KR101808192B1 - 7-아미노세팔로스포란산의 고농도 생산 재조합 아크레모니움 크리소제눔 균주의 제조방법 및 이 방법으로 제조된 균주 - Google Patents

7-아미노세팔로스포란산의 고농도 생산 재조합 아크레모니움 크리소제눔 균주의 제조방법 및 이 방법으로 제조된 균주 Download PDF

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KR101808192B1
KR101808192B1 KR1020160109365A KR20160109365A KR101808192B1 KR 101808192 B1 KR101808192 B1 KR 101808192B1 KR 1020160109365 A KR1020160109365 A KR 1020160109365A KR 20160109365 A KR20160109365 A KR 20160109365A KR 101808192 B1 KR101808192 B1 KR 101808192B1
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신용철
박철
윤상활
이현서
최연희
김은정
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Abstract

본 발명은 7-아미노세팔로스포란산의 고농도 생산 재조합 아크레모니움 크리소제눔 균주의 제조방법 및 이 방법으로 제조된 균주에 관한 것이며, 보다 상세하게는 7-ACA 고생산성 아크레모니움 크리소제눔 균주의 제조 방법, 이 방법으로 제조된 재조합 아크레모니움 크리소제눔 균주, 상기 균주를 7-ACA를 생산할 수 있는 조건에서 배양하고, 이를 회수하는 7-ACA의 생산방법 및 상기 제조방법에 이용된 아크레모니움 크리소제눔 균주에서의 단백질 고발현용 프로모터 및 아크레모니움 크리소제눔 균주에서의 CPC 아실라제 고발현용 발현 벡터에 관한 것이다. 본 발명의 방법에 의하면 재조합 아크레모니움 크리소제눔 균주를 제조하여 이의 발효를 통하여 7-ACA를 고농도로 제조할 수 있다.

Description

7-아미노세팔로스포란산의 고농도 생산 재조합 아크레모니움 크리소제눔 균주의 제조방법 및 이 방법으로 제조된 균주 {Methods for preparing recombinant Acremonium chrysogenum producing 7-aminocephalosporanic acid with high concentration and Acremonium chrysogenum prepared thereby}
본 발명은 7-아미노세팔로스포란산의 고농도 생산 재조합 아크레모니움 크리소제눔 균주의 제조방법 및 이 방법으로 제조된 균주에 관한 것이며, 보다 상세하게는 7-ACA 고생산성 아크레모니움 크리소제눔 균주의 제조 방법, 이 방법으로 제조된 재조합 아크레모니움 크리소제눔 균주, 상기 균주를 7-ACA를 생산할 수 있는 조건에서 배양하고, 이를 회수하는 7-ACA의 생산방법 및 상기 제조방법에 이용된 아크레모니움 크리소제눔 균주에서의 단백질 고발현용 프로모터 및 아크레모니움 크리소제눔 균주에서의 CPC 아실라제 고발현용 발현 벡터에 관한 것이다.
세팔로스포린 C(Cephalosporin C, 이하 "CPC"라고 약칭함)는 베타-락탐(beta-lactam)계 항생물질로서 사상균 곰팡이인 아크레모니움 크리소제눔(Acremonium chrysogenum)과 같은 일부 미생물에 의해서 생산된다. CPC는 그람음성 세균에 대해 세포벽 합성저해를 통해 항생활성을 나타내지만, 그 정도가 매우 미약하기 때문에 주로 반합성 세팔로스포린계 항생제(semi-synthetic cephalosporin antibiotics, 이하 "세파계 항생제"라고 약칭함)의 원료물질을 제조하는데 이용되고 있다. 특히, CPC로부터 아미노아디포일 곁가지(D-aminoadipoyl side chain)가 제거된 7-아미노세팔로스포란산(7-aminocephalosporanic acid, "7-ACA"이하 라고 약칭함)이 전세계 항생제 시장의 40% 이상을 점유하는 대부분의 세파계 항생제의 원료물질로 사용되고 있다.
CPC로부터 7-ACA를 제조하기 위한 종래의 공법으로서는 화학공법과 효소공법이 있다. 화학공법은 반응조건이 복잡하고, 환경 처리 비용이 많이 들기 때문에 현재 대부분의 기업은 환경 친화적인 효소공법으로 7-ACA를 생산하고 있다.
기존 기업들이 상업적으로 사용하였던 효소공법은 2단계로 구성되어 있었으나, 첫 번째 단계 반응 부산물의 피드-백 저해로 인한 낮은 수율로 경제성이 낮았다. 그러나, 최근에 고활성을 갖도록 개량된 CPC 아실라제를 이용한 1단계 효소법이 개발되어 현재 많은 기업이 상업용으로 1단계 효소를 사용하고 있다. (대한민국특허 출원번호 제10-2003-0055259호 및 대한민국특허 출원번호 제10-2013-0006483호)
아크레모니움 크리소제눔이 갖는 CPC 생합성 경로는 오래전부터 다양한 연구를 통해 그 전체 합성경로와 cofactor 및 일부의 2차 대사조절 메커니즘이 밝혀져 있다. CPC의 최초 전구체는 L-lysine으로부터 유도된 L-2-aminoadipate와 L-cysteine 및 L-valine의 3가지 물질이며, 이들 세 물질이 Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-Valine synthetase를 코딩하는 PcbAB에 의해 N-[(5S)-5-amino-5-carboxylpentanoyl]-L-cysteinyl-D-valine (이하 ACV로 약칭)로 전환되고, isopenicillin N synthase를 코딩하는 PcbC에 의해 이소페니실린 N(isopenicillin N, 이하 IPN으로 약칭)으로 전환된다. IPN은 isopenicillin N epimerase를 코딩하는 CefD1과 CefD2에 의해 페니실린 N(penicillin N, 이하 PN)으로 전환된 후 deacetoxycephalosporin C synthase를 코딩하는 CefEF 및 deacetylcephalosporin C를 코딩하는 CefG에 의해 디아세트옥시세팔로스포린 C (deaceoxylcephalosporin C, 이하 DO-CPC로 약칭) 및 디아세틸세팔로스포린 C (deacetylcephalosporin C, 이하 D-CPC로 약칭)를 거쳐 CPC로 전환된다.
현재 7-ACA의 생산은 아크레모니움 크리소제눔의 발효를 통해 생산된 CPC를 정제한 후 이를 1단계 효소법에 적용하여 이루어진다. 하지만, 발효를 통해 CPC를 생산 및 정제하여야 할 뿐 아니라, 대장균 등의 세균을 발효하여 효소를 생산 및 정제하고 이를 담체에 고정화한 후 1단계 효소법을 진행해야 하는 번거로움이 있었다.
이러한 다수의 단계를 통해야 하는 7-ACA 생산방법을 간소화하기 위하여 CPC로부터 7-ACA를 생산할 수 있는 효소를 CPC 생산균주인 아크레모니움 크리소제눔에 직접 도입하려는 시도가 다양한 연구 그룹에서 있어왔다.
하지만, 낮은 CPC 생산성 또는 7-ACA로의 낮은 전환율 때문에 사업화 수준의 생산성에 미치지 못하고 있는 실정이다.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
대한민국특허 출원번호 제 10-2003-0055259호 대한민국특허 출원번호 제 10-2013-0006483호
이에, 본 발명자들은 7-ACA의 효율적인 생산방법을 위해 지속적으로 연구하던 중 CPC의 생산성이 증대된 아크레모니움 크리소제눔 균주를 발현능이 향상된 프로모터가 포함된 CPC 아실라제의 발현벡터로 반복적으로 형질전환하는 경우, 높은 수준의 7-ACA 생산성을 가진 재조합 아크레모니움 크리소제눔 균주를 제조할 수 있음을 확인하여 발명을 완성하였다.
따라서, 본 발명의 목적은 7-ACA 고생산성 아크레모니움 크리소제눔 균주의 제조 방법을 제공하는 것이다.
본 발명의 다른 목적은 본 발명의 상기 제조 방법에 의해서 제조된 아크레모니움 크리소제눔 균주를 제공하는 것이다.
본 발명의 또다른 목적은 본 발명의 상기 균주를 7-ACA를 생산할 수 있는 조건에서 배양하는 단계; 및 배지에서 7-ACA를 회수하는 단계를 포함하는 7-ACA의 생산방법을 제공하는 것이다.
본 발명의 또다른 목적은 서열번호 10 내지 14로 이루어진 군에서 선택된 염기서열로 이루어진 아크레모니움 크리소제눔 균주에서의 단백질 고발현용 프로모터를 제공하는 것이다.
본 발명의 또다른 목적은 서열번호 15 내지 17로 이루어진 군에서 선택된 염기서열로 이루어진 아크레모니움 크리소제눔 균주에서의 CPC 아실라제 고발현용 발현 벡터를 제공하는 것이다.
상기한 본 발명의 목적을 달성하기 위해, 본 발명은
(a) CPC (cephalosporin C) 고생산성 아크레모니움 크리소제눔 (Acremonium chrysogenum) 균주를 제조하는 단계;
(b) 제1프로모터 및 이와 작동가능하게 연결된 CPC 아실라제를 암호화하는 폴리뉴클레오티드를 포함하는 제1발현벡터로 상기 (a) 단계의 균주를 형질전환 시키는 단계; 및
(c) 제2프로모터 및 이와 작동가능하게 연결된 상기 폴리뉴클레오티드를 포함하는 제2발현벡터로 상기 (b) 단계의 균주를 형질전환 시키는 단계를 포함하며,
상기 (c) 단계는 반복적으로 수행되는 것을 특징으로 하는 7-ACA 고생산성 아크레모니움 크리소제눔 균주의 제조 방법을 제공한다.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 본 발명의 방법에 의해서 제조된 아크레모니움 크리소제눔 균주를 제공한다.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 본 발명의 상기 균주를 7-ACA를 생산할 수 있는 조건에서 배양하는 단계; 및 배지에서 7-ACA를 회수하는 단계를 포함하는 7-ACA의 생산방법을 제공한다.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 서열번호 10 내지 14로 이루어진 군에서 선택된 염기서열로 이루어진 아크레모니움 크리소제눔 균주에서의 단백질 고발현용 프로모터를 제공한다.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은 서열번호 15 내지 17로 이루어진 군에서 선택된 염기서열로 이루어진 아크레모니움 크리소제눔 균주에서의 CPC 아실라제 고발현용 발현 벡터를 제공한다.
이하 본 발명에 대해 보다 상세히 설명한다.
본 발명의 7-ACA 고생산성 아크레모니움 크리소제눔 균주의 제조 방법은
(a) CPC 고생산성 아크레모니움 크리소제눔 (Acremonium chrysogenum) 균주를 제조하는 단계;
(b) 제1프로모터 및 이와 작동가능하게 연결된 CPC 아실라제를 암호화하는 폴리뉴클레오티드를 포함하는 제1발현벡터로 상기 (a) 단계의 균주를 형질전환 시키는 단계; 및
(c) 제2프로모터 및 이와 작동가능하게 연결된 상기 폴리뉴클레오티드를 포함하는 제2발현벡터로 상기 (b) 단계의 균주를 형질전환 시키는 단계를 포함하며,
상기 (c) 단계는 반복적으로 수행되는 것을 특징으로 한다.
본 발명의 제조방법에 의해 제조되는 균주는 CPC 고생산성의 특징과 더불어 7-ACA 고생산성의 특징을 가진다. 본 발명의 균주에 의해서 생산되는 CPC는 형질전환된 CPC 아실라제에 의해서 7-ACA로 전환되며, 그 전환효율은 매우 높은 특징을 가진다. 본 발명의 제조방법에 의해서 제조되는 7-ACA 고생산성 아크레모니움 크리소제눔 균주의 7-ACA 생산능은 15g/L이상, 16g/L이상, 17g/L이상, 18g/L이상, 19g/L이상, 20g/L이상, 21g/L이상, 22g/L이상, 23g/L이상, 24g/L이상, 25g/L이상, 26g/L이상, 27g/L이상, 28g/L이상, 29g/L이상, 30g/L이상, 31g/L이상, 32g/L이상, 33g/L이상일 수 있으며, 바람직하게는 20g/L이상, 가장 바람직하게는 30g/L이상일 수 있다. 생산능의 상한값은 아크레모니움 크리소제눔의 생육조건, 배지 내 원료물질의 양 등에 의해서 조절될 수 있으며, 본 발명이 속하는 기술분야에서 통상의 지식을 가진자라면 용이하게 설정할 수 있다.
이하에서 본 발명의 균주의 제조방법을 각 단계별로 보다 상세히 설명한다.
(a) 단계는 CPC 고생산성 아크레모니움 크리소제눔 (Acremonium chrysogenum) 균주를 제조하는 단계이다.
본 발명에서 “CPC 고생산성”이란 적절한 배양 조건에서 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 또는 35 g/L 이상, 바람직하게는 30, 31, 32, 33, 34 또는 35g/L 이상, 가장 바람직하게는 35g/L 이상의 CPC 생산능을 가지는 것을 나타낸다. 상기 “적절한 배양 조건”이란 상기 CPC 생산능을 나타낼 수 있도록 CPC 고생산성 아크레모니움 크리소제눔 균주를 특정의 배지 조성, 배양 온도, 배양 시간, pH 환경 하에서 배양하는 것을 의미한다. “적절한 배양 조건”은 다음의 기술에 따라서 당업자가 적절하게 설정할 수 있다.
형질전환된 숙주세포의 배양은 공지된 숙주세포 배양 방법 또는 이를 변형한 방법으로 행할 수 있다. 배양은 1 내지 5회의 종배양 및 본배양으로 나눌 수 있으나 이에 국한되지는 않는다. 배지조성은 형질전환된 재조합 숙주세포가 효율적으로 이용할 수 있는 탄소원, 질소원, 무기염 등을 포함하는 천연 배지 또는 합성 배지를 사용할 수 있다. 사용될 수 있는 탄소원은 글루코오스, 프럭토오스, 수크로오스, 콩가루, 땅콩가루, 밀가루, 옥수수가루, 덱스트린, 콘밀, 과당 시럽과 같은 탄수화물; 녹말, 녹말의 가수분해물; 아세트산 및 프로피온산과 같은 유기산; 에탄올, 프로판올, 글리세롤과 같은 알코올; 콩기름, 올리브기름, 카놀라유, 땅콩기름, 생선기름 등의 기름류 등을 포함한다. 질소원은 암모니아; 염화암모늄, 암모늄설페이트, 암모늄아세테이트 및 암모늄포스페이트와 같은 무기산 또는 유기산의 암모늄염; 펩톤, 육추출물(meat extract), 이스트추출물, 옥수수 침지액, 카제인 가수분해물, 대두추출물, 대두가수분해물; 다양한 발효된 세포 및 이들의 분해물 등을 포함한다. 아미노산으로, 글루타민산나트륨, 메티오닌, 라이신, 루이신, 시스테인, 발린 등을 포함한다. 무기염은 포타슘디하이드로젠 포스페이트, 다이포타슘하이드로젠 포스페이트, 마그네슘 포스페이트, 마그네슘 설페이트, 소디엄 클로라이드, 망간 설페이트, 구리 설페이트, 칼슘 카보네이트 등을 포함한다.
배양은 통상적으로 진탕배양 또는 회전기에 의한 회전에 의한 것과 같은 호기성 조건하에서 행한다. 배양 온도는 바람직하게는 5 내지 30℃의 범위에서 행하고, 배양시간은 일반적으로 1일 내지 20일, 바람직하게는 3일 내지 12일간 행한다. 배지의 pH는 배양 중에서 바람직하게는 3.0 내지 9.0의 범위를 유지한다. 배지의 pH는 무기 또는 유기산, 알칼리 용액, 우레아, 칼슘 카보네이트, 암모니아 등으로 조절할 수 있다. 배양 중에는 필요한 경우 형질전환체의 선별 및 오염의 방지를 위해 하이그로마이신 B, 제네티신, 플레오마이신, 베노밀, 암피실린, 스트렙토마이신, 클로람페니콜, 카나마이신 및 테트라사이클린과 같은 항생제를 첨가할 수 있다.
바람직하게는 본 발명의 CPC 고생산성 아크레모니움 크리소제눔 균주는 변이에 의해서 CPC 고생산성을 가지는 것일 수 있다.
본 발명의 일 실시예에서는 NTG (N-methyl-N'-nitro-N-nitrosoguanidine) 처리에 의해서 아크레모니움 크리소제눔 균주의 돌연변이를 유도하고, 이들 중 CPC 생산성이 높아진 균체를 선별하여 기탁하였다. 최종 선별된 E1 균주의 CPC 생산성은 약 35 g/L로 평가되었다.
따라서, 바람직하게는 본 발명의 CPC 고생산성 아크레모니움 크리소제눔 균주는 한국생명공학연구원 생물자원센터에 2016년 8월 18일자로 기탁된 아크레모니움 크리소제눔 E1 (기탁번호 KCTC13079BP)일 수 있다.
(b) 단계는 제1프로모터 및 이와 작동가능하게 연결된 CPC 아실라제를 암호화하는 폴리뉴클레오티드를 포함하는 제1발현벡터로 상기 (a) 단계의 균주를 형질전환시키는 단계이다.
본 명세서에서 '제1발현벡터'란 CPC 아실라제를 암호화하는 폴리뉴클레오타이드가 클로닝된 플라스미드, 바이러스 또는 기타 매개체, 바람직하게는 플라스미드 발현벡터를 의미한다. 본 발명에 따라 클로닝된 상기 폴리뉴클레오타이드 서열은 적절한 발현 조절 서열에 작동 가능하게 연결될 수 있으며, 상기 작동 가능하게 연결된 유전자 서열과 발현 조절서열은 선택 마커 및 복제 개시점(replication origin)을 같이 포함하고 있는 하나의 발현 벡터 내에 포함될 수 있다. '작동가능하게 연결(operably linked)' 된다는 것은 상기 폴리뉴클레오타이드 서열이 발현 조절 서열에 유전자 발현을 가능하게 하는 방식으로 연결된 것을 의미한다. 상기 '발현 조절 서열(expression control sequence)'이란 특정한 숙주세포에서 작동 가능하게 연결된 폴리뉴크레오티드 서열의 발현을 조절하는 DNA 서열을 의미한다. 그러한 조절 서열은 전사를 실시하기 위한 프로모터, 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리포좀 결합 부위를 코딩하는 서열, 및 전사 및 해독의 종결을 조절하는 서열 등으로 이루어진 군에서 선택된 하나 이상을 포함할 수 있다.
바람직하게는 본 발명의 제1발현벡터는 CPC 아실라제에 대한 유전자를 클로닝된 pB-HCX(서열번호 9, 도 1)일 수 있으며, 제1발현벡터에 의한 아크레모니움 크리소제눔 균주의 형질전환에 의한 결과는 종래의 CPC 아실라제 도입에 따른 7-ACA의 생산능에 비해서 큰 장점을 나타내지는 않으나, 이후 (c) 단계의 반복된 형질전환에 따른 생산능 증가의 기본 베이스가 되며, 이러한 점은 본 발명자들에 의해서 개발된 것이다.
본 발명에서 ‘프로모터’란 특정한 숙주 세포에서 작동 가능하게 연결된 핵산 서열의 발현을 조절하는 DNA 서열을 의미하며, ‘프로모터에 작동 가능하게 연결된다(operably linked)’는 것은 프로모터의 기능에 의해서 이후의 핵산 단편의 발현카세트가 암호화하는 단백질의 발현이 영향을 받는 것을 말한다. 상기 프로모터로는 모든 시간대에 상시적으로 목적 유전자의 발현을 유도하는 프로모터 (constitutive promoter) 또는 특정한 위치, 시기에 목적 유전자의 발현을 유도하는 프로모터(inducible promoter)를 사용할 수 있다. 바람직하게는 제1발현벡터에 포함된 ‘제1프로모터’는 아스퍼질러스 니둘란스의 gpdA 프로모터 (GenBank Z32698 염기서열의 1-2129 bp의 서열 또는 서열번호 9의 11410-13532 bp의 서열) 일 수 있다. 곰팡이 발현에 일반적으로 사용하는 프로모터로서 널리 알려진 pdc 프로모터, trpC 프로모터일수 있다. ‘발현 조절 서열’ 중 터미네이터 서열은 trpC 터미네이터일 수 있다.
상기 발현 벡터의 모벡터로 사용되는 벡터는 특별한 제한이 없으며, 이 발명이 속하는 기술분야에서 숙주세포로 사용되는 미생물에서의 발현을 위하여 통상적으로 사용되는 모든 플라스미드, 바이러스 또는 기타 매개체 등이 사용 가능하다. 예컨대, 상기 플라스미드에는 대장균 유래 플라스미드(pBR322, pBR325, pUC118 및 pUC119, pET-22b(+)), 바실러스 서브틸리스 유래 플라스미드(pUB110 및 pTP5) 및 효모 유래 플라스미드(YEp13, YEp24 및 YCp50), pBluescript 계열의 벡터 등이 있으며, 이에 제한되는 것은 아니다.
본 발명에서 ‘CPC 아실라제’는 야생형 또는 변이형 아실라제를 모두 포함하나, 바람직하게는 아미노산 서열의 경우 서열번호 19로 표시되는 아미노산서열 또는 이를 암호화하는 폴리뉴클레오티드 서열의 경우 서열번호 18로 이루어진 폴리뉴클레오티드 서열로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.
본 발명의 일실시예에서는 pBluescript II SK+ 클로닝 벡터에 항생제인 하이그로마이신 B (hygromycin B), 제네티신 (geneticin) 또는 플레오마이신 (phleomycin) 저항성을 갖는 마커유전자 카세트(cassette) 및 다중 도입을 목적으로 마커를 제거하기 위한 Flp 카세트를 도입한 것을 기본 프레임으로하여 CPC 아실라제에 대한 유전자를 클로닝하여 pB-HCX(서열번호 9, 도 1)를 제작하고 이를 E1균주에 형질전환하고, 그 중 생산성이 높은 균락을 E2 균주로 명명하여 선별하였다. E2 균주에 대한 7-ACA 발효생산성은 7일째 최대치로서 약 2 g/L로 평가되었다.
(c) 단계는 제2프로모터 및 이와 작동가능하게 연결된 상기 폴리뉴클레오티드를 포함하는 제2발현벡터로 상기 (b) 단계의 균주를 형질전환 시키는 단계이며, (c) 단계는 반복적으로 수행되는 것을 특징으로 한다.
(c) 단계에서의 발현벡터, 즉 제2발현벡터는 7-ACA의 생산능을 높이기 위하여 개량된 프로모터를 포함하며, 서열번호 15, 서열번호 16 또는 서열번호 17일 수 있다. 아울러, (c) 단계의 프로모터, 즉 제2프로모터는 서열번호 10, 서열번호 11, 서열번호 12, 서열번호 13 또는 서열번호 14의 프로모터 서열일 수 있다. 바람직하게는 제2프로모터는 서열번호 12의 프로모터 서열일 수 있다.
본 발명에서 ‘형질전환’은 본 발명의 제1발현벡터 또는 제2발현벡터를 숙주 세포로 도입하는 것이며, 이를 위하여 통상적으로 사용되는 모든 수단에 의하여 수행될 수 있다. 예컨대, 상기 발현벡터는 이에 제한되지는 않지만, 염화칼슘 (CaCl2) 및 열쇼크 (heat shock) 방법, 입자 총 충격법 (particle gun bombardment), 실리콘 탄화물 위스커 (Silicon carbide whiskers), 초음파 처리(sonication), 전기천공법 (electroporation), PEG (polyethylenglycol)에 의한 침전법 등의 방법으로 숙주세포에 도입될 수 있다.
본 발명의 형질전환의 대상이 되는 숙주세포는 아크레모니움 크리소제눔이며, 상기 (b) 단계에서의 숙주세포는 (a) 단계의 아크레모니움 크리소제눔일 수 있으며, (c) 단계에서의 숙주세포는 (b) 단계의 아크레모니움 크리소제눔 또는 n회차의 형질전환인 경우 n-1회의 형질전환이 이루어진 아크레모니움 크리소제눔일 수 있다.
(c) 단계의 숙주세포로의 형질전환은 반복적으로 수행되는 것이 특징이다. 형질전환 횟수는 2회 내지 8회가 바람직하며, 더 바람직하게는 4회 내지 7회, 한층 더 바람직하게는 5회 내지 6회, 가장 바람직하게는 5회 반복적으로 수행될 수 있다. 상기 형질전환 횟수가 2회 미만인 경우 충분한 수준의 7-ACA의 생산능을 가지는 균주를 제조할 수 없으며, 8회 초과인 경우 더 이상 7-ACA의 생산능의 증가가 이루어지지 않기 때문에 비경제적이다.
바람직하게는 (c) 단계의 반복적인 형질전환은 서열번호 15로 이루어진 발현벡터, 서열번호 16으로 이루어진 발현벡터, 서열번호 17로 이루어진 발현벡터, 서열번호 15로 이루어진 발현벡터, 서열번호 16으로 이루어진 발현벡터의 순으로 순차적으로 이루어질 수 있으며, 이와 같은 순차적/반복적 형질전환에 의해서 7-ACA의 생산능을 충분히 포화(saturation) 시킬 수 있다.
상기와 같은 5회의 형질전환에 추가적으로 1회 내지 3회의 형질전환을 더 수행할 수도 있으며, 이 경우 서열번호 15로 이루어진 발현벡터, 서열번호 16으로 이루어진 발현벡터 및 서열번호 17로 이루어진 발현벡터로 이루어진 군에서 임의로 선택하여 추가로 형질전환할 수 있다.
본 발명의 일실시예에서는 (b) 단계의 형질전환을 거친 균주에서 발현하는 CPC 아실라제는 발현량이 높지 않아 프로모터 튜닝 (promoter tuning)을 실시하여 개량하고, 이 돌연변이 프로모터의 서열을 각각 서열번호 10 내지 14로 나타내었다. 아울러, 이 돌연변이 프로모터를 가지며, CPC 아실라제를 발현하는 재조합 플라스미드 벡터를 각각 제조하여 이를 서열번호 15 내지 17로 나타내었다. 본 발명자들은 7-ACA의 생산성의 향상을 위해서 연구하던 중 반복형질전환이 7-ACA의 생산성을 향상시킨다는 점을 알아내었고, 이에 CX 유전자의 발현을 증대시키기 위해서 균주에 반복적으로 형질전환을 하였고, 7-ACA 생산성이 향상된 균주를 제조하였다.
한편, 본 발명은 본 발명의 제조 방법에 의해서 제조된 아크레모니움 크리소제눔 균주를 제공한다. 상기 균주는 발현능이 개선된 프로모터를 포함하는 발현 벡터로 5회 이상 반복적으로 형질전환된 균주인 특징이 있으며, 각 형질전환에 의해서 7-ACA 생산능이 증가되어 최종적으로 25g/L이상의 7-ACA 생산능을 가지는 특징이 있다. 바람직하게는 본 발명의 상기 균주의 7-ACA 생산능은 30g/L이상일 수 있다.
한편, 본 발명은
(a) 본 발명의 방법에 의해서 제조된 균주를 7-ACA를 생산할 수 있는 조건에서 배양하는 단계; 및
(b) 배지에서 7-ACA를 회수하는 단계를 포함하는 7-ACA의 생산방법을 제공한다.
7-ACA 생산방법의 (a) 단계는 본 발명의 방법에 의해서 제조된 균주를 7-ACA를 생산할 수 있는 조건에서 배양하는 단계이며, 7-ACA를 생산할 수 있는 조건은 CPC 생산에 있어서의 “적절한 배양 조건”과 동일 또는 유사하게 할 수 있다. 이 때 7-ACA 생산방법에의 “적절한 배양 조건”은 7-ACA의 생산능을 나타낼 수 있도록 본 발명의 방법에 의해서 제조된 아크레모니움 크리소제눔 균주를 특정의 배지 조성, 배양 온도, 배양 시간, pH 환경 하에서 배양하는 것을 의미한다.
7-ACA 생산방법의 (b) 단계는 배지에서 7-ACA를 회수하는 단계이며, 본 발명의 균주에 의해서 생산된 7-ACA를 균주 또는 이의 배양 배지로부터 분리, 수확, 정제 또는 수집시키는 것을 의미한다. 구체적으로, 7-ACA의 회수 방법은 특별히 이에 제한되지 않으나, 원심분리, 여과, 추출, 분무, 건조, 증방, 침전, 결정화, 전기영동, 분별용해(예를 들면 암모늄 설페이트 침전), 크로마토그래피(예를 들면 이온 교환, 친화성, 소수성 및 크기배제) 등의 방법을 사용할 수 있으며, 이는 당업자가 용이하게 구성할 수 있다.
한편, 본 발명은 아크레모니움 크리소제눔 균주에서의 단백질 고발현용 프로모터를 제공하며, 상기 프로모터는 서열번호 10, 서열번호 11, 서열번호 12, 서열번호 13 또는 서열번호 14의 염기서열로 이루어질 수 있다.
한편, 본 발명은 아크레모니움 크리소제눔 균주에서의 CPC 아실라제 고발현용 발현 벡터를 제공하며, 상기 발현 벡터는 서열번호 15, 서열번호 16 또는 서열번호 17의 염기서열로 이루어질 수 있다.
본 발명은 CPC 생산성이 높아지도록 돌연변이를 유발한 아크레모니움 크리소제눔을 제조하고, 강하게 발현하도록 조절한 프로모터와 CPC 아실라제를 포함하는 발현 벡터를 반복적으로 형질전환하여 7-ACA를 고농도로 생산할 수 있는 균주를 제조하는 방법 등을 제공한다. 본 발명의 방법에 의하면 재조합 아크레모니움 크리소제눔 균주를 제조하여 이의 발효를 통하여 7-ACA를 고농도로 제조할 수 있다.
도 1은 항생제 카세트, Flp 카세트 및 CX 카세트를 가진 재조합 플라스미드 벡터 pB-HCX의 구조이다.
도 2는 변이된 프로모터를 도입한 재조합 균주의 발효에 의한 7-ACA 생산성으로 발효 7일째의 결과를 나타낸다.
도 3은 야생형 gpdA 프로모터(A) 및 error prone PCR에 의해 변이된 프로모터의 1 ~ -283 부위에 대한 염기서열 분석 결과이다. (B) EP1 해당부위(서열번호 10), (C) EP2 부위(서열번호 11), (D) EP3 부위(서열번호 12), (E) EP4 부위(서열번호 13), (F) EP5 부위(서열번호 14). TSP는 전사 시작부위(transcription start point)를 나타낸다.
도 4는 반복적인 형질전환으로 7-ACA 생산성이 향상된 균주의 발효결과이다. E4: 2회 형질전환, E5: 3회 형질전환, E6: 4회 형질전환, E7: 5회 형질전환.
이하 본 발명을 상세히 설명한다.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
<실험방법>
효소 및 키트
클로닝방법으로서, PCR법에 의한 유전자의 증폭은 특별한 경우가 아닌 이상 DNA 폴리머라제 구입 시에 동봉된 메뉴얼에 따랐다. 일반적인 PCR일 경우 DNA 폴리머라제는 Pfu-X 폴리머라제 (Solgent, Korea), error prone PCR일 경우 Taq 폴리머라제 (Solgent, Korea)를 사용하였다. 제한효소, T4 DNA 리가제, Klenow fragment는 NEB (USA)로부터 구입하여 해당 효소의 메뉴얼에 따라 사용하였다. PCR 및 플라스미드 DNA의 정제, 아가로스 젤로부터의 추출 등 일련의 클로닝방법을 위해 QIAprep Spin Miniprep Kit, QIAquick PCR Purification Kit, QIAquick Gel Extraction Kit (Qiagen, 독일)를 사용하였다.
형질전환
클로닝을 위한 대장균 DH5alpha의 형질전환은 염화칼슘으로 세척 후 열충격을 주는 방식으로 하였으며, 상세하게는, 대장균을 LB에 접종하여 OD0.6까지 배양하고 원심분리(4℃, 4000 rpm)로 균체를 회수하여, 얼음으로 냉각한 0.1 M 염화칼슘용액으로 4회 세척하여 competent cell을 제조하였다. 준비한 100 내지 500 ng의 plasmid DNA를 100 μL의 competent cell과 섞은 후 얼음에서 30분간 방치하고, 42℃에서 30 초간 열충격을 주고 다시 얼음에서 2분간 방치 한 후 1 mL의 LB를 넣어 37℃ 1시간 배양 후 항생제를 갖는 LB 평판배지에 도말하였다.
균주의 발효
E1 균주를 비롯한 균주의 발효는 1차 종배양, 2차 종배양, 본배양의 단계로 진행하였다. 1차 종배양은 균락의 크기에 따라 4 내지 6 균락을 1차 종배양 배지 (콩가루 28.5 g/L, 옥수수침지액 25 mL/L, sucrose 35 g/L, 포도당 5 g/L, 탄산칼슘 5 g/L, 소포제 0.8 mL/L)에 접종 후 30℃, 200rpm에서 4 내지 5일 배양하였다. 2차 종배양은 1차 종배양액을 2차 종배양 배지(1차 종배양 배지와 동일하나 콩기름을 5 mL/L 추가함)가 담긴 발효조에 모두 접종 하고, 30℃, 35% DO, 400 rpm, 공기 1.0 vvm의 조건을 시작으로 균체 성장에 따라 6% 포도당을 feeding하고 교반속도를 단계적으로 올리면서 4 내지 5일 배양하였다. 본배양은 2차 종배양액 200 mL을 본 배양 배지(땅콩가루 23 g/L, 옥수수침지액 50 mL/L, 메티오닌 1.5 g/L, 덱스트린 70 g/L, 콘밀 35 g/L, 황산칼슘 13 g/L, 콩기름 60 mL/L, 황산암모늄 13 g/L, 과당 시럽 9 g/L, 탄산칼슘 10 g/L, 소포제 0.5 mL/L)가 담긴 발효조에 접종하고 28℃, 35% DO, 400 rpm, 공기 1.0 vvm의 조건을 시작으로 배양하였고, pH는 암모니아수를 이용하여 5.4 내지 5.7로 조절하였으며, 균체 성장에 따라 교반속도를 단계적으로 올리면서 배양하였다. 배양 2일 후에 배양온도를 28℃에서 25℃로 낮춘 후, 균체성장에 따라 5% 내지 10%의 콩기름을 단계적으로 공급하면서 5일 내지 7일 째 발효를 완료하였다.
균주의 CPC 생산성의 평가
균주의 CPC 또는 7-ACA의 생산성은 발효된 균주에 대해서 다음과 같이 HPLC를 통하여 평가하였다.
HPLC 분석을 위해 배양액을 원심분리하여 상등액 25 μL를 취한 뒤 3차 증류수 975 μL와 섞어서 40배 희석하였다. 희석한 배양액은 0.2 μm 필터로 여과한 후, Shimadzu LC10Avp로 분석하였다. 분석조건은 ZORBAX Eclipse Plus C18(Analytical 4.6 mm×250 mm, 5-Micron) 칼럼, 이동상 20 mM 암모늄 아세테이트:아세토니트릴(95:5), pH 7.0, 유속은 0.8 mL/분, 칼럼온도 40℃, UV 검출기 220 nm를 이용하였다.
<실시예 1>
NTG 처리에 의한 CPC (cephalosporin C) 생산능이 향상된 아크레모니움 크리소제눔 균주의 제조
NTG (N-methyl-N'-nitro-N-nitrosoguanidine) 용액은 아세톤에 NTG 10 mg/mL 농도로 녹여 stock solution을 만들었고, 이를 70℃에 저장하면서 사용하기 직전에 citrate buffer(22.32 g/L sodium citrate, 4.63 g/L citric acid, pH 5.5)로 희석하여 사용하였다.
아크레모니움 크리소제눔 (Acremonium chrysogenum) 균체 한 균락 (colony)을 LB배지(10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl)에 접종하여 3 내지 7일 동안 30℃, 200 rpm 조건에서 진탕배양하고 원심분리를 통해 회수한 후 citrate buffer로 2회 세척하고 NTG 용액을 첨가하여 30℃에서 30분 방치하였다. 이를 얼음에 위치하여 냉각시킨 후 원심분리하여 NTG 용액을 버리고, potassium phosphate buffer(107.13 g/L K2HPO4, 52.39 g/L KH2PO4)로 2회 세척한 후 동일 buffer를 이용하여 다양한 농도로 재현탁하였다. 이를 LB 평판배지(2% agar)에 도말하여 30℃에서 정치배양하면서 10일 내지 30일 후 나타난 균락을 계수하였다. NTG의 처리 농도는 NTG를 처리하지 않은 대조군과 비교하여 사멸율이 90% 내지 97%가 되는 농도로 사용하였다.
NTG 처리 후 생장한 균체에서 단일 균락 (colony)을 취하여 CPM 배지(60 g/L sucrose, 60 g/L soytone, 10 g/L D,L-메티오닌, 2 g/L CaCO3, 0.05 mL/L 소포제, pH 7.0)에 접종하였다. 균락의 접종은 LB 평판배지 상에 있는 균락을 메스를 이용하여 가로와 세로 5 내지 7 mm가 되게 자른 후 4 내지 6개를 취하여 접종하였다. 접종한 균체는 3일 내지 7일 동안 30℃, 200 rpm 조건에서 진탕배양하고 배양 상층액을 회수하여 생산된 CPC를 HPLC로 분석하였다. 이러한 방법으로 CPC 생산성이 높아진 균체를 선별한 후 상기한 방법을 다시 적용하여 반복하면서 CPC 생산성이 높아지는 균체를 지속적으로 선별하였다. 최종적으로 선발된 돌연변이 아크레모니움 크리소제눔 균주를 E1으로 명명하였고, 이를 한국생명공학연구원 생물자원센터에 2016년 8월 8일자로 기탁하였다 (기탁번호 KCTC13079BP). 최종 선별된 E1 균주의 CPC 생산성은 약 35 g/L로 평가되었다(결과미도시).
< 실시예 2>
E1 균주에 CPC 아실라제 도입에 의한 7- ACA 생산
<2-1> CPC 아실라제 발현 벡터의 디자인
본 단계에서 CPC (cephalosporin C)에서 7-ACA (7-aminocephalosporanic acid)로의 전환능을 갖는 유전자는 대한민국특허출원 제10-2013-0006483호에서 명시한 CPC 아실라제에 대한 유전자 (서열번호 18)를 사용하였다. 당 유전자를 발현시키기 위한 프로모터는 통상적으로 널리 알려진 아스퍼질러스 니둘란스의 gpdA 프로모터 및 trpC 터미네이터를 사용하였다(Punt PJ et al., Gene. 1990 Sep 1;93(1):101-109, Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase).
CPC 아실라제 유전자를 도입하기 위한 플라스미드 벡터는 pBluescript II SK+ 클로닝 벡터에 항생제인 hygromycin B, geneticin, phleomycin 저항성을 갖는 마커유전자 카세트(cassette) 및 다중 도입을 목적으로 마커를 제거하기 위한 Flp 카세트를 도입한 것을 기본 프레임으로 하였다.
마커유전자 카세트에서는 gpdA 프로모터와 trpC 터미네이터를 사용하였다.
마커 제거를 위한 FRT-Flp 재조합법은 플립파제(flippase)를 이용해 인접한 FRT 사이의 유전자를 제거하기 위한 방법으로 대장균의 유전자 다중 결손에 널리 쓰이고 있으며, 진균류에도 적용할 수 있다는 보고가 다수 있었다(Datsenko KA, Wanner BL, Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products; Yamada Y, Maeda M, Alshahni MM, Monod M, Staib P, Yamada T., Microbiology. 2014 Oct;160(Pt 10):2122-35, Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii; Bloemendal S, LD, Terfehr D, Kopke K, Kluge J, Teichert I, KU, J Biotechnol. 2014 Jan;169:51-62., Tools for advanced and targeted genetic manipulation of the β-lactam antibiotic producer Acremonium chrysogenum; Blatzer M, Gsaller F, Abt B, Schrettl M, Specht T, Haas H., J Biotechnol. 2014 Jan;169:82-6. doi: 10.1016/j.jbiotec.2013.11.003. Epub 2013 Nov 15, An endogenous promoter for conditional gene expression in Acremonium chrysogenum: the xylan and xylose inducible promoter xyl1).
본 발명자는 마커유전자 카세트를 두 FRT 부위의 사이에 위치시키고, 플립파제의 발현을 위해 아크레모니움 크리소제눔에 내재하면서 자일로스에 의해 유도되는 자일라제 유전자인 Xyl1의 프로모터 부위 (Pxyl)로서 상위 약 2,100 bp의 부위를 PCR로 증폭하여 도입하였고, 마커 제거 시에 자일로스가 존재하는 배지를 이용해 마커유전자 카세트가 효과적으로 제거될 수 있도록 구성하였다. 플립파제 유전자는 Barry Wanner 방법에서 사용했던 flp유전자의 아미노산 서열을 토대로 아크레모니움 크리소제눔의 코돈에 맞게 최적화하여 사용하였다.
<2-2> CPC 아실라제 발현벡터의 클로닝
프라이머 5‘-GCAAAGCTTCCTTGTATCTCTACACACAGGC-3’ (서열번호 20) 및 5’-GCTATCGATTACGTATTAATTAATCGAGTGGAGATGTGGAGTGG-3’ (서열번호 21)를 이용하여 pAN7-1(GenBank Z32698)로 부터 프로모터와 터미네이터를 포함하는 hygromycin B 저항성 유전자 카세트를 증폭하였다. 증폭한 유전자는 pBluescript II SK+(Stratagene, USA)를 SacI과 KpnI으로 절단하고 Klenow를 처리하여 Blunt 말단을 만든 부위로 삽입하여 pB-Hcast를 제작하였고, 또한 각각의 프라이머에 FRT를 포함하여 5‘-GCAAAGCTTCCTGCAGTTCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGCGCTTTCCTTGTATCTCTACACACAGGC-3’ (서열번호 22) 및 5‘-AAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGAACTGCAGGTCGAGTGGAGATGTGGAGTGG-3’ (서열번호 23)를 이용하여 pAN7-1로부터 상기와 동일 부위를 증폭하여 동일한 방법으로 pBluescript II SK+에 삽입하여 카세트 말단에 FRT 부위를 갖는 pB-HcastF(서열번호 1)를 제작하였다.
nptII 및 ble 유전자로 명명된 geneticin 및 phleomycin 저항성 유전자는 5‘ 말단 부위에는 gpdA 프로모터의 3’부위에 있는 SalI을 시작으로 하고, 3’말단 부위에 BamHI 제한부위를 넣어 유전자 합성하였다. nptII와 ble에 대한 합성유전자를 SalI과 BamHI으로 절단하고 pB-HcastF의 동일부위로 삽입하여, 각각 pB-GcastF(서열번호 2) 및 pB-PcastF(서열번호 3)를 구성하여 FRT를 사이에 위치한 항생제 마커 카세트를 완성하였다.
Flp 유전자의 발현을 위하여 Xyl1유전자의 프로모터 Pxyl1 (GenBank KF575626)을 아크레모니움 크리소제눔의 염색체 DNA로부터 프라이머 5‘-CGAAAGCTTGAGGCCGGACAAATTCAGCC-3’ (서열번호 24) 및 5‘-GGTGTCAGGGTGTTGAAGATGGG-3‘ (서열번호 25)를 이용하여 증폭하였다. Flp유전자는 5’부위에 Xyl 프로모터의 3‘방향으로 20 bp가 겹치도록 구성하여 유전자합성을 통해 확보하였다. Xyl 프로모터와 Flp 유전자는 overlapping PCR을 통하여 연결하였고, 이를 HindIII와 BamHI으로 처리한 후 pB-Hcast의 동일부위로 삽입하여 Flp카세트인 pB-Fcast(서열번호 4)를 완성하였다.
pB-Fcast로부터 PmeI, SmaI, SnaBI 제한부위를 가진 프라이머 5‘-GTTTAAACCCCGGGTACGTAAATTAACCCTCACTAAAGGG-3’ (서열번호 26) 및 T7 프라이머(5‘-TAATACGACTCACTATAGGGC-3‘, 서열번호 27)를 이용하여 Flp 카세트를 증폭하고 이를 각각 pB-HcastF, pB-GcastF, pB-PcastF의 HindIII를 blunt말단 처리를 한 부위에 삽입하여, 각각 pB-HF(서열번호 5), pB-GF(서열번호 6), pB-PF(서열번호 7)을 제작하였으며, 이들 각각의 다른 항생제를 가진 프레임을 바탕으로 CPC 아실라제를 도입하기 위한 플라스미드 벡터로 이용하였다.
CPC 아실라제의 염기서열은 대한민국특허출원 제10-2013-0006483호에 나타나 있으며, CX로 명명하기로 하였다. CX 유전자는 gpdA 프로모터의 3’부위에 있는 SalI을 시작으로 하고, 3’말단 부위에 BglII 제한부위를 가진 유전자를 합성하여 pB-Hcast의 SalI과 BamHI 부위로 삽입하여 CX카세트를 완성하였다 (pB-CXcast, 서열번호 8).
CX 카세트는 hygromycin B 항생제를 이용하기 위하여, pB-HF에 삽입하였다. pB-CXcast (서열번호 8)로부터 T3 (5’-AATTAACCCTCACTAAAGGG-3’, 서열번호 28)와 T7 프라이머를 이용하여 CX카세트를 증폭하고 이를 pB-HF의 SnaBI부위로 삽입하여 pB-HCX(서열번호 9, 도 1)를 제작하고 이를 E1균주에 형질전환하였다.
<2-3> CPC 아실라제 발현벡터 pB - HCX 의 E1 균주에의 형질전환
형질전환 방법은 일반적으로 알려진 진균류의 원형질체(protoplast)를 이용하는 방법을 본 발명에 맞게 변형하였다. E1 균주를 LB 평판배지에 도말하여 28℃에서 6 내지 8일 배양하여 균락을 얻은 후 이를 100 mL TB배지 (12 g/L 트립톤, 24 g/L 효모추출물, 9.4 g/L K2HPO4, 2.2 g/L KH2PO4, 4 g/L 글리세롤)를 넣고, LB 평판배지에서 균락을 취하여 접종하였다. 균락의 접종은 LB 평판배지 상에 있는 균락을 메스를 이용하여 한 변이 5 내지 7 mm가 되게 정사각형으로 자른 후 균락의 크기에 따라 4 내지 6개를 취하여 접종하고 28℃, 150 rpm 조건에서 3 내지 4일 배양하였다. 성장이 완료된 배양액을 4℃, 4,000 rpm에서 원심분리한 후 상등액을 버리고 0.6 M MgSO4 용액으로 1회 세척하였다.
원형질체 제작을 위해 균체 무게의 약 4배의 2%의 lysing enzyme (Sigma L1412)을 처리하여 30℃, 100 rpm에서 3시간 반응시켰다. 이 후 반응액에 동량의 separation buffer A(0.6 M sorbitol, 100 mM Tris-Cl, pH 7.0)를 overlay한 뒤, 1,800 g, 4℃ 조건으로 10분간 원심분리하였다. 상등액 및 경계면을 새 원심분리관에 옮겨 동량의 separation buffer B(1.2 M sorbitol, 100 mM Tris-Cl, pH 7.5)를 넣고 1,800 g, 4℃ 조건으로 10분간 원심분리하였다. 상등액을 버리고 침전물을 다시 MSC(1 M sorbitol, 10 mM MOPS, pH 6.5, 10 mM CaCl2)로 세척하였다. 현미경으로 관찰하여 형성된 원형질체를 계수한 후 형질전환을 위해 1×107개의 원형질체에 1 내지 5 μg DNA, 50 μL의 60% PEG용액(MSC에 polyethyleneglycol 6000을 60% 농도로 만듦)을 섞은 후, 얼음에서 20분 방치하였다. 다시 500 μL의 60% PEG 용액을 넣고 섞은 뒤 상온에서 20분 방치한 후, 항생제를 함유한 LB-sucrose 평판배지(0.8 M sucrose, 2% agar, 100 mg/L Hygromycin B, 또는 200 mg/L Geneticin, 또는 20 mg/L Phleomycin)에 도말하였다. 평판배지는 28℃에서 10일 내지 30일 동안 균락이 형성될 때까지 배양하였다.
자라나온 균락은 항생제를 함유한 LB 평판배지에 옮기고 28℃에서 7일 내지 10일 간 계대 배양하여 실제 성장 유무를 재확인하였다. 항생제 함유배지에서 잘 자라는 균락을 선별하여 배양하였고, 각 균락에 대한 7-ACA 생산성을 평가하였다. 각 균락마다 생산성 차이가 상이하였으며, 가장 생산성이 높은 균락을 E2 균주로 명명하여 선별하였다. E2 균주에 대한 7-ACA 발효생산성은 7일째 최대치로서 약 2 g/L로 평가되었다(결과미도시).
< 실시예 3>
반복 형질전환에 의한 7- ACA 생산성 향상
<3-1> 프로모터 튜닝 및 7- ACA 생산성 향상
E2 균주의 CPC 아실라제는 gpdA 프로모터로 발현되며, 발현량이 높지 않아 프로모터 튜닝 (promoter tuning)을 실시하여 프로모터의 강도를 향상시키고자 하였다.
프로모터 튜닝은 다양한 방법으로 프로모터의 작동부위를 무작위 돌연변이 시켜 프로모터 강도 향상을 모색하는 방법으로, 최근 합성생물학의 등장으로 활발히 연구되고 있는 기술이다(Alper H, Fischer C, Nevoigt E, Stephanopoulos G., Proc Natl Acad Sci U S A. 2005 Sep 6;102(36):12678-83. Epub 2005 Aug 25, Tuning genetic control through promoter engineering; Santos CN, Stephanopoulos G., Curr Opin Chem Biol. 2008 Apr;12(2):168-76., Combinatorial engineering of microbes for optimizing cellular phenotype, ; Dehli T, Solem C, Jensen PR., Subcell Biochem. 2012;64:181-201. Tunable promoters in synthetic and systems biology). 본 발명자는 에러 유발 PCR (error prone PCR) 방법으로 프로모터의 전사시작부위로부터 상위 283 bp 부위를 증폭하여 다양하게 돌연변이 된 부위를 포함하도록 프로모터 라이브러리를 구성하고 이를 E1 균주에 도입하여 생산성을 평가하였다.
gpdA 프로모터의 전사시작부위(transcription start site)로부터 상위 100 bp 부위와 상위 250 bp 부위의 gpd box 부위(Peter Jan Punt., Molecular Signals in Plant-Microbe Communications, D. P. S. Verma. ed, CRC Pre, Boca Raton, Florida, 1992, pp. 29-48, Functional elements in the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene of Aspergillus nidulans)를 포함하는 상위 283 bp 부위를 유력한 돌연변이 부위로 정하고 제한부위 EcoRI 및 NdeI을 도입한 프라이머 5‘-GCTGAATTCCCATCCGGCATCTGTAGGGCG-3’ (서열번호 29) 및 5‘-CGTCATATGGGAAGATGAATATACTGAAGATG-3’ (서열번호 30)와 pB-CXcast (서열번호 8) 플라스미드 벡터를 주형으로 하여 error prone PCR을 수행하였다. Error prone PCR 조건은 다음과 같이 하였다. PCR 반응액 총 부피를 100㎕로 하였고 1 ng 주형 DNA, 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 3.5 mM MgCl2, 0.05～0.075 mM MnCl2, 0.2 mM dATP, 0.2 mM dGTP, 1.0 mM dCTP, 1.0 mM dTTP, 0.3 μM의 각 프라이머, 5U의 Taq 폴리머라제를 사용하였다. PCR 반응 조건은 94℃ 5분, 94℃ 30초, 55℃ 30초, 72℃ 30초, 72℃ 10분 후 4℃ 냉각으로서 증폭 회수는 15 내지 20 회로 하였다. PCR 반응산물을 0.8% agarose gel에 전기영동하여 344 bp 부위를 추출 및 정제하여 에러가 유도된 DNA 단편의 pool을 제작하였다. 이때 대조구로서는 동일한 프라이머와 동일한 주형으로부터 일반조건의 PCR을 수행하여 상기와 같이 제조하였다.
이 DNA pool과 대조구 DNA단편을 도입하고자 하는 벡터는 상기 DNA 단편의 범위를 제외하는 나머지 부분의 pB-CXcast (서열번호 8)로서 제한 부위 에러가 유도된 DNA 단편을 도입하기 위하여 NdeI과 EcoRI 제한부위를 포함하는 프라이머 5‘-CGTCATATGCATCCAAGAACCTTTATTTCC-3’ (서열번호 31) 및 5‘-GCTGAATTCGCCAATTAGGGAGCTTACGC-3’ (서열번호 32)를 이용하여 pB-CXcast (서열번호 8) 플라스미드 벡터를 주형으로 증폭한 7,717 bp의 DNA 단편을 사용하였다. 각각 증폭한 에러 유발 DNA 단편의 pool과 벡터를 EcoRI과 NdeI을 이용하여 절단 및 연결하여 프로모터에 에러가 유발된 재조합 플라스미드 벡터를 완성하였다.
이 재조합 플라스미드 벡터의 pool을 대장균 DH5alpha에 형질전환하여 약 10,000개 대장균 균락의 라이브러리를 얻었다. 대장균 라이브러리 균락이 도말된 LB 평판배지에 100 μg/mL의 Ampicillin을 함유하는 LB 액체배지 1 mL을 넣어 균락을 모아 대장균 라이브러리 pool을 만든 후 총 플라스미드를 추출하여 재조합 플라스미드 라이브러리 pool을 제작하였다. 이 재조합 플라스미드 라이브러리 pool을 주형으로 T3와 T7 프라이머를 이용하여 돌연변이가 유발된 gpdA프로모터를 갖는 CX 카세트를 증폭하였고, 이를 실시예 2에서와 동일한 방법으로 pB-HF에 삽입하였다. 이렇게 만들어진 돌연변이가 유도된 프로모터를 갖는 라이브러리를 실시예 2에서와 동일한 방법으로 E1 균주에 도입하여 7-ACA 생산성을 평가에 이용하였다.
이렇게 평가된 재조합 균주 중 가장 높은 7-ACA 생산성을 보인 균주를 E3로 명명하였다. E3를 얻기 위한 후보로서 약 1,200의 균락을 시험관으로 배양하였으며, 이 중 7-ACA 생산성으로 상위 78개의 균락을 삼각플라스크에 배양하였다. 삼각플라스크 배양 결과로서 가장 7-ACA 생산성이 높았던 5 균락을 선별하여 발효 배양하였으며, 그 결과를 도 2에 나타내었다.
7-ACA 생산성 향상에 영향을 미친 변이된 프로모터의 서열을 확인하기 위해 다음과 같이 실시하였다. 5개 균주의 각 한 균락씩을 평판배지로부터 잘라내어 멸균된 3차 증류수로 4회 세척하고, 0.5 mL의 멸균 증류수에 재현탁하여 20분간 끓는 물에서 중탕하였다. 실온으로 냉각 후에 13,000 rpm에서 20분간 원심분리 후 상층액을 주형으로 프라이머 5‘-TAGCATGCCATTAACCTAGG-3’ (서열번호 33) 및 5‘-GGGTAGCTGTTAGTCAAGC-3’ (서열번호 34)를 이용하여 증폭한 후 T-Blunt Vector (Solgent, 한국)에 동봉된 메뉴얼에 따라 도입하여 대장균 DH5alpha에 형질전환하고 LB배지에 배양 및 플라스미드를 추출하여 염기서열 분석을 하였다. 염기서열 분석은 마크로젠(한국)에 의뢰하여 수행하였다. 상위 5개 균락에 대한 염기서열 분석결과는 도 3과 같으며, (B) 내지 (F)의 돌연변이 프로모터의 서열을 각각 서열번호 10 내지 14로 나타내었다 ((A)는 야생형 프로모터).
도 3의 (D)에서 나타낸 서열의 변이 프로모터 부위를 가지며 해당 균주에 도입되었던 재조합 플라스미드 벡터는 pB-HCXEP3 (서열번호 15)라 명명하였고, 이를 도입하여 7-ACA 발효생산성이 15 g/L를 나타낸 균주를 E3로 명명하였다.
<3-2> 반복 형질전환에 의한 7- ACA 생산성의 향상
본 발명자들은 7-ACA의 생산성의 향상을 위해서 연구하던 중 반복형질전환이 7-ACA의 생산성을 향상시킨다는 점을 알아내었다. 이에 CX 유전자의 발현을 증대시키기 위해서 E3 균주에 재 형질전환을 시도하였다. 도입되었던 EP3부위로 변이된 프로모터로 작동하는 CX 카세트를 앞서 제작했던 pB-GF(geneticin) 및 pB-PF(phleomycin)에 도입하여 항생제가 다른 두 재조합 플라스미드를 제작하였다.
상기 도 3에서 보여지는 염기서열 분석을 위해 EP3를 도입하여 제작했던 재조합 T-Blunt vector를 EcoRI과 NdeI으로 절단하여 약 330 bp의 DNA단편을 추출하였고, 이를 실시예 3에서 이용했던 pB-CXcast (서열번호 8) 플라스미드 벡터를 주형으로 증폭한 7,717 bp의 DNA 단편을 동일한 제한효소로 절단하여 연결하여 EP3 염기서열을 갖는 CX 카세트를 포함하는 플라스미드 벡터를 제조하였다. 이로부터 T3와 T7 프라이머를 이용하여 CX 카세트를 증폭한 후 이를 pB-GF 및 pB-PF의 SnaBI 부위에 도입하여 각각 pB-GCXEP3 (서열번호 16) 및 pB-PCXEP3(서열번호 17)를 제작하였다.
pB-HCXEP3가 도입된 E3균주에 pB-GCXEP3를 형질전환한 균주를 E4라 명명하였고, E4 균주에 pB-PCXEP3를 형질전환한 균주를 E5라 명명하였다.
E5균주의 경우 본 발명자가 보유한 세 종류의 항생제마커유전자를 모두 갖고 있으므로, 형질전환을 반복하기 위해서 마커유전자 카세트를 제거할 필요가 있었다. 이를 위해 E5균주를 2% 자일로스를 함유하고 항생제 LB 평판배지에 계대배양하여 7일 내지 30일 후 나온 균락으로부터 총 DNA를 추출하여 PCR을 통해 마커유전자 카세트의 결손 여부를 확인하였다. 이상의 단계를 반복하여 세 가지 항생제마커 유전자카세트가 모두 손실된 형질전환체를 선별한 후 pB-HCXEP3, pB-GCXEP3, pB-PCXEP3의 형질전환을 순차적으로 반복하였다. 반복적인 형질전환으로 7-ACA 생산성이 향상된 균주는 순차적으로 E6(4회 형질전환체), E7(5회 형질전환체), E8(6회 형질전환체)로 명명하였다. E7의 경우 E6에 비해 7-ACA 생산성이 증가하였으나 그 정도가 높지 않아 재도입의 효율이 낮았고, 또한 E8의 경우 7-ACA 생산성이 증가된 형질전환체가 선별되지 않았으므로 (유사한 수준의 생산성을 보임) E7 균주를 CPC 아실라제 도입에 대한 최종 균주로 선발하였다. E4 내지 E7의 7-ACA 발효 생산성을 도 4에 나타내었다.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
이상 살펴본 바와 같이, 본 발명은 CPC 생산성이 높아지도록 돌연변이를 유발한 아크레모니움 크리소제눔을 제조하고, 강하게 발현하도록 조절한 프로모터와 CPC 아실라제를 포함하는 발현 벡터를 반복적으로 형질전환하여 7-ACA를 고농도로 생산할 수 있는 균주를 제조하는 방법 등을 제공한다. 본 발명의 방법에 의하면 재조합 아크레모니움 크리소제눔 균주를 제조하여 이의 발효를 통하여 7-ACA를 고농도로 제조할 수 있어 산업상 이용가능성이 매우 우수하다.
한국생명공학연구원 생물자원센터 KCTC13079BP 20160818
<110> Amicogen Inc. <120> Methods for preparing recombinant Acremonium chrysogenum producing 7-aminocephalosporanic acid with high concentration and Acremonium chrysogenum prepared thereby <130> NP16-0100 <160> 34 <170> KopatentIn 2.0 <210> 1 <211> 7074 <212> DNA <213> Artificial Sequence <220> <223> pB-HcastF plasmid <400> 1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct tcctgcagtt 2220 cgaagttcct attctctaga aagtatagga acttcagagc gctttccttg tatctctaca 2280 cacaggctca aatcaataag aagaacggtt cgtctttttc gtttatatct tgcatcgtcc 2340 caaagctatt ggcgggatat tctgtttgca gttggctgac ttgaagtaat ctctgcagat 2400 ctttcgacac tgaaatacgt cgagcctgct ccgcttggaa gcggcgagga gcctcgtcct 2460 gtcacaacta ccaacatgga gtacgataag ggccagttcc gccagctcat taagagccag 2520 ttcatgggcg ttggcatgat ggccgtcatg catctgtact tcaagtacac caacgctctt 2580 ctgatccagt cgatcatccg ctgaaggcgc tttcgaatct ggttaagatc cacgtcttcg 2640 ggaagccagc gactggtgac ctccagcgtc cctttaaggc tgccaacagc tttctcagcc 2700 agggccagcc caagaccgac aaggcctccc tccagaacgc cgagaagaac tggaggggtg 2760 gtgtcaagga ggagtaagct ccttattgaa gtcggaggac ggagcggtgt caagaggata 2820 ttcttcgact ctgtattata gataagatga tgaggaattg gaggtagcat agcttcattt 2880 ggatttgctt tccaggctga gactctagct tggagcatag agggtccttt ggctttcaat 2940 attctcaagt atctcgagtt tgaacttatt ccctgtgaac cttttattca ccaatgagca 3000 ttggaatgaa catgaatctg aggactgcaa tcgccatgag gttttcgaaa tacatccgga 3060 tgtcgaaggc ttggggcacc tgcgttggtt gaatttagaa cgtggcacta ttgatcatcc 3120 gatagctctg caaagggcgt tgcacaatgc aagtcaaacg ttgctagcag ttccaggtgg 3180 aatgttatga tgagcattgt attaaatcag gagatatagc atgatctcta gttagctcac 3240 cacaaaagtc agacggcgta accaaaagtc acacaacaca agctgtaagg atttcggcac 3300 ggctacggaa gacggagaag ccaccttcag tggactcgag taccatttaa ttctatttgt 3360 gtttgatcga gacctaatac agcccctaca acgaccatca aagtcgtata gctaccagtg 3420 aggaagtgga ctcaaatcga cttcagcaac atctcctgga taaactttaa gcctaaacta 3480 tacagaataa gataggtgga gagcttatac cgagctccca aatctgtcca gatcatggtt 3540 gaccggtgcc tggatcttcc tatagaatca tccttattcg ttgacctagc tgattctgga 3600 gtgacccaga gggtcatgac ttgagcctaa aatccgccgc ctccaccatt tgtagaaaaa 3660 tgtgacgaac tcgtgagctc tgtacagtga ccggtgactc tttctggcat gcggagagac 3720 ggacggacgc agagagaagg gctgagtaat aagccactgg ccagacagct ctggcggctc 3780 tgaggtgcag tggatgatta ttaatccggg accggccgcc cctccgcccc gaagtggaaa 3840 ggctggtgtg cccctcgttg accaagaatc tattgcatca tcggagaata tggagcttca 3900 tcgaatcacc ggcagtaagc gaaggagaat gtgaagccag gggtgtatag ccgtcggcga 3960 aatagcatgc cattaaccta ggtacagaag tccaattgct tccgatctgg taaaagattc 4020 acgagatagt accttctccg aagtaggtag agcgagtacc cggcgcgtaa gctccctaat 4080 tggcccatcc ggcatctgta gggcgtccaa atatcgtgcc tctcctgctt tgcccggtgt 4140 atgaaaccgg aaaggccgct caggagctgg ccagcggcgc agaccgggaa cacaagctgg 4200 cagtcgaccc atccggtgct ctgcactcga cctgctgagg tccctcagtc cctggtaggc 4260 agctttgccc cgtctgtccg cccggtgtgt cggcggggtt gacaaggtcg ttgcgtcagt 4320 ccaacatttg ttgccatatt ttcctgctct ccccaccagc tgctcttttc ttttctcttt 4380 cttttcccat cttcagtata ttcatcttcc catccaagaa cctttatttc ccctaagtaa 4440 gtactttgct acatccatac tccatccttc ccatccctta ttcctttgaa cctttcagtt 4500 cgagctttcc cacttcatcg cagcttgact aacagctacc ccgcttgagc agacatcacc 4560 atgcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc 4620 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga 4680 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat 4740 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa 4800 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac 4860 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc 4920 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt 4980 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg 5040 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg 5100 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac 5160 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc 5220 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg 5280 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc 5340 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat 5400 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg 5460 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta 5520 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag 5580 agtagatgcc gaccgcggga tccacttaac gttactgaaa tcatcaaaca gcttgacgaa 5640 tctggatata agatcgttgg tgtcgatgtc agctccggag ttgagacaaa tggtgttcag 5700 gatctcgata agatacgttc atttgtccaa gcagcaaaga gtgccttcta gtgatttaat 5760 agctccatgt caacaagaat aaaacgcgtt ttcgggttta cctcttccag atacagctca 5820 tctgcaatgc attaatgcat tgactgcaac ctagtaacgc cttncaggct ccggcgaaga 5880 gaagaatagc ttagcagagc tattttcatt ttcgggagac gagatcaagc agatcaacgg 5940 tcgtcaagag acctacgaga ctgaggaatc cgctcttggc tccacgcgac tatatatttg 6000 tctctaattg tactttgaca tgctcctctt ctttactctg atagcttgac tatgaaaatt 6060 ccgtcaccag cncctgggtt cgcaaagata attgcatgtt tcttccttga actctcaagc 6120 ctacaggaca cacattcatc gtaggtataa acctcgaaat canttcctac taagatggta 6180 tacaatagta accatgcatg gttgcctagt gaatgctccg taacacccaa tacgccggcc 6240 gaaacttttt tacaactctc ctatgagtcg tttacccaga atgcacaggt acacttgttt 6300 agaggtaatc cttctttcta gaagtcctcg tgtactgtgt aagcgcccac tccacatctc 6360 cactcgacct gcagttcgaa gttcctattc tctagaaagt ataggaactt cagagcgctt 6420 tccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt 6480 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 6540 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 6600 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 6660 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 6720 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 6780 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 6840 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 6900 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 6960 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 7020 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttag 7074 <210> 2 <211> 6852 <212> DNA <213> Artificial Sequence <220> <223> pB-GcastF plasmid <400> 2 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct tcctgcagtt 2220 cgaagttcct attctctaga aagtatagga acttcagagc gctttccttg tatctctaca 2280 cacaggctca aatcaataag aagaacggtt cgtctttttc gtttatatct tgcatcgtcc 2340 caaagctatt ggcgggatat tctgtttgca gttggctgac ttgaagtaat ctctgcagat 2400 ctttcgacac tgaaatacgt cgagcctgct ccgcttggaa gcggcgagga gcctcgtcct 2460 gtcacaacta ccaacatgga gtacgataag ggccagttcc gccagctcat taagagccag 2520 ttcatgggcg ttggcatgat ggccgtcatg catctgtact tcaagtacac caacgctctt 2580 ctgatccagt cgatcatccg ctgaaggcgc tttcgaatct ggttaagatc cacgtcttcg 2640 ggaagccagc gactggtgac ctccagcgtc cctttaaggc tgccaacagc tttctcagcc 2700 agggccagcc caagaccgac aaggcctccc tccagaacgc cgagaagaac tggaggggtg 2760 gtgtcaagga ggagtaagct ccttattgaa gtcggaggac ggagcggtgt caagaggata 2820 ttcttcgact ctgtattata gataagatga tgaggaattg gaggtagcat agcttcattt 2880 ggatttgctt tccaggctga gactctagct tggagcatag agggtccttt ggctttcaat 2940 attctcaagt atctcgagtt tgaacttatt ccctgtgaac cttttattca ccaatgagca 3000 ttggaatgaa catgaatctg aggactgcaa tcgccatgag gttttcgaaa tacatccgga 3060 tgtcgaaggc ttggggcacc tgcgttggtt gaatttagaa cgtggcacta ttgatcatcc 3120 gatagctctg caaagggcgt tgcacaatgc aagtcaaacg ttgctagcag ttccaggtgg 3180 aatgttatga tgagcattgt attaaatcag gagatatagc atgatctcta gttagctcac 3240 cacaaaagtc agacggcgta accaaaagtc acacaacaca agctgtaagg atttcggcac 3300 ggctacggaa gacggagaag ccaccttcag tggactcgag taccatttaa ttctatttgt 3360 gtttgatcga gacctaatac agcccctaca acgaccatca aagtcgtata gctaccagtg 3420 aggaagtgga ctcaaatcga cttcagcaac atctcctgga taaactttaa gcctaaacta 3480 tacagaataa gataggtgga gagcttatac cgagctccca aatctgtcca gatcatggtt 3540 gaccggtgcc tggatcttcc tatagaatca tccttattcg ttgacctagc tgattctgga 3600 gtgacccaga gggtcatgac ttgagcctaa aatccgccgc ctccaccatt tgtagaaaaa 3660 tgtgacgaac tcgtgagctc tgtacagtga ccggtgactc tttctggcat gcggagagac 3720 ggacggacgc agagagaagg gctgagtaat aagccactgg ccagacagct ctggcggctc 3780 tgaggtgcag tggatgatta ttaatccggg accggccgcc cctccgcccc gaagtggaaa 3840 ggctggtgtg cccctcgttg accaagaatc tattgcatca tcggagaata tggagcttca 3900 tcgaatcacc ggcagtaagc gaaggagaat gtgaagccag gggtgtatag ccgtcggcga 3960 aatagcatgc cattaaccta ggtacagaag tccaattgct tccgatctgg taaaagattc 4020 acgagatagt accttctccg aagtaggtag agcgagtacc cggcgcgtaa gctccctaat 4080 tggcccatcc ggcatctgta gggcgtccaa atatcgtgcc tctcctgctt tgcccggtgt 4140 atgaaaccgg aaaggccgct caggagctgg ccagcggcgc agaccgggaa cacaagctgg 4200 cagtcgaccc atccggtgct ctgcactcga cctgctgagg tccctcagtc cctggtaggc 4260 agctttgccc cgtctgtccg cccggtgtgt cggcggggtt gacaaggtcg ttgcgtcagt 4320 ccaacatttg ttgccatatt ttcctgctct ccccaccagc tgctcttttc ttttctcttt 4380 cttttcccat cttcagtata ttcatcttcc catccaagaa cctttatttc ccctaagtaa 4440 gtactttgct acatccatac tccatccttc ccatccctta ttcctttgaa cctttcagtt 4500 cgagctttcc cacttcatcg cagcttgact aacagctacc ccgcttgagc agacatcacc 4560 atggggattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta 4620 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg 4680 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa 4740 ctccaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct 4800 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg 4860 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca 4920 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat 4980 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac 5040 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc 5100 gacggcgagg atctcgtcgt gacacatggc gatgcctgct tgccgaatat catggtggaa 5160 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag 5220 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc 5280 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt 5340 cttgacgagt tcttctgaag tagatgccga ccgcgggatc cacttaacgt tactgaaatc 5400 atcaaacagc ttgacgaatc tggatataag atcgttggtg tcgatgtcag ctccggagtt 5460 gagacaaatg gtgttcagga tctcgataag atacgttcat ttgtccaagc agcaaagagt 5520 gccttctagt gatttaatag ctccatgtca acaagaataa aacgcgtttt cgggtttacc 5580 tcttccagat acagctcatc tgcaatgcat taatgcattg actgcaacct agtaacgcct 5640 tncaggctcc ggcgaagaga agaatagctt agcagagcta ttttcatttt cgggagacga 5700 gatcaagcag atcaacggtc gtcaagagac ctacgagact gaggaatccg ctcttggctc 5760 cacgcgacta tatatttgtc tctaattgta ctttgacatg ctcctcttct ttactctgat 5820 agcttgacta tgaaaattcc gtcaccagcn cctgggttcg caaagataat tgcatgtttc 5880 ttccttgaac tctcaagcct acaggacaca cattcatcgt aggtataaac ctcgaaatca 5940 nttcctacta agatggtata caatagtaac catgcatggt tgcctagtga atgctccgta 6000 acacccaata cgccggccga aactttttta caactctcct atgagtcgtt tacccagaat 6060 gcacaggtac acttgtttag aggtaatcct tctttctaga agtcctcgtg tactgtgtaa 6120 gcgcccactc cacatctcca ctcgacctgc agttcgaagt tcctattctc tagaaagtat 6180 aggaacttca gagcgctttc caattcgccc tatagtgagt cgtattacgc gcgctcactg 6240 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 6300 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 6360 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc 6420 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 6480 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 6540 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 6600 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 6660 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 6720 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 6780 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 6840 cttacaattt ag 6852 <210> 3 <211> 6429 <212> DNA <213> Artificial Sequence <220> <223> pB-PcastF plasmid <400> 3 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct tcctgcagtt 2220 cgaagttcct attctctaga aagtatagga acttcagagc gctttccttg tatctctaca 2280 cacaggctca aatcaataag aagaacggtt cgtctttttc gtttatatct tgcatcgtcc 2340 caaagctatt ggcgggatat tctgtttgca gttggctgac ttgaagtaat ctctgcagat 2400 ctttcgacac tgaaatacgt cgagcctgct ccgcttggaa gcggcgagga gcctcgtcct 2460 gtcacaacta ccaacatgga gtacgataag ggccagttcc gccagctcat taagagccag 2520 ttcatgggcg ttggcatgat ggccgtcatg catctgtact tcaagtacac caacgctctt 2580 ctgatccagt cgatcatccg ctgaaggcgc tttcgaatct ggttaagatc cacgtcttcg 2640 ggaagccagc gactggtgac ctccagcgtc cctttaaggc tgccaacagc tttctcagcc 2700 agggccagcc caagaccgac aaggcctccc tccagaacgc cgagaagaac tggaggggtg 2760 gtgtcaagga ggagtaagct ccttattgaa gtcggaggac ggagcggtgt caagaggata 2820 ttcttcgact ctgtattata gataagatga tgaggaattg gaggtagcat agcttcattt 2880 ggatttgctt tccaggctga gactctagct tggagcatag agggtccttt ggctttcaat 2940 attctcaagt atctcgagtt tgaacttatt ccctgtgaac cttttattca ccaatgagca 3000 ttggaatgaa catgaatctg aggactgcaa tcgccatgag gttttcgaaa tacatccgga 3060 tgtcgaaggc ttggggcacc tgcgttggtt gaatttagaa cgtggcacta ttgatcatcc 3120 gatagctctg caaagggcgt tgcacaatgc aagtcaaacg ttgctagcag ttccaggtgg 3180 aatgttatga tgagcattgt attaaatcag gagatatagc atgatctcta gttagctcac 3240 cacaaaagtc agacggcgta accaaaagtc acacaacaca agctgtaagg atttcggcac 3300 ggctacggaa gacggagaag ccaccttcag tggactcgag taccatttaa ttctatttgt 3360 gtttgatcga gacctaatac agcccctaca acgaccatca aagtcgtata gctaccagtg 3420 aggaagtgga ctcaaatcga cttcagcaac atctcctgga taaactttaa gcctaaacta 3480 tacagaataa gataggtgga gagcttatac cgagctccca aatctgtcca gatcatggtt 3540 gaccggtgcc tggatcttcc tatagaatca tccttattcg ttgacctagc tgattctgga 3600 gtgacccaga gggtcatgac ttgagcctaa aatccgccgc ctccaccatt tgtagaaaaa 3660 tgtgacgaac tcgtgagctc tgtacagtga ccggtgactc tttctggcat gcggagagac 3720 ggacggacgc agagagaagg gctgagtaat aagccactgg ccagacagct ctggcggctc 3780 tgaggtgcag tggatgatta ttaatccggg accggccgcc cctccgcccc gaagtggaaa 3840 ggctggtgtg cccctcgttg accaagaatc tattgcatca tcggagaata tggagcttca 3900 tcgaatcacc ggcagtaagc gaaggagaat gtgaagccag gggtgtatag ccgtcggcga 3960 aatagcatgc cattaaccta ggtacagaag tccaattgct tccgatctgg taaaagattc 4020 acgagatagt accttctccg aagtaggtag agcgagtacc cggcgcgtaa gctccctaat 4080 tggcccatcc ggcatctgta gggcgtccaa atatcgtgcc tctcctgctt tgcccggtgt 4140 atgaaaccgg aaaggccgct caggagctgg ccagcggcgc agaccgggaa cacaagctgg 4200 cagtcgaccc atccggtgct ctgcactcga cctgctgagg tccctcagtc cctggtaggc 4260 agctttgccc cgtctgtccg cccggtgtgt cggcggggtt gacaaggtcg ttgcgtcagt 4320 ccaacatttg ttgccatatt ttcctgctct ccccaccagc tgctcttttc ttttctcttt 4380 cttttcccat cttcagtata ttcatcttcc catccaagaa cctttatttc ccctaagtaa 4440 gtactttgct acatccatac tccatccttc ccatccctta ttcctttgaa cctttcagtt 4500 cgagctttcc cacttcatcg cagcttgact aacagctacc ccgcttgagc agacatcacc 4560 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc 4620 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt 4680 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac 4740 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag 4800 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag 4860 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc 4920 gaggagcagg actgaagtag atgccgaccg cgggatccac ttaacgttac tgaaatcatc 4980 aaacagcttg acgaatctgg atataagatc gttggtgtcg atgtcagctc cggagttgag 5040 acaaatggtg ttcaggatct cgataagata cgttcatttg tccaagcagc aaagagtgcc 5100 ttctagtgat ttaatagctc catgtcaaca agaataaaac gcgttttcgg gtttacctct 5160 tccagataca gctcatctgc aatgcattaa tgcattgact gcaacctagt aacgccttnc 5220 aggctccggc gaagagaaga atagcttagc agagctattt tcattttcgg gagacgagat 5280 caagcagatc aacggtcgtc aagagaccta cgagactgag gaatccgctc ttggctccac 5340 gcgactatat atttgtctct aattgtactt tgacatgctc ctcttcttta ctctgatagc 5400 ttgactatga aaattccgtc accagcncct gggttcgcaa agataattgc atgtttcttc 5460 cttgaactct caagcctaca ggacacacat tcatcgtagg tataaacctc gaaatcantt 5520 cctactaaga tggtatacaa tagtaaccat gcatggttgc ctagtgaatg ctccgtaaca 5580 cccaatacgc cggccgaaac ttttttacaa ctctcctatg agtcgtttac ccagaatgca 5640 caggtacact tgtttagagg taatccttct ttctagaagt cctcgtgtac tgtgtaagcg 5700 cccactccac atctccactc gacctgcagt tcgaagttcc tattctctag aaagtatagg 5760 aacttcagag cgctttccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc 5820 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 5880 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 5940 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg 6000 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 6060 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 6120 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 6180 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 6240 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 6300 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg 6360 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt 6420 acaatttag 6429 <210> 4 <211> 7088 <212> DNA <213> Artificial Sequence <220> <223> pB-Fcast plasmid <400> 4 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct tgaggccgga 2220 caaattcagc ctgcaggcta aagtttctat tacatgatat ttgtcgtgga caagggcgtc 2280 tggggtaaaa aggaatgtgg gcagagagct ctcggcttaa agaccggctt cggagttcca 2340 gacccactcg aactgaaaac gatgattaga gataagtaga tctatttgga cagtataaga 2400 atcctagtcg gctagtattt ctccactttt actttaaaat tccccagagt aagggggcag 2460 gttcgtcgtc acgggtagcc tcgtctcgag gcatcgcgga tagcattatc ttggggtatc 2520 acgagtaact tcatcgtggc attatcgata ggtactgtaa gtactgtagg aacttatccg 2580 ggcatggata tgtataggta catacgatgc actaaaagtg gagaaatact agccgagtag 2640 agatcttata ccatccgaat agatttactc cttataatca aggggcaaca atcagttgtg 2700 acgatgtcgg ctgtgggagt gcagtttagg ggtcatcaaa gcacaggaaa acgtgttgta 2760 tggatgccta tcgtcttgac tattagaaag gaagagggga aaaaccctca tcaggagcga 2820 ttgtaagttc ggccatagta atgatattgg ccattaatgg acgtgtgtat gtatacatac 2880 acggttcatt tgccgcattc ggaccaaaac cgctgctgcc gcagacacag tgcttccatg 2940 gcctcgaaca accggttatt cttctcttcc atgtcctcgg gacgaaggaa atttcggagc 3000 cctaacagcc gtaccggact tgcgttaaca tggttgcgaa gaactaaaga ctgatggggt 3060 catgattggc agtgttgaca cttcatgtct tgaacctagc tagcagaaaa atgaatgctc 3120 tatgtgacac tggtagatct caacgcatca tgttccacct ttagtgtaaa cacggcaagc 3180 tgtccgcaac gtcggtacca acgtttgaac acacaacctt ctgccactcc aggagtttag 3240 cccacggctt actggatgaa acgtaatctc ccgctacccc ctcaattcca ttggggtcta 3300 gagtagtcgg aagaacggct gtcgccttgc gctgcggtta aagcacccac cgtggtggct 3360 actgcttctc gggaggagaa atcctgggta ctcgagcact tgcgctaggg gatgaaacat 3420 ccatggcata ttggcgaaag caatgcgcca tgttcggcgc ggcacatgac actcttgaga 3480 agccgtattc ttcgcaaact ttcctattta cattcagcct ctaactataa cacgatcgga 3540 aatgcctgaa cgagcccgcc ctcatgtgcc tagcgtcaaa caacgccaag cacacaacgc 3600 ctcctgctat tcttccctct tctcactgcc atcactgggc gcaagctgca atgtccttct 3660 cgaattcaag cgggcaaatc aagattggga aaatgcgcca tcttggtttg gatcttgatg 3720 tctatccgca tcaaggtctt ggtttggtcg tttatttagt agcaaaggca aatcaaaacg 3780 tgcgcgccaa gtgccagggt ccagactcca gactggagca tttagcctga atatcgtctc 3840 tagaatctgg tgctattatt caggatatag taactatgat ctactcgggc catgttcggc 3900 tcctccgcac ggccaagttg tctgccggcg tctggaccgg actcatggct tggcttctcc 3960 agggatagca tggcattggc aaaactctcc gccacgattc gagcatagct accgcagcca 4020 cgcggcattg tttccagatt gagatatgtc actattagcc ggaatgcaaa catcgagctg 4080 acctgcgcaa catctccatg agttccgaag tcgacctccg aagcaatatt ggtctagtat 4140 tacggatgat atagtggtcc ccggacccaa tgagccgtgt gttttttacc gcctttgctc 4200 tgaggattaa caagattcgt gcaattgggg actgtgcgga gtaactgggc tggtttactg 4260 ctttgccgac gaaagaaacg tataaacagc cgactcgtcc actgccactc gggacgatct 4320 cccaacccag acagcttccc agaacccatc ttcaacaccc tgacaccatg ccccagttcg 4380 acatcctatg caagactcct ccgaaggtgc tcgtccgcca gttcgtggag cgattcgagc 4440 gcccctcggg cgaaaagatc gccttgtgtg ccgctgagct tacctacctc tgctggatga 4500 tcacccacaa cggcaccgcc atcaagcggg ctacctttat gagttacaat accatcatta 4560 gcaactccct ctcgtttgac atcgtcaaca agtccctgca gttcaagtac aagacccaaa 4620 aggccaccat cctcgaggcc tcgctgaaga agctgatccc cgcatgggaa ttcactatca 4680 tcccctacta tggtcagaag catcagtctg acatcaccga cattgtaagc agtctccagc 4740 tccagtttga gtcctccgag gaggccgata aaggcaactc ccactccaag aagatgctca 4800 aggctttgct gagtgaaggc gagtccatct gggaaataac cgaaaagatc ttgaacagtt 4860 tcgagtacac cagccgcttc acgaagacca agacgctgta ccagttcctg ttcctcgcca 4920 cattcatcaa ttgcggacgc ttcagtgata tcaagaacgt ggacccgaag tcattcaagc 4980 tggttcagaa caagtacctc ggtgtcatca ttcagtgctt ggtcaccgag accaagacat 5040 ccgtcagccg tcacatctac ttcttttccg caagagggcg tatcgacccc ctagtgtacc 5100 tagacgaatt cctgcgcaac tcggagcccg tcctaaagcg cgtcaaccgt accggcaact 5160 ctagcagcaa caagcaggag taccagctct tgaaggataa cctcgtccga tcatacaaca 5220 aggcgctcaa gaagaacgct ccatacagca tctttgctat caagaatggg ccaaagtcac 5280 acatcggccg ccacctcatg acctcgtttc tgtcgatgaa gggcctcact gagcttacca 5340 acgttgttgg caactggtcc gataagcgcg ccagcgccgt ggcgcggacg acttacactc 5400 atcagatcac agctatccct gaccactact tcgccttggt cagtcgctat tacgcctacg 5460 acccaattag taaagagatg atcgctctca aggacgaaac caaccccatc gaggagtggc 5520 aacacatcga gcagcttaaa ggctccgccg agggctcgat ccggtacccc gcctggaacg 5580 gtatcatatc gcaggaggtc ttggattacc tgagcagtta tatcaatcgc cgcatatgat 5640 aaggatccac ttaacgttac tgaaatcatc aaacagcttg acgaatctgg atataagatc 5700 gttggtgtcg atgtcagctc cggagttgag acaaatggtg ttcaggatct cgataagata 5760 cgttcatttg tccaagcagc aaagagtgcc ttctagtgat ttaatagctc catgtcaaca 5820 agaataaaac gcgttttcgg gtttacctct tccagataca gctcatctgc aatgcattaa 5880 tgcattgact gcaacctagt aacgccttnc aggctccggc gaagagaaga atagcttagc 5940 agagctattt tcattttcgg gagacgagat caagcagatc aacggtcgtc aagagaccta 6000 cgagactgag gaatccgctc ttggctccac gcgactatat atttgtctct aattgtactt 6060 tgacatgctc ctcttcttta ctctgatagc ttgactatga aaattccgtc accagcncct 6120 gggttcgcaa agataattgc atgtttcttc cttgaactct caagcctaca ggacacacat 6180 tcatcgtagg tataaacctc gaaatcantt cctactaaga tggtatacaa tagtaaccat 6240 gcatggttgc ctagtgaatg ctccgtaaca cccaatacgc cggccgaaac ttttttacaa 6300 ctctcctatg agtcgtttac ccagaatgca caggtacact tgtttagagg taatccttct 6360 ttctagaagt cctcgtgtac tgtgtaagcg cccactccac atctccactc gattaattaa 6420 tacgtaatcg atagcccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg 6480 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 6540 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 6600 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg 6660 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 6720 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 6780 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 6840 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 6900 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 6960 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 7020 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta 7080 caatttag 7088 <210> 5 <211> 11368 <212> DNA <213> Artificial Sequence <220> <223> pB-HF plasmid <400> 5 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaaaccc 2220 cgggtacgta aattaaccct cactaaaggg aacaaaagct gggcaaagct tgaggccgga 2280 caaattcagc ctgcaggcta aagtttctat tacatgatat ttgtcgtgga caagggcgtc 2340 tggggtaaaa aggaatgtgg gcagagagct ctcggcttaa agaccggctt cggagttcca 2400 gacccactcg aactgaaaac gatgattaga gataagtaga tctatttgga cagtataaga 2460 atcctagtcg gctagtattt ctccactttt actttaaaat tccccagagt aagggggcag 2520 gttcgtcgtc acgggtagcc tcgtctcgag gcatcgcgga tagcattatc ttggggtatc 2580 acgagtaact tcatcgtggc attatcgata ggtactgtaa gtactgtagg aacttatccg 2640 ggcatggata tgtataggta catacgatgc actaaaagtg gagaaatact agccgagtag 2700 agatcttata ccatccgaat agatttactc cttataatca aggggcaaca atcagttgtg 2760 acgatgtcgg ctgtgggagt gcagtttagg ggtcatcaaa gcacaggaaa acgtgttgta 2820 tggatgccta tcgtcttgac tattagaaag gaagagggga aaaaccctca tcaggagcga 2880 ttgtaagttc ggccatagta atgatattgg ccattaatgg acgtgtgtat gtatacatac 2940 acggttcatt tgccgcattc ggaccaaaac cgctgctgcc gcagacacag tgcttccatg 3000 gcctcgaaca accggttatt cttctcttcc atgtcctcgg gacgaaggaa atttcggagc 3060 cctaacagcc gtaccggact tgcgttaaca tggttgcgaa gaactaaaga ctgatggggt 3120 catgattggc agtgttgaca cttcatgtct tgaacctagc tagcagaaaa atgaatgctc 3180 tatgtgacac tggtagatct caacgcatca tgttccacct ttagtgtaaa cacggcaagc 3240 tgtccgcaac gtcggtacca acgtttgaac acacaacctt ctgccactcc aggagtttag 3300 cccacggctt actggatgaa acgtaatctc ccgctacccc ctcaattcca ttggggtcta 3360 gagtagtcgg aagaacggct gtcgccttgc gctgcggtta aagcacccac cgtggtggct 3420 actgcttctc gggaggagaa atcctgggta ctcgagcact tgcgctaggg gatgaaacat 3480 ccatggcata ttggcgaaag caatgcgcca tgttcggcgc ggcacatgac actcttgaga 3540 agccgtattc ttcgcaaact ttcctattta cattcagcct ctaactataa cacgatcgga 3600 aatgcctgaa cgagcccgcc ctcatgtgcc tagcgtcaaa caacgccaag cacacaacgc 3660 ctcctgctat tcttccctct tctcactgcc atcactgggc gcaagctgca atgtccttct 3720 cgaattcaag cgggcaaatc aagattggga aaatgcgcca tcttggtttg gatcttgatg 3780 tctatccgca tcaaggtctt ggtttggtcg tttatttagt agcaaaggca aatcaaaacg 3840 tgcgcgccaa gtgccagggt ccagactcca gactggagca tttagcctga atatcgtctc 3900 tagaatctgg tgctattatt caggatatag taactatgat ctactcgggc catgttcggc 3960 tcctccgcac ggccaagttg tctgccggcg tctggaccgg actcatggct tggcttctcc 4020 agggatagca tggcattggc aaaactctcc gccacgattc gagcatagct accgcagcca 4080 cgcggcattg tttccagatt gagatatgtc actattagcc ggaatgcaaa catcgagctg 4140 acctgcgcaa catctccatg agttccgaag tcgacctccg aagcaatatt ggtctagtat 4200 tacggatgat atagtggtcc ccggacccaa tgagccgtgt gttttttacc gcctttgctc 4260 tgaggattaa caagattcgt gcaattgggg actgtgcgga gtaactgggc tggtttactg 4320 ctttgccgac gaaagaaacg tataaacagc cgactcgtcc actgccactc gggacgatct 4380 cccaacccag acagcttccc agaacccatc ttcaacaccc tgacaccatg ccccagttcg 4440 acatcctatg caagactcct ccgaaggtgc tcgtccgcca gttcgtggag cgattcgagc 4500 gcccctcggg cgaaaagatc gccttgtgtg ccgctgagct tacctacctc tgctggatga 4560 tcacccacaa cggcaccgcc atcaagcggg ctacctttat gagttacaat accatcatta 4620 gcaactccct ctcgtttgac atcgtcaaca agtccctgca gttcaagtac aagacccaaa 4680 aggccaccat cctcgaggcc tcgctgaaga agctgatccc cgcatgggaa ttcactatca 4740 tcccctacta tggtcagaag catcagtctg acatcaccga cattgtaagc agtctccagc 4800 tccagtttga gtcctccgag gaggccgata aaggcaactc ccactccaag aagatgctca 4860 aggctttgct gagtgaaggc gagtccatct gggaaataac cgaaaagatc ttgaacagtt 4920 tcgagtacac cagccgcttc acgaagacca agacgctgta ccagttcctg ttcctcgcca 4980 cattcatcaa ttgcggacgc ttcagtgata tcaagaacgt ggacccgaag tcattcaagc 5040 tggttcagaa caagtacctc ggtgtcatca ttcagtgctt ggtcaccgag accaagacat 5100 ccgtcagccg tcacatctac ttcttttccg caagagggcg tatcgacccc ctagtgtacc 5160 tagacgaatt cctgcgcaac tcggagcccg tcctaaagcg cgtcaaccgt accggcaact 5220 ctagcagcaa caagcaggag taccagctct tgaaggataa cctcgtccga tcatacaaca 5280 aggcgctcaa gaagaacgct ccatacagca tctttgctat caagaatggg ccaaagtcac 5340 acatcggccg ccacctcatg acctcgtttc tgtcgatgaa gggcctcact gagcttacca 5400 acgttgttgg caactggtcc gataagcgcg ccagcgccgt ggcgcggacg acttacactc 5460 atcagatcac agctatccct gaccactact tcgccttggt cagtcgctat tacgcctacg 5520 acccaattag taaagagatg atcgctctca aggacgaaac caaccccatc gaggagtggc 5580 aacacatcga gcagcttaaa ggctccgccg agggctcgat ccggtacccc gcctggaacg 5640 gtatcatatc gcaggaggtc ttggattacc tgagcagtta tatcaatcgc cgcatatgat 5700 aaggatccac ttaacgttac tgaaatcatc aaacagcttg acgaatctgg atataagatc 5760 gttggtgtcg atgtcagctc cggagttgag acaaatggtg ttcaggatct cgataagata 5820 cgttcatttg tccaagcagc aaagagtgcc ttctagtgat ttaatagctc catgtcaaca 5880 agaataaaac gcgttttcgg gtttacctct tccagataca gctcatctgc aatgcattaa 5940 tgcattgact gcaacctagt aacgccttnc aggctccggc gaagagaaga atagcttagc 6000 agagctattt tcattttcgg gagacgagat caagcagatc aacggtcgtc aagagaccta 6060 cgagactgag gaatccgctc ttggctccac gcgactatat atttgtctct aattgtactt 6120 tgacatgctc ctcttcttta ctctgatagc ttgactatga aaattccgtc accagcncct 6180 gggttcgcaa agataattgc atgtttcttc cttgaactct caagcctaca ggacacacat 6240 tcatcgtagg tataaacctc gaaatcantt cctactaaga tggtatacaa tagtaaccat 6300 gcatggttgc ctagtgaatg ctccgtaaca cccaatacgc cggccgaaac ttttttacaa 6360 ctctcctatg agtcgtttac ccagaatgca caggtacact tgtttagagg taatccttct 6420 ttctagaagt cctcgtgtac tgtgtaagcg cccactccac atctccactc gaccaattcg 6480 ccctatagtg agtcgtatta agcttcctgc agttcgaagt tcctattctc tagaaagtat 6540 aggaacttca gagcgctttc cttgtatctc tacacacagg ctcaaatcaa taagaagaac 6600 ggttcgtctt tttcgtttat atcttgcatc gtcccaaagc tattggcggg atattctgtt 6660 tgcagttggc tgacttgaag taatctctgc agatctttcg acactgaaat acgtcgagcc 6720 tgctccgctt ggaagcggcg aggagcctcg tcctgtcaca actaccaaca tggagtacga 6780 taagggccag ttccgccagc tcattaagag ccagttcatg ggcgttggca tgatggccgt 6840 catgcatctg tacttcaagt acaccaacgc tcttctgatc cagtcgatca tccgctgaag 6900 gcgctttcga atctggttaa gatccacgtc ttcgggaagc cagcgactgg tgacctccag 6960 cgtcccttta aggctgccaa cagctttctc agccagggcc agcccaagac cgacaaggcc 7020 tccctccaga acgccgagaa gaactggagg ggtggtgtca aggaggagta agctccttat 7080 tgaagtcgga ggacggagcg gtgtcaagag gatattcttc gactctgtat tatagataag 7140 atgatgagga attggaggta gcatagcttc atttggattt gctttccagg ctgagactct 7200 agcttggagc atagagggtc ctttggcttt caatattctc aagtatctcg agtttgaact 7260 tattccctgt gaacctttta ttcaccaatg agcattggaa tgaacatgaa tctgaggact 7320 gcaatcgcca tgaggttttc gaaatacatc cggatgtcga aggcttgggg cacctgcgtt 7380 ggttgaattt agaacgtggc actattgatc atccgatagc tctgcaaagg gcgttgcaca 7440 atgcaagtca aacgttgcta gcagttccag gtggaatgtt atgatgagca ttgtattaaa 7500 tcaggagata tagcatgatc tctagttagc tcaccacaaa agtcagacgg cgtaaccaaa 7560 agtcacacaa cacaagctgt aaggatttcg gcacggctac ggaagacgga gaagccacct 7620 tcagtggact cgagtaccat ttaattctat ttgtgtttga tcgagaccta atacagcccc 7680 tacaacgacc atcaaagtcg tatagctacc agtgaggaag tggactcaaa tcgacttcag 7740 caacatctcc tggataaact ttaagcctaa actatacaga ataagatagg tggagagctt 7800 ataccgagct cccaaatctg tccagatcat ggttgaccgg tgcctggatc ttcctataga 7860 atcatcctta ttcgttgacc tagctgattc tggagtgacc cagagggtca tgacttgagc 7920 ctaaaatccg ccgcctccac catttgtaga aaaatgtgac gaactcgtga gctctgtaca 7980 gtgaccggtg actctttctg gcatgcggag agacggacgg acgcagagag aagggctgag 8040 taataagcca ctggccagac agctctggcg gctctgaggt gcagtggatg attattaatc 8100 cgggaccggc cgcccctccg ccccgaagtg gaaaggctgg tgtgcccctc gttgaccaag 8160 aatctattgc atcatcggag aatatggagc ttcatcgaat caccggcagt aagcgaagga 8220 gaatgtgaag ccaggggtgt atagccgtcg gcgaaatagc atgccattaa cctaggtaca 8280 gaagtccaat tgcttccgat ctggtaaaag attcacgaga tagtaccttc tccgaagtag 8340 gtagagcgag tacccggcgc gtaagctccc taattggccc atccggcatc tgtagggcgt 8400 ccaaatatcg tgcctctcct gctttgcccg gtgtatgaaa ccggaaaggc cgctcaggag 8460 ctggccagcg gcgcagaccg ggaacacaag ctggcagtcg acccatccgg tgctctgcac 8520 tcgacctgct gaggtccctc agtccctggt aggcagcttt gccccgtctg tccgcccggt 8580 gtgtcggcgg ggttgacaag gtcgttgcgt cagtccaaca tttgttgcca tattttcctg 8640 ctctccccac cagctgctct tttcttttct ctttcttttc ccatcttcag tatattcatc 8700 ttcccatcca agaaccttta tttcccctaa gtaagtactt tgctacatcc atactccatc 8760 cttcccatcc cttattcctt tgaacctttc agttcgagct ttcccacttc atcgcagctt 8820 gactaacagc taccccgctt gagcagacat caccatgcct gaactcaccg cgacgtctgt 8880 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg 8940 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa 9000 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc 9060 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat 9120 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt 9180 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag 9240 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat 9300 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag 9360 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt 9420 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat 9480 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa 9540 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg 9600 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct 9660 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg 9720 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg 9780 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg 9840 acgccccagc actcgtccga gggcaaagga atagagtaga tgccgaccgc gggatccact 9900 taacgttact gaaatcatca aacagcttga cgaatctgga tataagatcg ttggtgtcga 9960 tgtcagctcc ggagttgaga caaatggtgt tcaggatctc gataagatac gttcatttgt 10020 ccaagcagca aagagtgcct tctagtgatt taatagctcc atgtcaacaa gaataaaacg 10080 cgttttcggg tttacctctt ccagatacag ctcatctgca atgcattaat gcattgactg 10140 caacctagta acgccttnca ggctccggcg aagagaagaa tagcttagca gagctatttt 10200 cattttcggg agacgagatc aagcagatca acggtcgtca agagacctac gagactgagg 10260 aatccgctct tggctccacg cgactatata tttgtctcta attgtacttt gacatgctcc 10320 tcttctttac tctgatagct tgactatgaa aattccgtca ccagcncctg ggttcgcaaa 10380 gataattgca tgtttcttcc ttgaactctc aagcctacag gacacacatt catcgtaggt 10440 ataaacctcg aaatcanttc ctactaagat ggtatacaat agtaaccatg catggttgcc 10500 tagtgaatgc tccgtaacac ccaatacgcc ggccgaaact tttttacaac tctcctatga 10560 gtcgtttacc cagaatgcac aggtacactt gtttagaggt aatccttctt tctagaagtc 10620 ctcgtgtact gtgtaagcgc ccactccaca tctccactcg acctgcagtt cgaagttcct 10680 attctctaga aagtatagga acttcagagc gctttccaat tcgccctata gtgagtcgta 10740 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 10800 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 10860 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg 10920 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 10980 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 11040 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 11100 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg 11160 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 11220 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt 11280 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 11340 taacaaaata ttaacgctta caatttag 11368 <210> 6 <211> 11146 <212> DNA <213> Artificial Sequence <220> <223> pB-GF plasmid <400> 6 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaaaccc 2220 cgggtacgta aattaaccct cactaaaggg aacaaaagct gggcaaagct tgaggccgga 2280 caaattcagc ctgcaggcta aagtttctat tacatgatat ttgtcgtgga caagggcgtc 2340 tggggtaaaa aggaatgtgg gcagagagct ctcggcttaa agaccggctt cggagttcca 2400 gacccactcg aactgaaaac gatgattaga gataagtaga tctatttgga cagtataaga 2460 atcctagtcg gctagtattt ctccactttt actttaaaat tccccagagt aagggggcag 2520 gttcgtcgtc acgggtagcc tcgtctcgag gcatcgcgga tagcattatc ttggggtatc 2580 acgagtaact tcatcgtggc attatcgata ggtactgtaa gtactgtagg aacttatccg 2640 ggcatggata tgtataggta catacgatgc actaaaagtg gagaaatact agccgagtag 2700 agatcttata ccatccgaat agatttactc cttataatca aggggcaaca atcagttgtg 2760 acgatgtcgg ctgtgggagt gcagtttagg ggtcatcaaa gcacaggaaa acgtgttgta 2820 tggatgccta tcgtcttgac tattagaaag gaagagggga aaaaccctca tcaggagcga 2880 ttgtaagttc ggccatagta atgatattgg ccattaatgg acgtgtgtat gtatacatac 2940 acggttcatt tgccgcattc ggaccaaaac cgctgctgcc gcagacacag tgcttccatg 3000 gcctcgaaca accggttatt cttctcttcc atgtcctcgg gacgaaggaa atttcggagc 3060 cctaacagcc gtaccggact tgcgttaaca tggttgcgaa gaactaaaga ctgatggggt 3120 catgattggc agtgttgaca cttcatgtct tgaacctagc tagcagaaaa atgaatgctc 3180 tatgtgacac tggtagatct caacgcatca tgttccacct ttagtgtaaa cacggcaagc 3240 tgtccgcaac gtcggtacca acgtttgaac acacaacctt ctgccactcc aggagtttag 3300 cccacggctt actggatgaa acgtaatctc ccgctacccc ctcaattcca ttggggtcta 3360 gagtagtcgg aagaacggct gtcgccttgc gctgcggtta aagcacccac cgtggtggct 3420 actgcttctc gggaggagaa atcctgggta ctcgagcact tgcgctaggg gatgaaacat 3480 ccatggcata ttggcgaaag caatgcgcca tgttcggcgc ggcacatgac actcttgaga 3540 agccgtattc ttcgcaaact ttcctattta cattcagcct ctaactataa cacgatcgga 3600 aatgcctgaa cgagcccgcc ctcatgtgcc tagcgtcaaa caacgccaag cacacaacgc 3660 ctcctgctat tcttccctct tctcactgcc atcactgggc gcaagctgca atgtccttct 3720 cgaattcaag cgggcaaatc aagattggga aaatgcgcca tcttggtttg gatcttgatg 3780 tctatccgca tcaaggtctt ggtttggtcg tttatttagt agcaaaggca aatcaaaacg 3840 tgcgcgccaa gtgccagggt ccagactcca gactggagca tttagcctga atatcgtctc 3900 tagaatctgg tgctattatt caggatatag taactatgat ctactcgggc catgttcggc 3960 tcctccgcac ggccaagttg tctgccggcg tctggaccgg actcatggct tggcttctcc 4020 agggatagca tggcattggc aaaactctcc gccacgattc gagcatagct accgcagcca 4080 cgcggcattg tttccagatt gagatatgtc actattagcc ggaatgcaaa catcgagctg 4140 acctgcgcaa catctccatg agttccgaag tcgacctccg aagcaatatt ggtctagtat 4200 tacggatgat atagtggtcc ccggacccaa tgagccgtgt gttttttacc gcctttgctc 4260 tgaggattaa caagattcgt gcaattgggg actgtgcgga gtaactgggc tggtttactg 4320 ctttgccgac gaaagaaacg tataaacagc cgactcgtcc actgccactc gggacgatct 4380 cccaacccag acagcttccc agaacccatc ttcaacaccc tgacaccatg ccccagttcg 4440 acatcctatg caagactcct ccgaaggtgc tcgtccgcca gttcgtggag cgattcgagc 4500 gcccctcggg cgaaaagatc gccttgtgtg ccgctgagct tacctacctc tgctggatga 4560 tcacccacaa cggcaccgcc atcaagcggg ctacctttat gagttacaat accatcatta 4620 gcaactccct ctcgtttgac atcgtcaaca agtccctgca gttcaagtac aagacccaaa 4680 aggccaccat cctcgaggcc tcgctgaaga agctgatccc cgcatgggaa ttcactatca 4740 tcccctacta tggtcagaag catcagtctg acatcaccga cattgtaagc agtctccagc 4800 tccagtttga gtcctccgag gaggccgata aaggcaactc ccactccaag aagatgctca 4860 aggctttgct gagtgaaggc gagtccatct gggaaataac cgaaaagatc ttgaacagtt 4920 tcgagtacac cagccgcttc acgaagacca agacgctgta ccagttcctg ttcctcgcca 4980 cattcatcaa ttgcggacgc ttcagtgata tcaagaacgt ggacccgaag tcattcaagc 5040 tggttcagaa caagtacctc ggtgtcatca ttcagtgctt ggtcaccgag accaagacat 5100 ccgtcagccg tcacatctac ttcttttccg caagagggcg tatcgacccc ctagtgtacc 5160 tagacgaatt cctgcgcaac tcggagcccg tcctaaagcg cgtcaaccgt accggcaact 5220 ctagcagcaa caagcaggag taccagctct tgaaggataa cctcgtccga tcatacaaca 5280 aggcgctcaa gaagaacgct ccatacagca tctttgctat caagaatggg ccaaagtcac 5340 acatcggccg ccacctcatg acctcgtttc tgtcgatgaa gggcctcact gagcttacca 5400 acgttgttgg caactggtcc gataagcgcg ccagcgccgt ggcgcggacg acttacactc 5460 atcagatcac agctatccct gaccactact tcgccttggt cagtcgctat tacgcctacg 5520 acccaattag taaagagatg atcgctctca aggacgaaac caaccccatc gaggagtggc 5580 aacacatcga gcagcttaaa ggctccgccg agggctcgat ccggtacccc gcctggaacg 5640 gtatcatatc gcaggaggtc ttggattacc tgagcagtta tatcaatcgc cgcatatgat 5700 aaggatccac ttaacgttac tgaaatcatc aaacagcttg acgaatctgg atataagatc 5760 gttggtgtcg atgtcagctc cggagttgag acaaatggtg ttcaggatct cgataagata 5820 cgttcatttg tccaagcagc aaagagtgcc ttctagtgat ttaatagctc catgtcaaca 5880 agaataaaac gcgttttcgg gtttacctct tccagataca gctcatctgc aatgcattaa 5940 tgcattgact gcaacctagt aacgccttnc aggctccggc gaagagaaga atagcttagc 6000 agagctattt tcattttcgg gagacgagat caagcagatc aacggtcgtc aagagaccta 6060 cgagactgag gaatccgctc ttggctccac gcgactatat atttgtctct aattgtactt 6120 tgacatgctc ctcttcttta ctctgatagc ttgactatga aaattccgtc accagcncct 6180 gggttcgcaa agataattgc atgtttcttc cttgaactct caagcctaca ggacacacat 6240 tcatcgtagg tataaacctc gaaatcantt cctactaaga tggtatacaa tagtaaccat 6300 gcatggttgc ctagtgaatg ctccgtaaca cccaatacgc cggccgaaac ttttttacaa 6360 ctctcctatg agtcgtttac ccagaatgca caggtacact tgtttagagg taatccttct 6420 ttctagaagt cctcgtgtac tgtgtaagcg cccactccac atctccactc gaccaattcg 6480 ccctatagtg agtcgtatta agcttcctgc agttcgaagt tcctattctc tagaaagtat 6540 aggaacttca gagcgctttc cttgtatctc tacacacagg ctcaaatcaa taagaagaac 6600 ggttcgtctt tttcgtttat atcttgcatc gtcccaaagc tattggcggg atattctgtt 6660 tgcagttggc tgacttgaag taatctctgc agatctttcg acactgaaat acgtcgagcc 6720 tgctccgctt ggaagcggcg aggagcctcg tcctgtcaca actaccaaca tggagtacga 6780 taagggccag ttccgccagc tcattaagag ccagttcatg ggcgttggca tgatggccgt 6840 catgcatctg tacttcaagt acaccaacgc tcttctgatc cagtcgatca tccgctgaag 6900 gcgctttcga atctggttaa gatccacgtc ttcgggaagc cagcgactgg tgacctccag 6960 cgtcccttta aggctgccaa cagctttctc agccagggcc agcccaagac cgacaaggcc 7020 tccctccaga acgccgagaa gaactggagg ggtggtgtca aggaggagta agctccttat 7080 tgaagtcgga ggacggagcg gtgtcaagag gatattcttc gactctgtat tatagataag 7140 atgatgagga attggaggta gcatagcttc atttggattt gctttccagg ctgagactct 7200 agcttggagc atagagggtc ctttggcttt caatattctc aagtatctcg agtttgaact 7260 tattccctgt gaacctttta ttcaccaatg agcattggaa tgaacatgaa tctgaggact 7320 gcaatcgcca tgaggttttc gaaatacatc cggatgtcga aggcttgggg cacctgcgtt 7380 ggttgaattt agaacgtggc actattgatc atccgatagc tctgcaaagg gcgttgcaca 7440 atgcaagtca aacgttgcta gcagttccag gtggaatgtt atgatgagca ttgtattaaa 7500 tcaggagata tagcatgatc tctagttagc tcaccacaaa agtcagacgg cgtaaccaaa 7560 agtcacacaa cacaagctgt aaggatttcg gcacggctac ggaagacgga gaagccacct 7620 tcagtggact cgagtaccat ttaattctat ttgtgtttga tcgagaccta atacagcccc 7680 tacaacgacc atcaaagtcg tatagctacc agtgaggaag tggactcaaa tcgacttcag 7740 caacatctcc tggataaact ttaagcctaa actatacaga ataagatagg tggagagctt 7800 ataccgagct cccaaatctg tccagatcat ggttgaccgg tgcctggatc ttcctataga 7860 atcatcctta ttcgttgacc tagctgattc tggagtgacc cagagggtca tgacttgagc 7920 ctaaaatccg ccgcctccac catttgtaga aaaatgtgac gaactcgtga gctctgtaca 7980 gtgaccggtg actctttctg gcatgcggag agacggacgg acgcagagag aagggctgag 8040 taataagcca ctggccagac agctctggcg gctctgaggt gcagtggatg attattaatc 8100 cgggaccggc cgcccctccg ccccgaagtg gaaaggctgg tgtgcccctc gttgaccaag 8160 aatctattgc atcatcggag aatatggagc ttcatcgaat caccggcagt aagcgaagga 8220 gaatgtgaag ccaggggtgt atagccgtcg gcgaaatagc atgccattaa cctaggtaca 8280 gaagtccaat tgcttccgat ctggtaaaag attcacgaga tagtaccttc tccgaagtag 8340 gtagagcgag tacccggcgc gtaagctccc taattggccc atccggcatc tgtagggcgt 8400 ccaaatatcg tgcctctcct gctttgcccg gtgtatgaaa ccggaaaggc cgctcaggag 8460 ctggccagcg gcgcagaccg ggaacacaag ctggcagtcg acccatccgg tgctctgcac 8520 tcgacctgct gaggtccctc agtccctggt aggcagcttt gccccgtctg tccgcccggt 8580 gtgtcggcgg ggttgacaag gtcgttgcgt cagtccaaca tttgttgcca tattttcctg 8640 ctctccccac cagctgctct tttcttttct ctttcttttc ccatcttcag tatattcatc 8700 ttcccatcca agaaccttta tttcccctaa gtaagtactt tgctacatcc atactccatc 8760 cttcccatcc cttattcctt tgaacctttc agttcgagct ttcccacttc atcgcagctt 8820 gactaacagc taccccgctt gagcagacat caccatgggg attgaacaag atggattgca 8880 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 8940 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 9000 tgtcaagacc gacctgtccg gtgccctgaa tgaactccag gacgaggcag cgcggctatc 9060 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 9120 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 9180 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 9240 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 9300 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 9360 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgacaca 9420 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 9480 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 9540 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 9600 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaagtagatg 9660 ccgaccgcgg gatccactta acgttactga aatcatcaaa cagcttgacg aatctggata 9720 taagatcgtt ggtgtcgatg tcagctccgg agttgagaca aatggtgttc aggatctcga 9780 taagatacgt tcatttgtcc aagcagcaaa gagtgccttc tagtgattta atagctccat 9840 gtcaacaaga ataaaacgcg ttttcgggtt tacctcttcc agatacagct catctgcaat 9900 gcattaatgc attgactgca acctagtaac gccttncagg ctccggcgaa gagaagaata 9960 gcttagcaga gctattttca ttttcgggag acgagatcaa gcagatcaac ggtcgtcaag 10020 agacctacga gactgaggaa tccgctcttg gctccacgcg actatatatt tgtctctaat 10080 tgtactttga catgctcctc ttctttactc tgatagcttg actatgaaaa ttccgtcacc 10140 agcncctggg ttcgcaaaga taattgcatg tttcttcctt gaactctcaa gcctacagga 10200 cacacattca tcgtaggtat aaacctcgaa atcanttcct actaagatgg tatacaatag 10260 taaccatgca tggttgccta gtgaatgctc cgtaacaccc aatacgccgg ccgaaacttt 10320 tttacaactc tcctatgagt cgtttaccca gaatgcacag gtacacttgt ttagaggtaa 10380 tccttctttc tagaagtcct cgtgtactgt gtaagcgccc actccacatc tccactcgac 10440 ctgcagttcg aagttcctat tctctagaaa gtataggaac ttcagagcgc tttccaattc 10500 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg 10560 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 10620 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 10680 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 10740 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 10800 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 10860 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg 10920 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 10980 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt 11040 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca 11100 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttag 11146 <210> 7 <211> 10723 <212> DNA <213> Artificial Sequence <220> <223> pB-PF plasmid <400> 7 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaaaccc 2220 cgggtacgta aattaaccct cactaaaggg aacaaaagct gggcaaagct tgaggccgga 2280 caaattcagc ctgcaggcta aagtttctat tacatgatat ttgtcgtgga caagggcgtc 2340 tggggtaaaa aggaatgtgg gcagagagct ctcggcttaa agaccggctt cggagttcca 2400 gacccactcg aactgaaaac gatgattaga gataagtaga tctatttgga cagtataaga 2460 atcctagtcg gctagtattt ctccactttt actttaaaat tccccagagt aagggggcag 2520 gttcgtcgtc acgggtagcc tcgtctcgag gcatcgcgga tagcattatc ttggggtatc 2580 acgagtaact tcatcgtggc attatcgata ggtactgtaa gtactgtagg aacttatccg 2640 ggcatggata tgtataggta catacgatgc actaaaagtg gagaaatact agccgagtag 2700 agatcttata ccatccgaat agatttactc cttataatca aggggcaaca atcagttgtg 2760 acgatgtcgg ctgtgggagt gcagtttagg ggtcatcaaa gcacaggaaa acgtgttgta 2820 tggatgccta tcgtcttgac tattagaaag gaagagggga aaaaccctca tcaggagcga 2880 ttgtaagttc ggccatagta atgatattgg ccattaatgg acgtgtgtat gtatacatac 2940 acggttcatt tgccgcattc ggaccaaaac cgctgctgcc gcagacacag tgcttccatg 3000 gcctcgaaca accggttatt cttctcttcc atgtcctcgg gacgaaggaa atttcggagc 3060 cctaacagcc gtaccggact tgcgttaaca tggttgcgaa gaactaaaga ctgatggggt 3120 catgattggc agtgttgaca cttcatgtct tgaacctagc tagcagaaaa atgaatgctc 3180 tatgtgacac tggtagatct caacgcatca tgttccacct ttagtgtaaa cacggcaagc 3240 tgtccgcaac gtcggtacca acgtttgaac acacaacctt ctgccactcc aggagtttag 3300 cccacggctt actggatgaa acgtaatctc ccgctacccc ctcaattcca ttggggtcta 3360 gagtagtcgg aagaacggct gtcgccttgc gctgcggtta aagcacccac cgtggtggct 3420 actgcttctc gggaggagaa atcctgggta ctcgagcact tgcgctaggg gatgaaacat 3480 ccatggcata ttggcgaaag caatgcgcca tgttcggcgc ggcacatgac actcttgaga 3540 agccgtattc ttcgcaaact ttcctattta cattcagcct ctaactataa cacgatcgga 3600 aatgcctgaa cgagcccgcc ctcatgtgcc tagcgtcaaa caacgccaag cacacaacgc 3660 ctcctgctat tcttccctct tctcactgcc atcactgggc gcaagctgca atgtccttct 3720 cgaattcaag cgggcaaatc aagattggga aaatgcgcca tcttggtttg gatcttgatg 3780 tctatccgca tcaaggtctt ggtttggtcg tttatttagt agcaaaggca aatcaaaacg 3840 tgcgcgccaa gtgccagggt ccagactcca gactggagca tttagcctga atatcgtctc 3900 tagaatctgg tgctattatt caggatatag taactatgat ctactcgggc catgttcggc 3960 tcctccgcac ggccaagttg tctgccggcg tctggaccgg actcatggct tggcttctcc 4020 agggatagca tggcattggc aaaactctcc gccacgattc gagcatagct accgcagcca 4080 cgcggcattg tttccagatt gagatatgtc actattagcc ggaatgcaaa catcgagctg 4140 acctgcgcaa catctccatg agttccgaag tcgacctccg aagcaatatt ggtctagtat 4200 tacggatgat atagtggtcc ccggacccaa tgagccgtgt gttttttacc gcctttgctc 4260 tgaggattaa caagattcgt gcaattgggg actgtgcgga gtaactgggc tggtttactg 4320 ctttgccgac gaaagaaacg tataaacagc cgactcgtcc actgccactc gggacgatct 4380 cccaacccag acagcttccc agaacccatc ttcaacaccc tgacaccatg ccccagttcg 4440 acatcctatg caagactcct ccgaaggtgc tcgtccgcca gttcgtggag cgattcgagc 4500 gcccctcggg cgaaaagatc gccttgtgtg ccgctgagct tacctacctc tgctggatga 4560 tcacccacaa cggcaccgcc atcaagcggg ctacctttat gagttacaat accatcatta 4620 gcaactccct ctcgtttgac atcgtcaaca agtccctgca gttcaagtac aagacccaaa 4680 aggccaccat cctcgaggcc tcgctgaaga agctgatccc cgcatgggaa ttcactatca 4740 tcccctacta tggtcagaag catcagtctg acatcaccga cattgtaagc agtctccagc 4800 tccagtttga gtcctccgag gaggccgata aaggcaactc ccactccaag aagatgctca 4860 aggctttgct gagtgaaggc gagtccatct gggaaataac cgaaaagatc ttgaacagtt 4920 tcgagtacac cagccgcttc acgaagacca agacgctgta ccagttcctg ttcctcgcca 4980 cattcatcaa ttgcggacgc ttcagtgata tcaagaacgt ggacccgaag tcattcaagc 5040 tggttcagaa caagtacctc ggtgtcatca ttcagtgctt ggtcaccgag accaagacat 5100 ccgtcagccg tcacatctac ttcttttccg caagagggcg tatcgacccc ctagtgtacc 5160 tagacgaatt cctgcgcaac tcggagcccg tcctaaagcg cgtcaaccgt accggcaact 5220 ctagcagcaa caagcaggag taccagctct tgaaggataa cctcgtccga tcatacaaca 5280 aggcgctcaa gaagaacgct ccatacagca tctttgctat caagaatggg ccaaagtcac 5340 acatcggccg ccacctcatg acctcgtttc tgtcgatgaa gggcctcact gagcttacca 5400 acgttgttgg caactggtcc gataagcgcg ccagcgccgt ggcgcggacg acttacactc 5460 atcagatcac agctatccct gaccactact tcgccttggt cagtcgctat tacgcctacg 5520 acccaattag taaagagatg atcgctctca aggacgaaac caaccccatc gaggagtggc 5580 aacacatcga gcagcttaaa ggctccgccg agggctcgat ccggtacccc gcctggaacg 5640 gtatcatatc gcaggaggtc ttggattacc tgagcagtta tatcaatcgc cgcatatgat 5700 aaggatccac ttaacgttac tgaaatcatc aaacagcttg acgaatctgg atataagatc 5760 gttggtgtcg atgtcagctc cggagttgag acaaatggtg ttcaggatct cgataagata 5820 cgttcatttg tccaagcagc aaagagtgcc ttctagtgat ttaatagctc catgtcaaca 5880 agaataaaac gcgttttcgg gtttacctct tccagataca gctcatctgc aatgcattaa 5940 tgcattgact gcaacctagt aacgccttnc aggctccggc gaagagaaga atagcttagc 6000 agagctattt tcattttcgg gagacgagat caagcagatc aacggtcgtc aagagaccta 6060 cgagactgag gaatccgctc ttggctccac gcgactatat atttgtctct aattgtactt 6120 tgacatgctc ctcttcttta ctctgatagc ttgactatga aaattccgtc accagcncct 6180 gggttcgcaa agataattgc atgtttcttc cttgaactct caagcctaca ggacacacat 6240 tcatcgtagg tataaacctc gaaatcantt cctactaaga tggtatacaa tagtaaccat 6300 gcatggttgc ctagtgaatg ctccgtaaca cccaatacgc cggccgaaac ttttttacaa 6360 ctctcctatg agtcgtttac ccagaatgca caggtacact tgtttagagg taatccttct 6420 ttctagaagt cctcgtgtac tgtgtaagcg cccactccac atctccactc gaccaattcg 6480 ccctatagtg agtcgtatta agcttcctgc agttcgaagt tcctattctc tagaaagtat 6540 aggaacttca gagcgctttc cttgtatctc tacacacagg ctcaaatcaa taagaagaac 6600 ggttcgtctt tttcgtttat atcttgcatc gtcccaaagc tattggcggg atattctgtt 6660 tgcagttggc tgacttgaag taatctctgc agatctttcg acactgaaat acgtcgagcc 6720 tgctccgctt ggaagcggcg aggagcctcg tcctgtcaca actaccaaca tggagtacga 6780 taagggccag ttccgccagc tcattaagag ccagttcatg ggcgttggca tgatggccgt 6840 catgcatctg tacttcaagt acaccaacgc tcttctgatc cagtcgatca tccgctgaag 6900 gcgctttcga atctggttaa gatccacgtc ttcgggaagc cagcgactgg tgacctccag 6960 cgtcccttta aggctgccaa cagctttctc agccagggcc agcccaagac cgacaaggcc 7020 tccctccaga acgccgagaa gaactggagg ggtggtgtca aggaggagta agctccttat 7080 tgaagtcgga ggacggagcg gtgtcaagag gatattcttc gactctgtat tatagataag 7140 atgatgagga attggaggta gcatagcttc atttggattt gctttccagg ctgagactct 7200 agcttggagc atagagggtc ctttggcttt caatattctc aagtatctcg agtttgaact 7260 tattccctgt gaacctttta ttcaccaatg agcattggaa tgaacatgaa tctgaggact 7320 gcaatcgcca tgaggttttc gaaatacatc cggatgtcga aggcttgggg cacctgcgtt 7380 ggttgaattt agaacgtggc actattgatc atccgatagc tctgcaaagg gcgttgcaca 7440 atgcaagtca aacgttgcta gcagttccag gtggaatgtt atgatgagca ttgtattaaa 7500 tcaggagata tagcatgatc tctagttagc tcaccacaaa agtcagacgg cgtaaccaaa 7560 agtcacacaa cacaagctgt aaggatttcg gcacggctac ggaagacgga gaagccacct 7620 tcagtggact cgagtaccat ttaattctat ttgtgtttga tcgagaccta atacagcccc 7680 tacaacgacc atcaaagtcg tatagctacc agtgaggaag tggactcaaa tcgacttcag 7740 caacatctcc tggataaact ttaagcctaa actatacaga ataagatagg tggagagctt 7800 ataccgagct cccaaatctg tccagatcat ggttgaccgg tgcctggatc ttcctataga 7860 atcatcctta ttcgttgacc tagctgattc tggagtgacc cagagggtca tgacttgagc 7920 ctaaaatccg ccgcctccac catttgtaga aaaatgtgac gaactcgtga gctctgtaca 7980 gtgaccggtg actctttctg gcatgcggag agacggacgg acgcagagag aagggctgag 8040 taataagcca ctggccagac agctctggcg gctctgaggt gcagtggatg attattaatc 8100 cgggaccggc cgcccctccg ccccgaagtg gaaaggctgg tgtgcccctc gttgaccaag 8160 aatctattgc atcatcggag aatatggagc ttcatcgaat caccggcagt aagcgaagga 8220 gaatgtgaag ccaggggtgt atagccgtcg gcgaaatagc atgccattaa cctaggtaca 8280 gaagtccaat tgcttccgat ctggtaaaag attcacgaga tagtaccttc tccgaagtag 8340 gtagagcgag tacccggcgc gtaagctccc taattggccc atccggcatc tgtagggcgt 8400 ccaaatatcg tgcctctcct gctttgcccg gtgtatgaaa ccggaaaggc cgctcaggag 8460 ctggccagcg gcgcagaccg ggaacacaag ctggcagtcg acccatccgg tgctctgcac 8520 tcgacctgct gaggtccctc agtccctggt aggcagcttt gccccgtctg tccgcccggt 8580 gtgtcggcgg ggttgacaag gtcgttgcgt cagtccaaca tttgttgcca tattttcctg 8640 ctctccccac cagctgctct tttcttttct ctttcttttc ccatcttcag tatattcatc 8700 ttcccatcca agaaccttta tttcccctaa gtaagtactt tgctacatcc atactccatc 8760 cttcccatcc cttattcctt tgaacctttc agttcgagct ttcccacttc atcgcagctt 8820 gactaacagc taccccgctt gagcagacat caccatggcc aagttgacca gtgccgttcc 8880 ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc ggctcgggtt 8940 ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt 9000 catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg tgtgggtgcg 9060 cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact tccgggacgc 9120 ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt tcgccctgcg 9180 cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgaa gtagatgccg 9240 accgcgggat ccacttaacg ttactgaaat catcaaacag cttgacgaat ctggatataa 9300 gatcgttggt gtcgatgtca gctccggagt tgagacaaat ggtgttcagg atctcgataa 9360 gatacgttca tttgtccaag cagcaaagag tgccttctag tgatttaata gctccatgtc 9420 aacaagaata aaacgcgttt tcgggtttac ctcttccaga tacagctcat ctgcaatgca 9480 ttaatgcatt gactgcaacc tagtaacgcc ttncaggctc cggcgaagag aagaatagct 9540 tagcagagct attttcattt tcgggagacg agatcaagca gatcaacggt cgtcaagaga 9600 cctacgagac tgaggaatcc gctcttggct ccacgcgact atatatttgt ctctaattgt 9660 actttgacat gctcctcttc tttactctga tagcttgact atgaaaattc cgtcaccagc 9720 ncctgggttc gcaaagataa ttgcatgttt cttccttgaa ctctcaagcc tacaggacac 9780 acattcatcg taggtataaa cctcgaaatc anttcctact aagatggtat acaatagtaa 9840 ccatgcatgg ttgcctagtg aatgctccgt aacacccaat acgccggccg aaactttttt 9900 acaactctcc tatgagtcgt ttacccagaa tgcacaggta cacttgttta gaggtaatcc 9960 ttctttctag aagtcctcgt gtactgtgta agcgcccact ccacatctcc actcgacctg 10020 cagttcgaag ttcctattct ctagaaagta taggaacttc agagcgcttt ccaattcgcc 10080 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga 10140 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 10200 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 10260 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 10320 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 10380 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 10440 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 10500 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 10560 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 10620 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 10680 atttaacgcg aattttaaca aaatattaac gcttacaatt tag 10723 <210> 8 <211> 8025 <212> DNA <213> Artificial Sequence <220> <223> pB-CXcast plasmid <400> 8 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct tccttgtatc 2220 tctacacaca ggctcaaatc aataagaaga acggttcgtc tttttcgttt atatcttgca 2280 tcgtcccaaa gctattggcg ggatattctg tttgcagttg gctgacttga agtaatctct 2340 gcagatcttt cgacactgaa atacgtcgag cctgctccgc ttggaagcgg cgaggagcct 2400 cgtcctgtca caactaccaa catggagtac gataagggcc agttccgcca gctcattaag 2460 agccagttca tgggcgttgg catgatggcc gtcatgcatc tgtacttcaa gtacaccaac 2520 gctcttctga tccagtcgat catccgctga aggcgctttc gaatctggtt aagatccacg 2580 tcttcgggaa gccagcgact ggtgacctcc agcgtccctt taaggctgcc aacagctttc 2640 tcagccaggg ccagcccaag accgacaagg cctccctcca gaacgccgag aagaactgga 2700 ggggtggtgt caaggaggag taagctcctt attgaagtcg gaggacggag cggtgtcaag 2760 aggatattct tcgactctgt attatagata agatgatgag gaattggagg tagcatagct 2820 tcatttggat ttgctttcca ggctgagact ctagcttgga gcatagaggg tcctttggct 2880 ttcaatattc tcaagtatct cgagtttgaa cttattccct gtgaaccttt tattcaccaa 2940 tgagcattgg aatgaacatg aatctgagga ctgcaatcgc catgaggttt tcgaaataca 3000 tccggatgtc gaaggcttgg ggcacctgcg ttggttgaat ttagaacgtg gcactattga 3060 tcatccgata gctctgcaaa gggcgttgca caatgcaagt caaacgttgc tagcagttcc 3120 aggtggaatg ttatgatgag cattgtatta aatcaggaga tatagcatga tctctagtta 3180 gctcaccaca aaagtcagac ggcgtaacca aaagtcacac aacacaagct gtaaggattt 3240 cggcacggct acggaagacg gagaagccac cttcagtgga ctcgagtacc atttaattct 3300 atttgtgttt gatcgagacc taatacagcc cctacaacga ccatcaaagt cgtatagcta 3360 ccagtgagga agtggactca aatcgacttc agcaacatct cctggataaa ctttaagcct 3420 aaactataca gaataagata ggtggagagc ttataccgag ctcccaaatc tgtccagatc 3480 atggttgacc ggtgcctgga tcttcctata gaatcatcct tattcgttga cctagctgat 3540 tctggagtga cccagagggt catgacttga gcctaaaatc cgccgcctcc accatttgta 3600 gaaaaatgtg acgaactcgt gagctctgta cagtgaccgg tgactctttc tggcatgcgg 3660 agagacggac ggacgcagag agaagggctg agtaataagc cactggccag acagctctgg 3720 cggctctgag gtgcagtgga tgattattaa tccgggaccg gccgcccctc cgccccgaag 3780 tggaaaggct ggtgtgcccc tcgttgacca agaatctatt gcatcatcgg agaatatgga 3840 gcttcatcga atcaccggca gtaagcgaag gagaatgtga agccaggggt gtatagccgt 3900 cggcgaaata gcatgccatt aacctaggta cagaagtcca attgcttccg atctggtaaa 3960 agattcacga gatagtacct tctccgaagt aggtagagcg agtacccggc gcgtaagctc 4020 cctaattggc ccatccggca tctgtagggc gtccaaatat cgtgcctctc ctgctttgcc 4080 cggtgtatga aaccggaaag gccgctcagg agctggccag cggcgcagac cgggaacaca 4140 agctggcagt cgacccatcc ggtgctctgc actcgacctg ctgaggtccc tcagtccctg 4200 gtaggcagct ttgccccgtc tgtccgcccg gtgtgtcggc ggggttgaca aggtcgttgc 4260 gtcagtccaa catttgttgc catattttcc tgctctcccc accagctgct cttttctttt 4320 ctctttcttt tcccatcttc agtatattca tcttcccatc caagaacctt tatttcccct 4380 aagtaagtac tttgctacat ccatactcca tccttcccat cccttattcc tttgaacctt 4440 tcagttcgag ctttcccact tcatcgcagc ttgactaaca gctaccccgc ttgagcagac 4500 atcaccatgg aaccgaccag caccccgcag gctccgatcg ctgcttataa accgcgtagc 4560 aacgaaatcc tgtgggatgg ttatggtgtt ccgcatatct atggtgttga cgctccgagc 4620 gctttttatg gttatggctg ggctcaggct cgtagccatg gtgataacat cctgcgtctg 4680 tatggtgaag ctcgtggtaa aggcgctgaa tattggggtc cggattacga acagacgacc 4740 gtttggctgc tgaccaacgg tgttccggaa cgtgctcagc agtggtatgc tcagcagagc 4800 ccggattttc gtgctaacct ggatgctttt gctgctggta tcaacgctta tgctcagcag 4860 aacccggatg atatcagccc ggaagttcgt caggttctgc cggttagcgg tgctgatgtt 4920 gttgctcatg ctcatcgtct gatgaacttt ctgtatgttg ctagcccggg tcgtaccctg 4980 ggtgaaggtg atccgccgga tctggctgat cagggtagca acagctgggc tgttgctccg 5040 ggtaaaaccg ctaacggtaa cgctctgctg ctgcagaacc ctcatctgag ctggaccacc 5100 gattatttta cctattatga agctcatctg gttaccccgg attttgaagt gtatggtgct 5160 acccagatcg gtctgccggt tatccgtgtt gcttttaacc agcgtatggg tatcaccaac 5220 accgttaacg gtatggttgg tgctaccaac tatcgtctga ccctgcagga tggtggttat 5280 ctgtatgatg gtcaggttcg tccgtttgaa cgtcgtcagg ctagctatcg tctgcgtcag 5340 gctgatggta ccaccgttga taaaccgctg gaaatccgtt ccagcgttca tggtccggtt 5400 tttgaacgtg ctgatggtac cgctgttgct gttcgtgttg ctggtctgga tcgtccgggt 5460 atgctggaac agacttttga tatgatcacc gctgatagct ttgatgatta tgaagctgct 5520 ctggctcgta tgcaggttcc gaccttgaac atcgtttatg ctgatcgtga aggtaccatc 5580 aactatagct ttaacggtgt tgctccgaaa cgtgctgaag gtgacatcgc tttttggcag 5640 ggtctggttc cgggtgatag cagccgttat ctgtggaccg aaacccatcc gctggatgat 5700 ctgccgcgtg ttaccaaccc gccgggtggt tttgttcaga acagcaacga tccgccgtgg 5760 accccgacct ggccggttac ctataccccg aaagattttc cgagctatct ggctccgcag 5820 accccgcata gcctgcgtgc tcagcagagc gttcgtctga tgagcgaaaa cgatgatctg 5880 accctggaac gttttatggc tctgcagctg agccatcgtg ctgttatggc tgatcgtacc 5940 ctgccggatc tgatcccggc tgctctgatc gatccggatc cggaagttca ggctgctgct 6000 cgtctgctgg ctgcgtggga tcgtgaattt accagcgata gccgtgctgc tctgctgttt 6060 gaagaatggg ctcgtctgtt tgctggtcag aactttgctg gtcaggctgg ctttgctacc 6120 ccgtggagcc tggataaacc ggttagcacc ccgtatggtc tgcgtgatcc gaaagctgct 6180 gttgatcagc tgcgtaccgc tatcgctaac accaaacgta aatatggtgc tatcgatcgt 6240 ccgtttggtg atgctagccg tatgatcctg aacgatgtta acgttccggg tgctgctggt 6300 tatggtaacc tgggtagctt tcgtgttttt acctggagcg atccggatga aaacggtgtg 6360 cgtaccccgg ttcatggtga aacctgggtt gctatgatcg aatttagcac cccggttcgt 6420 gcttatggtc tgatgagcta tggtaacagc cgtcagccgg gtaccaccca ttatagcgat 6480 cagatcgaac gtgttagccg tgctgatttt cgtgaactgc tgctgcgtcg tgaacaggtt 6540 gaagctgctg ttcaggaacg taccccgttt aactttaaac cgtgaagtag atgccgaccg 6600 cgagatccac ttaacgttac tgaaatcatc aaacagcttg acgaatctgg atataagatc 6660 gttggtgtcg atgtcagctc cggagttgag acaaatggtg ttcaggatct cgataagata 6720 cgttcatttg tccaagcagc aaagagtgcc ttctagtgat ttaatagctc catgtcaaca 6780 agaataaaac gcgttttcgg gtttacctct tccagataca gctcatctgc aatgcattaa 6840 tgcattgact gcaacctagt aacgccttnc aggctccggc gaagagaaga atagcttagc 6900 agagctattt tcattttcgg gagacgagat caagcagatc aacggtcgtc aagagaccta 6960 cgagactgag gaatccgctc ttggctccac gcgactatat atttgtctct aattgtactt 7020 tgacatgctc ctcttcttta ctctgatagc ttgactatga aaattccgtc accagcncct 7080 gggttcgcaa agataattgc atgtttcttc cttgaactct caagcctaca ggacacacat 7140 tcatcgtagg tataaacctc gaaatcantt cctactaaga tggtatacaa tagtaaccat 7200 gcatggttgc ctagtgaatg ctccgtaaca cccaatacgc cggccgaaac ttttttacaa 7260 ctctcctatg agtcgtttac ccagaatgca caggtacact tgtttagagg taatccttct 7320 ttctagaagt cctcgtgtac tgtgtaagcg cccactccac atctccactc gaccaattcg 7380 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg 7440 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 7500 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 7560 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 7620 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 7680 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 7740 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 7800 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 7860 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 7920 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 7980 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttag 8025 <210> 9 <211> 16598 <212> DNA <213> Artificial Sequence <220> <223> pB-HCX plasmid <400> 9 gtaaattaac cctcactaaa gggaacaaaa gctgggcaaa gcttgaggcc ggacaaattc 60 agcctgcagg ctaaagtttc tattacatga tatttgtcgt ggacaagggc gtctggggta 120 aaaaggaatg tgggcagaga gctctcggct taaagaccgg cttcggagtt ccagacccac 180 tcgaactgaa aacgatgatt agagataagt agatctattt ggacagtata agaatcctag 240 tcggctagta tttctccact tttactttaa aattccccag agtaaggggg caggttcgtc 300 gtcacgggta gcctcgtctc gaggcatcgc ggatagcatt atcttggggt atcacgagta 360 acttcatcgt ggcattatcg ataggtactg taagtactgt aggaacttat ccgggcatgg 420 atatgtatag gtacatacga tgcactaaaa gtggagaaat actagccgag tagagatctt 480 ataccatccg aatagattta ctccttataa tcaaggggca acaatcagtt gtgacgatgt 540 cggctgtggg agtgcagttt aggggtcatc aaagcacagg aaaacgtgtt gtatggatgc 600 ctatcgtctt gactattaga aaggaagagg ggaaaaaccc tcatcaggag cgattgtaag 660 ttcggccata gtaatgatat tggccattaa tggacgtgtg tatgtataca tacacggttc 720 atttgccgca ttcggaccaa aaccgctgct gccgcagaca cagtgcttcc atggcctcga 780 acaaccggtt attcttctct tccatgtcct cgggacgaag gaaatttcgg agccctaaca 840 gccgtaccgg acttgcgtta acatggttgc gaagaactaa agactgatgg ggtcatgatt 900 ggcagtgttg acacttcatg tcttgaacct agctagcaga aaaatgaatg ctctatgtga 960 cactggtaga tctcaacgca tcatgttcca cctttagtgt aaacacggca agctgtccgc 1020 aacgtcggta ccaacgtttg aacacacaac cttctgccac tccaggagtt tagcccacgg 1080 cttactggat gaaacgtaat ctcccgctac cccctcaatt ccattggggt ctagagtagt 1140 cggaagaacg gctgtcgcct tgcgctgcgg ttaaagcacc caccgtggtg gctactgctt 1200 ctcgggagga gaaatcctgg gtactcgagc acttgcgcta ggggatgaaa catccatggc 1260 atattggcga aagcaatgcg ccatgttcgg cgcggcacat gacactcttg agaagccgta 1320 ttcttcgcaa actttcctat ttacattcag cctctaacta taacacgatc ggaaatgcct 1380 gaacgagccc gccctcatgt gcctagcgtc aaacaacgcc aagcacacaa cgcctcctgc 1440 tattcttccc tcttctcact gccatcactg ggcgcaagct gcaatgtcct tctcgaattc 1500 aagcgggcaa atcaagattg ggaaaatgcg ccatcttggt ttggatcttg atgtctatcc 1560 gcatcaaggt cttggtttgg tcgtttattt agtagcaaag gcaaatcaaa acgtgcgcgc 1620 caagtgccag ggtccagact ccagactgga gcatttagcc tgaatatcgt ctctagaatc 1680 tggtgctatt attcaggata tagtaactat gatctactcg ggccatgttc ggctcctccg 1740 cacggccaag ttgtctgccg gcgtctggac cggactcatg gcttggcttc tccagggata 1800 gcatggcatt ggcaaaactc tccgccacga ttcgagcata gctaccgcag ccacgcggca 1860 ttgtttccag attgagatat gtcactatta gccggaatgc aaacatcgag ctgacctgcg 1920 caacatctcc atgagttccg aagtcgacct ccgaagcaat attggtctag tattacggat 1980 gatatagtgg tccccggacc caatgagccg tgtgtttttt accgcctttg ctctgaggat 2040 taacaagatt cgtgcaattg gggactgtgc ggagtaactg ggctggttta ctgctttgcc 2100 gacgaaagaa acgtataaac agccgactcg tccactgcca ctcgggacga tctcccaacc 2160 cagacagctt cccagaaccc atcttcaaca ccctgacacc atgccccagt tcgacatcct 2220 atgcaagact cctccgaagg tgctcgtccg ccagttcgtg gagcgattcg agcgcccctc 2280 gggcgaaaag atcgccttgt gtgccgctga gcttacctac ctctgctgga tgatcaccca 2340 caacggcacc gccatcaagc gggctacctt tatgagttac aataccatca ttagcaactc 2400 cctctcgttt gacatcgtca acaagtccct gcagttcaag tacaagaccc aaaaggccac 2460 catcctcgag gcctcgctga agaagctgat ccccgcatgg gaattcacta tcatccccta 2520 ctatggtcag aagcatcagt ctgacatcac cgacattgta agcagtctcc agctccagtt 2580 tgagtcctcc gaggaggccg ataaaggcaa ctcccactcc aagaagatgc tcaaggcttt 2640 gctgagtgaa ggcgagtcca tctgggaaat aaccgaaaag atcttgaaca gtttcgagta 2700 caccagccgc ttcacgaaga ccaagacgct gtaccagttc ctgttcctcg ccacattcat 2760 caattgcgga cgcttcagtg atatcaagaa cgtggacccg aagtcattca agctggttca 2820 gaacaagtac ctcggtgtca tcattcagtg cttggtcacc gagaccaaga catccgtcag 2880 ccgtcacatc tacttctttt ccgcaagagg gcgtatcgac cccctagtgt acctagacga 2940 attcctgcgc aactcggagc ccgtcctaaa gcgcgtcaac cgtaccggca actctagcag 3000 caacaagcag gagtaccagc tcttgaagga taacctcgtc cgatcataca acaaggcgct 3060 caagaagaac gctccataca gcatctttgc tatcaagaat gggccaaagt cacacatcgg 3120 ccgccacctc atgacctcgt ttctgtcgat gaagggcctc actgagctta ccaacgttgt 3180 tggcaactgg tccgataagc gcgccagcgc cgtggcgcgg acgacttaca ctcatcagat 3240 cacagctatc cctgaccact acttcgcctt ggtcagtcgc tattacgcct acgacccaat 3300 tagtaaagag atgatcgctc tcaaggacga aaccaacccc atcgaggagt ggcaacacat 3360 cgagcagctt aaaggctccg ccgagggctc gatccggtac cccgcctgga acggtatcat 3420 atcgcaggag gtcttggatt acctgagcag ttatatcaat cgccgcatat gataaggatc 3480 cacttaacgt tactgaaatc atcaaacagc ttgacgaatc tggatataag atcgttggtg 3540 tcgatgtcag ctccggagtt gagacaaatg gtgttcagga tctcgataag atacgttcat 3600 ttgtccaagc agcaaagagt gccttctagt gatttaatag ctccatgtca acaagaataa 3660 aacgcgtttt cgggtttacc tcttccagat acagctcatc tgcaatgcat taatgcattg 3720 actgcaacct agtaacgcct tncaggctcc ggcgaagaga agaatagctt agcagagcta 3780 ttttcatttt cgggagacga gatcaagcag atcaacggtc gtcaagagac ctacgagact 3840 gaggaatccg ctcttggctc cacgcgacta tatatttgtc tctaattgta ctttgacatg 3900 ctcctcttct ttactctgat agcttgacta tgaaaattcc gtcaccagcn cctgggttcg 3960 caaagataat tgcatgtttc ttccttgaac tctcaagcct acaggacaca cattcatcgt 4020 aggtataaac ctcgaaatca nttcctacta agatggtata caatagtaac catgcatggt 4080 tgcctagtga atgctccgta acacccaata cgccggccga aactttttta caactctcct 4140 atgagtcgtt tacccagaat gcacaggtac acttgtttag aggtaatcct tctttctaga 4200 agtcctcgtg tactgtgtaa gcgcccactc cacatctcca ctcgaccaat tcgccctata 4260 gtgagtcgta ttaagcttcc tgcagttcga agttcctatt ctctagaaag tataggaact 4320 tcagagcgct ttccttgtat ctctacacac aggctcaaat caataagaag aacggttcgt 4380 ctttttcgtt tatatcttgc atcgtcccaa agctattggc gggatattct gtttgcagtt 4440 ggctgacttg aagtaatctc tgcagatctt tcgacactga aatacgtcga gcctgctccg 4500 cttggaagcg gcgaggagcc tcgtcctgtc acaactacca acatggagta cgataagggc 4560 cagttccgcc agctcattaa gagccagttc atgggcgttg gcatgatggc cgtcatgcat 4620 ctgtacttca agtacaccaa cgctcttctg atccagtcga tcatccgctg aaggcgcttt 4680 cgaatctggt taagatccac gtcttcggga agccagcgac tggtgacctc cagcgtccct 4740 ttaaggctgc caacagcttt ctcagccagg gccagcccaa gaccgacaag gcctccctcc 4800 agaacgccga gaagaactgg aggggtggtg tcaaggagga gtaagctcct tattgaagtc 4860 ggaggacgga gcggtgtcaa gaggatattc ttcgactctg tattatagat aagatgatga 4920 ggaattggag gtagcatagc ttcatttgga tttgctttcc aggctgagac tctagcttgg 4980 agcatagagg gtcctttggc tttcaatatt ctcaagtatc tcgagtttga acttattccc 5040 tgtgaacctt ttattcacca atgagcattg gaatgaacat gaatctgagg actgcaatcg 5100 ccatgaggtt ttcgaaatac atccggatgt cgaaggcttg gggcacctgc gttggttgaa 5160 tttagaacgt ggcactattg atcatccgat agctctgcaa agggcgttgc acaatgcaag 5220 tcaaacgttg ctagcagttc caggtggaat gttatgatga gcattgtatt aaatcaggag 5280 atatagcatg atctctagtt agctcaccac aaaagtcaga cggcgtaacc aaaagtcaca 5340 caacacaagc tgtaaggatt tcggcacggc tacggaagac ggagaagcca ccttcagtgg 5400 actcgagtac catttaattc tatttgtgtt tgatcgagac ctaatacagc ccctacaacg 5460 accatcaaag tcgtatagct accagtgagg aagtggactc aaatcgactt cagcaacatc 5520 tcctggataa actttaagcc taaactatac agaataagat aggtggagag cttataccga 5580 gctcccaaat ctgtccagat catggttgac cggtgcctgg atcttcctat agaatcatcc 5640 ttattcgttg acctagctga ttctggagtg acccagaggg tcatgacttg agcctaaaat 5700 ccgccgcctc caccatttgt agaaaaatgt gacgaactcg tgagctctgt acagtgaccg 5760 gtgactcttt ctggcatgcg gagagacgga cggacgcaga gagaagggct gagtaataag 5820 ccactggcca gacagctctg gcggctctga ggtgcagtgg atgattatta atccgggacc 5880 ggccgcccct ccgccccgaa gtggaaaggc tggtgtgccc ctcgttgacc aagaatctat 5940 tgcatcatcg gagaatatgg agcttcatcg aatcaccggc agtaagcgaa ggagaatgtg 6000 aagccagggg tgtatagccg tcggcgaaat agcatgccat taacctaggt acagaagtcc 6060 aattgcttcc gatctggtaa aagattcacg agatagtacc ttctccgaag taggtagagc 6120 gagtacccgg cgcgtaagct ccctaattgg cccatccggc atctgtaggg cgtccaaata 6180 tcgtgcctct cctgctttgc ccggtgtatg aaaccggaaa ggccgctcag gagctggcca 6240 gcggcgcaga ccgggaacac aagctggcag tcgacccatc cggtgctctg cactcgacct 6300 gctgaggtcc ctcagtccct ggtaggcagc tttgccccgt ctgtccgccc ggtgtgtcgg 6360 cggggttgac aaggtcgttg cgtcagtcca acatttgttg ccatattttc ctgctctccc 6420 caccagctgc tcttttcttt tctctttctt ttcccatctt cagtatattc atcttcccat 6480 ccaagaacct ttatttcccc taagtaagta ctttgctaca tccatactcc atccttccca 6540 tcccttattc ctttgaacct ttcagttcga gctttcccac ttcatcgcag cttgactaac 6600 agctaccccg cttgagcaga catcaccatg cctgaactca ccgcgacgtc tgtcgagaag 6660 tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa 6720 tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc 6780 gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg 6840 attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc 6900 cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag 6960 ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc 7020 ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg 7080 attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc 7140 gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac 7200 ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg 7260 gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc 7320 ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat 7380 ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa 7440 ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc 7500 gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc 7560 gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc 7620 agcactcgtc cgagggcaaa ggaatagagt agatgccgac cgcgggatcc acttaacgtt 7680 actgaaatca tcaaacagct tgacgaatct ggatataaga tcgttggtgt cgatgtcagc 7740 tccggagttg agacaaatgg tgttcaggat ctcgataaga tacgttcatt tgtccaagca 7800 gcaaagagtg ccttctagtg atttaatagc tccatgtcaa caagaataaa acgcgttttc 7860 gggtttacct cttccagata cagctcatct gcaatgcatt aatgcattga ctgcaaccta 7920 gtaacgcctt ncaggctccg gcgaagagaa gaatagctta gcagagctat tttcattttc 7980 gggagacgag atcaagcaga tcaacggtcg tcaagagacc tacgagactg aggaatccgc 8040 tcttggctcc acgcgactat atatttgtct ctaattgtac tttgacatgc tcctcttctt 8100 tactctgata gcttgactat gaaaattccg tcaccagcnc ctgggttcgc aaagataatt 8160 gcatgtttct tccttgaact ctcaagccta caggacacac attcatcgta ggtataaacc 8220 tcgaaatcan ttcctactaa gatggtatac aatagtaacc atgcatggtt gcctagtgaa 8280 tgctccgtaa cacccaatac gccggccgaa acttttttac aactctccta tgagtcgttt 8340 acccagaatg cacaggtaca cttgtttaga ggtaatcctt ctttctagaa gtcctcgtgt 8400 actgtgtaag cgcccactcc acatctccac tcgacctgca gttcgaagtt cctattctct 8460 agaaagtata ggaacttcag agcgctttcc aattcgccct atagtgagtc gtattacgcg 8520 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 8580 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 8640 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc 8700 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 8760 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 8820 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 8880 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 8940 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 9000 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt 9060 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 9120 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 9180 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 9240 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 9300 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 9360 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 9420 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 9480 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 9540 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 9600 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 9660 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 9720 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 9780 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 9840 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 9900 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 9960 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 10020 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 10080 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 10140 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 10200 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 10260 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 10320 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 10380 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 10440 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 10500 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 10560 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 10620 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 10680 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 10740 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 10800 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 10860 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 10920 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 10980 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 11040 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg 11100 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 11160 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact 11220 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 11280 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc 11340 tgggcaaagc tgtttaaacc ccgggtacaa ttaaccctca ctaaagggaa caaaagctgg 11400 gcaaagcttc cttgtatctc tacacacagg ctcaaatcaa taagaagaac ggttcgtctt 11460 tttcgtttat atcttgcatc gtcccaaagc tattggcggg atattctgtt tgcagttggc 11520 tgacttgaag taatctctgc agatctttcg acactgaaat acgtcgagcc tgctccgctt 11580 ggaagcggcg aggagcctcg tcctgtcaca actaccaaca tggagtacga taagggccag 11640 ttccgccagc tcattaagag ccagttcatg ggcgttggca tgatggccgt catgcatctg 11700 tacttcaagt acaccaacgc tcttctgatc cagtcgatca tccgctgaag gcgctttcga 11760 atctggttaa gatccacgtc ttcgggaagc cagcgactgg tgacctccag cgtcccttta 11820 aggctgccaa cagctttctc agccagggcc agcccaagac cgacaaggcc tccctccaga 11880 acgccgagaa gaactggagg ggtggtgtca aggaggagta agctccttat tgaagtcgga 11940 ggacggagcg gtgtcaagag gatattcttc gactctgtat tatagataag atgatgagga 12000 attggaggta gcatagcttc atttggattt gctttccagg ctgagactct agcttggagc 12060 atagagggtc ctttggcttt caatattctc aagtatctcg agtttgaact tattccctgt 12120 gaacctttta ttcaccaatg agcattggaa tgaacatgaa tctgaggact gcaatcgcca 12180 tgaggttttc gaaatacatc cggatgtcga aggcttgggg cacctgcgtt ggttgaattt 12240 agaacgtggc actattgatc atccgatagc tctgcaaagg gcgttgcaca atgcaagtca 12300 aacgttgcta gcagttccag gtggaatgtt atgatgagca ttgtattaaa tcaggagata 12360 tagcatgatc tctagttagc tcaccacaaa agtcagacgg cgtaaccaaa agtcacacaa 12420 cacaagctgt aaggatttcg gcacggctac ggaagacgga gaagccacct tcagtggact 12480 cgagtaccat ttaattctat ttgtgtttga tcgagaccta atacagcccc tacaacgacc 12540 atcaaagtcg tatagctacc agtgaggaag tggactcaaa tcgacttcag caacatctcc 12600 tggataaact ttaagcctaa actatacaga ataagatagg tggagagctt ataccgagct 12660 cccaaatctg tccagatcat ggttgaccgg tgcctggatc ttcctataga atcatcctta 12720 ttcgttgacc tagctgattc tggagtgacc cagagggtca tgacttgagc ctaaaatccg 12780 ccgcctccac catttgtaga aaaatgtgac gaactcgtga gctctgtaca gtgaccggtg 12840 actctttctg gcatgcggag agacggacgg acgcagagag aagggctgag taataagcca 12900 ctggccagac agctctggcg gctctgaggt gcagtggatg attattaatc cgggaccggc 12960 cgcccctccg ccccgaagtg gaaaggctgg tgtgcccctc gttgaccaag aatctattgc 13020 atcatcggag aatatggagc ttcatcgaat caccggcagt aagcgaagga gaatgtgaag 13080 ccaggggtgt atagccgtcg gcgaaatagc atgccattaa cctaggtaca gaagtccaat 13140 tgcttccgat ctggtaaaag attcacgaga tagtaccttc tccgaagtag gtagagcgag 13200 tacccggcgc gtaagctccc taattggccc atccggcatc tgtagggcgt ccaaatatcg 13260 tgcctctcct gctttgcccg gtgtatgaaa ccggaaaggc cgctcaggag ctggccagcg 13320 gcgcagaccg ggaacacaag ctggcagtcg acccatccgg tgctctgcac tcgacctgct 13380 gaggtccctc agtccctggt aggcagcttt gccccgtctg tccgcccggt gtgtcggcgg 13440 ggttgacaag gtcgttgcgt cagtccaaca tttgttgcca tattttcctg ctctccccac 13500 cagctgctct tttcttttct ctttcttttc ccatcttcag tatattcatc ttcccatcca 13560 agaaccttta tttcccctaa gtaagtactt tgctacatcc atactccatc cttcccatcc 13620 cttattcctt tgaacctttc agttcgagct ttcccacttc atcgcagctt gactaacagc 13680 taccccgctt gagcagacat caccatggaa ccgaccagca ccccgcaggc tccgatcgct 13740 gcttataaac cgcgtagcaa cgaaatcctg tgggatggtt atggtgttcc gcatatctat 13800 ggtgttgacg ctccgagcgc tttttatggt tatggctggg ctcaggctcg tagccatggt 13860 gataacatcc tgcgtctgta tggtgaagct cgtggtaaag gcgctgaata ttggggtccg 13920 gattacgaac agacgaccgt ttggctgctg accaacggtg ttccggaacg tgctcagcag 13980 tggtatgctc agcagagccc ggattttcgt gctaacctgg atgcttttgc tgctggtatc 14040 aacgcttatg ctcagcagaa cccggatgat atcagcccgg aagttcgtca ggttctgccg 14100 gttagcggtg ctgatgttgt tgctcatgct catcgtctga tgaactttct gtatgttgct 14160 agcccgggtc gtaccctggg tgaaggtgat ccgccggatc tggctgatca gggtagcaac 14220 agctgggctg ttgctccggg taaaaccgct aacggtaacg ctctgctgct gcagaaccct 14280 catctgagct ggaccaccga ttattttacc tattatgaag ctcatctggt taccccggat 14340 tttgaagtgt atggtgctac ccagatcggt ctgccggtta tccgtgttgc ttttaaccag 14400 cgtatgggta tcaccaacac cgttaacggt atggttggtg ctaccaacta tcgtctgacc 14460 ctgcaggatg gtggttatct gtatgatggt caggttcgtc cgtttgaacg tcgtcaggct 14520 agctatcgtc tgcgtcaggc tgatggtacc accgttgata aaccgctgga aatccgttcc 14580 agcgttcatg gtccggtttt tgaacgtgct gatggtaccg ctgttgctgt tcgtgttgct 14640 ggtctggatc gtccgggtat gctggaacag acttttgata tgatcaccgc tgatagcttt 14700 gatgattatg aagctgctct ggctcgtatg caggttccga ccttgaacat cgtttatgct 14760 gatcgtgaag gtaccatcaa ctatagcttt aacggtgttg ctccgaaacg tgctgaaggt 14820 gacatcgctt tttggcaggg tctggttccg ggtgatagca gccgttatct gtggaccgaa 14880 acccatccgc tggatgatct gccgcgtgtt accaacccgc cgggtggttt tgttcagaac 14940 agcaacgatc cgccgtggac cccgacctgg ccggttacct ataccccgaa agattttccg 15000 agctatctgg ctccgcagac cccgcatagc ctgcgtgctc agcagagcgt tcgtctgatg 15060 agcgaaaacg atgatctgac cctggaacgt tttatggctc tgcagctgag ccatcgtgct 15120 gttatggctg atcgtaccct gccggatctg atcccggctg ctctgatcga tccggatccg 15180 gaagttcagg ctgctgctcg tctgctggct gcgtgggatc gtgaatttac cagcgatagc 15240 cgtgctgctc tgctgtttga agaatgggct cgtctgtttg ctggtcagaa ctttgctggt 15300 caggctggct ttgctacccc gtggagcctg gataaaccgg ttagcacccc gtatggtctg 15360 cgtgatccga aagctgctgt tgatcagctg cgtaccgcta tcgctaacac caaacgtaaa 15420 tatggtgcta tcgatcgtcc gtttggtgat gctagccgta tgatcctgaa cgatgttaac 15480 gttccgggtg ctgctggtta tggtaacctg ggtagctttc gtgtttttac ctggagcgat 15540 ccggatgaaa acggtgtgcg taccccggtt catggtgaaa cctgggttgc tatgatcgaa 15600 tttagcaccc cggttcgtgc ttatggtctg atgagctatg gtaacagccg tcagccgggt 15660 accacccatt atagcgatca gatcgaacgt gttagccgtg ctgattttcg tgaactgctg 15720 ctgcgtcgtg aacaggttga agctgctgtt caggaacgta ccccgtttaa ctttaaaccg 15780 tgaagtagat gccgaccgcg agatccactt aacgttactg aaatcatcaa acagcttgac 15840 gaatctggat ataagatcgt tggtgtcgat gtcagctccg gagttgagac aaatggtgtt 15900 caggatctcg ataagatacg ttcatttgtc caagcagcaa agagtgcctt ctagtgattt 15960 aatagctcca tgtcaacaag aataaaacgc gttttcgggt ttacctcttc cagatacagc 16020 tcatctgcaa tgcattaatg cattgactgc aacctagtaa cgccttncag gctccggcga 16080 agagaagaat agcttagcag agctattttc attttcggga gacgagatca agcagatcaa 16140 cggtcgtcaa gagacctacg agactgagga atccgctctt ggctccacgc gactatatat 16200 ttgtctctaa ttgtactttg acatgctcct cttctttact ctgatagctt gactatgaaa 16260 attccgtcac cagcncctgg gttcgcaaag ataattgcat gtttcttcct tgaactctca 16320 agcctacagg acacacattc atcgtaggta taaacctcga aatcanttcc tactaagatg 16380 gtatacaata gtaaccatgc atggttgcct agtgaatgct ccgtaacacc caatacgccg 16440 gccgaaactt ttttacaact ctcctatgag tcgtttaccc agaatgcaca ggtacacttg 16500 tttagaggta atccttcttt ctagaagtcc tcgtgtactg tgtaagcgcc cactccacat 16560 ctccactcga ccaattcgcc ctatagtgag tcgtatta 16598 <210> 10 <211> 284 <212> DNA <213> Artificial Sequence <220> <223> EP1 promoter <400> 10 tccaaatatc gtgcctctcc tgctttgcca ggtgtatgaa accggaaagg ccgctcagga 60 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 120 ctcgacctgc tgaggtccct cagtccctgg taggcagctt tgccccgtct gtccgcccgg 180 tgtgtcggcg gggttgacaa ggtcgttgcg tcagtccaac atttgttgcc atattttcct 240 gctctcctca ccagctgctc ttttcttttc tctttctttt ccca 284 <210> 11 <211> 284 <212> DNA <213> Artificial Sequence <220> <223> EP2 promoter <400> 11 tccaaatatc gtgcctctcc tgctttgctc ggtgtatgaa accggatagg ccgctcagga 60 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 120 ctcgacctgc tgaggtccct cagtccctgg taggcagctt taccccgtct gtccgcccgg 180 tgtgtcggcg gggttgacaa ggtcgttgcg tcagtccaac atttgttgcc atattttcct 240 gctctcccca ccagctgctc ttttcttttc tctttctttt ccca 284 <210> 12 <211> 284 <212> DNA <213> Artificial Sequence <220> <223> EP3 promoter <400> 12 tccaaatatc gtgcctctcc tgctttgcca ggtgtatgaa accggaaagg ccgctcagga 60 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 120 ctcgacctgc tgaggtccct cagtccctgg taggcagctt tgccccgtct gtccgcccgg 180 tgtgtcggcg gggttgacaa ggtcgttgcg tcagtccaac atttgttgcc atattttcct 240 gctttcccca ccagctgctc ttttcttttc tctttctttt ccca 284 <210> 13 <211> 284 <212> DNA <213> Artificial Sequence <220> <223> EP4 promoter <400> 13 tccaaatatc gttcctctcc tgctttgccc ggtgtatgaa accggaaagg ccgctcagga 60 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 120 ctcgacctgc tgaggtccct cagtccctgg taggcagctt tgccccgtct gtccgcccgg 180 tgtgtcggcg gggttgacaa ggtcgttgcg tctgtccaac atttgttgcc atattttcct 240 cctctcccta ccagctgctc ttttcttttc tctttctttt ccca 284 <210> 14 <211> 284 <212> DNA <213> Artificial Sequence <220> <223> EP5 promoter <400> 14 tccaaatatc gtgcctctcc tgctttgccc ggtgtatgaa accggaaagg ccgctcagga 60 gctggccagc ggcgcagacc gggaacacaa gctggcagtc gacccatccg gtgctctgca 120 ctcgacctgc tgaggtccct cattccctgg taggcagctt tgccccgtct gtccgcccgg 180 tgtgtcggcg gggttgacaa ggtcgttgcg tcagtccaac atttgttgcc atattttcct 240 gctctcccca ccagctgctc ttttcgtttc tctttctttt ccca 284 <210> 15 <211> 16610 <212> DNA <213> Artificial Sequence <220> <223> pB-HCXEP3 plasmid <400> 15 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaaaccc 2220 cgggtacaat taaccctcac taaagggaac aaaagctggg caaagcttcc ttgtatctct 2280 acacacaggc tcaaatcaat aagaagaacg gttcgtcttt ttcgtttata tcttgcatcg 2340 tcccaaagct attggcggga tattctgttt gcagttggct gacttgaagt aatctctgca 2400 gatctttcga cactgaaata cgtcgagcct gctccgcttg gaagcggcga ggagcctcgt 2460 cctgtcacaa ctaccaacat ggagtacgat aagggccagt tccgccagct cattaagagc 2520 cagttcatgg gcgttggcat gatggccgtc atgcatctgt acttcaagta caccaacgct 2580 cttctgatcc agtcgatcat ccgctgaagg cgctttcgaa tctggttaag atccacgtct 2640 tcgggaagcc agcgactggt gacctccagc gtccctttaa ggctgccaac agctttctca 2700 gccagggcca gcccaagacc gacaaggcct ccctccagaa cgccgagaag aactggaggg 2760 gtggtgtcaa ggaggagtaa gctccttatt gaagtcggag gacggagcgg tgtcaagagg 2820 atattcttcg actctgtatt atagataaga tgatgaggaa ttggaggtag catagcttca 2880 tttggatttg ctttccaggc tgagactcta gcttggagca tagagggtcc tttggctttc 2940 aatattctca agtatctcga gtttgaactt attccctgtg aaccttttat tcaccaatga 3000 gcattggaat gaacatgaat ctgaggactg caatcgccat gaggttttcg aaatacatcc 3060 ggatgtcgaa ggcttggggc acctgcgttg gttgaattta gaacgtggca ctattgatca 3120 tccgatagct ctgcaaaggg cgttgcacaa tgcaagtcaa acgttgctag cagttccagg 3180 tggaatgtta tgatgagcat tgtattaaat caggagatat agcatgatct ctagttagct 3240 caccacaaaa gtcagacggc gtaaccaaaa gtcacacaac acaagctgta aggatttcgg 3300 cacggctacg gaagacggag aagccacctt cagtggactc gagtaccatt taattctatt 3360 tgtgtttgat cgagacctaa tacagcccct acaacgacca tcaaagtcgt atagctacca 3420 gtgaggaagt ggactcaaat cgacttcagc aacatctcct ggataaactt taagcctaaa 3480 ctatacagaa taagataggt ggagagctta taccgagctc ccaaatctgt ccagatcatg 3540 gttgaccggt gcctggatct tcctatagaa tcatccttat tcgttgacct agctgattct 3600 ggagtgaccc agagggtcat gacttgagcc taaaatccgc cgcctccacc atttgtagaa 3660 aaatgtgacg aactcgtgag ctctgtacag tgaccggtga ctctttctgg catgcggaga 3720 gacggacgga cgcagagaga agggctgagt aataagccac tggccagaca gctctggcgg 3780 ctctgaggtg cagtggatga ttattaatcc gggaccggcc gcccctccgc cccgaagtgg 3840 aaaggctggt gtgcccctcg ttgaccaaga atctattgca tcatcggaga atatggagct 3900 tcatcgaatc accggcagta agcgaaggag aatgtgaagc caggggtgta tagccgtcgg 3960 cgaaatagca tgccattaac ctaggtacag aagtccaatt gcttccgatc tggtaaaaga 4020 ttcacgagat agtaccttct ccgaagtagg tagagcgagt acccggcgcg taagctccct 4080 aattggcgaa ttcccatccg gcatctgtag ggcgtccaaa tatcgtgcct ctcctgcttt 4140 gccaggtgta tgaaaccgga aaggccgctc aggagctggc cagcggcgca gaccgggaac 4200 acaagctggc agtcgaccca tccggtgctc tgcactcgac ctgctgaggt ccctcagtcc 4260 ctggtaggca gctttgcccc gtctgtccgc ccggtgtgtc ggcggggttg acaaggtcgt 4320 tgcgtcagtc caacatttgt tgccatattt tcctgctttc cccaccagct gctcttttct 4380 tttctctttc ttttcccatc ttcagtatat tcatcttccc atatgcatcc aagaaccttt 4440 atttccccta agtaagtact ttgctacatc catactccat ccttcccatc ccttattcct 4500 ttgaaccttt cagttcgagc tttcccactt catcgcagct tgactaacag ctaccccgct 4560 tgagcagaca tcaccatgga accgaccagc accccgcagg ctccgatcgc tgcttataaa 4620 ccgcgtagca acgaaatcct gtgggatggt tatggtgttc cgcatatcta tggtgttgac 4680 gctccgagcg ctttttatgg ttatggctgg gctcaggctc gtagccatgg tgataacatc 4740 ctgcgtctgt atggtgaagc tcgtggtaaa ggcgctgaat attggggtcc ggattacgaa 4800 cagacgaccg tttggctgct gaccaacggt gttccggaac gtgctcagca gtggtatgct 4860 cagcagagcc cggattttcg tgctaacctg gatgcttttg ctgctggtat caacgcttat 4920 gctcagcaga acccggatga tatcagcccg gaagttcgtc aggttctgcc ggttagcggt 4980 gctgatgttg ttgctcatgc tcatcgtctg atgaactttc tgtatgttgc tagcccgggt 5040 cgtaccctgg gtgaaggtga tccgccggat ctggctgatc agggtagcaa cagctgggct 5100 gttgctccgg gtaaaaccgc taacggtaac gctctgctgc tgcagaaccc tcatctgagc 5160 tggaccaccg attattttac ctattatgaa gctcatctgg ttaccccgga ttttgaagtg 5220 tatggtgcta cccagatcgg tctgccggtt atccgtgttg cttttaacca gcgtatgggt 5280 atcaccaaca ccgttaacgg tatggttggt gctaccaact atcgtctgac cctgcaggat 5340 ggtggttatc tgtatgatgg tcaggttcgt ccgtttgaac gtcgtcaggc tagctatcgt 5400 ctgcgtcagg ctgatggtac caccgttgat aaaccgctgg aaatccgttc cagcgttcat 5460 ggtccggttt ttgaacgtgc tgatggtacc gctgttgctg ttcgtgttgc tggtctggat 5520 cgtccgggta tgctggaaca gacttttgat atgatcaccg ctgatagctt tgatgattat 5580 gaagctgctc tggctcgtat gcaggttccg accttgaaca tcgtttatgc tgatcgtgaa 5640 ggtaccatca actatagctt taacggtgtt gctccgaaac gtgctgaagg tgacatcgct 5700 ttttggcagg gtctggttcc gggtgatagc agccgttatc tgtggaccga aacccatccg 5760 ctggatgatc tgccgcgtgt taccaacccg ccgggtggtt ttgttcagaa cagcaacgat 5820 ccgccgtgga ccccgacctg gccggttacc tataccccga aagattttcc gagctatctg 5880 gctccgcaga ccccgcatag cctgcgtgct cagcagagcg ttcgtctgat gagcgaaaac 5940 gatgatctga ccctggaacg ttttatggct ctgcagctga gccatcgtgc tgttatggct 6000 gatcgtaccc tgccggatct gatcccggct gctctgatcg atccggatcc ggaagttcag 6060 gctgctgctc gtctgctggc tgcgtgggat cgtgaattta ccagcgatag ccgtgctgct 6120 ctgctgtttg aagaatgggc tcgtctgttt gctggtcaga actttgctgg tcaggctggc 6180 tttgctaccc cgtggagcct ggataaaccg gttagcaccc cgtatggtct gcgtgatccg 6240 aaagctgctg ttgatcagct gcgtaccgct atcgctaaca ccaaacgtaa atatggtgct 6300 atcgatcgtc cgtttggtga tgctagccgt atgatcctga acgatgttaa cgttccgggt 6360 gctgctggtt atggtaacct gggtagcttt cgtgttttta cctggagcga tccggatgaa 6420 aacggtgtgc gtaccccggt tcatggtgaa acctgggttg ctatgatcga atttagcacc 6480 ccggttcgtg cttatggtct gatgagctat ggtaacagcc gtcagccggg taccacccat 6540 tatagcgatc agatcgaacg tgttagccgt gctgattttc gtgaactgct gctgcgtcgt 6600 gaacaggttg aagctgctgt tcaggaacgt accccgttta actttaaacc gtgaagtaga 6660 tgccgaccgc gagatccact taacgttact gaaatcatca aacagcttga cgaatctgga 6720 tataagatcg ttggtgtcga tgtcagctcc ggagttgaga caaatggtgt tcaggatctc 6780 gataagatac gttcatttgt ccaagcagca aagagtgcct tctagtgatt taatagctcc 6840 atgtcaacaa gaataaaacg cgttttcggg tttacctctt ccagatacag ctcatctgca 6900 atgcattaat gcattgactg caacctagta acgccttnca ggctccggcg aagagaagaa 6960 tagcttagca gagctatttt cattttcggg agacgagatc aagcagatca acggtcgtca 7020 agagacctac gagactgagg aatccgctct tggctccacg cgactatata tttgtctcta 7080 attgtacttt gacatgctcc tcttctttac tctgatagct tgactatgaa aattccgtca 7140 ccagcncctg ggttcgcaaa gataattgca tgtttcttcc ttgaactctc aagcctacag 7200 gacacacatt catcgtaggt ataaacctcg aaatcanttc ctactaagat ggtatacaat 7260 agtaaccatg catggttgcc tagtgaatgc tccgtaacac ccaatacgcc ggccgaaact 7320 tttttacaac tctcctatga gtcgtttacc cagaatgcac aggtacactt gtttagaggt 7380 aatccttctt tctagaagtc ctcgtgtact gtgtaagcgc ccactccaca tctccactcg 7440 accaattcgc cctatagtga gtcgtattag taaattaacc ctcactaaag ggaacaaaag 7500 ctgggcaaag cttgaggccg gacaaattca gcctgcaggc taaagtttct attacatgat 7560 atttgtcgtg gacaagggcg tctggggtaa aaaggaatgt gggcagagag ctctcggctt 7620 aaagaccggc ttcggagttc cagacccact cgaactgaaa acgatgatta gagataagta 7680 gatctatttg gacagtataa gaatcctagt cggctagtat ttctccactt ttactttaaa 7740 attccccaga gtaagggggc aggttcgtcg tcacgggtag cctcgtctcg aggcatcgcg 7800 gatagcatta tcttggggta tcacgagtaa cttcatcgtg gcattatcga taggtactgt 7860 aagtactgta ggaacttatc cgggcatgga tatgtatagg tacatacgat gcactaaaag 7920 tggagaaata ctagccgagt agagatctta taccatccga atagatttac tccttataat 7980 caaggggcaa caatcagttg tgacgatgtc ggctgtggga gtgcagttta ggggtcatca 8040 aagcacagga aaacgtgttg tatggatgcc tatcgtcttg actattagaa aggaagaggg 8100 gaaaaaccct catcaggagc gattgtaagt tcggccatag taatgatatt ggccattaat 8160 ggacgtgtgt atgtatacat acacggttca tttgccgcat tcggaccaaa accgctgctg 8220 ccgcagacac agtgcttcca tggcctcgaa caaccggtta ttcttctctt ccatgtcctc 8280 gggacgaagg aaatttcgga gccctaacag ccgtaccgga cttgcgttaa catggttgcg 8340 aagaactaaa gactgatggg gtcatgattg gcagtgttga cacttcatgt cttgaaccta 8400 gctagcagaa aaatgaatgc tctatgtgac actggtagat ctcaacgcat catgttccac 8460 ctttagtgta aacacggcaa gctgtccgca acgtcggtac caacgtttga acacacaacc 8520 ttctgccact ccaggagttt agcccacggc ttactggatg aaacgtaatc tcccgctacc 8580 ccctcaattc cattggggtc tagagtagtc ggaagaacgg ctgtcgcctt gcgctgcggt 8640 taaagcaccc accgtggtgg ctactgcttc tcgggaggag aaatcctggg tactcgagca 8700 cttgcgctag gggatgaaac atccatggca tattggcgaa agcaatgcgc catgttcggc 8760 gcggcacatg acactcttga gaagccgtat tcttcgcaaa ctttcctatt tacattcagc 8820 ctctaactat aacacgatcg gaaatgcctg aacgagcccg ccctcatgtg cctagcgtca 8880 aacaacgcca agcacacaac gcctcctgct attcttccct cttctcactg ccatcactgg 8940 gcgcaagctg caatgtcctt ctcgaattca agcgggcaaa tcaagattgg gaaaatgcgc 9000 catcttggtt tggatcttga tgtctatccg catcaaggtc ttggtttggt cgtttattta 9060 gtagcaaagg caaatcaaaa cgtgcgcgcc aagtgccagg gtccagactc cagactggag 9120 catttagcct gaatatcgtc tctagaatct ggtgctatta ttcaggatat agtaactatg 9180 atctactcgg gccatgttcg gctcctccgc acggccaagt tgtctgccgg cgtctggacc 9240 ggactcatgg cttggcttct ccagggatag catggcattg gcaaaactct ccgccacgat 9300 tcgagcatag ctaccgcagc cacgcggcat tgtttccaga ttgagatatg tcactattag 9360 ccggaatgca aacatcgagc tgacctgcgc aacatctcca tgagttccga agtcgacctc 9420 cgaagcaata ttggtctagt attacggatg atatagtggt ccccggaccc aatgagccgt 9480 gtgtttttta ccgcctttgc tctgaggatt aacaagattc gtgcaattgg ggactgtgcg 9540 gagtaactgg gctggtttac tgctttgccg acgaaagaaa cgtataaaca gccgactcgt 9600 ccactgccac tcgggacgat ctcccaaccc agacagcttc ccagaaccca tcttcaacac 9660 cctgacacca tgccccagtt cgacatccta tgcaagactc ctccgaaggt gctcgtccgc 9720 cagttcgtgg agcgattcga gcgcccctcg ggcgaaaaga tcgccttgtg tgccgctgag 9780 cttacctacc tctgctggat gatcacccac aacggcaccg ccatcaagcg ggctaccttt 9840 atgagttaca ataccatcat tagcaactcc ctctcgtttg acatcgtcaa caagtccctg 9900 cagttcaagt acaagaccca aaaggccacc atcctcgagg cctcgctgaa gaagctgatc 9960 cccgcatggg aattcactat catcccctac tatggtcaga agcatcagtc tgacatcacc 10020 gacattgtaa gcagtctcca gctccagttt gagtcctccg aggaggccga taaaggcaac 10080 tcccactcca agaagatgct caaggctttg ctgagtgaag gcgagtccat ctgggaaata 10140 accgaaaaga tcttgaacag tttcgagtac accagccgct tcacgaagac caagacgctg 10200 taccagttcc tgttcctcgc cacattcatc aattgcggac gcttcagtga tatcaagaac 10260 gtggacccga agtcattcaa gctggttcag aacaagtacc tcggtgtcat cattcagtgc 10320 ttggtcaccg agaccaagac atccgtcagc cgtcacatct acttcttttc cgcaagaggg 10380 cgtatcgacc ccctagtgta cctagacgaa ttcctgcgca actcggagcc cgtcctaaag 10440 cgcgtcaacc gtaccggcaa ctctagcagc aacaagcagg agtaccagct cttgaaggat 10500 aacctcgtcc gatcatacaa caaggcgctc aagaagaacg ctccatacag catctttgct 10560 atcaagaatg ggccaaagtc acacatcggc cgccacctca tgacctcgtt tctgtcgatg 10620 aagggcctca ctgagcttac caacgttgtt ggcaactggt ccgataagcg cgccagcgcc 10680 gtggcgcgga cgacttacac tcatcagatc acagctatcc ctgaccacta cttcgccttg 10740 gtcagtcgct attacgccta cgacccaatt agtaaagaga tgatcgctct caaggacgaa 10800 accaacccca tcgaggagtg gcaacacatc gagcagctta aaggctccgc cgagggctcg 10860 atccggtacc ccgcctggaa cggtatcata tcgcaggagg tcttggatta cctgagcagt 10920 tatatcaatc gccgcatatg ataaggatcc acttaacgtt actgaaatca tcaaacagct 10980 tgacgaatct ggatataaga tcgttggtgt cgatgtcagc tccggagttg agacaaatgg 11040 tgttcaggat ctcgataaga tacgttcatt tgtccaagca gcaaagagtg ccttctagtg 11100 atttaatagc tccatgtcaa caagaataaa acgcgttttc gggtttacct cttccagata 11160 cagctcatct gcaatgcatt aatgcattga ctgcaaccta gtaacgcctt ncaggctccg 11220 gcgaagagaa gaatagctta gcagagctat tttcattttc gggagacgag atcaagcaga 11280 tcaacggtcg tcaagagacc tacgagactg aggaatccgc tcttggctcc acgcgactat 11340 atatttgtct ctaattgtac tttgacatgc tcctcttctt tactctgata gcttgactat 11400 gaaaattccg tcaccagcnc ctgggttcgc aaagataatt gcatgtttct tccttgaact 11460 ctcaagccta caggacacac attcatcgta ggtataaacc tcgaaatcan ttcctactaa 11520 gatggtatac aatagtaacc atgcatggtt gcctagtgaa tgctccgtaa cacccaatac 11580 gccggccgaa acttttttac aactctccta tgagtcgttt acccagaatg cacaggtaca 11640 cttgtttaga ggtaatcctt ctttctagaa gtcctcgtgt actgtgtaag cgcccactcc 11700 acatctccac tcgaccaatt cgccctatag tgagtcgtat taagcttcct gcagttcgaa 11760 gttcctattc tctagaaagt ataggaactt cagagcgctt tccttgtatc tctacacaca 11820 ggctcaaatc aataagaaga acggttcgtc tttttcgttt atatcttgca tcgtcccaaa 11880 gctattggcg ggatattctg tttgcagttg gctgacttga agtaatctct gcagatcttt 11940 cgacactgaa atacgtcgag cctgctccgc ttggaagcgg cgaggagcct cgtcctgtca 12000 caactaccaa catggagtac gataagggcc agttccgcca gctcattaag agccagttca 12060 tgggcgttgg catgatggcc gtcatgcatc tgtacttcaa gtacaccaac gctcttctga 12120 tccagtcgat catccgctga aggcgctttc gaatctggtt aagatccacg tcttcgggaa 12180 gccagcgact ggtgacctcc agcgtccctt taaggctgcc aacagctttc tcagccaggg 12240 ccagcccaag accgacaagg cctccctcca gaacgccgag aagaactgga ggggtggtgt 12300 caaggaggag taagctcctt attgaagtcg gaggacggag cggtgtcaag aggatattct 12360 tcgactctgt attatagata agatgatgag gaattggagg tagcatagct tcatttggat 12420 ttgctttcca ggctgagact ctagcttgga gcatagaggg tcctttggct ttcaatattc 12480 tcaagtatct cgagtttgaa cttattccct gtgaaccttt tattcaccaa tgagcattgg 12540 aatgaacatg aatctgagga ctgcaatcgc catgaggttt tcgaaataca tccggatgtc 12600 gaaggcttgg ggcacctgcg ttggttgaat ttagaacgtg gcactattga tcatccgata 12660 gctctgcaaa gggcgttgca caatgcaagt caaacgttgc tagcagttcc aggtggaatg 12720 ttatgatgag cattgtatta aatcaggaga tatagcatga tctctagtta gctcaccaca 12780 aaagtcagac ggcgtaacca aaagtcacac aacacaagct gtaaggattt cggcacggct 12840 acggaagacg gagaagccac cttcagtgga ctcgagtacc atttaattct atttgtgttt 12900 gatcgagacc taatacagcc cctacaacga ccatcaaagt cgtatagcta ccagtgagga 12960 agtggactca aatcgacttc agcaacatct cctggataaa ctttaagcct aaactataca 13020 gaataagata ggtggagagc ttataccgag ctcccaaatc tgtccagatc atggttgacc 13080 ggtgcctgga tcttcctata gaatcatcct tattcgttga cctagctgat tctggagtga 13140 cccagagggt catgacttga gcctaaaatc cgccgcctcc accatttgta gaaaaatgtg 13200 acgaactcgt gagctctgta cagtgaccgg tgactctttc tggcatgcgg agagacggac 13260 ggacgcagag agaagggctg agtaataagc cactggccag acagctctgg cggctctgag 13320 gtgcagtgga tgattattaa tccgggaccg gccgcccctc cgccccgaag tggaaaggct 13380 ggtgtgcccc tcgttgacca agaatctatt gcatcatcgg agaatatgga gcttcatcga 13440 atcaccggca gtaagcgaag gagaatgtga agccaggggt gtatagccgt cggcgaaata 13500 gcatgccatt aacctaggta cagaagtcca attgcttccg atctggtaaa agattcacga 13560 gatagtacct tctccgaagt aggtagagcg agtacccggc gcgtaagctc cctaattggc 13620 ccatccggca tctgtagggc gtccaaatat cgtgcctctc ctgctttgcc cggtgtatga 13680 aaccggaaag gccgctcagg agctggccag cggcgcagac cgggaacaca agctggcagt 13740 cgacccatcc ggtgctctgc actcgacctg ctgaggtccc tcagtccctg gtaggcagct 13800 ttgccccgtc tgtccgcccg gtgtgtcggc ggggttgaca aggtcgttgc gtcagtccaa 13860 catttgttgc catattttcc tgctctcccc accagctgct cttttctttt ctctttcttt 13920 tcccatcttc agtatattca tcttcccatc caagaacctt tatttcccct aagtaagtac 13980 tttgctacat ccatactcca tccttcccat cccttattcc tttgaacctt tcagttcgag 14040 ctttcccact tcatcgcagc ttgactaaca gctaccccgc ttgagcagac atcaccatgc 14100 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg 14160 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc 14220 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt 14280 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca 14340 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc 14400 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg 14460 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat 14520 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa 14580 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt 14640 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg 14700 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg 14760 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc 14820 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg 14880 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg 14940 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg 15000 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag 15060 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagagta 15120 gatgccgacc gcgggatcca cttaacgtta ctgaaatcat caaacagctt gacgaatctg 15180 gatataagat cgttggtgtc gatgtcagct ccggagttga gacaaatggt gttcaggatc 15240 tcgataagat acgttcattt gtccaagcag caaagagtgc cttctagtga tttaatagct 15300 ccatgtcaac aagaataaaa cgcgttttcg ggtttacctc ttccagatac agctcatctg 15360 caatgcatta atgcattgac tgcaacctag taacgccttn caggctccgg cgaagagaag 15420 aatagcttag cagagctatt ttcattttcg ggagacgaga tcaagcagat caacggtcgt 15480 caagagacct acgagactga ggaatccgct cttggctcca cgcgactata tatttgtctc 15540 taattgtact ttgacatgct cctcttcttt actctgatag cttgactatg aaaattccgt 15600 caccagcncc tgggttcgca aagataattg catgtttctt ccttgaactc tcaagcctac 15660 aggacacaca ttcatcgtag gtataaacct cgaaatcant tcctactaag atggtataca 15720 atagtaacca tgcatggttg cctagtgaat gctccgtaac acccaatacg ccggccgaaa 15780 cttttttaca actctcctat gagtcgttta cccagaatgc acaggtacac ttgtttagag 15840 gtaatccttc tttctagaag tcctcgtgta ctgtgtaagc gcccactcca catctccact 15900 cgacctgcag ttcgaagttc ctattctcta gaaagtatag gaacttcaga gcgctttcca 15960 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg 16020 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 16080 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 16140 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 16200 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 16260 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 16320 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 16380 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt 16440 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 16500 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 16560 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag 16610 <210> 16 <211> 16388 <212> DNA <213> Artificial Sequence <220> <223> pB-GCXEP3 plasmid <400> 16 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaaaccc 2220 cgggtacaat taaccctcac taaagggaac aaaagctggg caaagcttcc ttgtatctct 2280 acacacaggc tcaaatcaat aagaagaacg gttcgtcttt ttcgtttata tcttgcatcg 2340 tcccaaagct attggcggga tattctgttt gcagttggct gacttgaagt aatctctgca 2400 gatctttcga cactgaaata cgtcgagcct gctccgcttg gaagcggcga ggagcctcgt 2460 cctgtcacaa ctaccaacat ggagtacgat aagggccagt tccgccagct cattaagagc 2520 cagttcatgg gcgttggcat gatggccgtc atgcatctgt acttcaagta caccaacgct 2580 cttctgatcc agtcgatcat ccgctgaagg cgctttcgaa tctggttaag atccacgtct 2640 tcgggaagcc agcgactggt gacctccagc gtccctttaa ggctgccaac agctttctca 2700 gccagggcca gcccaagacc gacaaggcct ccctccagaa cgccgagaag aactggaggg 2760 gtggtgtcaa ggaggagtaa gctccttatt gaagtcggag gacggagcgg tgtcaagagg 2820 atattcttcg actctgtatt atagataaga tgatgaggaa ttggaggtag catagcttca 2880 tttggatttg ctttccaggc tgagactcta gcttggagca tagagggtcc tttggctttc 2940 aatattctca agtatctcga gtttgaactt attccctgtg aaccttttat tcaccaatga 3000 gcattggaat gaacatgaat ctgaggactg caatcgccat gaggttttcg aaatacatcc 3060 ggatgtcgaa ggcttggggc acctgcgttg gttgaattta gaacgtggca ctattgatca 3120 tccgatagct ctgcaaaggg cgttgcacaa tgcaagtcaa acgttgctag cagttccagg 3180 tggaatgtta tgatgagcat tgtattaaat caggagatat agcatgatct ctagttagct 3240 caccacaaaa gtcagacggc gtaaccaaaa gtcacacaac acaagctgta aggatttcgg 3300 cacggctacg gaagacggag aagccacctt cagtggactc gagtaccatt taattctatt 3360 tgtgtttgat cgagacctaa tacagcccct acaacgacca tcaaagtcgt atagctacca 3420 gtgaggaagt ggactcaaat cgacttcagc aacatctcct ggataaactt taagcctaaa 3480 ctatacagaa taagataggt ggagagctta taccgagctc ccaaatctgt ccagatcatg 3540 gttgaccggt gcctggatct tcctatagaa tcatccttat tcgttgacct agctgattct 3600 ggagtgaccc agagggtcat gacttgagcc taaaatccgc cgcctccacc atttgtagaa 3660 aaatgtgacg aactcgtgag ctctgtacag tgaccggtga ctctttctgg catgcggaga 3720 gacggacgga cgcagagaga agggctgagt aataagccac tggccagaca gctctggcgg 3780 ctctgaggtg cagtggatga ttattaatcc gggaccggcc gcccctccgc cccgaagtgg 3840 aaaggctggt gtgcccctcg ttgaccaaga atctattgca tcatcggaga atatggagct 3900 tcatcgaatc accggcagta agcgaaggag aatgtgaagc caggggtgta tagccgtcgg 3960 cgaaatagca tgccattaac ctaggtacag aagtccaatt gcttccgatc tggtaaaaga 4020 ttcacgagat agtaccttct ccgaagtagg tagagcgagt acccggcgcg taagctccct 4080 aattggcgaa ttcccatccg gcatctgtag ggcgtccaaa tatcgtgcct ctcctgcttt 4140 gccaggtgta tgaaaccgga aaggccgctc aggagctggc cagcggcgca gaccgggaac 4200 acaagctggc agtcgaccca tccggtgctc tgcactcgac ctgctgaggt ccctcagtcc 4260 ctggtaggca gctttgcccc gtctgtccgc ccggtgtgtc ggcggggttg acaaggtcgt 4320 tgcgtcagtc caacatttgt tgccatattt tcctgctttc cccaccagct gctcttttct 4380 tttctctttc ttttcccatc ttcagtatat tcatcttccc atatgcatcc aagaaccttt 4440 atttccccta agtaagtact ttgctacatc catactccat ccttcccatc ccttattcct 4500 ttgaaccttt cagttcgagc tttcccactt catcgcagct tgactaacag ctaccccgct 4560 tgagcagaca tcaccatgga accgaccagc accccgcagg ctccgatcgc tgcttataaa 4620 ccgcgtagca acgaaatcct gtgggatggt tatggtgttc cgcatatcta tggtgttgac 4680 gctccgagcg ctttttatgg ttatggctgg gctcaggctc gtagccatgg tgataacatc 4740 ctgcgtctgt atggtgaagc tcgtggtaaa ggcgctgaat attggggtcc ggattacgaa 4800 cagacgaccg tttggctgct gaccaacggt gttccggaac gtgctcagca gtggtatgct 4860 cagcagagcc cggattttcg tgctaacctg gatgcttttg ctgctggtat caacgcttat 4920 gctcagcaga acccggatga tatcagcccg gaagttcgtc aggttctgcc ggttagcggt 4980 gctgatgttg ttgctcatgc tcatcgtctg atgaactttc tgtatgttgc tagcccgggt 5040 cgtaccctgg gtgaaggtga tccgccggat ctggctgatc agggtagcaa cagctgggct 5100 gttgctccgg gtaaaaccgc taacggtaac gctctgctgc tgcagaaccc tcatctgagc 5160 tggaccaccg attattttac ctattatgaa gctcatctgg ttaccccgga ttttgaagtg 5220 tatggtgcta cccagatcgg tctgccggtt atccgtgttg cttttaacca gcgtatgggt 5280 atcaccaaca ccgttaacgg tatggttggt gctaccaact atcgtctgac cctgcaggat 5340 ggtggttatc tgtatgatgg tcaggttcgt ccgtttgaac gtcgtcaggc tagctatcgt 5400 ctgcgtcagg ctgatggtac caccgttgat aaaccgctgg aaatccgttc cagcgttcat 5460 ggtccggttt ttgaacgtgc tgatggtacc gctgttgctg ttcgtgttgc tggtctggat 5520 cgtccgggta tgctggaaca gacttttgat atgatcaccg ctgatagctt tgatgattat 5580 gaagctgctc tggctcgtat gcaggttccg accttgaaca tcgtttatgc tgatcgtgaa 5640 ggtaccatca actatagctt taacggtgtt gctccgaaac gtgctgaagg tgacatcgct 5700 ttttggcagg gtctggttcc gggtgatagc agccgttatc tgtggaccga aacccatccg 5760 ctggatgatc tgccgcgtgt taccaacccg ccgggtggtt ttgttcagaa cagcaacgat 5820 ccgccgtgga ccccgacctg gccggttacc tataccccga aagattttcc gagctatctg 5880 gctccgcaga ccccgcatag cctgcgtgct cagcagagcg ttcgtctgat gagcgaaaac 5940 gatgatctga ccctggaacg ttttatggct ctgcagctga gccatcgtgc tgttatggct 6000 gatcgtaccc tgccggatct gatcccggct gctctgatcg atccggatcc ggaagttcag 6060 gctgctgctc gtctgctggc tgcgtgggat cgtgaattta ccagcgatag ccgtgctgct 6120 ctgctgtttg aagaatgggc tcgtctgttt gctggtcaga actttgctgg tcaggctggc 6180 tttgctaccc cgtggagcct ggataaaccg gttagcaccc cgtatggtct gcgtgatccg 6240 aaagctgctg ttgatcagct gcgtaccgct atcgctaaca ccaaacgtaa atatggtgct 6300 atcgatcgtc cgtttggtga tgctagccgt atgatcctga acgatgttaa cgttccgggt 6360 gctgctggtt atggtaacct gggtagcttt cgtgttttta cctggagcga tccggatgaa 6420 aacggtgtgc gtaccccggt tcatggtgaa acctgggttg ctatgatcga atttagcacc 6480 ccggttcgtg cttatggtct gatgagctat ggtaacagcc gtcagccggg taccacccat 6540 tatagcgatc agatcgaacg tgttagccgt gctgattttc gtgaactgct gctgcgtcgt 6600 gaacaggttg aagctgctgt tcaggaacgt accccgttta actttaaacc gtgaagtaga 6660 tgccgaccgc gagatccact taacgttact gaaatcatca aacagcttga cgaatctgga 6720 tataagatcg ttggtgtcga tgtcagctcc ggagttgaga caaatggtgt tcaggatctc 6780 gataagatac gttcatttgt ccaagcagca aagagtgcct tctagtgatt taatagctcc 6840 atgtcaacaa gaataaaacg cgttttcggg tttacctctt ccagatacag ctcatctgca 6900 atgcattaat gcattgactg caacctagta acgccttnca ggctccggcg aagagaagaa 6960 tagcttagca gagctatttt cattttcggg agacgagatc aagcagatca acggtcgtca 7020 agagacctac gagactgagg aatccgctct tggctccacg cgactatata tttgtctcta 7080 attgtacttt gacatgctcc tcttctttac tctgatagct tgactatgaa aattccgtca 7140 ccagcncctg ggttcgcaaa gataattgca tgtttcttcc ttgaactctc aagcctacag 7200 gacacacatt catcgtaggt ataaacctcg aaatcanttc ctactaagat ggtatacaat 7260 agtaaccatg catggttgcc tagtgaatgc tccgtaacac ccaatacgcc ggccgaaact 7320 tttttacaac tctcctatga gtcgtttacc cagaatgcac aggtacactt gtttagaggt 7380 aatccttctt tctagaagtc ctcgtgtact gtgtaagcgc ccactccaca tctccactcg 7440 accaattcgc cctatagtga gtcgtattag taaattaacc ctcactaaag ggaacaaaag 7500 ctgggcaaag cttgaggccg gacaaattca gcctgcaggc taaagtttct attacatgat 7560 atttgtcgtg gacaagggcg tctggggtaa aaaggaatgt gggcagagag ctctcggctt 7620 aaagaccggc ttcggagttc cagacccact cgaactgaaa acgatgatta gagataagta 7680 gatctatttg gacagtataa gaatcctagt cggctagtat ttctccactt ttactttaaa 7740 attccccaga gtaagggggc aggttcgtcg tcacgggtag cctcgtctcg aggcatcgcg 7800 gatagcatta tcttggggta tcacgagtaa cttcatcgtg gcattatcga taggtactgt 7860 aagtactgta ggaacttatc cgggcatgga tatgtatagg tacatacgat gcactaaaag 7920 tggagaaata ctagccgagt agagatctta taccatccga atagatttac tccttataat 7980 caaggggcaa caatcagttg tgacgatgtc ggctgtggga gtgcagttta ggggtcatca 8040 aagcacagga aaacgtgttg tatggatgcc tatcgtcttg actattagaa aggaagaggg 8100 gaaaaaccct catcaggagc gattgtaagt tcggccatag taatgatatt ggccattaat 8160 ggacgtgtgt atgtatacat acacggttca tttgccgcat tcggaccaaa accgctgctg 8220 ccgcagacac agtgcttcca tggcctcgaa caaccggtta ttcttctctt ccatgtcctc 8280 gggacgaagg aaatttcgga gccctaacag ccgtaccgga cttgcgttaa catggttgcg 8340 aagaactaaa gactgatggg gtcatgattg gcagtgttga cacttcatgt cttgaaccta 8400 gctagcagaa aaatgaatgc tctatgtgac actggtagat ctcaacgcat catgttccac 8460 ctttagtgta aacacggcaa gctgtccgca acgtcggtac caacgtttga acacacaacc 8520 ttctgccact ccaggagttt agcccacggc ttactggatg aaacgtaatc tcccgctacc 8580 ccctcaattc cattggggtc tagagtagtc ggaagaacgg ctgtcgcctt gcgctgcggt 8640 taaagcaccc accgtggtgg ctactgcttc tcgggaggag aaatcctggg tactcgagca 8700 cttgcgctag gggatgaaac atccatggca tattggcgaa agcaatgcgc catgttcggc 8760 gcggcacatg acactcttga gaagccgtat tcttcgcaaa ctttcctatt tacattcagc 8820 ctctaactat aacacgatcg gaaatgcctg aacgagcccg ccctcatgtg cctagcgtca 8880 aacaacgcca agcacacaac gcctcctgct attcttccct cttctcactg ccatcactgg 8940 gcgcaagctg caatgtcctt ctcgaattca agcgggcaaa tcaagattgg gaaaatgcgc 9000 catcttggtt tggatcttga tgtctatccg catcaaggtc ttggtttggt cgtttattta 9060 gtagcaaagg caaatcaaaa cgtgcgcgcc aagtgccagg gtccagactc cagactggag 9120 catttagcct gaatatcgtc tctagaatct ggtgctatta ttcaggatat agtaactatg 9180 atctactcgg gccatgttcg gctcctccgc acggccaagt tgtctgccgg cgtctggacc 9240 ggactcatgg cttggcttct ccagggatag catggcattg gcaaaactct ccgccacgat 9300 tcgagcatag ctaccgcagc cacgcggcat tgtttccaga ttgagatatg tcactattag 9360 ccggaatgca aacatcgagc tgacctgcgc aacatctcca tgagttccga agtcgacctc 9420 cgaagcaata ttggtctagt attacggatg atatagtggt ccccggaccc aatgagccgt 9480 gtgtttttta ccgcctttgc tctgaggatt aacaagattc gtgcaattgg ggactgtgcg 9540 gagtaactgg gctggtttac tgctttgccg acgaaagaaa cgtataaaca gccgactcgt 9600 ccactgccac tcgggacgat ctcccaaccc agacagcttc ccagaaccca tcttcaacac 9660 cctgacacca tgccccagtt cgacatccta tgcaagactc ctccgaaggt gctcgtccgc 9720 cagttcgtgg agcgattcga gcgcccctcg ggcgaaaaga tcgccttgtg tgccgctgag 9780 cttacctacc tctgctggat gatcacccac aacggcaccg ccatcaagcg ggctaccttt 9840 atgagttaca ataccatcat tagcaactcc ctctcgtttg acatcgtcaa caagtccctg 9900 cagttcaagt acaagaccca aaaggccacc atcctcgagg cctcgctgaa gaagctgatc 9960 cccgcatggg aattcactat catcccctac tatggtcaga agcatcagtc tgacatcacc 10020 gacattgtaa gcagtctcca gctccagttt gagtcctccg aggaggccga taaaggcaac 10080 tcccactcca agaagatgct caaggctttg ctgagtgaag gcgagtccat ctgggaaata 10140 accgaaaaga tcttgaacag tttcgagtac accagccgct tcacgaagac caagacgctg 10200 taccagttcc tgttcctcgc cacattcatc aattgcggac gcttcagtga tatcaagaac 10260 gtggacccga agtcattcaa gctggttcag aacaagtacc tcggtgtcat cattcagtgc 10320 ttggtcaccg agaccaagac atccgtcagc cgtcacatct acttcttttc cgcaagaggg 10380 cgtatcgacc ccctagtgta cctagacgaa ttcctgcgca actcggagcc cgtcctaaag 10440 cgcgtcaacc gtaccggcaa ctctagcagc aacaagcagg agtaccagct cttgaaggat 10500 aacctcgtcc gatcatacaa caaggcgctc aagaagaacg ctccatacag catctttgct 10560 atcaagaatg ggccaaagtc acacatcggc cgccacctca tgacctcgtt tctgtcgatg 10620 aagggcctca ctgagcttac caacgttgtt ggcaactggt ccgataagcg cgccagcgcc 10680 gtggcgcgga cgacttacac tcatcagatc acagctatcc ctgaccacta cttcgccttg 10740 gtcagtcgct attacgccta cgacccaatt agtaaagaga tgatcgctct caaggacgaa 10800 accaacccca tcgaggagtg gcaacacatc gagcagctta aaggctccgc cgagggctcg 10860 atccggtacc ccgcctggaa cggtatcata tcgcaggagg tcttggatta cctgagcagt 10920 tatatcaatc gccgcatatg ataaggatcc acttaacgtt actgaaatca tcaaacagct 10980 tgacgaatct ggatataaga tcgttggtgt cgatgtcagc tccggagttg agacaaatgg 11040 tgttcaggat ctcgataaga tacgttcatt tgtccaagca gcaaagagtg ccttctagtg 11100 atttaatagc tccatgtcaa caagaataaa acgcgttttc gggtttacct cttccagata 11160 cagctcatct gcaatgcatt aatgcattga ctgcaaccta gtaacgcctt ncaggctccg 11220 gcgaagagaa gaatagctta gcagagctat tttcattttc gggagacgag atcaagcaga 11280 tcaacggtcg tcaagagacc tacgagactg aggaatccgc tcttggctcc acgcgactat 11340 atatttgtct ctaattgtac tttgacatgc tcctcttctt tactctgata gcttgactat 11400 gaaaattccg tcaccagcnc ctgggttcgc aaagataatt gcatgtttct tccttgaact 11460 ctcaagccta caggacacac attcatcgta ggtataaacc tcgaaatcan ttcctactaa 11520 gatggtatac aatagtaacc atgcatggtt gcctagtgaa tgctccgtaa cacccaatac 11580 gccggccgaa acttttttac aactctccta tgagtcgttt acccagaatg cacaggtaca 11640 cttgtttaga ggtaatcctt ctttctagaa gtcctcgtgt actgtgtaag cgcccactcc 11700 acatctccac tcgaccaatt cgccctatag tgagtcgtat taagcttcct gcagttcgaa 11760 gttcctattc tctagaaagt ataggaactt cagagcgctt tccttgtatc tctacacaca 11820 ggctcaaatc aataagaaga acggttcgtc tttttcgttt atatcttgca tcgtcccaaa 11880 gctattggcg ggatattctg tttgcagttg gctgacttga agtaatctct gcagatcttt 11940 cgacactgaa atacgtcgag cctgctccgc ttggaagcgg cgaggagcct cgtcctgtca 12000 caactaccaa catggagtac gataagggcc agttccgcca gctcattaag agccagttca 12060 tgggcgttgg catgatggcc gtcatgcatc tgtacttcaa gtacaccaac gctcttctga 12120 tccagtcgat catccgctga aggcgctttc gaatctggtt aagatccacg tcttcgggaa 12180 gccagcgact ggtgacctcc agcgtccctt taaggctgcc aacagctttc tcagccaggg 12240 ccagcccaag accgacaagg cctccctcca gaacgccgag aagaactgga ggggtggtgt 12300 caaggaggag taagctcctt attgaagtcg gaggacggag cggtgtcaag aggatattct 12360 tcgactctgt attatagata agatgatgag gaattggagg tagcatagct tcatttggat 12420 ttgctttcca ggctgagact ctagcttgga gcatagaggg tcctttggct ttcaatattc 12480 tcaagtatct cgagtttgaa cttattccct gtgaaccttt tattcaccaa tgagcattgg 12540 aatgaacatg aatctgagga ctgcaatcgc catgaggttt tcgaaataca tccggatgtc 12600 gaaggcttgg ggcacctgcg ttggttgaat ttagaacgtg gcactattga tcatccgata 12660 gctctgcaaa gggcgttgca caatgcaagt caaacgttgc tagcagttcc aggtggaatg 12720 ttatgatgag cattgtatta aatcaggaga tatagcatga tctctagtta gctcaccaca 12780 aaagtcagac ggcgtaacca aaagtcacac aacacaagct gtaaggattt cggcacggct 12840 acggaagacg gagaagccac cttcagtgga ctcgagtacc atttaattct atttgtgttt 12900 gatcgagacc taatacagcc cctacaacga ccatcaaagt cgtatagcta ccagtgagga 12960 agtggactca aatcgacttc agcaacatct cctggataaa ctttaagcct aaactataca 13020 gaataagata ggtggagagc ttataccgag ctcccaaatc tgtccagatc atggttgacc 13080 ggtgcctgga tcttcctata gaatcatcct tattcgttga cctagctgat tctggagtga 13140 cccagagggt catgacttga gcctaaaatc cgccgcctcc accatttgta gaaaaatgtg 13200 acgaactcgt gagctctgta cagtgaccgg tgactctttc tggcatgcgg agagacggac 13260 ggacgcagag agaagggctg agtaataagc cactggccag acagctctgg cggctctgag 13320 gtgcagtgga tgattattaa tccgggaccg gccgcccctc cgccccgaag tggaaaggct 13380 ggtgtgcccc tcgttgacca agaatctatt gcatcatcgg agaatatgga gcttcatcga 13440 atcaccggca gtaagcgaag gagaatgtga agccaggggt gtatagccgt cggcgaaata 13500 gcatgccatt aacctaggta cagaagtcca attgcttccg atctggtaaa agattcacga 13560 gatagtacct tctccgaagt aggtagagcg agtacccggc gcgtaagctc cctaattggc 13620 ccatccggca tctgtagggc gtccaaatat cgtgcctctc ctgctttgcc cggtgtatga 13680 aaccggaaag gccgctcagg agctggccag cggcgcagac cgggaacaca agctggcagt 13740 cgacccatcc ggtgctctgc actcgacctg ctgaggtccc tcagtccctg gtaggcagct 13800 ttgccccgtc tgtccgcccg gtgtgtcggc ggggttgaca aggtcgttgc gtcagtccaa 13860 catttgttgc catattttcc tgctctcccc accagctgct cttttctttt ctctttcttt 13920 tcccatcttc agtatattca tcttcccatc caagaacctt tatttcccct aagtaagtac 13980 tttgctacat ccatactcca tccttcccat cccttattcc tttgaacctt tcagttcgag 14040 ctttcccact tcatcgcagc ttgactaaca gctaccccgc ttgagcagac atcaccatgg 14100 ggattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg 14160 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag 14220 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactcc 14280 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc 14340 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg 14400 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc 14460 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca 14520 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag 14580 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg 14640 gcgaggatct cgtcgtgaca catggcgatg cctgcttgcc gaatatcatg gtggaaaatg 14700 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca 14760 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc 14820 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg 14880 acgagttctt ctgaagtaga tgccgaccgc gggatccact taacgttact gaaatcatca 14940 aacagcttga cgaatctgga tataagatcg ttggtgtcga tgtcagctcc ggagttgaga 15000 caaatggtgt tcaggatctc gataagatac gttcatttgt ccaagcagca aagagtgcct 15060 tctagtgatt taatagctcc atgtcaacaa gaataaaacg cgttttcggg tttacctctt 15120 ccagatacag ctcatctgca atgcattaat gcattgactg caacctagta acgccttnca 15180 ggctccggcg aagagaagaa tagcttagca gagctatttt cattttcggg agacgagatc 15240 aagcagatca acggtcgtca agagacctac gagactgagg aatccgctct tggctccacg 15300 cgactatata tttgtctcta attgtacttt gacatgctcc tcttctttac tctgatagct 15360 tgactatgaa aattccgtca ccagcncctg ggttcgcaaa gataattgca tgtttcttcc 15420 ttgaactctc aagcctacag gacacacatt catcgtaggt ataaacctcg aaatcanttc 15480 ctactaagat ggtatacaat agtaaccatg catggttgcc tagtgaatgc tccgtaacac 15540 ccaatacgcc ggccgaaact tttttacaac tctcctatga gtcgtttacc cagaatgcac 15600 aggtacactt gtttagaggt aatccttctt tctagaagtc ctcgtgtact gtgtaagcgc 15660 ccactccaca tctccactcg acctgcagtt cgaagttcct attctctaga aagtatagga 15720 acttcagagc gctttccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg 15780 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 15840 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 15900 aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg 15960 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 16020 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 16080 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 16140 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 16200 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 16260 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 16320 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta 16380 caatttag 16388 <210> 17 <211> 15965 <212> DNA <213> Artificial Sequence <220> <223> pB-PCXEP3 plasmid <400> 17 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 120 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 180 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 240 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 300 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 420 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 480 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 540 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 600 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 660 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 720 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 780 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 840 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 900 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 960 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 1020 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 1080 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 1140 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 1200 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 1320 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 1380 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 1440 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 1500 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 1620 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 1680 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 1740 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 1800 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 1860 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 1920 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 1980 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 2040 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 2100 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 2160 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggcaaagct gtttaaaccc 2220 cgggtacaat taaccctcac taaagggaac aaaagctggg caaagcttcc ttgtatctct 2280 acacacaggc tcaaatcaat aagaagaacg gttcgtcttt ttcgtttata tcttgcatcg 2340 tcccaaagct attggcggga tattctgttt gcagttggct gacttgaagt aatctctgca 2400 gatctttcga cactgaaata cgtcgagcct gctccgcttg gaagcggcga ggagcctcgt 2460 cctgtcacaa ctaccaacat ggagtacgat aagggccagt tccgccagct cattaagagc 2520 cagttcatgg gcgttggcat gatggccgtc atgcatctgt acttcaagta caccaacgct 2580 cttctgatcc agtcgatcat ccgctgaagg cgctttcgaa tctggttaag atccacgtct 2640 tcgggaagcc agcgactggt gacctccagc gtccctttaa ggctgccaac agctttctca 2700 gccagggcca gcccaagacc gacaaggcct ccctccagaa cgccgagaag aactggaggg 2760 gtggtgtcaa ggaggagtaa gctccttatt gaagtcggag gacggagcgg tgtcaagagg 2820 atattcttcg actctgtatt atagataaga tgatgaggaa ttggaggtag catagcttca 2880 tttggatttg ctttccaggc tgagactcta gcttggagca tagagggtcc tttggctttc 2940 aatattctca agtatctcga gtttgaactt attccctgtg aaccttttat tcaccaatga 3000 gcattggaat gaacatgaat ctgaggactg caatcgccat gaggttttcg aaatacatcc 3060 ggatgtcgaa ggcttggggc acctgcgttg gttgaattta gaacgtggca ctattgatca 3120 tccgatagct ctgcaaaggg cgttgcacaa tgcaagtcaa acgttgctag cagttccagg 3180 tggaatgtta tgatgagcat tgtattaaat caggagatat agcatgatct ctagttagct 3240 caccacaaaa gtcagacggc gtaaccaaaa gtcacacaac acaagctgta aggatttcgg 3300 cacggctacg gaagacggag aagccacctt cagtggactc gagtaccatt taattctatt 3360 tgtgtttgat cgagacctaa tacagcccct acaacgacca tcaaagtcgt atagctacca 3420 gtgaggaagt ggactcaaat cgacttcagc aacatctcct ggataaactt taagcctaaa 3480 ctatacagaa taagataggt ggagagctta taccgagctc ccaaatctgt ccagatcatg 3540 gttgaccggt gcctggatct tcctatagaa tcatccttat tcgttgacct agctgattct 3600 ggagtgaccc agagggtcat gacttgagcc taaaatccgc cgcctccacc atttgtagaa 3660 aaatgtgacg aactcgtgag ctctgtacag tgaccggtga ctctttctgg catgcggaga 3720 gacggacgga cgcagagaga agggctgagt aataagccac tggccagaca gctctggcgg 3780 ctctgaggtg cagtggatga ttattaatcc gggaccggcc gcccctccgc cccgaagtgg 3840 aaaggctggt gtgcccctcg ttgaccaaga atctattgca tcatcggaga atatggagct 3900 tcatcgaatc accggcagta agcgaaggag aatgtgaagc caggggtgta tagccgtcgg 3960 cgaaatagca tgccattaac ctaggtacag aagtccaatt gcttccgatc tggtaaaaga 4020 ttcacgagat agtaccttct ccgaagtagg tagagcgagt acccggcgcg taagctccct 4080 aattggcgaa ttcccatccg gcatctgtag ggcgtccaaa tatcgtgcct ctcctgcttt 4140 gccaggtgta tgaaaccgga aaggccgctc aggagctggc cagcggcgca gaccgggaac 4200 acaagctggc agtcgaccca tccggtgctc tgcactcgac ctgctgaggt ccctcagtcc 4260 ctggtaggca gctttgcccc gtctgtccgc ccggtgtgtc ggcggggttg acaaggtcgt 4320 tgcgtcagtc caacatttgt tgccatattt tcctgctttc cccaccagct gctcttttct 4380 tttctctttc ttttcccatc ttcagtatat tcatcttccc atatgcatcc aagaaccttt 4440 atttccccta agtaagtact ttgctacatc catactccat ccttcccatc ccttattcct 4500 ttgaaccttt cagttcgagc tttcccactt catcgcagct tgactaacag ctaccccgct 4560 tgagcagaca tcaccatgga accgaccagc accccgcagg ctccgatcgc tgcttataaa 4620 ccgcgtagca acgaaatcct gtgggatggt tatggtgttc cgcatatcta tggtgttgac 4680 gctccgagcg ctttttatgg ttatggctgg gctcaggctc gtagccatgg tgataacatc 4740 ctgcgtctgt atggtgaagc tcgtggtaaa ggcgctgaat attggggtcc ggattacgaa 4800 cagacgaccg tttggctgct gaccaacggt gttccggaac gtgctcagca gtggtatgct 4860 cagcagagcc cggattttcg tgctaacctg gatgcttttg ctgctggtat caacgcttat 4920 gctcagcaga acccggatga tatcagcccg gaagttcgtc aggttctgcc ggttagcggt 4980 gctgatgttg ttgctcatgc tcatcgtctg atgaactttc tgtatgttgc tagcccgggt 5040 cgtaccctgg gtgaaggtga tccgccggat ctggctgatc agggtagcaa cagctgggct 5100 gttgctccgg gtaaaaccgc taacggtaac gctctgctgc tgcagaaccc tcatctgagc 5160 tggaccaccg attattttac ctattatgaa gctcatctgg ttaccccgga ttttgaagtg 5220 tatggtgcta cccagatcgg tctgccggtt atccgtgttg cttttaacca gcgtatgggt 5280 atcaccaaca ccgttaacgg tatggttggt gctaccaact atcgtctgac cctgcaggat 5340 ggtggttatc tgtatgatgg tcaggttcgt ccgtttgaac gtcgtcaggc tagctatcgt 5400 ctgcgtcagg ctgatggtac caccgttgat aaaccgctgg aaatccgttc cagcgttcat 5460 ggtccggttt ttgaacgtgc tgatggtacc gctgttgctg ttcgtgttgc tggtctggat 5520 cgtccgggta tgctggaaca gacttttgat atgatcaccg ctgatagctt tgatgattat 5580 gaagctgctc tggctcgtat gcaggttccg accttgaaca tcgtttatgc tgatcgtgaa 5640 ggtaccatca actatagctt taacggtgtt gctccgaaac gtgctgaagg tgacatcgct 5700 ttttggcagg gtctggttcc gggtgatagc agccgttatc tgtggaccga aacccatccg 5760 ctggatgatc tgccgcgtgt taccaacccg ccgggtggtt ttgttcagaa cagcaacgat 5820 ccgccgtgga ccccgacctg gccggttacc tataccccga aagattttcc gagctatctg 5880 gctccgcaga ccccgcatag cctgcgtgct cagcagagcg ttcgtctgat gagcgaaaac 5940 gatgatctga ccctggaacg ttttatggct ctgcagctga gccatcgtgc tgttatggct 6000 gatcgtaccc tgccggatct gatcccggct gctctgatcg atccggatcc ggaagttcag 6060 gctgctgctc gtctgctggc tgcgtgggat cgtgaattta ccagcgatag ccgtgctgct 6120 ctgctgtttg aagaatgggc tcgtctgttt gctggtcaga actttgctgg tcaggctggc 6180 tttgctaccc cgtggagcct ggataaaccg gttagcaccc cgtatggtct gcgtgatccg 6240 aaagctgctg ttgatcagct gcgtaccgct atcgctaaca ccaaacgtaa atatggtgct 6300 atcgatcgtc cgtttggtga tgctagccgt atgatcctga acgatgttaa cgttccgggt 6360 gctgctggtt atggtaacct gggtagcttt cgtgttttta cctggagcga tccggatgaa 6420 aacggtgtgc gtaccccggt tcatggtgaa acctgggttg ctatgatcga atttagcacc 6480 ccggttcgtg cttatggtct gatgagctat ggtaacagcc gtcagccggg taccacccat 6540 tatagcgatc agatcgaacg tgttagccgt gctgattttc gtgaactgct gctgcgtcgt 6600 gaacaggttg aagctgctgt tcaggaacgt accccgttta actttaaacc gtgaagtaga 6660 tgccgaccgc gagatccact taacgttact gaaatcatca aacagcttga cgaatctgga 6720 tataagatcg ttggtgtcga tgtcagctcc ggagttgaga caaatggtgt tcaggatctc 6780 gataagatac gttcatttgt ccaagcagca aagagtgcct tctagtgatt taatagctcc 6840 atgtcaacaa gaataaaacg cgttttcggg tttacctctt ccagatacag ctcatctgca 6900 atgcattaat gcattgactg caacctagta acgccttnca ggctccggcg aagagaagaa 6960 tagcttagca gagctatttt cattttcggg agacgagatc aagcagatca acggtcgtca 7020 agagacctac gagactgagg aatccgctct tggctccacg cgactatata tttgtctcta 7080 attgtacttt gacatgctcc tcttctttac tctgatagct tgactatgaa aattccgtca 7140 ccagcncctg ggttcgcaaa gataattgca tgtttcttcc ttgaactctc aagcctacag 7200 gacacacatt catcgtaggt ataaacctcg aaatcanttc ctactaagat ggtatacaat 7260 agtaaccatg catggttgcc tagtgaatgc tccgtaacac ccaatacgcc ggccgaaact 7320 tttttacaac tctcctatga gtcgtttacc cagaatgcac aggtacactt gtttagaggt 7380 aatccttctt tctagaagtc ctcgtgtact gtgtaagcgc ccactccaca tctccactcg 7440 accaattcgc cctatagtga gtcgtattag taaattaacc ctcactaaag ggaacaaaag 7500 ctgggcaaag cttgaggccg gacaaattca gcctgcaggc taaagtttct attacatgat 7560 atttgtcgtg gacaagggcg tctggggtaa aaaggaatgt gggcagagag ctctcggctt 7620 aaagaccggc ttcggagttc cagacccact cgaactgaaa acgatgatta gagataagta 7680 gatctatttg gacagtataa gaatcctagt cggctagtat ttctccactt ttactttaaa 7740 attccccaga gtaagggggc aggttcgtcg tcacgggtag cctcgtctcg aggcatcgcg 7800 gatagcatta tcttggggta tcacgagtaa cttcatcgtg gcattatcga taggtactgt 7860 aagtactgta ggaacttatc cgggcatgga tatgtatagg tacatacgat gcactaaaag 7920 tggagaaata ctagccgagt agagatctta taccatccga atagatttac tccttataat 7980 caaggggcaa caatcagttg tgacgatgtc ggctgtggga gtgcagttta ggggtcatca 8040 aagcacagga aaacgtgttg tatggatgcc tatcgtcttg actattagaa aggaagaggg 8100 gaaaaaccct catcaggagc gattgtaagt tcggccatag taatgatatt ggccattaat 8160 ggacgtgtgt atgtatacat acacggttca tttgccgcat tcggaccaaa accgctgctg 8220 ccgcagacac agtgcttcca tggcctcgaa caaccggtta ttcttctctt ccatgtcctc 8280 gggacgaagg aaatttcgga gccctaacag ccgtaccgga cttgcgttaa catggttgcg 8340 aagaactaaa gactgatggg gtcatgattg gcagtgttga cacttcatgt cttgaaccta 8400 gctagcagaa aaatgaatgc tctatgtgac actggtagat ctcaacgcat catgttccac 8460 ctttagtgta aacacggcaa gctgtccgca acgtcggtac caacgtttga acacacaacc 8520 ttctgccact ccaggagttt agcccacggc ttactggatg aaacgtaatc tcccgctacc 8580 ccctcaattc cattggggtc tagagtagtc ggaagaacgg ctgtcgcctt gcgctgcggt 8640 taaagcaccc accgtggtgg ctactgcttc tcgggaggag aaatcctggg tactcgagca 8700 cttgcgctag gggatgaaac atccatggca tattggcgaa agcaatgcgc catgttcggc 8760 gcggcacatg acactcttga gaagccgtat tcttcgcaaa ctttcctatt tacattcagc 8820 ctctaactat aacacgatcg gaaatgcctg aacgagcccg ccctcatgtg cctagcgtca 8880 aacaacgcca agcacacaac gcctcctgct attcttccct cttctcactg ccatcactgg 8940 gcgcaagctg caatgtcctt ctcgaattca agcgggcaaa tcaagattgg gaaaatgcgc 9000 catcttggtt tggatcttga tgtctatccg catcaaggtc ttggtttggt cgtttattta 9060 gtagcaaagg caaatcaaaa cgtgcgcgcc aagtgccagg gtccagactc cagactggag 9120 catttagcct gaatatcgtc tctagaatct ggtgctatta ttcaggatat agtaactatg 9180 atctactcgg gccatgttcg gctcctccgc acggccaagt tgtctgccgg cgtctggacc 9240 ggactcatgg cttggcttct ccagggatag catggcattg gcaaaactct ccgccacgat 9300 tcgagcatag ctaccgcagc cacgcggcat tgtttccaga ttgagatatg tcactattag 9360 ccggaatgca aacatcgagc tgacctgcgc aacatctcca tgagttccga agtcgacctc 9420 cgaagcaata ttggtctagt attacggatg atatagtggt ccccggaccc aatgagccgt 9480 gtgtttttta ccgcctttgc tctgaggatt aacaagattc gtgcaattgg ggactgtgcg 9540 gagtaactgg gctggtttac tgctttgccg acgaaagaaa cgtataaaca gccgactcgt 9600 ccactgccac tcgggacgat ctcccaaccc agacagcttc ccagaaccca tcttcaacac 9660 cctgacacca tgccccagtt cgacatccta tgcaagactc ctccgaaggt gctcgtccgc 9720 cagttcgtgg agcgattcga gcgcccctcg ggcgaaaaga tcgccttgtg tgccgctgag 9780 cttacctacc tctgctggat gatcacccac aacggcaccg ccatcaagcg ggctaccttt 9840 atgagttaca ataccatcat tagcaactcc ctctcgtttg acatcgtcaa caagtccctg 9900 cagttcaagt acaagaccca aaaggccacc atcctcgagg cctcgctgaa gaagctgatc 9960 cccgcatggg aattcactat catcccctac tatggtcaga agcatcagtc tgacatcacc 10020 gacattgtaa gcagtctcca gctccagttt gagtcctccg aggaggccga taaaggcaac 10080 tcccactcca agaagatgct caaggctttg ctgagtgaag gcgagtccat ctgggaaata 10140 accgaaaaga tcttgaacag tttcgagtac accagccgct tcacgaagac caagacgctg 10200 taccagttcc tgttcctcgc cacattcatc aattgcggac gcttcagtga tatcaagaac 10260 gtggacccga agtcattcaa gctggttcag aacaagtacc tcggtgtcat cattcagtgc 10320 ttggtcaccg agaccaagac atccgtcagc cgtcacatct acttcttttc cgcaagaggg 10380 cgtatcgacc ccctagtgta cctagacgaa ttcctgcgca actcggagcc cgtcctaaag 10440 cgcgtcaacc gtaccggcaa ctctagcagc aacaagcagg agtaccagct cttgaaggat 10500 aacctcgtcc gatcatacaa caaggcgctc aagaagaacg ctccatacag catctttgct 10560 atcaagaatg ggccaaagtc acacatcggc cgccacctca tgacctcgtt tctgtcgatg 10620 aagggcctca ctgagcttac caacgttgtt ggcaactggt ccgataagcg cgccagcgcc 10680 gtggcgcgga cgacttacac tcatcagatc acagctatcc ctgaccacta cttcgccttg 10740 gtcagtcgct attacgccta cgacccaatt agtaaagaga tgatcgctct caaggacgaa 10800 accaacccca tcgaggagtg gcaacacatc gagcagctta aaggctccgc cgagggctcg 10860 atccggtacc ccgcctggaa cggtatcata tcgcaggagg tcttggatta cctgagcagt 10920 tatatcaatc gccgcatatg ataaggatcc acttaacgtt actgaaatca tcaaacagct 10980 tgacgaatct ggatataaga tcgttggtgt cgatgtcagc tccggagttg agacaaatgg 11040 tgttcaggat ctcgataaga tacgttcatt tgtccaagca gcaaagagtg ccttctagtg 11100 atttaatagc tccatgtcaa caagaataaa acgcgttttc gggtttacct cttccagata 11160 cagctcatct gcaatgcatt aatgcattga ctgcaaccta gtaacgcctt ncaggctccg 11220 gcgaagagaa gaatagctta gcagagctat tttcattttc gggagacgag atcaagcaga 11280 tcaacggtcg tcaagagacc tacgagactg aggaatccgc tcttggctcc acgcgactat 11340 atatttgtct ctaattgtac tttgacatgc tcctcttctt tactctgata gcttgactat 11400 gaaaattccg tcaccagcnc ctgggttcgc aaagataatt gcatgtttct tccttgaact 11460 ctcaagccta caggacacac attcatcgta ggtataaacc tcgaaatcan ttcctactaa 11520 gatggtatac aatagtaacc atgcatggtt gcctagtgaa tgctccgtaa cacccaatac 11580 gccggccgaa acttttttac aactctccta tgagtcgttt acccagaatg cacaggtaca 11640 cttgtttaga ggtaatcctt ctttctagaa gtcctcgtgt actgtgtaag cgcccactcc 11700 acatctccac tcgaccaatt cgccctatag tgagtcgtat taagcttcct gcagttcgaa 11760 gttcctattc tctagaaagt ataggaactt cagagcgctt tccttgtatc tctacacaca 11820 ggctcaaatc aataagaaga acggttcgtc tttttcgttt atatcttgca tcgtcccaaa 11880 gctattggcg ggatattctg tttgcagttg gctgacttga agtaatctct gcagatcttt 11940 cgacactgaa atacgtcgag cctgctccgc ttggaagcgg cgaggagcct cgtcctgtca 12000 caactaccaa catggagtac gataagggcc agttccgcca gctcattaag agccagttca 12060 tgggcgttgg catgatggcc gtcatgcatc tgtacttcaa gtacaccaac gctcttctga 12120 tccagtcgat catccgctga aggcgctttc gaatctggtt aagatccacg tcttcgggaa 12180 gccagcgact ggtgacctcc agcgtccctt taaggctgcc aacagctttc tcagccaggg 12240 ccagcccaag accgacaagg cctccctcca gaacgccgag aagaactgga ggggtggtgt 12300 caaggaggag taagctcctt attgaagtcg gaggacggag cggtgtcaag aggatattct 12360 tcgactctgt attatagata agatgatgag gaattggagg tagcatagct tcatttggat 12420 ttgctttcca ggctgagact ctagcttgga gcatagaggg tcctttggct ttcaatattc 12480 tcaagtatct cgagtttgaa cttattccct gtgaaccttt tattcaccaa tgagcattgg 12540 aatgaacatg aatctgagga ctgcaatcgc catgaggttt tcgaaataca tccggatgtc 12600 gaaggcttgg ggcacctgcg ttggttgaat ttagaacgtg gcactattga tcatccgata 12660 gctctgcaaa gggcgttgca caatgcaagt caaacgttgc tagcagttcc aggtggaatg 12720 ttatgatgag cattgtatta aatcaggaga tatagcatga tctctagtta gctcaccaca 12780 aaagtcagac ggcgtaacca aaagtcacac aacacaagct gtaaggattt cggcacggct 12840 acggaagacg gagaagccac cttcagtgga ctcgagtacc atttaattct atttgtgttt 12900 gatcgagacc taatacagcc cctacaacga ccatcaaagt cgtatagcta ccagtgagga 12960 agtggactca aatcgacttc agcaacatct cctggataaa ctttaagcct aaactataca 13020 gaataagata ggtggagagc ttataccgag ctcccaaatc tgtccagatc atggttgacc 13080 ggtgcctgga tcttcctata gaatcatcct tattcgttga cctagctgat tctggagtga 13140 cccagagggt catgacttga gcctaaaatc cgccgcctcc accatttgta gaaaaatgtg 13200 acgaactcgt gagctctgta cagtgaccgg tgactctttc tggcatgcgg agagacggac 13260 ggacgcagag agaagggctg agtaataagc cactggccag acagctctgg cggctctgag 13320 gtgcagtgga tgattattaa tccgggaccg gccgcccctc cgccccgaag tggaaaggct 13380 ggtgtgcccc tcgttgacca agaatctatt gcatcatcgg agaatatgga gcttcatcga 13440 atcaccggca gtaagcgaag gagaatgtga agccaggggt gtatagccgt cggcgaaata 13500 gcatgccatt aacctaggta cagaagtcca attgcttccg atctggtaaa agattcacga 13560 gatagtacct tctccgaagt aggtagagcg agtacccggc gcgtaagctc cctaattggc 13620 ccatccggca tctgtagggc gtccaaatat cgtgcctctc ctgctttgcc cggtgtatga 13680 aaccggaaag gccgctcagg agctggccag cggcgcagac cgggaacaca agctggcagt 13740 cgacccatcc ggtgctctgc actcgacctg ctgaggtccc tcagtccctg gtaggcagct 13800 ttgccccgtc tgtccgcccg gtgtgtcggc ggggttgaca aggtcgttgc gtcagtccaa 13860 catttgttgc catattttcc tgctctcccc accagctgct cttttctttt ctctttcttt 13920 tcccatcttc agtatattca tcttcccatc caagaacctt tatttcccct aagtaagtac 13980 tttgctacat ccatactcca tccttcccat cccttattcc tttgaacctt tcagttcgag 14040 ctttcccact tcatcgcagc ttgactaaca gctaccccgc ttgagcagac atcaccatgg 14100 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt 14160 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg 14220 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca 14280 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg 14340 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt 14400 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg 14460 agcaggactg aagtagatgc cgaccgcggg atccacttaa cgttactgaa atcatcaaac 14520 agcttgacga atctggatat aagatcgttg gtgtcgatgt cagctccgga gttgagacaa 14580 atggtgttca ggatctcgat aagatacgtt catttgtcca agcagcaaag agtgccttct 14640 agtgatttaa tagctccatg tcaacaagaa taaaacgcgt tttcgggttt acctcttcca 14700 gatacagctc atctgcaatg cattaatgca ttgactgcaa cctagtaacg ccttncaggc 14760 tccggcgaag agaagaatag cttagcagag ctattttcat tttcgggaga cgagatcaag 14820 cagatcaacg gtcgtcaaga gacctacgag actgaggaat ccgctcttgg ctccacgcga 14880 ctatatattt gtctctaatt gtactttgac atgctcctct tctttactct gatagcttga 14940 ctatgaaaat tccgtcacca gcncctgggt tcgcaaagat aattgcatgt ttcttccttg 15000 aactctcaag cctacaggac acacattcat cgtaggtata aacctcgaaa tcanttccta 15060 ctaagatggt atacaatagt aaccatgcat ggttgcctag tgaatgctcc gtaacaccca 15120 atacgccggc cgaaactttt ttacaactct cctatgagtc gtttacccag aatgcacagg 15180 tacacttgtt tagaggtaat ccttctttct agaagtcctc gtgtactgtg taagcgccca 15240 ctccacatct ccactcgacc tgcagttcga agttcctatt ctctagaaag tataggaact 15300 tcagagcgct ttccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg 15360 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 15420 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 15480 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg 15540 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 15600 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 15660 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 15720 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 15780 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 15840 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 15900 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa 15960 tttag 15965 <210> 18 <211> 2079 <212> DNA <213> Wild-type Gl-7-ACA acylase gene of Pseudomonas sp. GK16 <400> 18 atggaaccga ccagcacccc gcaggctccg atcgctgctt ataaaccgcg tagcaacgaa 60 atcctgtggg atggttatgg tgttccgcat atctatggtg ttgacgctcc gagcgctttt 120 tatggttatg gctgggctca ggctcgtagc catggtgata acatcctgcg tctgtatggt 180 gaagctcgtg gtaaaggcgc tgaatattgg ggtccggatt acgaacagac gaccgtttgg 240 ctgctgacca acggtgttcc ggaacgtgct cagcagtggt atgctcagca gagcccggat 300 tttcgtgcta acctggatgc ttttgctgct ggtatcaacg cttatgctca gcagaacccg 360 gatgatatca gcccggaagt tcgtcaggtt ctgccggtta gcggtgctga tgttgttgct 420 catgctcatc gtctgatgaa ctttctgtat gttgctagcc cgggtcgtac cctgggtgaa 480 ggtgatccgc cggatctggc tgatcagggt agcaacagct gggctgttgc tccgggtaaa 540 accgctaacg gtaacgctct gctgctgcag aaccctcatc tgagctggac caccgattat 600 tttacctatt atgaagctca tctggttacc ccggattttg aaatctatgg tgctacccag 660 atcggtctgc cggttatccg ttttgctttt aaccagcgta tgggtatcac caacaccgtt 720 aacggtatgg ttggtgctac caactatcgt ctgaccctgc aggatggtgg ttatctgtat 780 gatggtcagg ttcgtccgtt tgaacgtcgt caggctagct atcgtctgcg tcaggctgat 840 ggtaccaccg ttgataaacc gctggaaatc cgttccagcg ttcatggtcc ggtttttgaa 900 cgtgctgatg gtaccgctgt tgctgttcgt gttgctggtc tggatcgtcc gggtatgctg 960 gaacagtatt ttgatatgat caccgctgat agctttgatg attatgaagc tgctctggct 1020 cgtatgcagg ttccgacctt taacatcgtt tatgctgatc gtgaaggtac catcaactat 1080 agctttaacg gtgttgctcc gaaacgtgct gaaggtgaca tcgctttttg gcagggtctg 1140 gttccgggtg atagcagccg ttatctgtgg accgaaaccc atccgctgga tgatctgccg 1200 cgtgttacca acccgccggg tggttttgtt cagaacagca acgatccgcc gtggaccccg 1260 acctggccgg ttacctatac cccgaaagat tttccgagct atctggctcc gcagaccccg 1320 catagcctgc gtgctcagca gagcgttcgt ctgatgagcg aaaacgatga tctgaccctg 1380 gaacgtttta tggctctgca gctgagccat cgtgctgtta tggctgatcg taccctgccg 1440 gatctgatcc cggctgctct gatcgatccg gatccggaag ttcaggctgc tgctcgtctg 1500 ctggctgcgt gggatcgtga atttaccagc gatagccgtg ctgctctgct gtttgaagaa 1560 tgggctcgtc tgtttgctgg tcagaacttt gctggtcagg ctggctttgc taccccgtgg 1620 agcctggata aaccggttag caccccgtat ggtgttcgtg atccgaaagc tgctgttgat 1680 cagctgcgta ccgctatcgc taacaccaaa cgtaaatatg gtgctatcga tcgtccgttt 1740 ggtgatgcta gccgtatgat cctgaacgat gttaacgttc cgggtgctgc tggttatggt 1800 aacctgggta gctttcgtgt ttttacctgg agcgatccgg atgaaaacgg tgtgcgtacc 1860 ccggttcatg gtgaaacctg ggttgctatg atcgaattta gcaccccggt tcgtgcttat 1920 ggtctgatga gctatggtaa cagccgtcag ccgggtacca cccattatag cgatcagatc 1980 gaacgtgtta gccgtgctga ttttcgtgaa ctgctgctgc gtcgtgaaca ggttgaagct 2040 gctgttcagg aacgtacccc gtttaacttt aaaccgtga 2079 <210> 19 <211> 692 <212> PRT <213> Amino acid sequence coded by nucleotide of sequence No.18 <400> 19 Met Glu Pro Thr Ser Thr Pro Gln Ala Pro Ile Ala Ala Tyr Lys Pro 1 5 10 15 Arg Ser Asn Glu Ile Leu Trp Asp Gly Tyr Gly Val Pro His Ile Tyr 20 25 30 Gly Val Asp Ala Pro Ser Ala Phe Tyr Gly Tyr Gly Trp Ala Gln Ala 35 40 45 Arg Ser His Gly Asp Asn Ile Leu Arg Leu Tyr Gly Glu Ala Arg Gly 50 55 60 Lys Gly Ala Glu Tyr Trp Gly Pro Asp Tyr Glu Gln Thr Thr Val Trp 65 70 75 80 Leu Leu Thr Asn Gly Val Pro Glu Arg Ala Gln Gln Trp Tyr Ala Gln 85 90 95 Gln Ser Pro Asp Phe Arg Ala Asn Leu Asp Ala Phe Ala Ala Gly Ile 100 105 110 Asn Ala Tyr Ala Gln Gln Asn Pro Asp Asp Ile Ser Pro Glu Val Arg 115 120 125 Gln Val Leu Pro Val Ser Gly Ala Asp Val Val Ala His Ala His Arg 130 135 140 Leu Met Asn Phe Leu Tyr Val Ala Ser Pro Gly Arg Thr Leu Gly Glu 145 150 155 160 Gly Asp Pro Pro Asp Leu Ala Asp Gln Gly Ser Asn Ser Trp Ala Val 165 170 175 Ala Pro Gly Lys Thr Ala Asn Gly Asn Ala Leu Leu Leu Gln Asn Pro 180 185 190 His Leu Ser Trp Thr Thr Asp Tyr Phe Thr Tyr Tyr Glu Ala His Leu 195 200 205 Val Thr Pro Asp Phe Glu Ile Tyr Gly Ala Thr Gln Ile Gly Leu Pro 210 215 220 Val Ile Arg Phe Ala Phe Asn Gln Arg Met Gly Ile Thr Asn Thr Val 225 230 235 240 Asn Gly Met Val Gly Ala Thr Asn Tyr Arg Leu Thr Leu Gln Asp Gly 245 250 255 Gly Tyr Leu Tyr Asp Gly Gln Val Arg Pro Phe Glu Arg Arg Gln Ala 260 265 270 Ser Tyr Arg Leu Arg Gln Ala Asp Gly Thr Thr Val Asp Lys Pro Leu 275 280 285 Glu Ile Arg Ser Ser Val His Gly Pro Val Phe Glu Arg Ala Asp Gly 290 295 300 Thr Ala Val Ala Val Arg Val Ala Gly Leu Asp Arg Pro Gly Met Leu 305 310 315 320 Glu Gln Tyr Phe Asp Met Ile Thr Ala Asp Ser Phe Asp Asp Tyr Glu 325 330 335 Ala Ala Leu Ala Arg Met Gln Val Pro Thr Phe Asn Ile Val Tyr Ala 340 345 350 Asp Arg Glu Gly Thr Ile Asn Tyr Ser Phe Asn Gly Val Ala Pro Lys 355 360 365 Arg Ala Glu Gly Asp Ile Ala Phe Trp Gln Gly Leu Val Pro Gly Asp 370 375 380 Ser Ser Arg Tyr Leu Trp Thr Glu Thr His Pro Leu Asp Asp Leu Pro 385 390 395 400 Arg Val Thr Asn Pro Pro Gly Gly Phe Val Gln Asn Ser Asn Asp Pro 405 410 415 Pro Trp Thr Pro Thr Trp Pro Val Thr Tyr Thr Pro Lys Asp Phe Pro 420 425 430 Ser Tyr Leu Ala Pro Gln Thr Pro His Ser Leu Arg Ala Gln Gln Ser 435 440 445 Val Arg Leu Met Ser Glu Asn Asp Asp Leu Thr Leu Glu Arg Phe Met 450 455 460 Ala Leu Gln Leu Ser His Arg Ala Val Met Ala Asp Arg Thr Leu Pro 465 470 475 480 Asp Leu Ile Pro Ala Ala Leu Ile Asp Pro Asp Pro Glu Val Gln Ala 485 490 495 Ala Ala Arg Leu Leu Ala Ala Trp Asp Arg Glu Phe Thr Ser Asp Ser 500 505 510 Arg Ala Ala Leu Leu Phe Glu Glu Trp Ala Arg Leu Phe Ala Gly Gln 515 520 525 Asn Phe Ala Gly Gln Ala Gly Phe Ala Thr Pro Trp Ser Leu Asp Lys 530 535 540 Pro Val Ser Thr Pro Tyr Gly Val Arg Asp Pro Lys Ala Ala Val Asp 545 550 555 560 Gln Leu Arg Thr Ala Ile Ala Asn Thr Lys Arg Lys Tyr Gly Ala Ile 565 570 575 Asp Arg Pro Phe Gly Asp Ala Ser Arg Met Ile Leu Asn Asp Val Asn 580 585 590 Val Pro Gly Ala Ala Gly Tyr Gly Asn Leu Gly Ser Phe Arg Val Phe 595 600 605 Thr Trp Ser Asp Pro Asp Glu Asn Gly Val Arg Thr Pro Val His Gly 610 615 620 Glu Thr Trp Val Ala Met Ile Glu Phe Ser Thr Pro Val Arg Ala Tyr 625 630 635 640 Gly Leu Met Ser Tyr Gly Asn Ser Arg Gln Pro Gly Thr Thr His Tyr 645 650 655 Ser Asp Gln Ile Glu Arg Val Ser Arg Ala Asp Phe Arg Glu Leu Leu 660 665 670 Leu Arg Arg Glu Gln Val Glu Ala Ala Val Gln Glu Arg Thr Pro Phe 675 680 685 Asn Phe Lys Pro 690 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer for cloning hygromycin B resistance gene cassette <400> 20 gcaaagcttc cttgtatctc tacacacagg c 31 <210> 21 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for cloning hygromycin B resistance gene cassette <400> 21 gctatcgatt acgtattaat taatcgagtg gagatgtgga gtgg 44 <210> 22 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> forward primer for hygromycin B + FRP <400> 22 gcaaagcttc ctgcagttcg aagttcctat tctctagaaa gtataggaac ttcagagcgc 60 tttccttgta tctctacaca caggc 85 <210> 23 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for hygromycin B + FRP <400> 23 aaagcgctct gaagttccta tactttctag agaataggaa cttcgaactg caggtcgagt 60 ggagatgtgg agtgg 75 <210> 24 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer for promoter Pxyl1 <400> 24 cgaaagcttg aggccggaca aattcagcc 29 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for promoter Pxyl1 <400> 25 ggtgtcaggg tgttgaagat ggg 23 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Flp <400> 26 gtttaaaccc cgggtacgta aattaaccct cactaaaggg 40 <210> 27 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Flp <400> 27 taatacgact cactataggg c 21 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> T3 primer <400> 28 aattaaccct cactaaaggg 20 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for error prone PCR <400> 29 gctgaattcc catccggcat ctgtagggcg 30 <210> 30 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for error prone PCR <400> 30 cgtcatatgg gaagatgaat atactgaaga tg 32 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for cloning mutated primer <400> 31 cgtcatatgc atccaagaac ctttatttcc 30 <210> 32 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for cloning mutated primer <400> 32 gctgaattcg ccaattaggg agcttacgc 29 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for transforming E. coli DH5alpha <400> 33 tagcatgcca ttaacctagg 20 <210> 34 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for transforming E. coli DH5alpha <400> 34 gggtagctgt tagtcaagc 19

Claims (17)

  1. (a) CPC (cephalosporin C) 고생산성 아크레모니움 크리소제눔 (Acremonium chrysogenum) 균주를 제조하는 단계;
    (b) 제1프로모터 및 이와 작동가능하게 연결된 CPC 아실라제를 암호화하는 폴리뉴클레오티드를 포함하는 제1발현벡터로 상기 (a) 단계의 균주를 형질전환 시키는 단계; 및
    (c) 서열번호 10 내지 14로 이루어진 군에서 선택된 염기서열로 이루어진 제2프로모터 및 이와 작동가능하게 연결된 상기 폴리뉴클레오티드를 포함하는 제2발현벡터로 상기 (b) 단계의 균주를 형질전환 시키는 단계를 포함하며,
    상기 (c) 단계는 반복적으로 수행되는 것을 특징으로 하는 7-ACA 고생산성 아크레모니움 크리소제눔 균주의 제조 방법.
  2. 제1항에 있어서 상기 (a) 단계의 CPC 고생산성 아크레모니움 크리소제눔 균주는 30 g/L 이상의 CPC 생산능을 가지는 것을 특징으로 하는 방법.
  3. 제1항에 있어서, 상기 (a) 단계의 CPC 고생산성 아크레모니움 크리소제눔 균주는 KCTC13079BP의 기탁번호를 가지는 균주인 것을 특징으로 하는 방법.
  4. 제1항에 있어서, 상기 CPC 아실라제는 서열번호 19로 표시되는 아미노산서열로 이루어진 것을 특징으로 하는 방법.
  5. 제1항에 있어서, 상기 제1발현벡터는 서열번호 9의 염기서열로 이루어진 것을 특징으로 하는 방법
  6. 삭제
  7. 제1항에 있어서, 상기 제2발현벡터는 서열번호 15 내지 서열번호 17로 이루어진 군에서 하나이상 선택된 것임을 특징으로 하는 방법.
  8. 제1항에 있어서, 상기 (c) 단계의 반복은 5회이상 반복인 것을 특징으로 하는 방법.
  9. 제1항에 있어서, 상기 (c) 단계의 형질전환은 서열번호 15로 이루어진 발현벡터, 서열번호 16으로 이루어진 발현벡터, 서열번호 17로 이루어진 발현벡터, 서열번호 15로 이루어진 발현벡터, 서열번호 16으로 이루어진 발현벡터의 순으로 순차적으로 이루어지는 것을 특징으로 하는 방법.
  10. 제9항에 있어서, 상기 형질전환은 서열번호 15로 이루어진 발현벡터, 서열번호 16으로 이루어진 발현벡터 및 서열번호 17로 이루어진 발현벡터로 이루어진 군에서 선택된 발현벡터로 추가로 형질전환되는 것을 특징으로 하는 방법.
  11. 제1항에 있어서, 상기 7-ACA 고생산성 아크레모니움 크리소제눔 균주의 7-ACA 생산능은 15g/L이상인 것을 특징으로 하는 방법.
  12. 제1항 내지 제5항 및 제7항 내지 제11항 중 어느 한 항의 방법에 의해서 제조된 아크레모니움 크리소제눔 균주.
  13. 제12항에 있어서, 상기 균주의 7-ACA 생산능은 15g/L이상인 것을 특징으로 하는 균주.
  14. 제13항에 있어서, 상기 균주의 7-ACA 생산능은 30g/L이상인 것을 특징으로 하는 균주.
  15. (a) 제12항의 균주를 7-ACA를 생산할 수 있는 조건에서 배양하는 단계; 및
    (b) 배지에서 7-ACA를 회수하는 단계를 포함하는 7-ACA의 생산방법.
  16. 서열번호 10 내지 14로 이루어진 군에서 선택된 염기서열로 이루어진 아크레모니움 크리소제눔 균주에서의 단백질 고발현용 프로모터.
  17. 서열번호 15 내지 17로 이루어진 군에서 선택된 염기서열로 이루어진 아크레모니움 크리소제눔 균주에서의 CPC 아실라제 고발현용 발현 벡터.
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