KR20090126458A - 오이 모자이크 바이러스의 감염성 클론을 이용한 식물체내에서의 외래 유전자 과발현 방법 - Google Patents

Abstract

본 발명은 오이 모자이크 바이러스(Cucumber mosaic virus; CMV)의 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법에 관한 것으로서, 1) 오이 모자이크 바이러스(Cucumber mosaic virus; CMV)의 RNA1a 유전자를 포함하는 재조합 벡터를 제조하는 단계; 2) CMV의 RNA2a 유전자와 2b 유전자를 포함하고 상기 2b 유전자의 3' 말단에 외래 유전자를 삽입할 수 있는 제한 효소 부위를 포함하는 발현벡터에 외래유전자를 삽입하여 재조합 벡터를 제조하는 단계; 3) CMV의 RNA3a 유전자와 코트 단백질(coat protein; CP)을 코딩하는 유전자를 포함하는 재조합 벡터를 제조하는 단계; 4) 상기 1), 2) 및 3) 단계에서 제조된 각각의 재조합 벡터로 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens)를 각각 형질전환하여 각각의 형질전환체를 얻는 단계; 및 5) 상기 각각의 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens) 형질전환체를 혼합하고 혼합액을 식물체에 도입하는 단계를 포함하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법을 제공한다.

오이 모자이크 바이러스(CMV), 외래 유전자 과발현, 아그로-인필트레이션

Description

오이 모자이크 바이러스의 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법{Methods for over-expression of foreign genes in plant using infectious clones of Cucumber mosaic virus}

본 발명은 오이 모자이크 바이러스(Cucumber mosaic virus; CMV)의 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법에 관한 것이다.

일반적으로 외래 유전자가 다른 세포에 삽입되어 하나의 독립적인 복제가능단위(replicon)를 형성하거나 혹은 동종 재조합 (homologous recombination)에 의해 도입된 세포의 게놈 속으로 통합(integration)되어 계속적인 복제가 가능하고, 유전자 발현이 됨으로써 새로운 형질을 나타내도록 하는 것을 형질전환이라고 한다. 이와 같이 외래 유전자의 도입이 가능해 짐에 따라 원하는 유전자를 원하는 세포에서 대량으로 발현할 수 있게 되었으며, 이러한 재조합 DNA 기술(recombinant DNA technology)은 현대 유전공학 발전의 핵심이라 할 수 있다. 예를 들어 원하는 단백질을 대량으로 생산하기 위하여 대장균 등의 박테리아에 원하는 유전자를 도입하거나 동물 또는 식물 세포에 도입하는 방법, 동물 또는 식물 세포가 특정 병원체에 저항성을 갖도록 하는 유전자를 도입하여 형질전환체를 제조하는 방법, 유용 물 질을 생산하는 세포의 대사 조절을 통해 보다 많은 산물을 생산하도록 하기 위하여 조절 유전자를 삽입하는 방법 또는 질병의 치료를 위해 목적하는 유전자를 포함하는 벡터로 세포를 형질전환시키는 방법 등이 널리 이용되고 있다.

이 중 식물 세포의 형질전환에 이용되는 대표적인 방법은 아그로박테리움(Agrobacterium)을 이용하는 방법이다. 아그로박테리움은 Ti 플라스미드(tumour inducing plasmid)를 포함하고 있는데, 일단 이 세균이 식물 세포와 접촉하면 약 23kb 크기의 T-DNA라고 불리는 단편이 Ti 플라스미드로부터 식물 세포의 식물의 염색체 중 임의의 위치로 통합되어 들어가게 된다. T-DNA가 식물 세포 게놈으로 통합되는 데에는 상기 T-DNA의 양쪽 끝에 위치한 약 23 bp 크기의 두 개의 경계부위 (왼쪽 보더(left border), 오른쪽 보더(right border))가 중요한 역할을 한다. 이러한 성질을 이용하여 Ti 플라스미드에서 T-DNA의 통합에 필수적인 왼쪽 및 오른쪽 경계부위만을 남겨두고 나머지 T-DNA 대신에 목적하는 외래유전자를 대체시켜 형질전환용 재조합 플라스미드를 제작하고, 이를 아그로박테리움에 도입한 후 숙주식물과 아그로박테리움을 공동배양하는 방법으로 식물의 형질전환에 이용한다.

상기와 같이 아그로박테리움을 사용하는 경우에는 재조합 플라스미드로 형질전환된 애그로박테리움과 식물체 절편을 공동 배양함으로써 상처 부위 등을 통해 재조합 플라스미드가 식물 세포로 전이되게 된다. 아그로-인필트레이션(Agro-infiltration) 방법도 이 중 하나이다. 이외에도 T-DNA 부위를 포함하지 않는 벡터를 이용할 수도 있는데, 이 경우에는 외래 유전자의 도입을 위해 전기 천공법 (electroporation), 미세입자 충격법(microparticle bombardment) 또는 폴리에틸렌 글리콜 침전법(polyethylene glycol-mediated uptake) 등의 방법이 식물 세포를 형질전환시키는 데 이용될 수 있다. 그러나, 이러한 방법에 의해 모든 식물 세포의 형질전환을 효율적으로 이룰 수 있는 것은 아니다. 일반적으로 식물 세포는 동물 세포에 비해 형질전환 효율이 낮으며, 이용될 수 있는 숙주세포도 제한되는 것으로 알려져 있다.

또한 이처럼 외래유전자를 도입하여 신기능의 작물을 개발함에 있어서 외래유전자 도입에 따른 효과를 가시적으로 얻기 위해서는 도입유전자의 발현량을 일정 이상 증대시켜야 할 뿐만 아니라, 도입유전자의 발현을 안정적으로 유지시켜야 한다. 그러나 유전자 침묵현상(gene silencing)과 같은 문제가 발생하여 유용한 도입유전자의 안정적인 발현을 안정적으로 증대시키기 곤란하다는 문제가 빈번하게 발생하고 있다. 어떤 식물 바이러스는 유전자 침묵현상을 차단하는 단백질을 암호하는 것으로 알려져 있는데, 포티 바이러스(poty virus)의 HC-Pro(helper component-proteinase) 단백질은 유전자침묵현상이 유지되는 것을 방해하고, 오이 모자이크 바이러스의 2b 단백질은 새로 생겨나는 잎에 유전자 침묵현상이 일어나는 것을 차단한다. 또한 감자바이러스 X의 p25 단백질은 유전자침묵현상이 식물체 전체로 퍼지는 것을 차단하는 것으로 알려져 있다(The Plant Journal (2002), 29(5), 555~567).

오이 모자이크 바이러스(Cucumber mosaic virus; CMV)는 1916년 처음으로 보고되었다(Doolittle, S, The mosaic diseases of cucurbits, U.S.D.A. Agri. Bull (1961) 879:69). CMV는 직경 28-30nm인 공모양 3분절 게놈의 다입자성 바이러스로 서, 오이(Cucumis sativus) 등에서 잎에 모자이크 증상을 나타내고, 과실은 작아지고 기형이 되어 수확량은 물론 상품가치가 크게 떨어지게 한다. CMV는 매우 넓은 범위의 숙주 범위를 가지며, 세계 전역에 분포되어 있다.

한국특허등록번호 10-679715호(등록일 2007. 01. 31)는 한국산 오이모자이크 바이러스 유래 2b유전자를 이용한 식물체내에서의 외래 유전자의 발현을 증대시키는 방법에 관한 것으로서, 보다 상세하게는 서열번호 1로 기재되는 염기서열로 이루어지는 유전자를 식물체 내에서 발현시키면 그 식물체에 동시에 도입된 다른 유용한 유전자의 발현을 증대시켜 유용유전자의 발현에 의한 단백질의 발현량, 발현기간 및 안정성이 증대 되도록 하는 한국산 오이모자이크 바이러스 유래 2b유전자를 이용한 식물체내에서의 외래 유전자의 발현을 증대시키는 방법이 개시되어 있다.

따라서 본 발명자들은 오이 모자이크 바이러스의 감염성 클론을 식물체에 도입하여 감염성 CMV 활성이 재구성됨을 확인하였다. 또한 CMV 감염성 클론을 이용하여 외래 유전자를 과량 발현할 수 있는 벡터를 개발하였는데, 이는 외래 유전자를 식물체에 전달하여 식물체 전신에서의 발현이 가능하도록 제작되었다. 이를 검증하기 위해 CMV 과발현 벡터에 GFP 유전자를 도입하고, 아그로-인필트레이션(Agro-infiltration)을 통해 식물체에 전달하여, 식물체 내에서 GFP 단백질이 발현됨을 확인하고 본 발명을 완성하였다.

본 발명은 오이 모자이크 바이러스(Cucumber mosaic virus; CMV)의 감염성 클론을 이용하여 식물체 내에서 외래 유전자를 과발현할 수 있는 벡터를 제조하고 외래유전자를 과발현할 수 있는 방법을 제공하는 것을 목적으로 한다.

상기한 목적을 달성하기 위해서, 본 발명은 1) 오이 모자이크 바이러스(Cucumber mosaic virus; CMV)의 RNA1a 유전자를 포함하는 재조합 벡터를 제조하는 단계; 2) CMV의 RNA2a 유전자와 2b 유전자를 포함하고 상기 2b 유전자의 3' 말단에 외래 유전자를 삽입할 수 있는 제한 효소 부위를 포함하는 발현벡터에 외래유전자를 삽입하여 재조합 벡터를 제조하는 단계; 3) CMV의 RNA3a 유전자와 코트 단백질(coat protein; CP)을 코딩하는 유전자를 포함하는 재조합 벡터를 제조하는 단계; 4) 상기 1), 2) 및 3) 단계에서 제조된 각각의 재조합 벡터로 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens)를 각각 형질전환하여 각각의 형질전환체를 얻는 단계; 및 5) 상기 각각의 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens) 형질전환체를 혼합하고 혼합액을 식물체에 도입하는 단계를 포함하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법을 제공한다. 상세하게는 상기 4 단계 이후에 5) 상기 각각의 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens) 형질전환체를 배양하고 원심분리하여 현탁하는 단계; 6) 상기 각각의 현탁액을 같은 부피비율로 혼합하는 단계; 7) 상기 혼합액을 식물체에 인필트레이션(infiltration)하는 단계; 및 8) 상기 인필트레이션(infiltration)된 식물체에서 상기 외래유전자의 발현을 확인하는 단계를 포함하는 것을 특징으로 한다. 보다 상세하게는 상기 식물체는 니코티아나 벤타미아나(Nicotiana benthamiana)인 것을 특징으로 한다.

본 발명의 제 1의 양태는 1) 오이 모자이크 바이러스(Cucumber mosaic virus; CMV)의 RNA1a 유전자를 포함하는 재조합 벡터를 제조하는 단계; 2) CMV의 RNA2a 유전자와 2b 유전자를 포함하고 상기 2b 유전자의 3' 말단에 외래 유전자를 삽입할 수 있는 제한 효소 부위를 포함하는 발현벡터에 외래유전자를 삽입하여 재조합 벡터를 제조하는 단계; 3) CMV의 RNA3a 유전자와 코트 단백질(coat protein; CP)을 코딩하는 유전자를 포함하는 재조합 벡터를 제조하는 단계; 4) 상기 1), 2) 및 3) 단계에서 제조된 각각의 재조합 벡터로 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens)를 각각 형질전환하여 각각의 형질전환체를 얻는 단계; 및 5) 상기 각각의 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens) 형질전환체를 혼합하고 혼합액을 식물체에 도입하는 단계를 포함하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법을 제공한다. 상세하게는 상기 4 단계 이후에 5) 상기 각각의 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens) 형질전환체를 배양하고 원심분리하여 현탁하는 단계; 6) 상기 각각의 현탁액을 같은 부피비율로 혼합하는 단계; 7) 상기 혼합액을 식물체에 인필트레이 션(infiltration)하는 단계; 및 8) 상기 인필트레이션(infiltration)된 식물체에서 상기 외래유전자의 발현을 확인하는 단계를 포함하는 것을 특징으로 한다. 보다 상세하게는 상기 식물체는 니코티아나 벤타미아나(Nicotiana benthamiana)인 것을 특징으로 한다.

보다 상세하게는 상기 1) 단계에서 제조되는 상기 재조합 벡터는 서열번호 1 또는 서열번호 4인 것을 특징으로 하고, 상기 2) 단계의 상기 발현벡터는 서열번호 5인 것을 특징으로 하고, 상기 3) 단계에서 제조되는 상기 재조합 벡터는 서열번호 3 또는 서열번호 6인 것을 특징으로 하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법을 제공한다.

상기 CMV의 유전체(genome)는 3개의 양성 센스 단일 가닥(positive-sense single-stranded) RNA들(RNA1, RNA2 및 RNA3)과 RNA3으로부터 파생된 RNA4가 있다. RNA1 및 RNA2는 병원성을 지니고 있고, 바이러스 복제와 관련된 각각 1a 및 2a 단백질을 코딩하고 있다. RNA3은 3a 단백질과 코트 단백질(coat protein; CP)을 코딩하고 있다. 3a 단백질은 세포 대 세포 이동 기능과 관련되어 있다. 코트 단백질은 RNA3으로부터 전사된 서브게놈(subgenome) RNA(RNA4)로부터 번역된다. 또한 CMV RNA2의 3'말단에 있는 2b 유전자가 암호하는 2b 단백질은 유전자 침묵현상(gene silencing)를 저해한다.

상기 '유전자 침묵 현상(gene silencing)'은 식물체 내에 외래 유전자가 도 입되면 그 유전자가 과도하게 축적되는 것을 막아 발현되지 않는 현상이며, 가장 대표적인 현상은 침묵유전자의 RNA 전사체는 만들어지지만 RNA가 세포 내에 축적되지 않는 전사 후 유전자 침묵현상(post-transcriptional gene silencing; PTGS)를 예로 들 수 있다.

상기 외래유전자는 식물체 내에서 발현되기를 원하는 유용 유전자로서, 이에는 제초제 저항성 유전자, 질병 저항성 유전자, 살충제 저항성 유전자, 곡식 또는 과실의 품질에 영향을 주는 유전자, 스크리닝 가능한 표식 유전자, 환경 또는 스트레스 저항성 유전자 등이 포함될 수 있다. 보다 상세하게는 제초제 글리포세이트(Glyphosate)의 저항성 유전자인 5-에놀피루빌시키메이트 3-포스페이트 신타아제(5-Enolpyruvylshikimate 3-phosphate synthase; EPSP synthase), 제초제 비알라포스(바스타)(bialaphos(Basta))의 저항성 유전자인 포스피노트리신 아세틸트랜스퍼라제(phosphinothricin acetyltransferase), 바이러스 저항성 단백질인 코트 단백질(coat protein)을 코딩하는 유전자, 해충 저항성 유전자인 바실러스 투린지엔시스(Bacillus thuringiensis)로부터 분리한 Bt 독소(Bt toxin) 생성 유전자, 과실 완숙 방지를 위한 1-아미노사이클로프로판-1-카르복실릭산 디아미나아제(1-aminocyclopropane-1-carboxylic acid(ACC) deaminase) 유전자, 바이러스 저항성 식물을 용이하게 선발할 수 있는 식물체 세포의 괴사유발 유전자인 INF1(Phytophthora infestans elicitin gene) 유전자 등이다.

본 발명에서 사용되는 '아그로-인필트레이션(Agro-infiltration)'라는 용어는 식물체 내로 유전자를 도입하여 발현시키거나 식물체 내에서 원하는 단백질을 생산하기 위한 방법을 의미한다. 이동시키고자 하는 유전자를 가진 아그로박테리움의 현탁액을 바늘이 없는 주사기를 사용해서 압력을 줌으로써 식물의 잎에 주입하여 식물체 세포로 원하는 유전자를 이동시키는 방법이다. 이는 전통적인 식물 형질전환과 비교하면 신속하고 편리하다는 장점이 있다.

본 발명에서 사용되는 '벡터'라는 용어는 또다른 핵산을 그것에 결합시켜 이송시킬 수 있는 핵산 분자를 의미한다. 바람직한 벡터는 그것이 결합된 핵산을 자기 복제 및 발현시킬 수 있는 벡터이다.

본 발명에서 사용되는 '발현벡터'란 용어는 상기 벡터에 의해 운반되는 각 재조합형 유전자에 의해 암호화되는 단백질을 합성시킬 수 있는 플라스미드, 코스미드 또는 파아지를 포함한다.

본 발명에서 사용되는 '형질전환'이란 용어는 외래 DNA 또는 RNA가 세포에 흡수되어 세포의 유전형이 변화되는 것을 말한다. 숙주세포는 이에 제한되는 것은 아니나, 식물세포, 원핵세포, 효모세포, 곤충세포를 포함한다.

이하, 실시예에 의하여 본 발명을 더욱 상세히 설명하고자 한다.

단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.

< 실시예 1 > 오이 모자이크 바이러스의 감염활성을 식물체에서 재현시킬 수 있는 재조합 벡터의 설계 및 제조

오이 모자이크 바이러스(Cucumber mosaic virus; CMV)의 감염성 클론을 운반하기 위한 벡터로서 pSNU1 벡터를 이용하였다(도 1). pSNU1 벡터는 T-DNA의 왼쪽 보더(left border), 꽃양배추 모자이크 바이러스(Cauliflower mosaic virus; CaMV)의 더블 35S 프로모터, 멀티플 클로닝 사이트(multiple cloning site; MCS; EcoRI, XbaI, StuI, NcoI, BamHI, KpnI, SacI, MluI, XhoI, SrfI, SmaI, SpeI), cis-cleaving ribozyme sequence(Rz), NOS terminator(NOS poly-A), T-DNA의 오른쪽 보더(right border)를 순서대로 포함하고 있다. 따라서 아그로박테리움(Agrobacterium)에 형질전환하여 아그로-인필트레이션(Agro-infiltration)을 통해 식물에 원하는 유전자를 전달할 수 있다. 또한 pSNU1 벡터는 카나마이신(Kanamycin) 저항성 유전자를 포함하고 있다.

T-DNA를 바탕으로 한 아그로-인필트레이션(Agro-infiltration)이 가능한 CMV 감염성 클론을 제작하기 위해, 국내 포장의 팥으로부터 분리한 CMV 분리체(isolate)을 이용하였다. 해당 CMV 분리체(isolate)에 의해 감염된 팥으로부터 전체 RNA를 추출하였다. 이 전체 RNA로부터 프라이머 (5'- GGAATTCTGGTCTCCTTTTRGAGRCC-3'(서열번호 7) 혹은 5'-AGGGATCCTGGTCTCCTTTTRGAGRCC-3'(서열번호 8))를 이용하여 역전사 반응을 통해 CMV RNA1, RNA2, RNA3에 대한 cDNA를 합성하였다. 합성한 cDNA로부터 CMV RNA1을 증폭하기 위해 프라이머 (5'-AGGATCCGTTTATTTACAAGAGCGTACGG-3'(서열번호 9)와 5'-AGGGATCCTGGTCTCCTTTTRGAGRCC-3'(서열번호 10))을 이용하여 PCR 반응을 실시하였다. CMV RNA2를 증폭하기 위해서는 프라이머 (5'-AGGGATCCGTTTATTTACAAGAGCGTACGG-3'(서열번호 11)와 5'-GACTAGTGGTCTCCTTTTRGAGRCC-3'(서열번호 12))를, CMV RNA3를 증폭하기 위해서는 프라이머 (5'-AGGGATCCGTAATCTTACCACTGTG-3'(서열번호 13)와 5'-AGGGATCCTGGTCTCCTTTTRGAGRCC-3'(서열번호 14))을 이용하였다. 각 프라이머들을 이용하여 94℃에서 3분간 변성시킨 후, 94℃에서 30초, 55℃에서 30초, 72℃에서 3분을 1주기로 35주기 반복한 후, 72℃에서 10분간 반응시켜 CMV 각 RNA의 염기서열을 함유한 DNA 절편을 증폭하였다.

pSNU1 벡터에 클로닝하기 위해 CMV RNA1 PCR 산물을 BamHI으로 처리하여 벡터의 해당 위치에 클로닝하였으며 CMV RNA2의 경우 BamHI과 SpeI, CMV RNA3의 경우 BamHI 제한 효소를 이용하여 클로닝하였다. 그 결과 얻게된 CMV RNA1이 클로닝 된 벡터를 pCkpaR1(서열번호 1), CMV RNA2가 클로닝 된 벡터를 pCkpaR2(서열번호 2), CMV RNA3이 클로닝 된 벡터를 pCkpaR3(서열번호 3)이라 명명하였다(도 2).

pCkpaR1(서열번호 1), pCkpaR2(서열번호 2), pCkpaR3(서열번호 3) 유전자를 포함하는 균주는 2008년 5월 21일 농업생명공학연구원에 각각 기탁번호 KACC 95081P, KACC 95082P, KACC 95083P로 기탁되었다.

< 실시예 2 > 아그로 - 인필트레이션(Agro-infiltration)을 통한 감염성 클론의 접종 및 감염성 확인

각 클론의 플라스미드 DNA를 이용하여 전기충격(electroporation) 방법을 통해 아그로박테리움 균주(Agrobacterium strain) GV2260을 형질 전환하였다. pCkpaR1, pCkpaR2 및 pCkpaR3로 각각 형질 전환된 아그로박테리움(Agrobacterium)을 5ml의 YEP 액체 배지 (50mg/ml의 카나마이신(kanamycin)과 50mg/ml의 리팜피신(rifampicin) 포함)에서 30℃의 조건에서 16시간 동안 진탕배양한 후 1차 배양액 1ml을 50ml의 새 YEP 배지 (50mg/ml의 카나마이신(kanamycin), 50mg/ml의 리팜피신(rifampicin)과 20uM의 아세토시린존(acetosyringon) 포함)에 접종하여 30℃의 조건에서 6시간 동안 진탕배양하였다. 배양액을 4800G에서 10분간 원심분리하여 아그로박테리움(Agrobacterium)을 침전시키고 MMA 인필트레이션(infiltration) 버퍼 (MS salts, 10mM MES, pH5.6, 200uM 아세토시린존(acetosyringon))에 600nm의 파장에서 O.D. 1의 농도로 다시 현탁시켰다. 그 다음 현탁액을 30℃의 조건에서 2시간 동안 진탕배양하였다. pCkpaR1, pCkpaR2 및 pCkpaR3로 각각 형질 전환된 아그로박테리움(Agrobacterium) 배양액을 같은 비율로 섞은 후 니코티아나 벤타미아나(Nicotiana benthamiana)의 잎의 배축면에 1ml 주사기를 이용하여 압력으로 인플트레이션(infiltration) 하였다. 아그로-인필트레이션(Agro-infiltration) 후 5-8 일 후 RT-PCR 및 병징의 발달 관찰을 통해 CMV DNA 클론의 감염성을 확인하였다(도 3). 그 결과, CMV 감염성 클론을 아그로-인필트레이션(Agro-infiltration)을 통해 니코티아나 벤타미아나(Nicotiana benthamiana) 식물에 접종한 후 7일 후 상엽으로부터 CMV에 의해 나타나는 병징을 관찰할 수 있었다(도 4).

또한 CMV 감염성 클론을 아그로-인필트레이션(Agro-infiltration)을 통해 니코티아나 벤타미아나(Nicotiana benthamiana) 식물에 접종한 후 7일 후 상엽으로부터 전체 RNA를 추출한 후 CMV RNA3에 특이적인 프라이머 (5'-TCCCTGTTGAGCCCCCTTACTTT-3'(서열번호 15) 와 5'-CAACTCAGATCCCGCCACAGA-3'(서열번호 16))를 이용하여 RT-PCR을 수행하여 젤 전기영동(gel electrophoresis)을 통해 PCR 산물을 확인하였다. PCR 조건은 94℃에서 2분간 변성시킨 후, 94℃에서 20초, 60℃에서 30초, 72℃에서 1분을 1주기로 35주기 반복한 후, 72℃에서 10분간 반응시켰다.

확인결과 CMV 감염성 클론을 접종한 식물에 대해서 예상되는 크기의 PCR 산물을 확인할 수 있었으며, 따라서 해당 식물이 CMV에 의해 전신 감염(systemic infection)이 되었음을 확인할 수 있었다(도 5).

< 실시예 3 > 오이 모자이크 바이러스의 감염성 클론을 이용하여 식물체에서 외래 유전자를 과발현시킬 수 있는 재조합 벡터의 설계 및 제조

운반 벡터로는 실시예 1과 같은 pSNU1 벡터를 사용하였다(도 1).

T-DNA를 바탕으로한 아그로-인필트레이션(Agro-infilration)을 통해 외래 유전자의 과발현이 가능한 벡터를 제작하기 위해 CMV Fny 및 Y 균주을 이용하였다. 해당 바이러스를 증폭하기 위해 CMV Fny RNA1의 경우 프라이머(5'-GGAATTCGTTTATTTACAAGAGCGTACGG-3'(서열번호 17)와 5'-AGGGATCCTGGTCTCCTTTTRGAGRCC-3'(서열번호 18))를 이용하여 PCR 반응을 실시하였다. CMV Y 균주 RNA2를 증폭하기 위해서는 프라이머(5'-AGGGATCCGTTTATTTACAAGAGCGTACGG-3'(서열번호 19)와 5'-AGGGATCCTGGTCTCCTTTTRGAGRCC-3'(서열번호 20))를, CMV Fny RNA3를 증폭하기 위해서는 프라이머(5'-GGAATTCGTAATCTTACCACTGTG-3'(서열번호 21)와 5'-GGAATTCTGGTCTCCTTTTRGAGRCC-3'(서열번호 22))를 이용하였다. pSNU1 벡터에 클로닝하기 위해 CMV Fny RNA1 PCR 산물을 EcoRI과 BamHI으로 처리하여 벡터의 해당 위치에 클로닝하였으며 CMV Y RNA2의 경우 BamHI, CMV Fny RNA3의 경우 EcoRI 제한 효소를 이용하여 클로닝하였다. CMV의 RNA2의 경우 서브게노믹 프로모터(subgenomic promoter)로부터 2b 유전자를 발현한다. 2b 유전자는 유전자 침묵 현상을 억제하는 침묵현상 억제자(silencing suppressor)의 기능을 갖는 것으로 알려져 있다. 또한 이전 연구에 따르면 2b 유전자의 발현을 억제하더라도 바이러스의 전신성(systemic) 감염에는 영향을 주지 않는 것으로 보고되었다. 따라서 2b 유전자의 서브게노믹 프로모터(subgenomic promoter)를 이용하여 외래 유전자를 발현하는 구조체를 만들기 위해서 2b 유전자에 위치 지정 돌연변이(site-directed mutagenesis)를 통해 종결 코돈(stop codon)과 외래 유전자를 삽입하기 위한 클로 닝 제한효소 사이트 (StuI과 MluI)를 삽입하였다. 또한 2b 유전자 부분에 GFP를 삽입하기 위해 PCR을 통해 GFP 유전자 (프라이머, 5'-CTAGTAAAGGAGAAGAACTTTTCAC-3'(서열번호 23) 및 5'-ATCATTTGTATAGTTCATCCATGC-3'(서열번호 24))를 증폭한 후 2b 유전자 부분의 StuI 클로닝 사이트에 삽입하여 바이러스의 전신성(systemic) 감염을 통해 식물체 내에서 GFP의 발현이 가능하도록 제작하였다. 그 결과 얻게 된 CMV Fny RNA1이 클로닝 된 벡터를 pCfnyR1(서열번호 4), 종결 코돈(stop codon)을 도입한 CMV Y RNA2가 클로닝 된 벡터를 pCYR2△2b(서열번호 5), pCYR2△2b에 GFP를 삽입한 벡터를 pCYR2△2b-GFP(서열번호 25), CMV Fny RNA3이 클로닝 된 벡터를 pCfnyR3(서열번호 6)이라 명명하였다(도 6).

CMV RNA1,2,3을 증폭하기 위한 PCR 조건은 94℃에서 3분간 변성 후, 94℃에서 20초, 60℃에서 30초, 72℃에서 3분을 1주기로 35주기 반복한 후, 72℃에서 10분간 반응하였다. GFP 절편을 증폭하기 위한 PCR 조건은 94℃에서 3분간 변성 후, 94℃에서 20초, 55℃에서 30초, 72℃에서 1분을 1주기로 35주기 반복한 후, 72℃에서 10분간 반응하였다.

< 실시예 4 > 아그로 - 인필트레이션(Agro-infiltration)을 통한 접종 및 유전자 발현 확인

각 클론의 플라스미드 DNA를 이용하여 전기충격(electroporation) 방법을 통해 아그로박테리움 균주(Agrobacterium strain) GV2260을 형질 전환하였다. pCfnyR1, pCYR2△2b-GFP 및 pCfnyR3로 각각 형질 전환된 아그로박테리 움(Agrobacterium)을 실시예 2와 동일한 방법으로 인필트레이션(infiltration) 하였다(도 3). 상기 벡터들로 각각 형질 전환된 아그로박테리움 균주들을 아그로-인필트레이션(Agro-infiltration)을 통해 니코티아나 벤타미아나(Nicotiana benthamiana) 식물에 접종한 후 6일 후, 상엽으로부터 CMV에 의해 나타나는 병징을 관찰할 수 있었다. 또한 상엽을 형광 현미경(Carl Zeiss DE/Axioplan II )으로 관찰한 결과 GFP가 강하게 발현됨을 확인하였다(도 7).

본 발명은 오이 모자이크 바이러스의 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법에 관한 것이다. 식물체 내의 외래유전자 도입에 따른 효과를 가시적으로 얻기 위해서는 도입유전자의 발현량을 일정 이상 증대시켜야 할 뿐만 아니라, 도입유전자의 발현을 안정적으로 유지시켜야 한다. 그러나 유전자 침묵현상(gene silencing)과 같은 문제가 발생하여 유용한 도입유전자의 안정적인 발현을 안정적으로 증대시키기 곤란하다는 문제가 빈번하게 발생하고 있다. 본 발명은 유전자 침묵현상을 제해하는 CMV 2b 단백질을 코딩하는 2b유전자를 활용하여 식물체 내에서 유용단백질을 과발현할 수 있는 시스템을 개발함으로써, 유용단백질을 식물체 내에서 간편하고 저비용으로 대량 생산하는 분자농업(molecular farming)에서 유용하게 활용될 수 있다. 또한 이러한 활용도가 높은 식물 과발현 시스템 개발은 식물의 발달, 분화에 관여하는 여러 가지 현상을 규명하거나 유용유전자의 도입을 통한 신 기능성 품종을 개발하는 기술로 활용될 수 있다.

도 1은 pSNU1 벡터의 구조 모식도를 나타낸다.

멀티플 클로닝 사이트(multiple cloning site) 부분:1-68bp, cis-cleaving 리보자임 서열(Rz):69-119bp, NOS 폴리-A:184-436bp, T-보더(오른쪽):474-499bp, pVS1 sta:1540-2540bp, pVS1 rep:3223-4223bp, pBR322 bom:4543-4803bp, pBR322 ori:4943-5223bp, 카나마이신(Kanamycin) 저항 유전자(aadA):5514-6308bp, T-보더(왼쪽):6733-6758bp, 꽃양배추 모자이크 바이러스(Cauliflower mosaic virus; CaMV) 35S 듀플리케이트 프로모터:6877-7628bp

도 2는 오이 모자이크 바이러스(Cucumber mosaic virus; CMV)의 감염성 클론의 구조 모식도를 나타낸다. A) CMV RNA1이 클로닝된 pCkpaR1, B) CMV RNA2가 클로닝된 pCkpaR2, C) CMV RNA3이 클로닝된 pCkpaR3; CP: 코트 단백질(coat protein).

도 3은 아그로-인필트레이션(agro-infiltration)을 통한 감염성 클론의 접종 방법에 대한 모식도를 나타낸다.

도 4는 CMV에 감염된 니코티아나 벤타미아나(Nicotiana benthamiana) 식물체의 병징 발현 사진을 나타낸다.

도 5는 RT-PCR 기법을 이용하여 CMV 감염성 여부를 확인한 젤 사진이다.

레인 1: 사이즈 마커(size marker), 레인 2: 대조구(mock), 레인 3: 아그로-인필트레이션(agro-infiltration)을 통해 CMV 감염성 클론이 접종된 실험구(상엽), 레인 4: 아그로-인필트레이션(agro-infiltration)을 통해 CMV 감염성 클론이 접종된 실험구(접종된 잎)

도 6은 CMV의 감염성 클론을 이용한 외래 유전자 과발현 벡터의 구조 모식도를 나타낸다. A) CMV Fny RNA1이 클로닝된 pCfnyR1, B) 한 CMV Y RNA2가 클로닝된 pCYR2△2b, C) pCYR2△2b에 GFP를 삽입한 pCYR2△2b-GFP; D) CMV Fny RNA3이 클로닝된 pCfnyR3, CP: 코트 단백질(coat protein).

도 7은 형질전환된 니코티아나 벤타미아나(Nicotiana benthamiana) 식물체에서 GFP 유전자가 과발현되는 것을 보여준다.

<110> Seoul National University Industry Foundation <120> Methods for over-expression of foreign genes in plant using infectious clones of Cucumber mosaic virus <160> 25 <170> KopatentIn 1.71 <210> 1 <211> 10993 <212> DNA <213> Artificial Sequence <220> <223> pCkpaR1 <400> 1 gaattctcta gaaggcctcc atggggatcc gtttatttac aagagcgtac ggttcaatcc 60 ctgcctcctc tgtaaaatta ccctttgaaa acctctcttt tctaatcttt tcttagtaat 120 tcctatggcg acgtcctcgt tcagtatcaa tgaattggta gcctcccacg gcgataaagg 180 actactcgcg accgccctcg ttgataaaac agctcatgag cagctcgagg agcaattaca 240 acatcaacgt agaggccgta aggtctacat tcggaacgtt ctgagtgtaa aggattccga 300 agtcatccgg aatcggtatg gaggaaagta cgacctccat cttacccagc aggagtttgc 360 tccccacggc ctagctggtg ccctccgctt gtgtgaaact ctcgattgtc tagactcttt 420 cccttcatcg ggtctgcggc aggacctcgt cttagacttc ggaggaagtt gggtcacaca 480 ttacctccgc ggacacaacg tacactgctg ttccccttgt ttgggtatcc gcgataagat 540 gcgccacgcg gaacgtttaa tgaacatgcg caagatcatc ttgaacgatc cacaacagtt 600 cgatggtcga cagccggatt tttgcactca tccagctgct gattgcaaag tacaagctca 660 ctttgctata tctattcatg gaggttatga tatgggcttt agaggattat gtgaggcgat 720 gaacgctcac ggaaccacta ttttgaaggg aacgatgatg ttcgatggtg cgatgatgtt 780 tgacgaccaa ggcacgatac ccgaacttaa ttgccagtgg agaaaaatta ggagcgcatt 840 ctctgaagct gaagacgtca catctctgtc tggtaaaatt gattcctctg gattcaccgg 900 tgtgcgtaag ttcaagactt tggttgcttt tgattttatc aatgagtcta ccatgtcgta 960 cgttcatgat tggaagaata taaaatcctt tctaacggac cagacttact cgtataaagg 1020 gatgacttat ggcattgaac gatgtgtcat taatgcgggt atcatgacgt ataagataat 1080 cggtgtgcct gggatgtgtc cgcccgaact cattcgacat tgtatttggt tcccctctat 1140 taaagattat gttggtttaa agattcccgc gtcgcaggac ttggttaagt ggaaaacagt 1200 gcgtgtttta atgtcaacat tacgtgaacc agaacagatt gctatgaggt gttacaacga 1260 taagaaagcg tggatggaac aattcaaggt tatcctgggt gttttgtctg cgaaatcatc 1320 taccattgtc attaatggta tgtccatgca atctggtgag cgtatagata ttaatgatta 1380 ccattatatt ggtttcgcta ttcttctgca cacaaaaatg aagtatgaac aactgggaaa 1440 aatgtatgat atgtggaatg cttcgagcat atcgaagtgg tttgcagcgt tgactcgacc 1500 gctgcgtgtg tttttctcca gtgttgttca cgcactgttc ccgactttga gaccccgcga 1560 ggaaaaagaa ttcctgatta agctctccac cttcgttact tttaatgaag agtgctcatt 1620 tgatggtgga gaggaatggg acgttatatc atctgctgca tatgttgcta cgcaggctgt 1680 taccgatggg aagattttgg ctacgcagaa agctgagaag cttgctgaga agcttgcaca 1740 acccgtgatt gaggtatcgg acagacctga ggtgtcatct ccaacacctg atgatacagc 1800 tgatgtttgt ggaaaagagc aagaagtttc ggaacttgac tctttatcag ctcagacacg 1860 ttcccccatc accagaattg ctgaaagggc tactgctatg ttagagtatg ccgcttatga 1920 gaaacagtta cacgacacca cagtgtctaa tctgaaacgt atttggaaca tggcgggcgg 1980 tgatgataag agaaattccc ttgagggtaa tctgaagttc gttttcgata cgtatttcac 2040 tgttgatccc atggtgaata ttcatttctc cacgggtcgg tggatgcgtc ctgtgcccga 2100 agggattatc tattctgttg gttataatga acgcggttta ggtccgaagt cggatggaga 2160 gctttacatt gtcaacagag agtgcgtgat ttgcaatagc gagtctttgt ctgctatcac 2220 tcgttctctt caggccccga ccggaaccat tagtcaagtc gatggggttg ccggttgcgg 2280 gaagacaacg gcaataaaat ccatttttga gccatccact gacatgatcg ttaccgcgaa 2340 caagaaatcc gcccaagatg ttcgtatggc actgtttaaa tcatcggatt ccaaagaagc 2400 ttgcaccttt gttcgtaccg ccgattctgt cctacttaat gaatgtccga ctgttagtag 2460 ggttttaatt gatgaggttg tgctgcttca ctttggtcaa ttgtgtgccg tgatgtctaa 2520 gctgaaggct gtgcgggcta tatgtttcgg ggattcggag caaattgcct tttcttctcg 2580 cgacgcctcg tttgacatgc gtttccctaa gattattcct gatgaaacta gtgatgcgga 2640 tactacattc cgtagcccac aagacgttgt gccgcttgtg cgtttaatgg ctacgaaggc 2700 tcttccgaaa ggaactcatt cgaaatacac gaaatgggtt tctcaatcta aagtgaaaag 2760 atctattaca tctcgtgcca tcgctagcgt gacattggtt gatctggatt cttccaggtt 2820 ttacattacg atgacccaag ctgataaggc ctcactgatt tcaagagcga aagagatgaa 2880 tctaccaaag actttctggg acgaaaggat taaaactgtg catgagtctc aaggcatttc 2940 tgaagatcat gttactttgg taagattaaa gagcacaaag tgtgacctgt tcaagaaatt 3000 ttcatattgt cttgttgctt tgacgagaca caaggtcaca ttccgctacg agtactgcgg 3060 tgtactgaac ggcgatttaa tcgccgaatg tattgctcgt gcttagcgga ctccctcctt 3120 gtgaggcggg aggtgagttg gcagtattgc tataaactgt ctgaagtcac taaatacatt 3180 tgtagtgaac gggttgtcca tccagcttac ggctaaaatg gtcagttgta gaaaaatcta 3240 cgccggaggc ttacaagtct ccgaggcgcc tttgaaacca tctcctaggt ttcttcggaa 3300 ggacttcggt ccgtgtactt ctagcacaag atgctagttt cagggtacag gtgccccccc 3360 cgcttggtgg ggcctctaaa aggagaccag gatccggtac cgagctcacg cgtctcgagg 3420 cccgggcact agtgtctgta cttatatcag tacactgacg agtccctaaa ggacgaaacg 3480 ggagaacgct agccaccacc accaccacca cgtgtgaatt acaggtgacc agctcgaatt 3540 tccccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc 3600 ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt 3660 aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt 3720 aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt 3780 catctatgtt actagatcgg gaattaaact atcagtgttt gacaggatat attggcgggt 3840 aaacctaaga gaaaagagcg tttattagaa taacggatat ttaaaagggc gtgaaaaggt 3900 ttatccgttc gtccatttgt atgtgcatgc caaccacagg gttcccctcg ggatcaaagt 3960 actttgatcc aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg 4020 tcttctgaaa acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc 4080 ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag 4140 aaccggagac attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc 4200 gtcagcaccg acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc 4260 accaagctgt tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg 4320 atgcttgacc acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc 4380 cgcagcaccc gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg 4440 cgtagcctgg cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc 4500 gtgttcgccg gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg 4560 cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca 4620 cagatcgcgc acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct 4680 gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg 4740 acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac 4800 gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg 4860 gccaggacga accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt 4920 acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt 4980 tgtctgatgc caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc 5040 gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt 5100 atatgatgcg atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt 5160 acttaaccag aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct 5220 gcaactcgcc ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga 5280 ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat 5340 tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca 5400 ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca 5460 gccaagccct tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat 5520 tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac 5580 gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc 5640 ccgtatcacg cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga 5700 atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc 5760 aaaactcatt tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt 5820 gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg 5880 ccagccatga agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg 5940 tacgcggtac gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta 6000 ccagagtaaa tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg 6060 catggaaaat caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac 6120 gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac 6180 ccccaagccc gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta caaatcggcg 6240 cggcgctggg tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac 6300 gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca 6360 aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg 6420 acgagcaacc agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca 6480 gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga 6540 tccgctacga gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca 6600 gtgtgtggga ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc 6660 gataccggga agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg 6720 tactcaagtt ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct 6780 gcattcggtt aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc 6840 gcctggtgac ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg 6900 aaaccgggcg gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca 6960 cagaaggcaa gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg 7020 gcatcggccg ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat 7080 ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt 7140 tcaccgtgcg caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg 7200 cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat 7260 ccgccggttc ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag 7320 gtcgaaaagg tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg 7380 ggaaccggaa cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt 7440 aagtgactga tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta 7500 ttaaaactct taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag 7560 agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc 7620 ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag 7680 ggcgcggaca agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc 7740 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 7800 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 7860 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact 7920 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa 7980 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca 8040 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 8100 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 8160 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 8220 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 8280 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 8340 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 8400 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 8460 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 8520 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 8580 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 8640 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 8700 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 8760 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 8820 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact 8880 aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca 8940 gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga 9000 cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc 9060 cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga 9120 caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa 9180 atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt 9240 aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga 9300 tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt 9360 catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc 9420 agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata 9480 gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca 9540 tttttaaata taggttttca ttttctccca ccagcttata taccttagca ggagacattc 9600 cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc 9660 tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga 9720 agctaattat aacaagacga actccaattc actgttcctt gcattctaaa accttaaata 9780 ccagaaaaca gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga 9840 gccgattttg aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca 9900 tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg 9960 aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg 10020 tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg 10080 agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa 10140 cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt aattcctagg 10200 ccaccatgtt gggcccggcg cgccaagctt gcatgcctgc aggtcaacat ggtggagcac 10260 gacactctcg tctactccaa gaatatcaaa gatacagtct cagaagacca gagggctatt 10320 gagacttttc aacaaagggt aatatcggga aacctcctcg gattccattg cccagctatc 10380 tgtcacttca tcgaaaggac agtagaaaag gaagatggct tctacaaatg ccatcattgc 10440 gataaaggaa aggctatcgt tcaagatgcc tctaccgaca gtggtcccaa agatggaccc 10500 ccacccacga ggaacatcgt ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg 10560 gattgatgtg atggtcaaca tggtggagca cgacactctc gtctactcca agaatatcaa 10620 agatacagtc tcagaagacc agagggctat tgagactttt caacaaaggg taatatcggg 10680 aaacctcctc ggattccatt gcccagctat ctgtcacttc atcgaaagga cagtagaaaa 10740 ggaagatggc ttctacaaat gccatcattg cgataaagga aaggctatcg ttcaagatgc 10800 ctctaccgac agtggtccca aagatggacc cccacccacg aggaacatcg tggaaaaaga 10860 agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca ctgacgtaag 10920 ggatgacgca caatcccact atccttcgca agacccttcc tctatataag gaagttcatt 10980 tcatttggag agg 10993 <210> 2 <211> 10643 <212> DNA <213> Artificial Sequence <220> <223> pCkpaR2 <400> 2 gaattctcta gaaggcctcc atggggatcc gtttatttac aagagcgtac ggttcaaccc 60 ctgcctcccc tgtaaaactc cctagactga aaactttctt tctagtatct tttctatggc 120 ttcccttgcc cccacattct cactagccaa tcttttgaac ggtagttacg gtgtcgacac 180 tcccgaggat gtggaacgtt tgcgatctga gcaacgtgaa gaggctgctg cggcctgtcg 240 taactacagg cccctacccg ctgtggatgt cagcgagagt gtcacagagg acgcacattc 300 cctccgaact cctgacggag ctcccgctga agcggtgtct gatgagtttg taacttatgg 360 tgctgaagat taccttgaaa aatctgatga tgagctcctt gtcgcttttg agacgatggt 420 caaacccatg cgtatcggac aactatggtg ccccgcgttt aataaatgtt cttttatttc 480 cagcattgct atggccagag ctttgttgtt ggcacctaga acatccaacc gaaccatgaa 540 gtgttttgaa gacctggtcg cggctattta cactaaatcc gatttctatt atggtgaaga 600 gtgtgaagcc gacgacgttc agatggatat ctcgtctcgc gatgtacccg gttattcttt 660 cgaaccgtgg tcccgaacgt ctggattcga accgccgccc atttgtgaag cgtgcgacat 720 gatcatgtac cagtgcccgt gttttgattt caatgcttta aagaaatcgt gcgctgagag 780 gactttcgct gatgactatg ttattgaagg tttagatggt gttgttgata atgcgactct 840 gttgtcgaat ttgggtccat ttttggtacc cgtgaagtgt caatatgaaa aatgtccaac 900 accaaccctc gcgattcctc cagatttaaa tcgtgctact gatcgtgttg atattaattt 960 agttcaatcc atttgtgact cgactctgcc cactcatagt aattacgacg actcttttca 1020 tcaagtgttt gtcgaaagtg cagactactc tatagatctg gatcatgtta gacttcgaca 1080 gtctgatctt atcgcaaaaa ttccagattc agggcatatg ataccggttt tgaacaccgg 1140 gagcggtcac aagagagtag gtacaacgaa ggaggtcctt acagcaatta agaaacgtaa 1200 tgctgacgtt ccagagctag gtgattccgt taatctgtct agattgagta aagctgtggc 1260 tgagagattc ttcatttcat acatcaatgg taactctcta gcgtccagta actttgtcaa 1320 tgtcgttagt aacttccacg attacatgga aaaatggaag tcctcaggtc tttcttatga 1380 tgatctccca gatcttcatg ctgagaatct gcagttttat gatcacatga taaaatctga 1440 tgtgaagcct gtggtgagcg acacactcaa tatcgacaga ccggttccag ctactataac 1500 gtatcataag aagagtataa cctcccagtt ctcaccgtta ttcacagcgc tatttgagcg 1560 cttccagaga tgccttcgag aacgtattat tcttcctgtt ggtaagattt catcccttga 1620 gatggcagga tttgatgtca aaaacaagca ctgcctcgaa attgatctgt ctaagtttga 1680 taaatctcag ggtgaattcc atttgctaat tcaggaacat attttgaatg gtctaggatg 1740 tccagctccg ataactaagt ggtggtgtga ttttcaccga ttctcttaca ttagagaccg 1800 tagagctggt gttggtatgc ctattagttt ccagagacga actggtgatg cattcactta 1860 ttttggcaat accattgtca ccatggcaga gtttgcctgg tgttacgaca ccgaccaatt 1920 cgaaaagctt ttattctcgg gcgatgactc tctaggattt tcattgcttc cccctgttgg 1980 cgatccgagt aaattcacga ctcttttcaa catggaagct aaggtgatgg aacctgcagt 2040 accatatatt tgttcgaagt tcttactctc tgacgagttc ggtaatacat tttccgttcc 2100 agatccattg cgcgaggttc agcggttagg aacaaagaaa attccctatt ctgacaatga 2160 tgaattcttg tttgctcact tcatgagctt tgttgatcga ttgaagtttt tggaccgaat 2220 gtctcagtcg tgcatcgatc agctttcgat tttctttgaa ttgaaataca agaagtctgg 2280 ggaagaggct gctttaatgt taggcgcctt taagaaatat accgctaatt ttcagtccta 2340 caaggaactc tattatttag atcgtcgtca gtgcgaattg atcaattcgt tttgtagtac 2400 agagttcagg gttgagcgtg taaattccaa taaacagcga aagaaatatg gaattgaacg 2460 taggtgcaat gacaaacgtc gaactccaac tggctcgtat ggtggaggcg aagaagcaga 2520 gacgaagatc tcacaagcag aatcgacggg aacgaggtca caaaagtccc agcgagagag 2580 cgcgttcaaa tctcagactg ttccgcttcc taccgttcta tcaagtagat ggttcggaac 2640 tgatagggtc ggaccgcctt gtgaacgcgg cggagttacc cgagcctgag gcctctcgtt 2700 tagagttatc ggcggaagac catgagtttg acgatacgga ttggttcgcc ggtaacgaat 2760 gggcggaagg tgctttctga aacctcccct tccgcaactc cctccggtca cgtggcggga 2820 gctgagttgg cagtattgct ataaactgtc tgaagtcact aaacacattg tggtgaacgg 2880 gttgtccatc cagcttacgg ctaaaatggt cagtcgtaga gaaatctaca ccagcagact 2940 tgcaagtctc tgaggcacct ttgaaaccat ctcctaggtt tcttcggaag gactccggtc 3000 cgtgtacttc tagcacaacg tgctagtttt agggtacggg tgccccccca ctttcgtggg 3060 gcctctaaag gagaccaact agtgtctgta cttatatcag tacactgacg agtccctaaa 3120 ggacgaaacg ggagaacgct agccaccacc accaccacca cgtgtgaatt acaggtgacc 3180 agctcgaatt tccccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct 3240 gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata 3300 attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa 3360 ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg 3420 cgcgcggtgt catctatgtt actagatcgg gaattaaact atcagtgttt gacaggatat 3480 attggcgggt aaacctaaga gaaaagagcg tttattagaa taacggatat ttaaaagggc 3540 gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg gttcccctcg 3600 ggatcaaagt actttgatcc aacccctccg ctgctatagt gcagtcggct tctgacgttc 3660 agtgcagccg tcttctgaaa acgacatgtc gcacaagtcc taagttacgc gacaggctgc 3720 cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa agtagaatac 3780 ttgcgactag aaccggagac attacgccat gaacaagagc gccgccgctg gcctgctggg 3840 ctatgcccgc gtcagcaccg acgaccagga cttgaccaac caacgggccg aactgcacgc 3900 ggccggctgc accaagctgt tttccgagaa gatcaccggc accaggcgcg accgcccgga 3960 gctggccagg atgcttgacc acctacgccc tggcgacgtt gtgacagtga ccaggctaga 4020 ccgcctggcc cgcagcaccc gcgacctact ggacattgcc gagcgcatcc aggaggccgg 4080 cgcgggcctg cgtagcctgg cagagccgtg ggccgacacc accacgccgg ccggccgcat 4140 ggtgttgacc gtgttcgccg gcattgccga gttcgagcgt tccctaatca tcgaccgcac 4200 ccggagcggg cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc gccctaccct 4260 caccccggca cagatcgcgc acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa 4320 agaggcggct gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag 4380 cgaggaagtg acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac 4440 cgaggccgac gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg 4500 caccaggacg gccaggacga accgtttttc attaccgaag agatcgaggc ggagatgatc 4560 gcggccgggt acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc 4620 ctggccggtt tgtctgatgc caagctggcg gcctggccgg ccagcttggc cgctgaagaa 4680 accgagcgcc gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg 4740 gtcgctgcgt atatgatgcg atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg 4800 ttatcgctgt acttaaccag aaaggcgggt caggcaagac gaccatcgca acccatctag 4860 cccgcgccct gcaactcgcc ggggccgatg ttctgttagt cgattccgat ccccagggca 4920 gtgcccgcga ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc 4980 gcccgacgat tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg 5040 gagcgcccca ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga 5100 ttccggtgca gccaagccct tacgacatat gggccaccgc cgacctggtg gagctggtta 5160 agcagcgcat tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga 5220 tcaaaggcac gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac gagctgccca 5280 ttcttgagtc ccgtatcacg cagcgcgtga gctacccagg cactgccgcc gccggcacaa 5340 ccgttcttga atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg 5400 aaattaaatc aaaactcatt tgagttaatg aggtaaagag aaaatgagca aaagcacaaa 5460 cacgctaagt gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt tggccagcct 5520 ggcagacacg ccagccatga agcgggtcaa ctttcagttg ccggcggagg atcacaccaa 5580 gctgaagatg tacgcggtac gccaaggcaa gaccattacc gagctgctat ctgaatacat 5640 cgcgcagcta ccagagtaaa tgagcaaatg aataaatgag tagatgaatt ttagcggcta 5700 aaggaggcgg catggaaaat caagaacaac caggcaccga cgccgtggaa tgccccatgt 5760 gtggaggaac gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg 5820 gcactggaac ccccaagccc gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta 5880 caaatcggcg cggcgctggg tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc 5940 cagcggcaac gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc ggccgctgat 6000 cgaatccgca aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat taggaagccg 6060 cccaagggcg acgagcaacc agattttttc gttccgatgc tctatgacgt gggcacccgc 6120 gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct 6180 ggcgaggtga tccgctacga gcttccagac gggcacgtag aggtttccgc agggccggcc 6240 ggcatggcca gtgtgtggga ttacgacctg gtactgatgg cggtttccca tctaaccgaa 6300 tccatgaacc gataccggga agggaaggga gacaagcccg gccgcgtgtt ccgtccacac 6360 gttgcggacg tactcaagtt ctgccggcga gccgatggcg gaaagcagaa agacgacctg 6420 gtagaaacct gcattcggtt aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc 6480 aagaacggcc gcctggtgac ggtatccgag ggtgaagcct tgattagccg ctacaagatc 6540 gtaaagagcg aaaccgggcg gccggagtac atcgagatcg agctagctga ttggatgtac 6600 cgcgagatca cagaaggcaa gaacccggac gtgctgacgg ttcaccccga ttactttttg 6660 atcgatcccg gcatcggccg ttttctctac cgcctggcac gccgcgccgc aggcaaggca 6720 gaagccagat ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg agagttcaag 6780 aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg acctgccgga gtacgatttg 6840 aaggaggagg cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag 6900 ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc tagggcaaat tgccctagca 6960 ggggaaaaag gtcgaaaagg tctctttcct gtggatagca cgtacattgg gaacccaaag 7020 ccgtacattg ggaaccggaa cccgtacatt gggaacccaa agccgtacat tgggaaccgg 7080 tcacacatgt aagtgactga tataaaagag aaaaaaggcg atttttccgc ctaaaactct 7140 ttaaaactta ttaaaactct taaaacccgc ctggcctgtg cataactgtc tggccagcgc 7200 acagccgaag agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct acgccccgcc 7260 gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct acggccaggc 7320 aatctaccag ggcgcggaca agccgcgccg tcgccactcg accgccggcg cccacatcaa 7380 ggcaccctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 7440 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc 7500 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg 7560 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg 7620 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct 7680 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 7740 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 7800 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 7860 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 7920 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 7980 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 8040 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 8100 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 8160 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 8220 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 8280 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 8340 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 8400 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 8460 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgcat 8520 tctaggtact aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc 8580 ttgatcccca gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg 8640 atcgaccgga cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga 8700 tcaataaagc cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg 8760 tgggaaaaga caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc 8820 ggatctttaa atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta 8880 ttcagtaagt aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc 8940 gatatgtcga tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca 9000 gggctttgtt catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc 9060 agattgctcc agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct 9120 tccagccata gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg 9180 ctgtccgtca tttttaaata taggttttca ttttctccca ccagcttata taccttagca 9240 ggagacattc cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc 9300 ggtgatattc tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga 9360 taccccaaga agctaattat aacaagacga actccaattc actgttcctt gcattctaaa 9420 accttaaata ccagaaaaca gctttttcaa agttgttttc aaagttggcg tataacatag 9480 tatcgacgga gccgattttg aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt 9540 acaatcaaca tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc 9600 cgttcttccg aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc 9660 gctgacgccg tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg 9720 ccggtcgggg agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac 9780 gcttagacaa cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt 9840 aattcctagg ccaccatgtt gggcccggcg cgccaagctt gcatgcctgc aggtcaacat 9900 ggtggagcac gacactctcg tctactccaa gaatatcaaa gatacagtct cagaagacca 9960 gagggctatt gagacttttc aacaaagggt aatatcggga aacctcctcg gattccattg 10020 cccagctatc tgtcacttca tcgaaaggac agtagaaaag gaagatggct tctacaaatg 10080 ccatcattgc gataaaggaa aggctatcgt tcaagatgcc tctaccgaca gtggtcccaa 10140 agatggaccc ccacccacga ggaacatcgt ggaaaaagaa gacgttccaa ccacgtcttc 10200 aaagcaagtg gattgatgtg atggtcaaca tggtggagca cgacactctc gtctactcca 10260 agaatatcaa agatacagtc tcagaagacc agagggctat tgagactttt caacaaaggg 10320 taatatcggg aaacctcctc ggattccatt gcccagctat ctgtcacttc atcgaaagga 10380 cagtagaaaa ggaagatggc ttctacaaat gccatcattg cgataaagga aaggctatcg 10440 ttcaagatgc ctctaccgac agtggtccca aagatggacc cccacccacg aggaacatcg 10500 tggaaaaaga agacgttcca accacgtctt caaagcaagt ggattgatgt gatatctcca 10560 ctgacgtaag ggatgacgca caatcccact atccttcgca agacccttcc tctatataag 10620 gaagttcatt tcatttggag agg 10643 <210> 3 <211> 9849 <212> DNA <213> Artificial Sequence <220> <223> pCkpaR3 <400> 3 gaattctcta gaaggcctcc atggggatcc gtaatcttac cactgtgtgt gtgcgtgtgt 60 gtgtgtcgag tcgtgttgtc cgcacatttg agtcgtgttg tccgcacatt ttattttatc 120 tttgtacagt gtgttagatt tcccgaggca tggctttcca aggtaccagt aggactttaa 180 ctcaacagtc ctcagcggct acgtctgacg atcttcaaaa gatattattt agccctgaag 240 ccattaagaa aatggctact gagtgtgacc taggccggca tcattggatg cgcgctgata 300 atgctatttc agtccggccc ctcgttcccg aagtaaccca cggtcgtatt gcttccttct 360 ttaaatctgg atatgatgtt ggtgaattgt gctcaaaagg atacatgagc gtccctcaag 420 tgttgtgtgc tgttactcga acggtttcca ctgatgctga agggtccttg agaatttact 480 tagctgatct aggtgacaag gagttatctc ctatagatgg acaatgcgtt tcgttacata 540 accatgatct tcccgctttg gtgtctttcc aaccgacgta tgactgtcct atggaaacag 600 ttgggaatcg caagcggtgt tttgctgtcg ttatcgaaag acatggttac attgggtata 660 ccggtaccac agctagcgtg tgtagtaatt ggcaagcaag gttttcttct aagaataaca 720 actacactca tatcgcagct gggaagactc tagtactgcc tttcaacaga ttagctgagc 780 aaacaaaacc gtcagctgtc gctcgcctgt tgaagtcgca attgaacaac attgaatctt 840 cgcaatatct gttaacgaac gcgaagatta atcagaatgc gcgcagtgag tccgaggaat 900 taaatgttga gagccctccc gccgcaatcg ggagttcttc cgcgtcccgc tccgaagcct 960 tcagaccgca ggtggttaac ggtctttagc actttggtgc gtattagtat ataagtattt 1020 gtgagtctgt acataatact atatctatag tgtcctgtgt gagttgatac agtagacatc 1080 tgtgacgcga tgccgtgttg agaagggaac acatctggtt ttagtaagcc tacatcatag 1140 ttttgaggtt caattcctct tactccctgt tgagcccctt actttctcat ggatgcttct 1200 ccgcgagatt gcgttattgt ctactgacta tttagagagt gtgtgtgctg tgttttctct 1260 tttgtgtcgt agaattgagt cgagtcatgg acaaatctga atcaaccagt gctggtcgta 1320 accgtcgacg tcgtccgcgt cgtggttccc gctccgcccc ctcctccgcg gatgccacct 1380 ttagagtctt gtcgcagcag ctttcgcgac ttaataagac gttgtcagct ggtcgtccaa 1440 ctattaacca cccaaccttt gtagggagtg agcgttgtaa acctggatac acgttcacat 1500 ctattaccgt aaagccacca aaaatagacc gcgggtctta ttatggtaaa aggttgctat 1560 tacctgattc agtcacagaa tatgataaga aacttgtttc gcgcattcaa attcaagtta 1620 atcctttgcc gaaatttgat tctaccgtgt gggtgacagt ccgtaaagtt cctgcctcct 1680 cggacttatc cgttgccgcc atctctgcta tgtttgcgga cggagcctca ccggtactgg 1740 tttatcagta tgccgcatct ggagtccaag ctaacaacaa actgttgtat gatctttcgg 1800 cgatgcgcgc tgatataggc gacatgagaa agtacgccgt cctcgtgtac tcaaaagacg 1860 atgcactcga gacggacgag ctggtacttc atgttgacgt cgagcaccaa cgcattccca 1920 cgtctggggt gctcccagta tgattctgtg ttttccagaa ccctccctcc gatttctgtg 1980 gcgggagctg agttgtcagt tctgctataa actgtctgaa gtcactaaac gttttacggt 2040 gaacgggttg tccatccagc ttacggctaa aatggtcagt cgtggagaaa tccacgccag 2100 cagacttaca agtctctgag gcgcctttga aaccatctcc taggtttctt tggaaggact 2160 tcggtccgtg tacttctagc acaatgtgct agtttcaggg tacgggtgcc cccccacttt 2220 cgtgggggcc tctaaaagga gaccaggatc cggtaccgag ctcacgcgtc tcgaggcccg 2280 ggcactagtg tctgtactta tatcagtaca ctgacgagtc cctaaaggac gaaacgggag 2340 aacgctagcc accaccacca ccaccacgtg tgaattacag gtgaccagct cgaatttccc 2400 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 2460 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 2520 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 2580 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 2640 tatgttacta gatcgggaat taaactatca gtgtttgaca ggatatattg gcgggtaaac 2700 ctaagagaaa agagcgttta ttagaataac ggatatttaa aagggcgtga aaaggtttat 2760 ccgttcgtcc atttgtatgt gcatgccaac cacagggttc ccctcgggat caaagtactt 2820 tgatccaacc cctccgctgc tatagtgcag tcggcttctg acgttcagtg cagccgtctt 2880 ctgaaaacga catgtcgcac aagtcctaag ttacgcgaca ggctgccgcc ctgccctttt 2940 cctggcgttt tcttgtcgcg tgttttagtc gcataaagta gaatacttgc gactagaacc 3000 ggagacatta cgccatgaac aagagcgccg ccgctggcct gctgggctat gcccgcgtca 3060 gcaccgacga ccaggacttg accaaccaac gggccgaact gcacgcggcc ggctgcacca 3120 agctgttttc cgagaagatc accggcacca ggcgcgaccg cccggagctg gccaggatgc 3180 ttgaccacct acgccctggc gacgttgtga cagtgaccag gctagaccgc ctggcccgca 3240 gcacccgcga cctactggac attgccgagc gcatccagga ggccggcgcg ggcctgcgta 3300 gcctggcaga gccgtgggcc gacaccacca cgccggccgg ccgcatggtg ttgaccgtgt 3360 tcgccggcat tgccgagttc gagcgttccc taatcatcga ccgcacccgg agcgggcgcg 3420 aggccgccaa ggcccgaggc gtgaagtttg gcccccgccc taccctcacc ccggcacaga 3480 tcgcgcacgc ccgcgagctg atcgaccagg aaggccgcac cgtgaaagag gcggctgcac 3540 tgcttggcgt gcatcgctcg accctgtacc gcgcacttga gcgcagcgag gaagtgacgc 3600 ccaccgaggc caggcggcgc ggtgccttcc gtgaggacgc attgaccgag gccgacgccc 3660 tggcggccgc cgagaatgaa cgccaagagg aacaagcatg aaaccgcacc aggacggcca 3720 ggacgaaccg tttttcatta ccgaagagat cgaggcggag atgatcgcgg ccgggtacgt 3780 gttcgagccg cccgcgcacg tctcaaccgt gcggctgcat gaaatcctgg ccggtttgtc 3840 tgatgccaag ctggcggcct ggccggccag cttggccgct gaagaaaccg agcgccgccg 3900 tctaaaaagg tgatgtgtat ttgagtaaaa cagcttgcgt catgcggtcg ctgcgtatat 3960 gatgcgatga gtaaataaac aaatacgcaa ggggaacgca tgaaggttat cgctgtactt 4020 aaccagaaag gcgggtcagg caagacgacc atcgcaaccc atctagcccg cgccctgcaa 4080 ctcgccgggg ccgatgttct gttagtcgat tccgatcccc agggcagtgc ccgcgattgg 4140 gcggccgtgc gggaagatca accgctaacc gttgtcggca tcgaccgccc gacgattgac 4200 cgcgacgtga aggccatcgg ccggcgcgac ttcgtagtga tcgacggagc gccccaggcg 4260 gcggacttgg ctgtgtccgc gatcaaggca gccgacttcg tgctgattcc ggtgcagcca 4320 agcccttacg acatatgggc caccgccgac ctggtggagc tggttaagca gcgcattgag 4380 gtcacggatg gaaggctaca agcggccttt gtcgtgtcgc gggcgatcaa aggcacgcgc 4440 atcggcggtg aggttgccga ggcgctggcc gggtacgagc tgcccattct tgagtcccgt 4500 atcacgcagc gcgtgagcta cccaggcact gccgccgccg gcacaaccgt tcttgaatca 4560 gaacccgagg gcgacgctgc ccgcgaggtc caggcgctgg ccgctgaaat taaatcaaaa 4620 ctcatttgag ttaatgaggt aaagagaaaa tgagcaaaag cacaaacacg ctaagtgccg 4680 gccgtccgag cgcacgcagc agcaaggctg caacgttggc cagcctggca gacacgccag 4740 ccatgaagcg ggtcaacttt cagttgccgg cggaggatca caccaagctg aagatgtacg 4800 cggtacgcca aggcaagacc attaccgagc tgctatctga atacatcgcg cagctaccag 4860 agtaaatgag caaatgaata aatgagtaga tgaattttag cggctaaagg aggcggcatg 4920 gaaaatcaag aacaaccagg caccgacgcc gtggaatgcc ccatgtgtgg aggaacgggc 4980 ggttggccag gcgtaagcgg ctgggttgtc tgccggccct gcaatggcac tggaaccccc 5040 aagcccgagg aatcggcgtg acggtcgcaa accatccggc ccggtacaaa tcggcgcggc 5100 gctgggtgat gacctggtgg agaagttgaa ggccgcgcag gccgcccagc ggcaacgcat 5160 cgaggcagaa gcacgccccg gtgaatcgtg gcaagcggcc gctgatcgaa tccgcaaaga 5220 atcccggcaa ccgccggcag ccggtgcgcc gtcgattagg aagccgccca agggcgacga 5280 gcaaccagat tttttcgttc cgatgctcta tgacgtgggc acccgcgata gtcgcagcat 5340 catggacgtg gccgttttcc gtctgtcgaa gcgtgaccga cgagctggcg aggtgatccg 5400 ctacgagctt ccagacgggc acgtagaggt ttccgcaggg ccggccggca tggccagtgt 5460 gtgggattac gacctggtac tgatggcggt ttcccatcta accgaatcca tgaaccgata 5520 ccgggaaggg aagggagaca agcccggccg cgtgttccgt ccacacgttg cggacgtact 5580 caagttctgc cggcgagccg atggcggaaa gcagaaagac gacctggtag aaacctgcat 5640 tcggttaaac accacgcacg ttgccatgca gcgtacgaag aaggccaaga acggccgcct 5700 ggtgacggta tccgagggtg aagccttgat tagccgctac aagatcgtaa agagcgaaac 5760 cgggcggccg gagtacatcg agatcgagct agctgattgg atgtaccgcg agatcacaga 5820 aggcaagaac ccggacgtgc tgacggttca ccccgattac tttttgatcg atcccggcat 5880 cggccgtttt ctctaccgcc tggcacgccg cgccgcaggc aaggcagaag ccagatggtt 5940 gttcaagacg atctacgaac gcagtggcag cgccggagag ttcaagaagt tctgtttcac 6000 cgtgcgcaag ctgatcgggt caaatgacct gccggagtac gatttgaagg aggaggcggg 6060 gcaggctggc ccgatcctag tcatgcgcta ccgcaacctg atcgagggcg aagcatccgc 6120 cggttcctaa tgtacggagc agatgctagg gcaaattgcc ctagcagggg aaaaaggtcg 6180 aaaaggtctc tttcctgtgg atagcacgta cattgggaac ccaaagccgt acattgggaa 6240 ccggaacccg tacattggga acccaaagcc gtacattggg aaccggtcac acatgtaagt 6300 gactgatata aaagagaaaa aaggcgattt ttccgcctaa aactctttaa aacttattaa 6360 aactcttaaa acccgcctgg cctgtgcata actgtctggc cagcgcacag ccgaagagct 6420 gcaaaaagcg cctacccttc ggtcgctgcg ctccctacgc cccgccgctt cgcgtcggcc 6480 tatcgcggcc gctggccgct caaaaatggc tggcctacgg ccaggcaatc taccagggcg 6540 cggacaagcc gcgccgtcgc cactcgaccg ccggcgccca catcaaggca ccctgcctcg 6600 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag 6660 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg 6720 gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct 6780 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc 6840 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga 6900 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 6960 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 7020 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 7080 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 7140 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 7200 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 7260 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 7320 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 7380 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 7440 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 7500 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 7560 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 7620 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 7680 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgcattcta ggtactaaaa 7740 caattcatcc agtaaaatat aatattttat tttctcccaa tcaggcttga tccccagtaa 7800 gtcaaaaaat agctcgacat actgttcttc cccgatatcc tccctgatcg accggacgca 7860 gaaggcaatg tcataccact tgtccgccct gccgcttctc ccaagatcaa taaagccact 7920 tactttgcca tctttcacaa agatgttgct gtctcccagg tcgccgtggg aaaagacaag 7980 ttcctcttcg ggcttttccg tctttaaaaa atcatacagc tcgcgcggat ctttaaatgg 8040 agtgtcttct tcccagtttt cgcaatccac atcggccaga tcgttattca gtaagtaatc 8100 caattcggct aagcggctgt ctaagctatt cgtataggga caatccgata tgtcgatgga 8160 gtgaaagagc ctgatgcact ccgcatacag ctcgataatc ttttcagggc tttgttcatc 8220 ttcatactct tccgagcaaa ggacgccatc ggcctcactc atgagcagat tgctccagcc 8280 atcatgccgt tcaaagtgca ggacctttgg aacaggcagc tttccttcca gccatagcat 8340 catgtccttt tcccgttcca catcataggt ggtcccttta taccggctgt ccgtcatttt 8400 taaatatagg ttttcatttt ctcccaccag cttatatacc ttagcaggag acattccttc 8460 cgtatctttt acgcagcggt atttttcgat cagttttttc aattccggtg atattctcat 8520 tttagccatt tattatttcc ttcctctttt ctacagtatt taaagatacc ccaagaagct 8580 aattataaca agacgaactc caattcactg ttccttgcat tctaaaacct taaataccag 8640 aaaacagctt tttcaaagtt gttttcaaag ttggcgtata acatagtatc gacggagccg 8700 attttgaaac cgcggtgatc acaggcagca acgctctgtc atcgttacaa tcaacatgct 8760 accctccgcg agatcatccg tgtttcaaac ccggcagctt agttgccgtt cttccgaata 8820 gcatcggtaa catgagcaaa gtctgccgcc ttacaacggc tctcccgctg acgccgtccc 8880 ggactgatgg gctgcctgta tcgagtggtg attttgtgcc gagctgccgg tcggggagct 8940 gttggctggc tggtggcagg atatattgtg gtgtaaacaa attgacgctt agacaactta 9000 ataacacatt gcggacgttt ttaatgtact gaattaacgc cgaattaatt cctaggccac 9060 catgttgggc ccggcgcgcc aagcttgcat gcctgcaggt caacatggtg gagcacgaca 9120 ctctcgtcta ctccaagaat atcaaagata cagtctcaga agaccagagg gctattgaga 9180 cttttcaaca aagggtaata tcgggaaacc tcctcggatt ccattgccca gctatctgtc 9240 acttcatcga aaggacagta gaaaaggaag atggcttcta caaatgccat cattgcgata 9300 aaggaaaggc tatcgttcaa gatgcctcta ccgacagtgg tcccaaagat ggacccccac 9360 ccacgaggaa catcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt 9420 gatgtgatgg tcaacatggt ggagcacgac actctcgtct actccaagaa tatcaaagat 9480 acagtctcag aagaccagag ggctattgag acttttcaac aaagggtaat atcgggaaac 9540 ctcctcggat tccattgccc agctatctgt cacttcatcg aaaggacagt agaaaaggaa 9600 gatggcttct acaaatgcca tcattgcgat aaaggaaagg ctatcgttca agatgcctct 9660 accgacagtg gtcccaaaga tggaccccca cccacgagga acatcgtgga aaaagaagac 9720 gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga cgtaagggat 9780 gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag ttcatttcat 9840 ttggagagg 9849 <210> 4 <211> 10967 <212> DNA <213> Artificial Sequence <220> <223> pCfnyR1 <400> 4 gaattcgttt atttacaaga gcgtacggtt caatccctgc ctcccctgta aaactaccct 60 ttgaaaacct ctctttctta atcttttctt tgtaattcct atggcgacgt cctcgttcaa 120 catcaatgaa ttggtagcct cccacggcga taaaggacta ctcgcgaccg ccctcgttga 180 taagacagct catgagcagc tcgaggagca attacagcat caacgtagag gccgtaaggt 240 ctacatccgg aacgttttgg gtgtaaagga ttccgaggtc atccggaatc ggtatggagg 300 aaagtacgac ctccatctta cccagcagga gtttgctccc cacggcctag ctggtgccct 360 ccgcttgtgt gaaactctcg attgtctaga ctctttccct tcatcaggtc tgcggcagga 420 cctcgtctta gacttcggag gaagttgggt cacacattac ctccgcggac ataacgtaca 480 ctgttgttcc ccttgtttgg gtatccgcga taagatgcgc catgcggaac gcttaatgaa 540 catgcgcaag atcatcttga acgatccaca acagttcgat ggtcggcagc cggatttctg 600 cactcaaccg gctgcggatt gcaaagtaca agcccacttt gctatatcta ttcatggagg 660 ttatgatatg ggctttagag gattatgtga agcgatgaat gctcacggaa ccactatttt 720 gaagggaacg atgatgttcg atggtgcgat gatgtttgac gaccaaggtg taatacccga 780 acttaattgt cagtggagga agatcaggag tgctttctcc gaaactgaag acgtcacacc 840 actggttggt aagcttaatt ccacagtttt ctcccgcgtg cggaagttca agacgatggt 900 agcttttgat ttcatcaacg agtctactat gtcttatgtt catgattggg agaatataaa 960 atcttttctt acggaccaga cttattcgta ccgagggatg acttacggta tcgaacgctg 1020 cgttatccac gctggtatta tgacgtacaa gattatcggt gtacctggga tgtgcccacc 1080 cgaactcatt cgacattgta tttggttccc ctctattaaa gactatgttg gtctaaagat 1140 ccccgcgtcg caggatttgg ttgagtggaa aacagtgcgg attttaacgt caacattacg 1200 tgagactgaa gagattgcta tgaggtgtta taatgataag aaagcgtgga tggaacagtt 1260 caaggttatc ctaggtgttc tatccgcgaa atcatctacc attgttatta atggtatgtc 1320 catgcaatct ggcgagcgaa tagacattaa tgattatcac tacatcggtt tcgccattct 1380 tcttcacaca aaaatgaagt acgaacaact tggaaaaatg tatgatatgt ggaatgcttc 1440 gagtatttcg aagtggtttg ccgcgttgac tcgtccgctg cgtgtgtttt tctccagtgt 1500 tgttcacgca ctattcccga ctttgagacc ccgcgaggaa aaagaatttt tgatcaagct 1560 ctccaccttc gtgactttta atgaagagtg ctcatttgac ggtggagagg aatgggacgt 1620 gatatcatcc gctgcatacg ttgctacgca ggctgttacc gatgggaaga ttttggctgc 1680 gcagaaagcc gagaagcttg ctgagaagct tgcacaaccc gtgagtgagg tatcggacag 1740 tcctgagacg tcatctcaaa cgcctgatga tactgctgat gtttgtggaa gggagcgaga 1800 ggtttcggaa ctcgactccc tatcagctca gacacgttcc cccatcacta gagttgctga 1860 aagggctact gctatgttag agtatgccgc ttatgagaaa caattgcacg acactacagt 1920 gtctaattta aaacgtattt ggaacatggc gggcggtgat gacaaaagaa actccctcga 1980 gggtaatttg aagtttgttt tcgatacgta ctttaccgtt gatcctatgg tgaacattca 2040 tttctccacg ggtcggtgga tgcgtcctgt gcccgagggt attgtttatt ctgtcggtta 2100 taatgaacgc ggtttaggtc cgaagtctga tggagagctt tacattgtca atagtgaatg 2160 cgtgatctgt aacagtgaat ctttatccac tgtcacgcgt tctcttcaag ctccaaccgg 2220 gaccattagt caagttgacg gagttgctgg ttgtgggaaa accacggcaa ttaaatccat 2280 ttttgagccg tccactgaca tgatcgttac cgcgaacaag aagtccgccc aagatgtacg 2340 tatggcactt ttcaaatcgt cggattccaa agaagcttgc acctttgttc gaacagccga 2400 ttctgtccta cttaatgaat gtccgactgt tagtagggtt ttggttgacg aggtcgttct 2460 gctacacttt ggtcaattat gtgccgtcat gtctaagttg aaggccgtgc gagctatatg 2520 ttttggggat tcggagcaga ttgccttttc ctcgcgtgat gcttcatttg acatgcgttt 2580 ctctaagatt attcctgatg aaactagtga tgctgacacc acattccgta gcccacaaga 2640 tgttgtaccg cttgtgcgtt taatggctac gaaggccctt ccgaaaggaa cccattcaaa 2700 atacacgaaa tgggtttctc aatctaaagt gaaaagatct gttacatctc gtgccatcgc 2760 tagtgtgaca ttggttgact tggattcttc caggttctat ataacgatga cccaagctga 2820 taaggcctca ctgatttcaa gggcgaaaga gatgaatcta ccaaagactt tctggaatga 2880 aaggattaaa accgtgcatg agtctcaggg tatctccgaa gaccacgtta ctttggtaag 2940 attaaagagt acaaaatgtg acctgtttaa acagttttct tattgtctcg ttgcactgac 3000 gagacacaag gtcacattcc gctacgagta ctgtggtgta ttgaacggcg atttaatcgc 3060 cgaatgtgtt gctcgtgctt agcggtctcc ctcttcgggc gggatctgag ttggcggtaa 3120 tctgcaaacc gtctgaagtc actaaacaca ttgtgtggtg aacgggttgt ccatccagct 3180 aacggctaaa atggtcagtc gtggagaaat ccacgccagt agacttacaa gtctctgagg 3240 cacctttgaa accatctcct aggtttcttc ggaaggactt cggtccgtgt acttctagca 3300 caacgtgcta gtttcagggt acgggtgccc cccactttcg tgggggtctc taaaaggaga 3360 ccaggatccg gtaccgagct cacgcgtctc gaggcccggg cactagtgtc tgtacttata 3420 tcagtacact gacgagtccc taaaggacga aacgggagaa cgctagccac caccaccacc 3480 accacgtgtg aattacaggt gaccagctcg aatttccccg atcgttcaaa catttggcaa 3540 taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg 3600 ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg 3660 gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag 3720 cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tcgggaatta 3780 aactatcagt gtttgacagg atatattggc gggtaaacct aagagaaaag agcgtttatt 3840 agaataacgg atatttaaaa gggcgtgaaa aggtttatcc gttcgtccat ttgtatgtgc 3900 atgccaacca cagggttccc ctcgggatca aagtactttg atccaacccc tccgctgcta 3960 tagtgcagtc ggcttctgac gttcagtgca gccgtcttct gaaaacgaca tgtcgcacaa 4020 gtcctaagtt acgcgacagg ctgccgccct gcccttttcc tggcgttttc ttgtcgcgtg 4080 ttttagtcgc ataaagtaga atacttgcga ctagaaccgg agacattacg ccatgaacaa 4140 gagcgccgcc gctggcctgc tgggctatgc ccgcgtcagc accgacgacc aggacttgac 4200 caaccaacgg gccgaactgc acgcggccgg ctgcaccaag ctgttttccg agaagatcac 4260 cggcaccagg cgcgaccgcc cggagctggc caggatgctt gaccacctac gccctggcga 4320 cgttgtgaca gtgaccaggc tagaccgcct ggcccgcagc acccgcgacc tactggacat 4380 tgccgagcgc atccaggagg ccggcgcggg cctgcgtagc ctggcagagc cgtgggccga 4440 caccaccacg ccggccggcc gcatggtgtt gaccgtgttc gccggcattg ccgagttcga 4500 gcgttcccta atcatcgacc gcacccggag cgggcgcgag gccgccaagg cccgaggcgt 4560 gaagtttggc ccccgcccta ccctcacccc ggcacagatc gcgcacgccc gcgagctgat 4620 cgaccaggaa ggccgcaccg tgaaagaggc ggctgcactg cttggcgtgc atcgctcgac 4680 cctgtaccgc gcacttgagc gcagcgagga agtgacgccc accgaggcca ggcggcgcgg 4740 tgccttccgt gaggacgcat tgaccgaggc cgacgccctg gcggccgccg agaatgaacg 4800 ccaagaggaa caagcatgaa accgcaccag gacggccagg acgaaccgtt tttcattacc 4860 gaagagatcg aggcggagat gatcgcggcc gggtacgtgt tcgagccgcc cgcgcacgtc 4920 tcaaccgtgc ggctgcatga aatcctggcc ggtttgtctg atgccaagct ggcggcctgg 4980 ccggccagct tggccgctga agaaaccgag cgccgccgtc taaaaaggtg atgtgtattt 5040 gagtaaaaca gcttgcgtca tgcggtcgct gcgtatatga tgcgatgagt aaataaacaa 5100 atacgcaagg ggaacgcatg aaggttatcg ctgtacttaa ccagaaaggc gggtcaggca 5160 agacgaccat cgcaacccat ctagcccgcg ccctgcaact cgccggggcc gatgttctgt 5220 tagtcgattc cgatccccag ggcagtgccc gcgattgggc ggccgtgcgg gaagatcaac 5280 cgctaaccgt tgtcggcatc gaccgcccga cgattgaccg cgacgtgaag gccatcggcc 5340 ggcgcgactt cgtagtgatc gacggagcgc cccaggcggc ggacttggct gtgtccgcga 5400 tcaaggcagc cgacttcgtg ctgattccgg tgcagccaag cccttacgac atatgggcca 5460 ccgccgacct ggtggagctg gttaagcagc gcattgaggt cacggatgga aggctacaag 5520 cggcctttgt cgtgtcgcgg gcgatcaaag gcacgcgcat cggcggtgag gttgccgagg 5580 cgctggccgg gtacgagctg cccattcttg agtcccgtat cacgcagcgc gtgagctacc 5640 caggcactgc cgccgccggc acaaccgttc ttgaatcaga acccgagggc gacgctgccc 5700 gcgaggtcca ggcgctggcc gctgaaatta aatcaaaact catttgagtt aatgaggtaa 5760 agagaaaatg agcaaaagca caaacacgct aagtgccggc cgtccgagcg cacgcagcag 5820 caaggctgca acgttggcca gcctggcaga cacgccagcc atgaagcggg tcaactttca 5880 gttgccggcg gaggatcaca ccaagctgaa gatgtacgcg gtacgccaag gcaagaccat 5940 taccgagctg ctatctgaat acatcgcgca gctaccagag taaatgagca aatgaataaa 6000 tgagtagatg aattttagcg gctaaaggag gcggcatgga aaatcaagaa caaccaggca 6060 ccgacgccgt ggaatgcccc atgtgtggag gaacgggcgg ttggccaggc gtaagcggct 6120 gggttgtctg ccggccctgc aatggcactg gaacccccaa gcccgaggaa tcggcgtgac 6180 ggtcgcaaac catccggccc ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag 6240 aagttgaagg ccgcgcaggc cgcccagcgg caacgcatcg aggcagaagc acgccccggt 6300 gaatcgtggc aagcggccgc tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc 6360 ggtgcgccgt cgattaggaa gccgcccaag ggcgacgagc aaccagattt tttcgttccg 6420 atgctctatg acgtgggcac ccgcgatagt cgcagcatca tggacgtggc cgttttccgt 6480 ctgtcgaagc gtgaccgacg agctggcgag gtgatccgct acgagcttcc agacgggcac 6540 gtagaggttt ccgcagggcc ggccggcatg gccagtgtgt gggattacga cctggtactg 6600 atggcggttt cccatctaac cgaatccatg aaccgatacc gggaagggaa gggagacaag 6660 cccggccgcg tgttccgtcc acacgttgcg gacgtactca agttctgccg gcgagccgat 6720 ggcggaaagc agaaagacga cctggtagaa acctgcattc ggttaaacac cacgcacgtt 6780 gccatgcagc gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa 6840 gccttgatta gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag 6900 atcgagctag ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg 6960 acggttcacc ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg 7020 gcacgccgcg ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc 7080 agtggcagcg ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca 7140 aatgacctgc cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc 7200 atgcgctacc gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag 7260 atgctagggc aaattgccct agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat 7320 agcacgtaca ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac 7380 ccaaagccgt acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa 7440 ggcgattttt ccgcctaaaa ctctttaaaa cttattaaaa ctcttaaaac ccgcctggcc 7500 tgtgcataac tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg 7560 tcgctgcgct ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca 7620 aaaatggctg gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca 7680 ctcgaccgcc ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga 7740 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg 7800 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat 7860 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag 7920 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 7980 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 8040 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 8100 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 8160 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 8220 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 8280 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 8340 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 8400 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 8460 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 8520 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 8580 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 8640 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 8700 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 8760 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 8820 cgttaaggga ttttggtcat gcattctagg tactaaaaca attcatccag taaaatataa 8880 tattttattt tctcccaatc aggcttgatc cccagtaagt caaaaaatag ctcgacatac 8940 tgttcttccc cgatatcctc cctgatcgac cggacgcaga aggcaatgtc ataccacttg 9000 tccgccctgc cgcttctccc aagatcaata aagccactta ctttgccatc tttcacaaag 9060 atgttgctgt ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg cttttccgtc 9120 tttaaaaaat catacagctc gcgcggatct ttaaatggag tgtcttcttc ccagttttcg 9180 caatccacat cggccagatc gttattcagt aagtaatcca attcggctaa gcggctgtct 9240 aagctattcg tatagggaca atccgatatg tcgatggagt gaaagagcct gatgcactcc 9300 gcatacagct cgataatctt ttcagggctt tgttcatctt catactcttc cgagcaaagg 9360 acgccatcgg cctcactcat gagcagattg ctccagccat catgccgttc aaagtgcagg 9420 acctttggaa caggcagctt tccttccagc catagcatca tgtccttttc ccgttccaca 9480 tcataggtgg tccctttata ccggctgtcc gtcattttta aatataggtt ttcattttct 9540 cccaccagct tatatacctt agcaggagac attccttccg tatcttttac gcagcggtat 9600 ttttcgatca gttttttcaa ttccggtgat attctcattt tagccattta ttatttcctt 9660 cctcttttct acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca 9720 attcactgtt ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt 9780 tttcaaagtt ggcgtataac atagtatcga cggagccgat tttgaaaccg cggtgatcac 9840 aggcagcaac gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg 9900 tttcaaaccc ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt 9960 ctgccgcctt acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc 10020 gagtggtgat tttgtgccga gctgccggtc ggggagctgt tggctggctg gtggcaggat 10080 atattgtggt gtaaacaaat tgacgcttag acaacttaat aacacattgc ggacgttttt 10140 aatgtactga attaacgccg aattaattcc taggccacca tgttgggccc ggcgcgccaa 10200 gcttgcatgc ctgcaggtca acatggtgga gcacgacact ctcgtctact ccaagaatat 10260 caaagataca gtctcagaag accagagggc tattgagact tttcaacaaa gggtaatatc 10320 gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcgaaa ggacagtaga 10380 aaaggaagat ggcttctaca aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga 10440 tgcctctacc gacagtggtc ccaaagatgg acccccaccc acgaggaaca tcgtggaaaa 10500 agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgatggtc aacatggtgg 10560 agcacgacac tctcgtctac tccaagaata tcaaagatac agtctcagaa gaccagaggg 10620 ctattgagac ttttcaacaa agggtaatat cgggaaacct cctcggattc cattgcccag 10680 ctatctgtca cttcatcgaa aggacagtag aaaaggaaga tggcttctac aaatgccatc 10740 attgcgataa aggaaaggct atcgttcaag atgcctctac cgacagtggt cccaaagatg 10800 gacccccacc cacgaggaac atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 10860 aagtggattg atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt 10920 cgcaagaccc ttcctctata taaggaagtt catttcattt ggagagg 10967 <210> 5 <211> 10613 <212> DNA <213> Artificial Sequence <220> <223> pCYR2 2b <400> 5 gaattctcta gaaggcctcc atggggatcc gtttatttac aagagcgtac ggttcaaccc 60 ctgcctcccc tgtaaaactc cctagacttg aaacttcttc tttctagtat cttttctatg 120 gctttccccg cccccgcatt ctcactagcc aatcttttga acggcagtta cggtgtcgac 180 actcccgagg atgtggaacg tttgcgatct gagcaacgcg aagaggctgc tgcggcttgt 240 cgtaattaca ggcccctacc cgctgtggat gtcagcgaga gtgtcacaga ggacgcgcat 300 tccctccgaa ctcctgacgg agctcccgct gaagcggtgt ctgatgagtt tgtaacttat 360 ggtgctgaag attaccttga aaaatctgat gatgagctcc ttgtcgcttt tgagacgatg 420 gtcaaaccca tgcgtatcgg acaactatgg tgccctgcgt ttaataaatg ttcttttatt 480 tccagcattg ctatggccag agctttgttg ttggcaccta gaacatccca ccgaaccatg 540 aagtgttttg aagacctggt cgcggctatt tacactaaat ctgatttcta ttacagtgaa 600 gagtgtgaag ccgacgacgc tcagatagat atctcgtctc gcgatgtacc cggttattct 660 ttcgaaccgt ggtcccgaac gtctggattt gaaccgccgc ccatttgtga agcgtgcgac 720 atgatcatgt accagtgccc gtgttttgat ttcaatgctt taaagaaatc gtgcgctgag 780 aggactttcg ctgatgatta tgttattgaa ggtttagatg gtgttgttga taatgcgact 840 ctgttgtcga atttaggtcc atttttggta cctgtgaaat gtcaatatga aaaatgtcca 900 acaccaacca tcgcgattcc tccggattta aatcgtgcta ctgatcgtgt tgatatcaat 960 ttagttcaat ccatttgtga ctcgactctg cccactcata gtaattacga cgactctttt 1020 catcaagtgt tcgtcgaaag tgcagactac tctatagatc tggatcatgt tagacttcga 1080 cagtctgatc ttatcgcaaa aattccagat tcagggcata tgataccggt tctgaacacc 1140 gggagcggtc acaagagagt aggtacaacg aaggaggtcc ttacagcaat taagaaacgt 1200 aatgctgacg ttccagagct aggtgattcc gttaatctgt ctagattgag taaagctgtg 1260 gctgagagat tctttatttc atacatcaat ggtaactctc tagcatccag taactttgtc 1320 aatgtcgtta gtaatttcca cgattacatg gaaaagtgga agtcctcagg tctttcttat 1380 gatgatcttc cggatcttca tgctgagaat ttgcagtttt atgaccacat gataaaatcc 1440 gatgtgaaac ctgtggtgag cgacacactc aatatcgaca gaccggttcc agctactata 1500 acgtatcata agaagagtat aacctcccag ttctcaccgt tattcacagc gctattcgag 1560 cgcttccaga gatgccttcg agaacgcatt attcttcctg ttggtaagat ttcatccctt 1620 gagatggcag gatttgatgt caaaaacaaa cactgcctcg agattgattt gtctaagttt 1680 gataagtctc aaggtgaatt tcacttgcta atccaggaac acattttgaa tggtctagga 1740 tgtccagctc cgataactaa gtggtggtgt gatttccacc gattctctta catcagagac 1800 cgtagagctg gtgttggtat gcctattagt ttccagagac gaactggtga tgcattcact 1860 tattttggca ataccattgt caccatggct gagtttgcct ggtgttatga caccgaccaa 1920 ttcgaaaagc ttttattctc aggcgatgat tctctaggat tttcactgct tccccctgtt 1980 ggtgacccga gtaaattcac aactcttttc aacatggaag ccaaggtgat ggaacctgca 2040 gtaccatata tttgttcgaa gttcttactc tctgacgagt tcggtaacac attttccgtt 2100 ccagatccat tgcgcgaggt tcagcggtta ggaacaaaga aaattcccta ttctgacaat 2160 gatgaattct tgtttgctca cttcatgagc tttgttgatc gattgaagtt tttggaccga 2220 atgtctcagt cgtgtatcga tcaactttcg attttcttcg aattgaaata caagaagtct 2280 ggggaagagg ctgctttaat gctaggcgcc tttaagaagt ataccgctaa tttccagtcc 2340 tacaaagaac tctattattc agatcgtcgt cagtgcgaat tgatcaattc gttttgtagt 2400 acagagttca gggttgagcg tgtaaattcc aataaacagc gaaagaaata tggaattgaa 2460 cgtaggtgca atgacaaacg tcgaactcca actggctcgt atggtggagg cgaagaagca 2520 gagacgaagg tctcacaaac agaatcgacg ggaacgaggt cacaaaagtc ccagcgagag 2580 agcgcgttca aatctcagac tgttccgctt cctaccgttc tatcaagtag atggttcgga 2640 actgacaggg tcatgccgcc atgcgaacgt ggcggagtca cccgagcctg aggcctgacg 2700 cgtgtacgta aacctcccct tccgcatctc cctccggttt tctgtggcgg gagctgagtt 2760 ggcagtattg ctataaactg tctgaagtca ctaaacacat tgtggtgaac gggttgtcca 2820 tccagcttac ggctaaaatg gtcagtcgta gagaaatcta cgccagcaga cttacaagtc 2880 tctgaggcac ctttgaaacc atctcctagg tttcttcgga aggacttcgg tccgtgtact 2940 tctagcacaa cgtgctagtt tcagggtacg ggtgcccccc actttcgtgg ggcctccaaa 3000 aggagaccag gatccggtac cgagctcacg cgtctcgagg cccgggcact agtgtctgta 3060 cttatatcag tacactgacg agtccctaaa ggacgaaacg ggagaacgct agccaccacc 3120 accaccacca cgtgtgaatt acaggtgacc agctcgaatt tccccgatcg ttcaaacatt 3180 tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa 3240 tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg 3300 agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 3360 atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg 3420 gaattaaact atcagtgttt gacaggatat attggcgggt aaacctaaga gaaaagagcg 3480 tttattagaa taacggatat ttaaaagggc gtgaaaaggt ttatccgttc gtccatttgt 3540 atgtgcatgc caaccacagg gttcccctcg ggatcaaagt actttgatcc aacccctccg 3600 ctgctatagt gcagtcggct tctgacgttc agtgcagccg tcttctgaaa acgacatgtc 3660 gcacaagtcc taagttacgc gacaggctgc cgccctgccc ttttcctggc gttttcttgt 3720 cgcgtgtttt agtcgcataa agtagaatac ttgcgactag aaccggagac attacgccat 3780 gaacaagagc gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg acgaccagga 3840 cttgaccaac caacgggccg aactgcacgc ggccggctgc accaagctgt tttccgagaa 3900 gatcaccggc accaggcgcg accgcccgga gctggccagg atgcttgacc acctacgccc 3960 tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc gcgacctact 4020 ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg cagagccgtg 4080 ggccgacacc accacgccgg ccggccgcat ggtgttgacc gtgttcgccg gcattgccga 4140 gttcgagcgt tccctaatca tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg 4200 aggcgtgaag tttggccccc gccctaccct caccccggca cagatcgcgc acgcccgcga 4260 gctgatcgac caggaaggcc gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg 4320 ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg aggccaggcg 4380 gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac gccctggcgg ccgccgagaa 4440 tgaacgccaa gaggaacaag catgaaaccg caccaggacg gccaggacga accgtttttc 4500 attaccgaag agatcgaggc ggagatgatc gcggccgggt acgtgttcga gccgcccgcg 4560 cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc caagctggcg 4620 gcctggccgg ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa aaggtgatgt 4680 gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat 4740 aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt 4800 caggcaagac gaccatcgca acccatctag cccgcgccct gcaactcgcc ggggccgatg 4860 ttctgttagt cgattccgat ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag 4920 atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac gtgaaggcca 4980 tcggccggcg cgacttcgta gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt 5040 ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca gccaagccct tacgacatat 5100 gggccaccgc cgacctggtg gagctggtta agcagcgcat tgaggtcacg gatggaaggc 5160 tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg 5220 ccgaggcgct ggccgggtac gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga 5280 gctacccagg cactgccgcc gccggcacaa ccgttcttga atcagaaccc gagggcgacg 5340 ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc aaaactcatt tgagttaatg 5400 aggtaaagag aaaatgagca aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg 5460 cagcagcaag gctgcaacgt tggccagcct ggcagacacg ccagccatga agcgggtcaa 5520 ctttcagttg ccggcggagg atcacaccaa gctgaagatg tacgcggtac gccaaggcaa 5580 gaccattacc gagctgctat ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg 5640 aataaatgag tagatgaatt ttagcggcta aaggaggcgg catggaaaat caagaacaac 5700 caggcaccga cgccgtggaa tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa 5760 gcggctgggt tgtctgccgg ccctgcaatg gcactggaac ccccaagccc gaggaatcgg 5820 cgtgacggtc gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg 5880 gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc 5940 cccggtgaat cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg 6000 gcagccggtg cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc 6060 gttccgatgc tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt 6120 ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac 6180 gggcacgtag aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg 6240 gtactgatgg cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga 6300 gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga 6360 gccgatggcg gaaagcagaa agacgacctg gtagaaacct gcattcggtt aaacaccacg 6420 cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag 6480 ggtgaagcct tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac 6540 atcgagatcg agctagctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac 6600 gtgctgacgg ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac 6660 cgcctggcac gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac 6720 gaacgcagtg gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc 6780 gggtcaaatg acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc 6840 ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg 6900 gagcagatgc tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg tctctttcct 6960 gtggatagca cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt 7020 gggaacccaa agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaaagag 7080 aaaaaaggcg atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc 7140 ctggcctgtg cataactgtc tggccagcgc acagccgaag agctgcaaaa agcgcctacc 7200 cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc 7260 cgctcaaaaa tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg 7320 tcgccactcg accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt tcggtgatga 7380 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 7440 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc 7500 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca 7560 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 7620 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 7680 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 7740 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 7800 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 7860 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 7920 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 7980 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 8040 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 8100 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 8160 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 8220 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 8280 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 8340 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 8400 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 8460 aactcacgtt aagggatttt ggtcatgcat tctaggtact aaaacaattc atccagtaaa 8520 atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa aaatagctcg 8580 acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggc aatgtcatac 8640 cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt gccatctttc 8700 acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc ttcgggcttt 8760 tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc ttcttcccag 8820 ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc ggctaagcgg 8880 ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa gagcctgatg 8940 cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata ctcttccgag 9000 caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg ccgttcaaag 9060 tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc cttttcccgt 9120 tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata taggttttca 9180 ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc ttttacgcag 9240 cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc catttattat 9300 ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat aacaagacga 9360 actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca gctttttcaa 9420 agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg aaaccgcggt 9480 gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca 9540 tccgtgtttc aaacccggca gcttagttgc cgttcttccg aatagcatcg gtaacatgag 9600 caaagtctgc cgccttacaa cggctctccc gctgacgccg tcccggactg atgggctgcc 9660 tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg agctgttggc tggctggtgg 9720 caggatatat tgtggtgtaa acaaattgac gcttagacaa cttaataaca cattgcggac 9780 gtttttaatg tactgaatta acgccgaatt aattcctagg ccaccatgtt gggcccggcg 9840 cgccaagctt gcatgcctgc aggtcaacat ggtggagcac gacactctcg tctactccaa 9900 gaatatcaaa gatacagtct cagaagacca gagggctatt gagacttttc aacaaagggt 9960 aatatcggga aacctcctcg gattccattg cccagctatc tgtcacttca tcgaaaggac 10020 agtagaaaag gaagatggct tctacaaatg ccatcattgc gataaaggaa aggctatcgt 10080 tcaagatgcc tctaccgaca gtggtcccaa agatggaccc ccacccacga ggaacatcgt 10140 ggaaaaagaa gacgttccaa ccacgtcttc aaagcaagtg gattgatgtg atggtcaaca 10200 tggtggagca cgacactctc gtctactcca agaatatcaa agatacagtc tcagaagacc 10260 agagggctat tgagactttt caacaaaggg taatatcggg aaacctcctc ggattccatt 10320 gcccagctat ctgtcacttc atcgaaagga cagtagaaaa ggaagatggc ttctacaaat 10380 gccatcattg cgataaagga aaggctatcg ttcaagatgc ctctaccgac agtggtccca 10440 aagatggacc cccacccacg aggaacatcg tggaaaaaga agacgttcca accacgtctt 10500 caaagcaagt ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact 10560 atccttcgca agacccttcc tctatataag gaagttcatt tcatttggag agg 10613 <210> 6 <211> 9850 <212> DNA <213> Artificial Sequence <220> <223> pCfnyR3 <400> 6 gaattcgtaa tcttaccact gtgtgtgtgc gtgtgtgtgt gtcgagtcgt gttgtccgca 60 catttgagtc gtgctgtccg cacatatttt atcttttggg tacagtgtgt tagatttccc 120 gaggcatggc tttccaaggt accagtagga ctttaactca acagtcctca gcggctacgt 180 ctgacgatct tcaaaagata ttatttagcc ctgaagccat taagaaaatg gctactgagt 240 gtgacctagg ccggcatcat tggatgcgcg ctgataatgc tatttcagtc cggcccctcg 300 ttcccgaagt aacccacggt cgtattgctt ccttctttaa gtctggatat gatgttggtg 360 aattatgctc aaaaggatac atgagtgtcc ctcaagtgtt atgtgctgtt actcgaacag 420 tttccactga tgctgaaggg tctttgagaa tttacttagc tgatctgggc gacaaggagt 480 tatctcccat agatgggcaa tgcgtttcgt tacataacca tgatcttccc gctttggtgt 540 ctttccaacc gacgtatgat tgtcctatgg aaacagttgg gaatcgtaag cggtgttttg 600 ctgtcgttat cgaaagacat ggttacattg ggtataccgg taccacagct agcgtgtgta 660 gtaattggca agcaaggttt tcatccaaga ataacaacta cactcatatc gcagctggga 720 agactctagt actgcctttc aacagattag ctgagcaaac aaaaccgtca gctgtcgctc 780 gcctgttgaa gtcgcaattg aacaacattg aatcttcgca atatttgtta acgaatgcga 840 agattaatca aaatgcgcgc agtgagtccg aggatttaaa tgttgagagc cctcccgccg 900 caatcgggag ttcttccgcg tcccgctccg aagccttcag accgcaggtg gttaacggtc 960 tttagcactt tggtgcgtat tagtatataa gtatttgtga gtctgtacat aatactatat 1020 ctatagtgtc ctgtgtgagt tgatacagta gacatctgtg acgcgatgcc gtgttgagaa 1080 gggaacacat ctggttttag taagcctaca tcatagtttt gaggttcaat tcctcttact 1140 ccctgttgag ccccttactt tctcatggat gcttctccgc gagattgcgt tattgtctac 1200 tgactatata gagagtgttt gtgctgtgtt ttctcttttg tgtcgtagaa ttgagtcgag 1260 tcatggacaa atctgaatca accagtgctg gtcgtaaccg tcgacgtcgt ccgcgtcgtg 1320 gttcccgctc cgccccctcc tccgcggatg ctaactttag agtcttgtcg cagcagcttt 1380 cgcgacttaa taagacgtta gcagctggtc gtccaactat taaccaccca acctttgtag 1440 ggagtgaacg ctgtagacct gggtacacgt tcacatctat taccctaaag ccaccaaaaa 1500 tagaccgtgg gtcttattac ggtaaaaggt tgttactacc tgattcagtc acggaatatg 1560 ataagaagct tgtttcgcgc attcaaattc gagttaatcc tttgccgaaa tttgattcta 1620 ccgtgtgggt gacagtccgt aaagttcctg cctcctcgga cttatccgtt gccgccatct 1680 ctgctatgtt cgcggacgga gcctcaccgg tactggttta tcagtatgcc gcatctggag 1740 tccaagccaa caacaaactg ttgtatgatc tttcggcgat gcgcgctgat ataggtgaca 1800 tgagaaagta cgccgtcctc gtgtattcaa aagacgatgc gctcgagacg gacgagctag 1860 tacttcatgt tgacatcgag caccaacgca ttcccacatc tggagtgctc ccagtctgat 1920 tccgtgttcc cagaatcctc cctccgatct ctgtggcggg acgtgagttg gcagttctgc 1980 tataaactgt ctgaagtcac taaacgtttt ttacggtgaa cgggttgtcc atccagctta 2040 cggctaaaat ggtcagtcgt ggagaaatcc acgccagcag atttacaaat ctctgaggcg 2100 cctttgaaac catctcctag gtttcttcgg aaggacttcg gtccgtgtac ctctagcaca 2160 acgtgctagt ttcagggtac gggtgccccc ccactttcgt gggggcctcc aaaaggagac 2220 cagaattctc tagaaggcct ccatggggat ccggtaccga gctcacgcgt ctcgaggccc 2280 gggcactagt gtctgtactt atatcagtac actgacgagt ccctaaagga cgaaacggga 2340 gaacgctagc caccaccacc accaccacgt gtgaattaca ggtgaccagc tcgaatttcc 2400 ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 2460 cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 2520 gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 2580 acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 2640 ctatgttact agatcgggaa ttaaactatc agtgtttgac aggatatatt ggcgggtaaa 2700 cctaagagaa aagagcgttt attagaataa cggatattta aaagggcgtg aaaaggttta 2760 tccgttcgtc catttgtatg tgcatgccaa ccacagggtt cccctcggga tcaaagtact 2820 ttgatccaac ccctccgctg ctatagtgca gtcggcttct gacgttcagt gcagccgtct 2880 tctgaaaacg acatgtcgca caagtcctaa gttacgcgac aggctgccgc cctgcccttt 2940 tcctggcgtt ttcttgtcgc gtgttttagt cgcataaagt agaatacttg cgactagaac 3000 cggagacatt acgccatgaa caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc 3060 agcaccgacg accaggactt gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc 3120 aagctgtttt ccgagaagat caccggcacc aggcgcgacc gcccggagct ggccaggatg 3180 cttgaccacc tacgccctgg cgacgttgtg acagtgacca ggctagaccg cctggcccgc 3240 agcacccgcg acctactgga cattgccgag cgcatccagg aggccggcgc gggcctgcgt 3300 agcctggcag agccgtgggc cgacaccacc acgccggccg gccgcatggt gttgaccgtg 3360 ttcgccggca ttgccgagtt cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc 3420 gaggccgcca aggcccgagg cgtgaagttt ggcccccgcc ctaccctcac cccggcacag 3480 atcgcgcacg cccgcgagct gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca 3540 ctgcttggcg tgcatcgctc gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg 3600 cccaccgagg ccaggcggcg cggtgccttc cgtgaggacg cattgaccga ggccgacgcc 3660 ctggcggccg ccgagaatga acgccaagag gaacaagcat gaaaccgcac caggacggcc 3720 aggacgaacc gtttttcatt accgaagaga tcgaggcgga gatgatcgcg gccgggtacg 3780 tgttcgagcc gcccgcgcac gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt 3840 ctgatgccaa gctggcggcc tggccggcca gcttggccgc tgaagaaacc gagcgccgcc 3900 gtctaaaaag gtgatgtgta tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata 3960 tgatgcgatg agtaaataaa caaatacgca aggggaacgc atgaaggtta tcgctgtact 4020 taaccagaaa ggcgggtcag gcaagacgac catcgcaacc catctagccc gcgccctgca 4080 actcgccggg gccgatgttc tgttagtcga ttccgatccc cagggcagtg cccgcgattg 4140 ggcggccgtg cgggaagatc aaccgctaac cgttgtcggc atcgaccgcc cgacgattga 4200 ccgcgacgtg aaggccatcg gccggcgcga cttcgtagtg atcgacggag cgccccaggc 4260 ggcggacttg gctgtgtccg cgatcaaggc agccgacttc gtgctgattc cggtgcagcc 4320 aagcccttac gacatatggg ccaccgccga cctggtggag ctggttaagc agcgcattga 4380 ggtcacggat ggaaggctac aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg 4440 catcggcggt gaggttgccg aggcgctggc cgggtacgag ctgcccattc ttgagtcccg 4500 tatcacgcag cgcgtgagct acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc 4560 agaacccgag ggcgacgctg cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa 4620 actcatttga gttaatgagg taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc 4680 ggccgtccga gcgcacgcag cagcaaggct gcaacgttgg ccagcctggc agacacgcca 4740 gccatgaagc gggtcaactt tcagttgccg gcggaggatc acaccaagct gaagatgtac 4800 gcggtacgcc aaggcaagac cattaccgag ctgctatctg aatacatcgc gcagctacca 4860 gagtaaatga gcaaatgaat aaatgagtag atgaatttta gcggctaaag gaggcggcat 4920 ggaaaatcaa gaacaaccag gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg 4980 cggttggcca ggcgtaagcg gctgggttgt ctgccggccc tgcaatggca ctggaacccc 5040 caagcccgag gaatcggcgt gacggtcgca aaccatccgg cccggtacaa atcggcgcgg 5100 cgctgggtga tgacctggtg gagaagttga aggccgcgca ggccgcccag cggcaacgca 5160 tcgaggcaga agcacgcccc ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag 5220 aatcccggca accgccggca gccggtgcgc cgtcgattag gaagccgccc aagggcgacg 5280 agcaaccaga ttttttcgtt ccgatgctct atgacgtggg cacccgcgat agtcgcagca 5340 tcatggacgt ggccgttttc cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc 5400 gctacgagct tccagacggg cacgtagagg tttccgcagg gccggccggc atggccagtg 5460 tgtgggatta cgacctggta ctgatggcgg tttcccatct aaccgaatcc atgaaccgat 5520 accgggaagg gaagggagac aagcccggcc gcgtgttccg tccacacgtt gcggacgtac 5580 tcaagttctg ccggcgagcc gatggcggaa agcagaaaga cgacctggta gaaacctgca 5640 ttcggttaaa caccacgcac gttgccatgc agcgtacgaa gaaggccaag aacggccgcc 5700 tggtgacggt atccgagggt gaagccttga ttagccgcta caagatcgta aagagcgaaa 5760 ccgggcggcc ggagtacatc gagatcgagc tagctgattg gatgtaccgc gagatcacag 5820 aaggcaagaa cccggacgtg ctgacggttc accccgatta ctttttgatc gatcccggca 5880 tcggccgttt tctctaccgc ctggcacgcc gcgccgcagg caaggcagaa gccagatggt 5940 tgttcaagac gatctacgaa cgcagtggca gcgccggaga gttcaagaag ttctgtttca 6000 ccgtgcgcaa gctgatcggg tcaaatgacc tgccggagta cgatttgaag gaggaggcgg 6060 ggcaggctgg cccgatccta gtcatgcgct accgcaacct gatcgagggc gaagcatccg 6120 ccggttccta atgtacggag cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc 6180 gaaaaggtct ctttcctgtg gatagcacgt acattgggaa cccaaagccg tacattggga 6240 accggaaccc gtacattggg aacccaaagc cgtacattgg gaaccggtca cacatgtaag 6300 tgactgatat aaaagagaaa aaaggcgatt tttccgccta aaactcttta aaacttatta 6360 aaactcttaa aacccgcctg gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc 6420 tgcaaaaagc gcctaccctt cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc 6480 ctatcgcggc cgctggccgc tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc 6540 gcggacaagc cgcgccgtcg ccactcgacc gccggcgccc acatcaaggc accctgcctc 6600 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 6660 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 6720 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc 6780 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac 6840 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg 6900 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 6960 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 7020 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 7080 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 7140 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 7200 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 7260 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 7320 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 7380 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 7440 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 7500 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 7560 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 7620 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 7680 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgcattct aggtactaaa 7740 acaattcatc cagtaaaata taatatttta ttttctccca atcaggcttg atccccagta 7800 agtcaaaaaa tagctcgaca tactgttctt ccccgatatc ctccctgatc gaccggacgc 7860 agaaggcaat gtcataccac ttgtccgccc tgccgcttct cccaagatca ataaagccac 7920 ttactttgcc atctttcaca aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa 7980 gttcctcttc gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg 8040 gagtgtcttc ttcccagttt tcgcaatcca catcggccag atcgttattc agtaagtaat 8100 ccaattcggc taagcggctg tctaagctat tcgtataggg acaatccgat atgtcgatgg 8160 agtgaaagag cctgatgcac tccgcataca gctcgataat cttttcaggg ctttgttcat 8220 cttcatactc ttccgagcaa aggacgccat cggcctcact catgagcaga ttgctccagc 8280 catcatgccg ttcaaagtgc aggacctttg gaacaggcag ctttccttcc agccatagca 8340 tcatgtcctt ttcccgttcc acatcatagg tggtcccttt ataccggctg tccgtcattt 8400 ttaaatatag gttttcattt tctcccacca gcttatatac cttagcagga gacattcctt 8460 ccgtatcttt tacgcagcgg tatttttcga tcagtttttt caattccggt gatattctca 8520 ttttagccat ttattatttc cttcctcttt tctacagtat ttaaagatac cccaagaagc 8580 taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc ttaaatacca 8640 gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat cgacggagcc 8700 gattttgaaa ccgcggtgat cacaggcagc aacgctctgt catcgttaca atcaacatgc 8760 taccctccgc gagatcatcc gtgtttcaaa cccggcagct tagttgccgt tcttccgaat 8820 agcatcggta acatgagcaa agtctgccgc cttacaacgg ctctcccgct gacgccgtcc 8880 cggactgatg ggctgcctgt atcgagtggt gattttgtgc cgagctgccg gtcggggagc 8940 tgttggctgg ctggtggcag gatatattgt ggtgtaaaca aattgacgct tagacaactt 9000 aataacacat tgcggacgtt tttaatgtac tgaattaacg ccgaattaat tcctaggcca 9060 ccatgttggg cccggcgcgc caagcttgca tgcctgcagg tcaacatggt ggagcacgac 9120 actctcgtct actccaagaa tatcaaagat acagtctcag aagaccagag ggctattgag 9180 acttttcaac aaagggtaat atcgggaaac ctcctcggat tccattgccc agctatctgt 9240 cacttcatcg aaaggacagt agaaaaggaa gatggcttct acaaatgcca tcattgcgat 9300 aaaggaaagg ctatcgttca agatgcctct accgacagtg gtcccaaaga tggaccccca 9360 cccacgagga acatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat 9420 tgatgtgatg gtcaacatgg tggagcacga cactctcgtc tactccaaga atatcaaaga 9480 tacagtctca gaagaccaga gggctattga gacttttcaa caaagggtaa tatcgggaaa 9540 cctcctcgga ttccattgcc cagctatctg tcacttcatc gaaaggacag tagaaaagga 9600 agatggcttc tacaaatgcc atcattgcga taaaggaaag gctatcgttc aagatgcctc 9660 taccgacagt ggtcccaaag atggaccccc acccacgagg aacatcgtgg aaaaagaaga 9720 cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat atctccactg acgtaaggga 9780 tgacgcacaa tcccactatc cttcgcaaga cccttcctct atataaggaa gttcatttca 9840 tttggagagg 9850 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggaattctgg tctccttttr gagrcc 26 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 agggatcctg gtctcctttt rgagrcc 27 <210> 9 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 aggatccgtt tatttacaag agcgtacgg 29 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 agggatcctg gtctcctttt rgagrcc 27 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 agggatccgt ttatttacaa gagcgtacgg 30 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gactagtggt ctccttttrg agrcc 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 agggatccgt aatcttacca ctgtg 25 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 agggatcctg gtctcctttt rgagrcc 27 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 tccctgttga gcccccttac ttt 23 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 caactcagat cccgccacag a 21 <210> 17 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ggaattcgtt tatttacaag agcgtacgg 29 <210> 18 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 agggatcctg gtctcctttt rgagrcc 27 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 agggatccgt ttatttacaa gagcgtacgg 30 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 agggatcctg gtctcctttt rgagrcc 27 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ggaattcgta atcttaccac tgtg 24 <210> 22 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 ggaattctgg tctccttttr gagrcc 26 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 ctagtaaagg agaagaactt ttcac 25 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 atcatttgta tagttcatcc atgc 24 <210> 25 <211> 11330 <212> DNA <213> Artificial Sequence <220> <223> pCYR2 2b-GFP <400> 25 gaattctcta gaaggcctcc atggggatcc gtttatttac aagagcgtac ggttcaaccc 60 ctgcctcccc tgtaaaactc cctagacttg aaacttcttc tttctagtat cttttctatg 120 gctttccccg cccccgcatt ctcactagcc aatcttttga acggcagtta cggtgtcgac 180 actcccgagg atgtggaacg tttgcgatct gagcaacgcg aagaggctgc tgcggcttgt 240 cgtaattaca ggcccctacc cgctgtggat gtcagcgaga gtgtcacaga ggacgcgcat 300 tccctccgaa ctcctgacgg agctcccgct gaagcggtgt ctgatgagtt tgtaacttat 360 ggtgctgaag attaccttga aaaatctgat gatgagctcc ttgtcgcttt tgagacgatg 420 gtcaaaccca tgcgtatcgg acaactatgg tgccctgcgt ttaataaatg ttcttttatt 480 tccagcattg ctatggccag agctttgttg ttggcaccta gaacatccca ccgaaccatg 540 aagtgttttg aagacctggt cgcggctatt tacactaaat ctgatttcta ttacagtgaa 600 gagtgtgaag ccgacgacgc tcagatagat atctcgtctc gcgatgtacc cggttattct 660 ttcgaaccgt ggtcccgaac gtctggattt gaaccgccgc ccatttgtga agcgtgcgac 720 atgatcatgt accagtgccc gtgttttgat ttcaatgctt taaagaaatc gtgcgctgag 780 aggactttcg ctgatgatta tgttattgaa ggtttagatg gtgttgttga taatgcgact 840 ctgttgtcga atttaggtcc atttttggta cctgtgaaat gtcaatatga aaaatgtcca 900 acaccaacca tcgcgattcc tccggattta aatcgtgcta ctgatcgtgt tgatatcaat 960 ttagttcaat ccatttgtga ctcgactctg cccactcata gtaattacga cgactctttt 1020 catcaagtgt tcgtcgaaag tgcagactac tctatagatc tggatcatgt tagacttcga 1080 cagtctgatc ttatcgcaaa aattccagat tcagggcata tgataccggt tctgaacacc 1140 gggagcggtc acaagagagt aggtacaacg aaggaggtcc ttacagcaat taagaaacgt 1200 aatgctgacg ttccagagct aggtgattcc gttaatctgt ctagattgag taaagctgtg 1260 gctgagagat tctttatttc atacatcaat ggtaactctc tagcatccag taactttgtc 1320 aatgtcgtta gtaatttcca cgattacatg gaaaagtgga agtcctcagg tctttcttat 1380 gatgatcttc cggatcttca tgctgagaat ttgcagtttt atgaccacat gataaaatcc 1440 gatgtgaaac ctgtggtgag cgacacactc aatatcgaca gaccggttcc agctactata 1500 acgtatcata agaagagtat aacctcccag ttctcaccgt tattcacagc gctattcgag 1560 cgcttccaga gatgccttcg agaacgcatt attcttcctg ttggtaagat ttcatccctt 1620 gagatggcag gatttgatgt caaaaacaaa cactgcctcg agattgattt gtctaagttt 1680 gataagtctc aaggtgaatt tcacttgcta atccaggaac acattttgaa tggtctagga 1740 tgtccagctc cgataactaa gtggtggtgt gatttccacc gattctctta catcagagac 1800 cgtagagctg gtgttggtat gcctattagt ttccagagac gaactggtga tgcattcact 1860 tattttggca ataccattgt caccatggct gagtttgcct ggtgttatga caccgaccaa 1920 ttcgaaaagc ttttattctc aggcgatgat tctctaggat tttcactgct tccccctgtt 1980 ggtgacccga gtaaattcac aactcttttc aacatggaag ccaaggtgat ggaacctgca 2040 gtaccatata tttgttcgaa gttcttactc tctgacgagt tcggtaacac attttccgtt 2100 ccagatccat tgcgcgaggt tcagcggtta ggaacaaaga aaattcccta ttctgacaat 2160 gatgaattct tgtttgctca cttcatgagc tttgttgatc gattgaagtt tttggaccga 2220 atgtctcagt cgtgtatcga tcaactttcg attttcttcg aattgaaata caagaagtct 2280 ggggaagagg ctgctttaat gctaggcgcc tttaagaagt ataccgctaa tttccagtcc 2340 tacaaagaac tctattattc agatcgtcgt cagtgcgaat tgatcaattc gttttgtagt 2400 acagagttca gggttgagcg tgtaaattcc aataaacagc gaaagaaata tggaattgaa 2460 cgtaggtgca atgacaaacg tcgaactcca actggctcgt atggtggagg cgaagaagca 2520 gagacgaagg tctcacaaac agaatcgacg ggaacgaggt cacaaaagtc ccagcgagag 2580 agcgcgttca aatctcagac tgttccgctt cctaccgttc tatcaagtag atggttcgga 2640 actgacaggg tcatgccgcc atgcgaacgt ggcggagtca cccgagcctg aggctagtaa 2700 aggagaagaa cttttcactg gagttgtccc aattcttgtt gaattagatg gtgatgttaa 2760 tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat gcaacatacg gaaaacttac 2820 ccttaaattt atttgcacta ctggaaaact acctgttccg tggccaacac ttgtcactac 2880 tttctcttat ggtgttcaat gcttttcaag atacccagat catatgaagc ggcacgactt 2940 cttcaagagc gccatgcctg agggatacgt gcaggagagg accatcttct tcaaggacga 3000 cgggaactac aagacacgtg ctgaagtcaa gtttgaggga gacaccctcg tcaacaggat 3060 cgagcttaag ggaatcgatt tcaaggagga cggaaacatc ctcggccaca agttggaata 3120 caactacaac tcccacaacg tatacatcat ggccgacaag caaaagaacg gcatcaaagc 3180 caacttcaag acccgccaca acatcgaaga cggcggcgtg caactcgctg atcattatca 3240 acaaaatact ccaattggcg atggccctgt ccttttacca gacaaccatt acctgtccac 3300 acaatctgcc ctttcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt 3360 tgtaacagct gctgggatta cacatggcat ggatgaacta tacaaatgat cctgacgcgt 3420 gtacgtaaac ctccccttcc gcatctccct ccggttttct gtggcgggag ctgagttggc 3480 agtattgcta taaactgtct gaagtcacta aacacattgt ggtgaacggg ttgtccatcc 3540 agcttacggc taaaatggtc agtcgtagag aaatctacgc cagcagactt acaagtctct 3600 gaggcacctt tgaaaccatc tcctaggttt cttcggaagg acttcggtcc gtgtacttct 3660 agcacaacgt gctagtttca gggtacgggt gccccccact ttcgtggggc ctccaaaagg 3720 agaccaggat ccggtaccga gctcacgcgt ctcgaggccc gggcactagt gtctgtactt 3780 atatcagtac actgacgagt ccctaaagga cgaaacggga gaacgctagc caccaccacc 3840 accaccacgt gtgaattaca ggtgaccagc tcgaatttcc ccgatcgttc aaacatttgg 3900 caataaagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt 3960 ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga 4020 tgggttttta tgattagagt cccgcaatta tacatttaat acgcgataga aaacaaaata 4080 tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa 4140 ttaaactatc agtgtttgac aggatatatt ggcgggtaaa cctaagagaa aagagcgttt 4200 attagaataa cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg 4260 tgcatgccaa ccacagggtt cccctcggga tcaaagtact ttgatccaac ccctccgctg 4320 ctatagtgca gtcggcttct gacgttcagt gcagccgtct tctgaaaacg acatgtcgca 4380 caagtcctaa gttacgcgac aggctgccgc cctgcccttt tcctggcgtt ttcttgtcgc 4440 gtgttttagt cgcataaagt agaatacttg cgactagaac cggagacatt acgccatgaa 4500 caagagcgcc gccgctggcc tgctgggcta tgcccgcgtc agcaccgacg accaggactt 4560 gaccaaccaa cgggccgaac tgcacgcggc cggctgcacc aagctgtttt ccgagaagat 4620 caccggcacc aggcgcgacc gcccggagct ggccaggatg cttgaccacc tacgccctgg 4680 cgacgttgtg acagtgacca ggctagaccg cctggcccgc agcacccgcg acctactgga 4740 cattgccgag cgcatccagg aggccggcgc gggcctgcgt agcctggcag agccgtgggc 4800 cgacaccacc acgccggccg gccgcatggt gttgaccgtg ttcgccggca ttgccgagtt 4860 cgagcgttcc ctaatcatcg accgcacccg gagcgggcgc gaggccgcca aggcccgagg 4920 cgtgaagttt ggcccccgcc ctaccctcac cccggcacag atcgcgcacg cccgcgagct 4980 gatcgaccag gaaggccgca ccgtgaaaga ggcggctgca ctgcttggcg tgcatcgctc 5040 gaccctgtac cgcgcacttg agcgcagcga ggaagtgacg cccaccgagg ccaggcggcg 5100 cggtgccttc cgtgaggacg cattgaccga ggccgacgcc ctggcggccg ccgagaatga 5160 acgccaagag gaacaagcat gaaaccgcac caggacggcc aggacgaacc gtttttcatt 5220 accgaagaga tcgaggcgga gatgatcgcg gccgggtacg tgttcgagcc gcccgcgcac 5280 gtctcaaccg tgcggctgca tgaaatcctg gccggtttgt ctgatgccaa gctggcggcc 5340 tggccggcca gcttggccgc tgaagaaacc gagcgccgcc gtctaaaaag gtgatgtgta 5400 tttgagtaaa acagcttgcg tcatgcggtc gctgcgtata tgatgcgatg agtaaataaa 5460 caaatacgca aggggaacgc atgaaggtta tcgctgtact taaccagaaa ggcgggtcag 5520 gcaagacgac catcgcaacc catctagccc gcgccctgca actcgccggg gccgatgttc 5580 tgttagtcga ttccgatccc cagggcagtg cccgcgattg ggcggccgtg cgggaagatc 5640 aaccgctaac cgttgtcggc atcgaccgcc cgacgattga ccgcgacgtg aaggccatcg 5700 gccggcgcga cttcgtagtg atcgacggag cgccccaggc ggcggacttg gctgtgtccg 5760 cgatcaaggc agccgacttc gtgctgattc cggtgcagcc aagcccttac gacatatggg 5820 ccaccgccga cctggtggag ctggttaagc agcgcattga ggtcacggat ggaaggctac 5880 aagcggcctt tgtcgtgtcg cgggcgatca aaggcacgcg catcggcggt gaggttgccg 5940 aggcgctggc cgggtacgag ctgcccattc ttgagtcccg tatcacgcag cgcgtgagct 6000 acccaggcac tgccgccgcc ggcacaaccg ttcttgaatc agaacccgag ggcgacgctg 6060 cccgcgaggt ccaggcgctg gccgctgaaa ttaaatcaaa actcatttga gttaatgagg 6120 taaagagaaa atgagcaaaa gcacaaacac gctaagtgcc ggccgtccga gcgcacgcag 6180 cagcaaggct gcaacgttgg ccagcctggc agacacgcca gccatgaagc gggtcaactt 6240 tcagttgccg gcggaggatc acaccaagct gaagatgtac gcggtacgcc aaggcaagac 6300 cattaccgag ctgctatctg aatacatcgc gcagctacca gagtaaatga gcaaatgaat 6360 aaatgagtag atgaatttta gcggctaaag gaggcggcat ggaaaatcaa gaacaaccag 6420 gcaccgacgc cgtggaatgc cccatgtgtg gaggaacggg cggttggcca ggcgtaagcg 6480 gctgggttgt ctgccggccc tgcaatggca ctggaacccc caagcccgag gaatcggcgt 6540 gacggtcgca aaccatccgg cccggtacaa atcggcgcgg cgctgggtga tgacctggtg 6600 gagaagttga aggccgcgca ggccgcccag cggcaacgca tcgaggcaga agcacgcccc 6660 ggtgaatcgt ggcaagcggc cgctgatcga atccgcaaag aatcccggca accgccggca 6720 gccggtgcgc cgtcgattag gaagccgccc aagggcgacg agcaaccaga ttttttcgtt 6780 ccgatgctct atgacgtggg cacccgcgat agtcgcagca tcatggacgt ggccgttttc 6840 cgtctgtcga agcgtgaccg acgagctggc gaggtgatcc gctacgagct tccagacggg 6900 cacgtagagg tttccgcagg gccggccggc atggccagtg tgtgggatta cgacctggta 6960 ctgatggcgg tttcccatct aaccgaatcc atgaaccgat accgggaagg gaagggagac 7020 aagcccggcc gcgtgttccg tccacacgtt gcggacgtac tcaagttctg ccggcgagcc 7080 gatggcggaa agcagaaaga cgacctggta gaaacctgca ttcggttaaa caccacgcac 7140 gttgccatgc agcgtacgaa gaaggccaag aacggccgcc tggtgacggt atccgagggt 7200 gaagccttga ttagccgcta caagatcgta aagagcgaaa ccgggcggcc ggagtacatc 7260 gagatcgagc tagctgattg gatgtaccgc gagatcacag aaggcaagaa cccggacgtg 7320 ctgacggttc accccgatta ctttttgatc gatcccggca tcggccgttt tctctaccgc 7380 ctggcacgcc gcgccgcagg caaggcagaa gccagatggt tgttcaagac gatctacgaa 7440 cgcagtggca gcgccggaga gttcaagaag ttctgtttca ccgtgcgcaa gctgatcggg 7500 tcaaatgacc tgccggagta cgatttgaag gaggaggcgg ggcaggctgg cccgatccta 7560 gtcatgcgct accgcaacct gatcgagggc gaagcatccg ccggttccta atgtacggag 7620 cagatgctag ggcaaattgc cctagcaggg gaaaaaggtc gaaaaggtct ctttcctgtg 7680 gatagcacgt acattgggaa cccaaagccg tacattggga accggaaccc gtacattggg 7740 aacccaaagc cgtacattgg gaaccggtca cacatgtaag tgactgatat aaaagagaaa 7800 aaaggcgatt tttccgccta aaactcttta aaacttatta aaactcttaa aacccgcctg 7860 gcctgtgcat aactgtctgg ccagcgcaca gccgaagagc tgcaaaaagc gcctaccctt 7920 cggtcgctgc gctccctacg ccccgccgct tcgcgtcggc ctatcgcggc cgctggccgc 7980 tcaaaaatgg ctggcctacg gccaggcaat ctaccagggc gcggacaagc cgcgccgtcg 8040 ccactcgacc gccggcgccc acatcaaggc accctgcctc gcgcgtttcg gtgatgacgg 8100 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 8160 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc 8220 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag 8280 cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga 8340 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 8400 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 8460 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 8520 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 8580 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 8640 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 8700 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 8760 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 8820 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 8880 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 8940 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 9000 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 9060 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 9120 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 9180 tcacgttaag ggattttggt catgcattct aggtactaaa acaattcatc cagtaaaata 9240 taatatttta ttttctccca atcaggcttg atccccagta agtcaaaaaa tagctcgaca 9300 tactgttctt ccccgatatc ctccctgatc gaccggacgc agaaggcaat gtcataccac 9360 ttgtccgccc tgccgcttct cccaagatca ataaagccac ttactttgcc atctttcaca 9420 aagatgttgc tgtctcccag gtcgccgtgg gaaaagacaa gttcctcttc gggcttttcc 9480 gtctttaaaa aatcatacag ctcgcgcgga tctttaaatg gagtgtcttc ttcccagttt 9540 tcgcaatcca catcggccag atcgttattc agtaagtaat ccaattcggc taagcggctg 9600 tctaagctat tcgtataggg acaatccgat atgtcgatgg agtgaaagag cctgatgcac 9660 tccgcataca gctcgataat cttttcaggg ctttgttcat cttcatactc ttccgagcaa 9720 aggacgccat cggcctcact catgagcaga ttgctccagc catcatgccg ttcaaagtgc 9780 aggacctttg gaacaggcag ctttccttcc agccatagca tcatgtcctt ttcccgttcc 9840 acatcatagg tggtcccttt ataccggctg tccgtcattt ttaaatatag gttttcattt 9900 tctcccacca gcttatatac cttagcagga gacattcctt ccgtatcttt tacgcagcgg 9960 tatttttcga tcagtttttt caattccggt gatattctca ttttagccat ttattatttc 10020 cttcctcttt tctacagtat ttaaagatac cccaagaagc taattataac aagacgaact 10080 ccaattcact gttccttgca ttctaaaacc ttaaatacca gaaaacagct ttttcaaagt 10140 tgttttcaaa gttggcgtat aacatagtat cgacggagcc gattttgaaa ccgcggtgat 10200 cacaggcagc aacgctctgt catcgttaca atcaacatgc taccctccgc gagatcatcc 10260 gtgtttcaaa cccggcagct tagttgccgt tcttccgaat agcatcggta acatgagcaa 10320 agtctgccgc cttacaacgg ctctcccgct gacgccgtcc cggactgatg ggctgcctgt 10380 atcgagtggt gattttgtgc cgagctgccg gtcggggagc tgttggctgg ctggtggcag 10440 gatatattgt ggtgtaaaca aattgacgct tagacaactt aataacacat tgcggacgtt 10500 tttaatgtac tgaattaacg ccgaattaat tcctaggcca ccatgttggg cccggcgcgc 10560 caagcttgca tgcctgcagg tcaacatggt ggagcacgac actctcgtct actccaagaa 10620 tatcaaagat acagtctcag aagaccagag ggctattgag acttttcaac aaagggtaat 10680 atcgggaaac ctcctcggat tccattgccc agctatctgt cacttcatcg aaaggacagt 10740 agaaaaggaa gatggcttct acaaatgcca tcattgcgat aaaggaaagg ctatcgttca 10800 agatgcctct accgacagtg gtcccaaaga tggaccccca cccacgagga acatcgtgga 10860 aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgatg gtcaacatgg 10920 tggagcacga cactctcgtc tactccaaga atatcaaaga tacagtctca gaagaccaga 10980 gggctattga gacttttcaa caaagggtaa tatcgggaaa cctcctcgga ttccattgcc 11040 cagctatctg tcacttcatc gaaaggacag tagaaaagga agatggcttc tacaaatgcc 11100 atcattgcga taaaggaaag gctatcgttc aagatgcctc taccgacagt ggtcccaaag 11160 atggaccccc acccacgagg aacatcgtgg aaaaagaaga cgttccaacc acgtcttcaa 11220 agcaagtgga ttgatgtgat atctccactg acgtaaggga tgacgcacaa tcccactatc 11280 cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagagg 11330

Claims (8)

1) 오이 모자이크 바이러스(Cucumber mosaic virus; CMV)의 RNA1a 유전자를 포함하는 재조합 벡터를 제조하는 단계;

2) CMV의 RNA2a 유전자와 2b 유전자를 포함하고 상기 2b 유전자의 3' 말단에 외래 유전자를 삽입할 수 있는 제한 효소 부위를 포함하는 발현벡터에 외래유전자를 삽입하여 재조합 벡터를 제조하는 단계;

3) CMV의 RNA3a 유전자와 코트 단백질(coat protein; CP)을 코딩하는 유전자를 포함하는 재조합 벡터를 제조하는 단계;

4) 상기 1), 2) 및 3) 단계에서 제조된 각각의 재조합 벡터로 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens)를 각각 형질전환하여 각각의 형질전환체를 얻는 단계; 및

5) 상기 각각의 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens) 형질전환체를 혼합하고 혼합액을 식물체에 도입하는 단계를 포함하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법.

1) 오이 모자이크 바이러스(Cucumber mosaic virus; CMV)의 RNA1a 유전자를 포함하는 재조합 벡터를 제조하는 단계;

2) CMV의 RNA2a 유전자와 2b 유전자를 포함하고 상기 2b 유전자의 3' 말단에 외래 유전자를 삽입할 수 있는 제한 효소 부위를 포함하는 발현벡터에 외래유전자 를 삽입하여 재조합 벡터를 제조하는 단계;

3) CMV의 RNA3a 유전자와 코트 단백질(coat protein; CP)을 코딩하는 유전자를 포함하는 재조합 벡터를 제조하는 단계;

4) 상기 1), 2) 및 3) 단계에서 제조된 각각의 재조합 벡터로 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens)를 각각 형질전환하여 각각의 형질전환체를 얻는 단계;

5) 상기 각각의 아그로박테리움 투메파시엔스(Agrobacterium tumefaciens) 형질전환체를 배양하고 원심분리하여 현탁하는 단계;

6) 상기 각각의 현탁액을 같은 부피비율로 혼합하는 단계;

7) 상기 혼합액을 식물체에 인필트레이션(infiltration)하는 단계; 및

8) 상기 인필트레이션(infiltration)된 식물체에서 상기 외래유전자의 발현을 확인하는 단계를 포함하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법.

제 1 항 또는 제 2 항에 있어서, 상기 1) 단계에서 제조되는 상기 재조합 벡터는 서열번호 1 또는 서열번호 4인 것을 특징으로 하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법.

제 1 항 또는 제 2 항에 있어서, 상기 2) 단계의 상기 발현벡터는 서열번호 5인 것을 특징으로 하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법.

제 1 항 또는 제 2 항에 있어서, 상기 3) 단계에서 제조되는 상기 재조합 벡터는 서열번호 3 또는 서열번호 6인 것을 특징으로 하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법.

제 1 항 또는 제 2 항에 있어서, 상기 식물체는 니코티아나 벤타미아나(Nicotiana benthamiana)인 것을 특징으로 하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법.

제 1 항 또는 제 2 항에 있어서, 상기 외래유전자는 제초제 저항성 유전자, 질병 저항성 유전자, 살충제 저항성 유전자, 곡식 또는 과실의 품질에 영향을 주는 유전자, 스크리닝 가능한 표식 유전자 및 환경 또는 스트레스 저항성 유전자로 구성된 그룹에서 선택되는 것을 특징으로 하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법.

제 1 항 또는 제 2 항에 있어서, 상기 외래유전자는 제초제 글리포세이트(Glyphosate)의 저항성 유전자인 5-에놀피루빌시키메이트 3-포스페이트 신타아제(5-Enolpyruvylshikimate 3-phosphate synthase; EPSP synthase), 제초제 비알라포스(바스타)(bialaphos(Basta))의 저항성 유전자인 포스피노트리신 아세틸트랜스 퍼라제(phosphinothricin acetyltransferase), 바이러스 저항성 단백질인 코트 단백질(coat protein)을 코딩하는 유전자, 해충 저항성 유전자인 바실러스 투린지엔시스(Bacillus thuringiensis)로부터 분리한 Bt 독소(Bt toxin) 생성 유전자 및 과실 완숙 방지를 위한 1-아미노사이클로프로판-1-카르복실릭산 디아미나아제(1-aminocyclopropane-1-carboxylic acid(ACC) deaminase) 유전자, 바이러스 저항성 식물을 용이하게 선발할 수 있는 식물체 세포의 괴사유발 유전자인 INF1(Phytophthora infestans elicitin gene) 유전자로 구성된 그룹에서 선택되는 것을 특징으로 하는 CMV 감염성 클론을 이용한 식물체 내에서의 외래 유전자 과발현 방법.

Images