacccacatg ttcattgcag ct 22 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 4.9kb forward primer <400> 46 cactcagtat gagagtctta g 21 <210> 47 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 4.9kb reverse primer <400> 47 ctgttcagtt cattgatgat ttc 23 <210> 48 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 5.4kb forward primer <400> 48 tttggttctg ctgtgccata a 21 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 5.4kb reverse primer <400> 49 gtagagctta gagttcaact c 21 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 5.3kb forward primer <400> 50 cactcaggat gagattctct a 21 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 5.3kb reverse primer <400> 51 aactgattga tgactactat gtt 23 <210> 52 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 4.7kb forward primer <400> 52 agatctgaca ccttctaatt ac 22 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 4.7kb reverse primer <400> 53 gtgtattcct gcacagcaac 20 <210> 54 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 4.4kb forward primer <400> 54 gtcaaactgc cttctagagt c 21 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 4.4kb reverse primer <400> 55 gtagacttat gtgaagctct g 21 <210> 56 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 promoter 5.5kb forward primer <400> 56 ggatccggct gtcgttttgt tatgatt 27 <210> 57 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 promoter 5.5kb reverse primer <400> 57 ctcgagaact aaaaggcaca gggaact 27 <210> 58 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 3' arm 4.3kb forward primer <400> 58 ctcgagttac aattcagtgt ggggaat 27 <210> 59 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> pig aS1 3' arm 4.3kb reverse primer <400> 59 gcggccgcca gctttattac aggcagagg 29 <210> 60 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> forward primer for first point mutation <400> 60 tatatactac atcttccggg tccaatcatc tgttgatgg 39 <210> 61 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for first point mutation <400> 61 ccatcaacag atgattggac ccggaagatg tagtatata 39 <210> 62 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer for second point mutation <400> 62 aagacgtggc ttgggtccca cgttgctgt 29 <210> 63 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for second point mutation <400> 63 acagcaacgt gggacccaag ccacgtctt 29 <210> 64 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for hEPO <400> 64 ggatcctgtg gtcacccggc gcgc 24 <210> 65 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for hEPO <400> 65 gatatcccat gggacaggct ggcgct 26 <210> 66 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for WPRE <400> 66 gatatctctg ttcctgttaa tcaacctc 28 <210> 67 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for WPRE <400> 67 gcggccgcga gcccgaggcg aaacag 26 <210> 68 <211> 2331 <212> DNA <213> Homo sapiens <220> <221> gene <222> (1)..(2331) <223> DNA of hEPO <400> 68 tgtggtcacc cggcgcgccc caggtcgctg agggaccccg gccaggcgcg gagatggggg 60 tgcacggtga gtactcgcgg gctgggcgct cccgcccgcc cgggtccctg tttgagcggg 120 gatttagcgc cccggctatt ggccaggagg tggctgggtt caaggaccgg cgacttgtca 180 aggaccccgg aagggggagg ggggtggggc agcctccacg tgccagcggg gacttggggg 240 agtccttggg gatggcaaaa acctgacctg tgaaggggac acagtttggg ggttgagggg 300 aagaaggttt ggggggttct gctgtgccag tggagaggaa gctgataagc tgataacctg 360 ggcgctggag ccaccactta tctgccagag gggaagcctc tgtcacacca ggattgaagt 420 ttggccggag aagtggatgc tggtagcctg ggggtggggt gtgcacacgg cagcaggatt 480 gaatgaaggc cagggaggca gcacctgagt gcttgcatgg ttggggacag gaaggacgag 540 ctggggcaga gacgtgggga tgaaggaagc tgtccttcca cagccaccct tctccctccc 600 cgcctgactc tcagcctggc tatctgttct agaatgtcct gcctggctgt ggcttctcct 660 gtccctgctg tcgctccctc tgggcctccc agtcctgggc gccccaccac gcctcatctg 720 tgacagccga gtcctggaga ggtacctctt ggaggccaag gaggccgaga atatcacggt 780 gagacccctt ccccagcaca ttccacagaa ctcacgctca gggcttcagg gaactcctcc 840 cagatccagg aacctggcac ttggtttggg gtggagttgg gaagctagac actgcccccc 900 tacataagaa taagtctggt ggccccaaac catacctgga aactaggcaa ggagcaaagc 960 cagcagatcc tacgcctgtg gccagggcca gagccttcag ggacccttga ctccccgggc 1020 tgtgtgcatt tcagacgggc tgtgctgaac actgcagctt gaatgagaat atcactgtcc 1080 cagacaccaa agttaatttc tatgcctgga agaggatgga ggtgagttcc tttttttttt 1140 tttttccttt cttttggaga atctcatttg cgagcctgat tttggatgaa agggagaatg 1200 atcgagggaa aggtaaaatg gagcagcaga gatgaggctg cctgggcgca gaggctcacg 1260 tctataatcc caggctgaga tggccgagat gggagaattg cttgagccct ggagtttcag 1320 accaacctag gcagcatagt gagatccccc atctctacaa acatttaaaa aaattagtca 1380 ggtgaagtgg tgcatggtgg tagtcccaga tatttggaag gctgaggcgg gaggatcgct 1440 tgagcccagg aatttgaggc tgcagtgagc tgtgatcaca ccactgcact ccagcctcag 1500 tgacagagtg aggccctgtc tcaaaaaaga aaagaaaaaa gaaaaataat gagggctgta 1560 tggaatacgt tcattattca ttcactcact cactcactca ttcattcatt cattcattca 1620 acaagtctta ttgcatacct tctgtttgct cagcttggtg cttggggctg ctgaggggca 1680 ggagggagag ggtgacatgg gtcagctgac tcccagagtc cactccctgt aggtcgggca 1740 gcaggccgta gaagtctggc agggcctggc cctgctgtcg gaagctgtcc tgcggggcca 1800 ggccctgttg gtcaactctt cccagccgtg ggagcccctg cagctgcatg tggataaagc 1860 cgtcagtggc cttcgcagcc tcaccactct gcttcgggct ctgggagccc aggtgagtag 1920 gagcggacac ttctgcttgc cctttctgta agaaggggag aagggtcttg ctaaggagta 1980 caggaactgt ccgtattcct tccctttctg tggcactgca gcgacctcct gttttctcct 2040 tggcagaagg aagccatctc ccctccagat gcggcctcag ctgctccact ccgaacaatc 2100 actgctgaca ctttccgcaa actcttccga gtctactcca atttcctccg gggaaagctg 2160 aagctgtaca caggggaggc ctgcaggaca ggggacagat gaccaggtgt gtccacctgg 2220 gcatatccac cacctccctc accaacattg cttgtgccac accctccccc gccactcctg 2280 aaccccgtcg aggggctctc agctcagcgc cagcctgtcc catggctcga g 2331 <210> 69 <211> 619 <212> DNA <213> Woodchuck hepatitis B virus <220> <221> misc_signal <222> (1)..(619) <223> DNA of WPRE <400> 69 tctgttcctg ttaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 60 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 120 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 180 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 240 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 300 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 360 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt 420 ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 480 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 540 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 600 cctgtttcgc ctcgggctc 619 <210> 70 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer for promoter <400> 70 ggatccggct gtcgttttgt tatgatt 27 <210> 71 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for promoter <400> 71 ggatccaact aaaaggcaca gggaact 27 <210> 72 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer for 3' arm <400> 72 gcggccgctt acaattcagt gtggggaat 29 <210> 73 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for 3' arm <400> 73 gcggccgcca gctttattac aggcagagg 29 <210> 74 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> first forward primer for aS1 hEPO <400> 74 gtgttgacaa ccatgggggt gcacggtgag tactc 35 <210> 75 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> second forward primer for aS1 hEPO <400> 75 gatatctttt cttatatagg tgttgacaac catggggg 38 <210> 76 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for aS1 hEPO <400> 76 gaattcatgg gacaggctgg cgctga 26 <210> 77 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for promoter <400> 77 gtcgacagct gcaatgaaca tgtgggtg 28 <210> 78 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for promoter <400> 78 gatatccaaa ataaaaattt aggtctgaca g 31 <210> 79 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for 3' arm <400> 79 gcggccgcat ggcatatgga agttcccagg 30 <210> 80 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for 3' arm <400> 80 ccgcggtggg aacttccata tgccat 26 <210> 81 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> neo forward primer <400> 81 gcggccgcgc gcgtcaggtg gcac 24 <210> 82 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> neo reverse primer <400> 82 cgatcggacg ctcagtggaa cgaaaactc 29 <210> 83 <211> 1902 <212> DNA <213> Artificial Sequence <220> <223> neo gene sequence <400> 83 gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct 60 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 120 attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg 180 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 240 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 300 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 360 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 420 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 480 cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac 540 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact 600 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc 660 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg 720 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg 780 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt 840 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc 900 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag 960 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg 1020 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc 1080 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt 1140 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg 1200 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt 1260 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct 1320 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg 1380 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga 1440 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag 1500 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc 1560 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg 1620 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg 1680 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc 1740 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga 1800 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 1860 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tc 1902 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for EPO <400> 84 caaggaggcc gagaatatca 20 <210> 85 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for EPO <400> 85 aagtgtcagc agtgattgtt cg 22 <210> 86 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Neo <400> 86 gctacccgtg atattgctga a 21 <210> 87 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Neo <400> 87 caacaccgtg cgttttattc t 21 <210> 88 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for human beta actin <400> 88 cgtgggccgc cctaggcacc a 21 <210> 89 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for human beta actin <400> 89 ttggccttag ggttcagggg gg 22 <210> 90 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for mouse beta actin <400> 90 tgtgatggtg ggaatgggtc ag 22 <210> 91 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for mouse beta actin <400> 91 tttgatgtca cgcacgattt tcc 23 <210> 92 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for EPO-WPRE <400> 92 aactcttccg agtctactcc a 21 <210> 93 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for EPO-WPRE <400> 93 ctcctcataa agagacagca ac 22 <210> 94 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for WPRE-3'arm <400> 94 ttcctgttaa tcaacctctg g 21 <210> 95 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for WPRE-3'arm <400> 95 taccaaaggc cataattgtg g 21

Claims (25)

  1. 프로모터, 및 3' UTR 부위를 포함하는 서열번호 1의 염기서열을 갖는 돼지 알파에스 1 카제인 유전자.
  2. 서열번호 2의 염기서열을 포함하는 돼지 알파에스 1 카제인 유전자 프로모터.
  3. 서열번호 2의 염기서열, 서열번호 3의 염기서열, 및 서열번호 4의 염기서열 중에서 선택된 하나 이상의 염기서열을 포함하는 발현 벡터.
  4. 제3항에 있어서, 상기 벡터는 서열번호 5 또는 서열번호 6 중에서 선택된 하나 이상의 염기서열을 추가로 갖는 것인 발현벡터.
  5. 제3항에 있어서, 상기 벡터는 선택적 표지 유전자, 인슐레이터, 및 WPRE(woodchuck hepatitus virus posttranscriptional regulatory element) 중에서 선택된 하나 이상이 추가된 것인 발현벡터.
  6. 제5항에 있어서, 상기 벡터는 도 2의 개열지도를 갖는 것인 발현 벡터.
  7. 제6항에 있어서, 상기 벡터는 기탁번호 KCTC 11324BP로 기탁된 pBC1-pig αS1 casein인 발현 벡터.
  8. 제3항 내지 제5항 중 어느 한 항에 있어서, 프로모터 염기서열의 3' 쪽으로 목적단백질을 암호화하는 염기서열을 추가로 갖는 발현 벡터.
  9. 제8항에 있어서, 상기 목적단백질은 인간EPO(erythropoietin)인 발현 벡터.
  10. 제9항에 있어서, 상기 벡터는 도 3의 개열지도를 갖는 것인 발현 벡터.
  11. 제10항에 있어서, 상기 벡터는 기탁번호 KCTC 11325BP로 기탁된 pBC1-pig αS1 casein+hEPO-WPRE인 발현 벡터.
  12. 제4항에 있어서, 상기 발현벡터는 낙인벡터(knock-in vector)인 발현벡터.
  13. 제12항에 있어서, 상기 낙인벡터는 선별유전자가 추가된 것인 발현 벡터.
  14. 제13항에 있어서, 상기 낙인벡터는 도 4의 개열지도를 갖는 것인 발현 벡터.
  15. 제14항에 있어서, 상기 낙인벡터는 기탁번호 KCTC 11326BP로 기탁된 Pig α S1 casein-hEPO knock in인 발현 벡터.
  16. 제3항 내지 제5항 중 어느 한 항의 발현 벡터를 도입시켜 형질전환시킨 동물의 체세포.
  17. 제3항 내지 제5항 중 어느 한 항의 발현 벡터가 주입된 동물 수정란.
  18. 제17항에 있어서, 상기 주입은 미세주입법에 의한 것인 동물 수정란.
  19. 제17항의 동물 수정란을 이식시켜 얻은 형질전환동물.
  20. 제19항에 있어서, 상기 형질전환동물은 돼지, 생쥐, 소, 양, 및 염소 중에서 선택된 하나인 형질전환동물.
  21. 제17항의 동물수정란을 대리모의 난관 내에 이식한 후, 대리모로부터 형질전환동물을 얻고, 상기 형질전환동물의 유즙으로부터 목적단백질을 제조하는 제조방법.
  22. 제3항 내지 제5항 중 어느 한 항의 발현 벡터를 도입시켜 형질전환시킨 동물의 체세포를 탈핵된 난자에 핵이식하여 만든 동물 수정란.
  23. 제22항의 수정란을 이식시켜 얻은 형질전환동물.
  24. 제23항에 있어서, 상기 형질전환동물은 돼지, 생쥐, 소, 양, 및 염소 중에서 선택된 하나인 형질전환동물.
  25. 제3항 내지 제5항 중 어느 한 항의 발현 벡터를 도입시켜 형질전환된 동물의 체세포를 탈핵된 난자에 핵이식하여 만든 동물 수정란을 대리모 동물에 이식하고, 상기 대리모 동물로부터 형질전환동물을 얻고, 상기 형질전환동물의 유즙으로부터 목적단백질을 생산하는 단계를 포함하는 단백질의 제조방법.
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