KR102528337B1 - 정의된 서열 및 길이의 dna 단일 가닥 분자의 확장 가능한 생명공학적 생산 - Google Patents

정의된 서열 및 길이의 dna 단일 가닥 분자의 확장 가능한 생명공학적 생산 Download PDF

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KR102528337B1
KR102528337B1 KR1020197008132A KR20197008132A KR102528337B1 KR 102528337 B1 KR102528337 B1 KR 102528337B1 KR 1020197008132 A KR1020197008132 A KR 1020197008132A KR 20197008132 A KR20197008132 A KR 20197008132A KR 102528337 B1 KR102528337 B1 KR 102528337B1
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플로리안 프라에토리우스
헨드릭 디에츠
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Abstract

본 발명은 (1) 위유전자 핵산을 제공하는 단계; (2) 위유전자 핵산을 벡터 속으로 통합시키고, 박테리아 세포를 상기 벡터로 형질 전환시키고, 박테리아 배양 조건 하에서 상기 벡터로부터 전구체 ssDNA를 생성시키는 단계; (3) 박테리아 배양물로부터 전구체 ssDNA를 분리하는 단계; (4) 자가-절단 DNA 서열이 활성화되는 반응 조건 하에서 전구체 ssDNA를 소화하는 단계; 및 (5) 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)을 분리 및 수득하는 단계를 포함하는, DNA 단일 가닥 분자의 재조합적 생산을 위한 방법에 관한 것이다. 본 발명의 방법은 DNA 단일 가닥 분자의 대량 생산에 적합하다. 본 발명은 또한 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)의 용도, 특히 DNA 나노 기술에서의 또는 연구 도구로서의 용도에 관한 것이다.

Description

정의된 서열 및 길이의 DNA 단일 가닥 분자의 확장 가능한 생명공학적 생산
본 발명은 DNA 단일 가닥 분자의 재조합적 생산 방법에 관한 것으로서, 상기 방법은 (1) 위유전자 핵산을 제공하는 단계; (2) 위유전자 핵산을 벡터 속으로 통합시키고, 박테리아 세포를 상기 벡터로 형질 전환시키고, 그리고 박테리아 배양 조건 하에서 상기 벡터로부터 전구체 ssDNA를 생성시키는 단계; (3) 박테리아 배양물로부터 전구체 ssDNA를 분리하는 단계; (4) 자가-절단 DNA 서열이 활성화되는 반응 조건 하에서 전구체 ssDNA를 소화하는 단계; 및 (5) 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)을 분리하고 수득하는 단계를 포함한다. 본 발명의 방법은 DNA 단일 가닥 분자의 대량 생산에 적합하다. 본 발명은 또한 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)의 사용, 특히 DNA 나노 기술에서의 또는 연구 도구로서의 사용에 관한 것이다.
DNA 올리고뉴클레오타이드는 생명 과학 및 의학에서 다양한 응용을 위하여 필요하다. 올리고뉴클레오타이드는 예를 들어 중합효소 연쇄 반응(PCR) 또는 실시간 PCR(RT-PCR) 또는 정량적 PCR(Q-PCR) 각각을 수행하기 위한 프라이머로서 요구된다. 또한, 올리고뉴클레오타이드는 새로운 유전자의 합성을 위한 출발 물질로서 필요하다. DNA 올리고뉴클레오타이드에 대한 증가하는 수요는, 원하는 서열의 DNA 올리고뉴클레오타이드를 가장 작은 양으로 신속하게 생산하는 것을 주로 전문적으로 다루는 산업을 이끌었다. DNA 올리고뉴클레오타이드는 전형적으로 고체 상에 순환 화학적 합성 공정에서 염기에서 염기로(base by base) 생산된다. 이 공정은 현미경 비드 상에서 또는 칩 표면상의 잉크젯 방법으로 수행될 수 있다. 염기 부가 반응은 100% 효율로 진행되지 않기 때문에, 어떠한 길이의 DNA 단일 가닥도 생산할 수 없다. 수율은 표적 DNA 올리고뉴클레오타이드의 원하는 길이로 지수적으로 감소한다. 일반적으로 최대 100개 염기 길이 분자가 정당한 노력으로 생산될 수 있다. 일부 회사는 최대 200 염기의 길이를 갖는 DNA 올리고뉴클레오타이드의 (비용 집약적인) 합성을 제공한다. 또한, DNA 올리고뉴클레오타이드의 합성은 통상적으로 밀리그램 규모로 제한되며, 이는 지금까지 대부분의 적용에 충분하다.
현재, 사용자-정의 서열을 갖는 단일 가닥 DNA 올리고뉴클레오타이드를 대량 으로(> 그램 규모) 및/또는 더 긴 길이로(> 200 염기 길이) 요구하는 새로운 기술이 개발되었다. 이러한 요구는 DNA 올리고뉴클레오타이드의 합성을 위한 기존의 공정으로는 충족될 수 없다. 특히 DNA 나노 기술에서, 사용자-정의 서열을 갖는 단일 가닥 DNA 올리고뉴클레오타이드는 "건축 재료"로서 대량으로 필요하다. DNA 나노 기술은 잠재적인 의학적 또는 심지어 화학적/물리적 관련성을 가진 새로운 약물 전달체 및 추가 나노입자의 생성을 위한 특별한 가능성을 약속한다(Jones et al., 2015). 그러나, 필요한 출발 물질(즉, DNA 올리고뉴클레오타이드)은 현재 확장 가능한 방식으로 이용 가능하지 않다. 또한, 촉매 활성 DNA 서열(DNAzyme) 및 DNA 앱타머(DNA aptamer)는 진단으로서의, 치료 및 감지에서의 응용과 관련하여 증가하는 관심을 얻고 있다(Krug et al., 2015; Keefe et al., 2010; Ng et al., 2006; Torabi et al., 2015). 이러한 응용을 위해, DNA 올리고뉴클레오타이드의 대량 생산은 큰 관심사이다.
본 발명에 따르면, 이러한 목적은 DNA 단일 가닥 분자의 재조합 생산 방법에 의해 해결되며, 이는 다음의 단계를 포함한다:
(1) 위유전자(pseudogene) 핵산을 제공하는 단계,
여기서 상기 위유전자 핵산은 적어도 하나의 표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열 및 각각의 표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열에 인접하는 두 개의 자가-절단 DNA 서열을 포함하는 핵산임,
(2) 위유전자 핵산을 벡터 속으로 통합시키고, 박테리아 세포를 상기 벡터로 형질전환시키고, 그리고 박테리아 배양 조건 하에서 상기 벡터로부터 전구체 ssDNA를 생성시키는 단계,
여기서 상기 전구체 ssDNA는 위유전자 핵산을 포함함;
(3) 박테리아 배양물로부터 전구체 ssDNA를 분리하는 단계;
(4) 자가-절단 DNA 서열이 활성화되는 반응 조건 하에서 전구체 ssDNA를 소화시키는 단계; 및
(5) 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)을 분리하고, 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)을 수득하는 단계.
본 발명에 따르면, 이 목적은 본 발명의 방법에서 수득된 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)을 다음에 사용하는 것에 의하여 해결된다:
- DNA 나노 기술에서,
- 연구 도구로서,
- 진단용 프로브로서,
- 유전자 합성에서,
- 유전자 요법에서,
- 분자 감지에서/분자 센서로서.
본 발명이 하기에 보다 상세하게 기술되기 전에, 본 발명은 이들이 다양할 수 있으므로 본 명세서에 기술된 특정 방법론, 프로토콜 및 시약에 한정되지 않는다는 것을 이해해야 한다. 본 명세서에서 사용되는 용어는 오직 특정 구현예를 설명하기 위한 것이며, 오직 첨부된 청구항에 의해서만 제한될 것인 본 발명의 범위를 제한하려는 의도가 아님을 이해해야 한다. 다르게 정의되지 않는 한, 본 명세서에서 사용되는 모든 기술 및 과학 용어는, 본 분야의 일반 기술자 중 하나에 의하여 일반적으로 이해되는 것과 동일한 의미를 갖는다. 본 발명의 목적을 위해, 본 명세서에 인용된 모든 문헌은 참조에 의하여 그 전체로서 통합된다.
농도, 양 및 다른 수치 데이터는 범위 형식으로 본 명세서에 표현되거나 제시될 수 있다. 이러한 범위 형식은 단지 편의 및 간략화를 위해 사용된 것이므로 범위의 한계로서 명시적으로 언급된 수치 값을 포함할 뿐만 아니라, 모든 개별 수치 값 또는 그 범위 이내에 포괄되는 하부-범위들을, 마치 각각의 수치 값 및 하위-범위들이 명시적으로 언급된 것처럼 포함하는 것으로 유연하게 해석되어야 함을 이해해야 한다. 예시로서, "50 내지 3,000 뉴클레오타이드"의 수치 범위는 50 내지 3,000의 명시적으로 언급된 값뿐만 아니라, 지시된 범위 이내의 개별 값 및 하위- 범위들을 또한 포함하는 것으로 해석되어야 한다. 따라서 이 수치 범위 안에는 50, 51, 52... 2,998, 2,999, 3,000과 같은 개별 값들, 그리고 5 내지 150, 100 내지 250, 200 내지 500, 500 내지 2,500 등과 같은 하위 범위들이 포함된다. 이러한 동일한 원리가 "적어도 하나"와 같이 오직 하나의 수치 값을 언급하는 범위에도 적용된다. 또한, 그러한 해석은 기술된 범위 또는 특성의 폭에 관계없이 적용되어야 한다.
ssDNA를 생산하는 방법
본 발명은 DNA 단일 가닥 분자, 특히 단일 가닥 DNA 올리고뉴클레오타이드 및 폴리펩타이드의 재조합적 생산 방법을 제공한다.
상기 방법은 다음의 단계를 포함한다:
(1) 위유전자(pseudogene) 핵산을 제공하는 단계,
여기서 상기 위유전자 핵산은 적어도 하나의 표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열 및 각각의 표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열에 인접하는 두 개의 자가-절단 DNA 서열을 포함하는 핵산임;
(2) 위유전자 핵산을 벡터 속으로 통합시키고, 박테리아 세포를 상기 벡터로 형질전환시키고, 그리고 박테리아 배양 조건 하에서 상기 벡터로부터 전구체 ssDNA를 생성시키는 단계,
여기서 상기 전구체 ssDNA는 위유전자 핵산을 포함함;
(3) 박테리아 배양물로부터 전구체 ssDNA를 분리하는 단계;
(4) 자가-절단 DNA 서열이 활성화되는 반응 조건 하에서 전구체 ssDNA를 소화시키는 단계; 및
(5) 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)을 분리 및 수득하는 단계.
(1) 위유전자 핵산의 설계 및 제공
"위유전자(pseudogene) 핵산"은 적어도 하나의 표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열 및 각 표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열에 인접하는 두 개의 자가-절단 DNA 서열을 포함하는 핵산이다.
바람직하게는, 위유전자 핵산은 2개, 3개, 4개 또는 약 50개를 초과하는 표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열과 같은, 하나 또는 다수의 표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열을 포함한다.
표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열은 그들의 길이뿐만 아니라 그들의 서열에 있어서도 동일 또는 상이할 수 있다.
바람직하게는, 표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열은 화학적으로 합성된 올리고뉴클레오타이드가 이용 가능한 범위를 커버 및 초과하면서, 약 20 뉴클레오타이드 내지 약 수천 뉴클레오타이드 범위의 길이를 갖는다.
바람직하게는, 위유전자 핵산 내의 표적 DNA 올리고- 또는 폴리뉴클레오타이드 서열의 각각에 인접하는 자가-절단 DNA 서열은 자가-절단 데옥시리보자임 또는 DNA자임이며, 예를 들어
- Zn2 +- 의존성 DNA자임,
예컨대, I-R3 및 문헌(Gu et al., 2013)에 기술된 다른 변이체들, 그뿐만 아니라 문헌(Gu et al., 2013)에 기술된 것들로부터 유래한 변이체들.
데옥시리보자임(DNA자임이라고도 함)는 화학 반응을 촉매할 수 있는 구조를 형성하는 DNA 분자이다(Breaker, 1997). 자가-처리 반응을 촉매하는 DNA가 있다 (Carmi et al., 1996). 이러한 데옥시리보자임은 특정 반응 조건에 노출될 때, 고유한 촉매 활성에 기초하여 변형된 DNA 구조물을 생성하기 위해 활용될 수 있다. 예를 들어, 다양하게 유도된 진화 전략을 사용하여 창출된 산화(Carmi et al., 1996), 탈푸린화(Sheppard et al., 2000), 또는 가수분해(Chandra et al., 2009 참조) 메커니즘을 채택한 조작된 자가-절단 데옥시리보자임이 있다.
최근에, 높은 속도 및 서열 특이성을 가지면서 DNA를 가수 분해하는 조작된 자가-절단 데옥시리보자임의 2가지 클래스가 기술되었다(Gu et al., 2013). 그러한 한가지 데옥시리보자임은, I-R3으로 명명된 것인데(도 3 참조), 1개 또는 2개의 이중 가닥 하부 구조가 측면에 위치하는 17개의 뉴클레오타이드로 구성된 작은 촉매 코어를 수반한다. 이 데옥시리보자임 클래스의 대표자는 거의 중성인 pH 및 밀리몰 농도의 Zn2 + 존재 하에서 배양될 때, DNA 가수분해에 대하여 ~ 1분-1 (~ 40초의 반감기)의 관찰 속도 상수(kobs)를 나타낸다. 이 데옥시리보자임은 3 '산소(oxygen)와 ApA 결합의 인(phosphorus) 중심 사이에 있는 포스포에스테르 결합을 절단하여 5' 포스페이트기를 갖는 3' 절단 단편을 생성한다.
P1- TAGTTGAGCTGT- P2 - P3 - P2'- ACGTTGAAG - P1'
또는
P2'- ACGTTGAAG - P1- P3 - P1'- TAGTTGAGCTGT - P2
여기서 TAGTTGAGCTGT는 SEQ ID NO. 1이고, ACGTTGAAG는 SEQ ID NO. 2이다;
그리고 여기서 P3는 임의의 서열의 스페이서이고, 여기서 P1 및 P1'은 DNA 이중 나선을 형성하는 2개의 상보적인 서열이다. P2와 P2'에 대해서도 동일하다.
P1 - [SEQ ID NO. 1] - P2 - P3 - P2' - [SEQ ID NO. 2] - P1'
P2'- [SEQ ID NO. 2] - P1- P3 - P1'- [SEQ ID NO. 1] - P2
본 발명에서 사용되는 자가-절단 DNA 서열(즉, 자가-절단 데옥시리보자임 또는 DNA자임)은 정의된 반응 조건 하에서만 활성화된다(및 자가-절단을 촉매한다).
예를 들어, Zn2 + 의존성 DNA자임, 예컨대 I-R3은 Zn 이온의 첨가를 요구한다.
위유전자 핵산은 통상적인/확립된 유전자 합성 방법에 의해 합성될 수 있다.
예를 들어, 이는 주문되어 상업적 공급업체가 합성할 수 있다.
(2) 박테리아 배양물 중에서 전구체 ssDNA의 생산
단계 (2)에서, 위유전자 핵산은 벡터 속으로 통합된다. 상기 벡터는 형질 전환을 통해 박테리아 세포 속으로 도입되고, 전구체 ssDNA는 박테리아 배양 조건하에서 상기 벡터로부터 생산된다.
몇몇 구현예에서, 벡터는 2개, 3개, 4개 또는 그 이상의 벡터와 같은 적어도 하나의 벡터이다.
"전구체 단일 가닥 DNA" 또는 "전구체 ssDNA"는 단일 가닥 형태의 위유전자 핵산 및 벡터 백본을 포함한다.
바람직하게는, 박테리아는 대장균, 특히 DH5알파, XL-1블루 또는 JM109와 같은 K12-유도된 대장균 안전 균주들로부터 선택된다.
바람직한 구현예에서, 단계 (2)의 벡터는 파지미드이다.
일부 구현예에서, 파지미드는 2개, 3개, 4개 또는 그 이상의 파지미드와 같은 하나 이상의 파지미드이다.
상기 (적어도 하나의) 파지미드는 플라스미드 백본을 포함하고, 선택적으로 다음과 같은 추가의 성분(들)을 포함할 수 있다:
- 포장 서열(a packaging sequence),
- 세포 분열 동안 파지미드의 증식을 보장하는 성분(들),
- 선택 마커(a selection marker), 전형적으로 항생제 내성 유전자.
상기 구현예에서, 추가로 하기와 같은 추가 성분(들)을 포함하는 헬퍼 플라스미드 또는 헬퍼 파지가 사용된다:
- 박테리오파지, 예컨대 M13 박테리오파지의 단백질을 코딩하는 유전자,
- 세포 분열 동안 헬퍼 플라스미드의 증식을 보장하는 성분(들),
- 선택 마커, 전형적으로 항생제 내성 유전자.
이 구현예에서, (적어도 하나의) 파지미드 및 헬퍼 플라스미드 또는 헬퍼 파지 둘 모두가, 바람직하게는 동시 형질 전환을 통해 박테리아 세포 속으로 도입된다.
이 구현예에서, 벡터가 파지미드(및 헬퍼 플라스미드를 또한 사용하는)인 경우, 파지미드는 롤링 서클 증폭(RCA)을 통해 박테리아 세포 내에서 증폭된다. 하나 이상의 파지미드를 이용하는 구현예에 대해서도 동일하게 적용된다.
롤링 서클 증폭(RCA)은 파지미드의 단일 가닥 형태인 파지미드 ssDNA를 초래한다. 상기 파지미드 ssDNA는 이 구현예에서 "전구체 ssDNA"이다.
상기 파지미드 ssDNA는 바람직하게는 세포로부터 주변 매질로 분비되는 세포 표면상의 파지-유사 입자 속으로 바람직하게 포장된다.
파지-유사 입자는 정상 파지 게놈 대신에 파지미드 ssDNA를 함유한다.
예를 들어, 헬퍼 플라스미드가 M13 박테리오파지의 단백질을 코딩하는 유전자를 함유하는 일 구현예에서, 세포 용해없이 숙주 세포로부터 분비되는 M13 박테리오파지-유사 입자가 형성된다.
(3) 박테리아 배양물로부터 전구체 ssDNA를 수득
전구체 ssDNA는 박테리아 배양물로부터 분리된다.
상기 분리 또는 정제는 당업계에 공지된 통상적인/확립된 방법을 사용하여 수행될 수 있으며,
예컨대 원심분리, 침전, 용제-기반 추출, 크로마토그래피 방법, 또는 이들의 조합이 있다.
하나의 구현예에서, (적어도 하나의) 파지미드 및 헬퍼 플라스미드가 사용되는 경우, 분리 또는 정제는 다음을 포함한다:
- 세포 배양물로부터, 바람직하게는 배양 상등액으로부터 파지-유사 입자를 추출하는 것,
예를 들어 세포를 펠렛화하고, 파지-유사 입자를 상등액으로부터 제거/추출하는 것을 통해서 수행함, 예를 들어 침전(예를 들어, PEG를 사용)에 의함,
- 파지-유사 입자로부터 파지미드 ssDNA를 추출하는 것,
예를 들어 단백질 코트의 화학적 용해 및 ssDNA의 에탄올 침전을 통해서 수행함.
예를 들어, ssDNA의 다운스트림 공정은 다음 단계를 포함할 수 있다: 숙주 세포 및 세포 외 생성된 파지-유사 입자의 분리, PEG-매개 파지 침전, 화학적 파지 용해 및 에탄올로 ssDNA 침전.
참고문헌은 예를 들어 Kick et al., 2015.
(4) 전구체 ssDNA의 자동촉매적 소화
그 다음, 분리된 전구체 ssDNA는 자동촉매적 방식으로 소화된다, 즉 자가-절단 DNA 서열은 전구체 ssDNA를 절단할 것이다.
상기 소화는 자가-절단 DNA 서열이 활성화되는 반응 조건 하에서 수행된다.
일 구현예에서, Zn2 +-의존성 DNA자임, 예컨대 I-R3이 이용되는 경우, 단계 (4)의 상기 소화는 Zn2 + 이온의 첨가에 의하여 촉발되는데, 즉 자가-절단 DNA 서열이 활성화되는 반응 조건은 Zn2+ 이온의 존재를 요구한다.
(5) 표적 단일 가닥 DNA 분자(들)의 수득
다음으로, 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드는 특히 단계 (4)의 부산물인 자가-절단 DNA 서열로부터 분리될 것이다.
상기 분리 또는 정제는 당업계에 공지된 통상적인/확립된 방법을 사용하여 수행될 수 있으며,
예컨대
침전, 예컨대 에탄올 및 초산칼륨, 또는 폴리에틸렌 글리콜을 사용하는 것,
크로마토 그래피,
또는 이들의 조합이 있다.
마지막으로, 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)이 얻어진다.
(6) 추가 단계들
일 구현예에서, 본 발명의 방법은 다음의 추가 단계를 포함하는 것이다:
(6) 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이(들)의 추가 처리.
예를 들어, 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)은 DNA 오리가미 구조물, 타일-기반 DNA 나노구조물 또는 결정형 DNA 나노 물질로 자가-조립 및/또는 폴딩될 수 있다. 대안적으로는, 그들은 다른 분자를 결합, 검출 또는 가공하기 위한 앱타머 또는 DN자임으로서 사용될 수 있다.
- ssDNA 분자의 대량 생산
일 구현예에서, 박테리아 배양은 교반-탱크 생물 반응기와 같은 생물 반응기 중에서 수행된다.
이러한 구현예는 그램 규모의 표적 ssDNA 분자의 생산을 허용한다.
따라서, 단일 가닥 DNA 분자의 대량 생산이 가능하다.
얻어진 ssDNA 분자의 용도
본 발명은 DNA 나노 기술에서 본 발명의 방법으로 얻은 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드의 용도를 제공한다.
예를 들어, 표적 단일 가닥 DNA 올리고뉴클레오타이드(들) 및 폴리뉴클레오타이드(들)는 DNA 오리가미 구조, 즉 스캐폴드 가닥 및 몇몇 스테이플 가닥으로 자가-조립될 수 있도록 설계 될 수 있다.
DNA 오리가미 구조는 다음과 같을 수 있다:
DNA 나노 막대, 본원의 실시예 1에 기술된 바와 같은 것;
DNA 나노 기공, 유럽 특허 출원 제2 695 949호에 기술된 바와 같은 것;
DNA 헬릭스 튜브, 본원의 실시예 3에 기술된 바와 같은 것;
DNA 포인터 개체, 본원의 실시예 3 또는 문헌(Bai et al., 2012)에 기술된 바와 같은 것;
치료학적으로 활성인 DNA 나노 구조물 또는 약물 전달 비히클
위치결정 장치(positioning devices),
나노 센서.
본 발명은 본 발명의 방법에서 수득한 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)의 연구 도구로서의 용도를 제공한다.
본 발명은 본 발명의 방법에서 수득한 표적 단일 가닥 DNA 올리고- 또는 폴리펩타이드(들)의 진단용 프로브로서의 용도를 제공한다.
본 발명은 본 발명의 방법에서 수득한 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)의 유전자 요법에서의 용도를 제공한다.
본 발명은 본 발명의 방법에서 수득한 표적 단일 가닥 DNA 올리고- 또는 폴리뉴클레오타이드(들)의 분자 감지에서의/분자 센서로서의 용도를 제공한다.
바람직한 구현예의 추가 설명
본 발명자들은 박테리아에서 단일 가닥 DNA 올리고뉴클레오타이드의 재조합적 생산을 위한 생명공학적 방법을 제시한다. 상기 방법으로, 수천 개까지 달하는 염기 길이를 갖는 단일 가닥 DNA 분자가 임의의 확장 가능한 양으로 제조될 수 있다. 이 방법으로 인해 DNA-기반 나노구조물의 대량 생산이 현실적으로 가능하게 된다.
상기 방법은 도 1의 흐름도에 도시된 바와 같이 7개의 단계를 포함한다.
단계 (1)에서, 특별하게 조절된 형태로 생성될 DNA 뉴클레오타이드 서열을 포함하는 위유전자가 설계되거나 구성된다. 이 형태는, 하기에서 보다 상세하게 설명되는데, 본 발명의 방법에 필수적이다.
단계 (2)에서, 위유전자는 현재/확립된 유전자 합성 방법을 통해 생성된다.
다음으로, 위유전자는 벡터 또는 플라스미드 속으로 통합되고, 확장 가능한 방식으로 액체 박테리아(대장균) 배양물 중에서 "연결된 전구체 ssDNA"로서 생성된다(단계 (3)).
그 다음, 결합된 전구체 ssDNA를 정제하고 분리한다(단계 (4)).
단계 (5)에서, 연결된 전구체 ssDNA는 자동촉매 방식으로 소화되며, 여기서 상기 소화는 본 발명의 방법의 중요한 하나의 특징이다.
상기 소화로부터 생성된 표적 단일 가닥 DNA 올리고뉴클레오타이드는 통상적 인 방법을 사용하여 단리된다(단계 (6)).
그 다음, 표적 단일 가닥 DNA 올리고뉴클레오타이드는 더 큰 스케일의 DNA 오리가미 구조의 생성을 위한 것과 같이, 추가로 가공될 수 있다(단계 (7): 다운 스트림 적용).
- 연결된 전구체 ssDNA의 생명공학적 생산
두 개의 DNA 플라스미드는 동시 형질 전환을 통해 대장균 세포 속으로 도입된다(도 2). "헬퍼 플라스미드"는 M13 박테리오파지의 단백질을 위한 유전자와 대장균(E. coli) 세포 분열 동안 딸 세포 속으로 헬퍼 플라스미드의 전파를 보장하는 추가 정보(플라스미드 백본)를 포함한다. "파지미드"(= 파지-플라스미드)는 특별하게 조절된 형태로 생성될 DNA 올리고뉴클레오타이드 서열("연결된 전구체 ssDNA") 및, 다른 것들 중에서, 세포 분열 동안 파지미드의 증식을 보장하는 추가 정보를 포함한다.
처음에는 두 개의 플라스미드 각각을 복제를 통해 생성할 필요가 있다. 연결된 전구체 ssDNA와 함께 파지미드를 생성하기 위해서는, 통상의 유전자 합성을 미리 수행할 필요가 있다.
세포 내에서, 파지미드는 증폭된다(롤링 서클 증폭을 통해). 동시에, M13 파지 단백질이 생산된다.
단일 가닥 파지미드는 파지 단백질에 의해 인식되고 파지 입자로 포장되는 특별한 포장 서열을 함유한다. 결과적으로, 세포는 정상 파지 게놈 대신에 파지미드의 단일 가닥 형태를 포함하는 파지-유사 입자를 분비하고, 그리하여 DNA 올리고뉴클레오타이드 서열을 생성시킨다.
파지 입자는 세포가 펠렛화 된 후 상등액으로부터 분리될 수 있다. 파지 입자에 중에 함유된 DNA는 정제될 수 있다.
상기 기술한 생명공학 프로세스는 박테리아 배양물 중에서 단백질의 재조합 생산이 그러하듯이 확장 가능하다(Kick et al., 2015).
- 연결된 전구체 ssDNA의 추가 처리
도 3은 상기 특별히 조절된 형태의 위유전자의 구조, 즉 "연결된 전구체 ssDNA"를 보여준다.
생산될 DNA 올리고뉴클레오타이드 서열의 양쪽 말단에 촉매적으로 활성있는 DNA 서열( "I-R3")이 측면 위치한다(Gu et al. (2013) 참조). 이들 DNA자임은 자가-절단 효과를 갖는다. 즉, 상기 DNA자임은 2가 아연 양이온의 첨가 후 및 적절한 반응 조건 하에서, 백본 가수분해를 촉매한다(도 3의 삽입도에서 검은 화살표로 나타낸 위치).
따라서, 연결된 전구체 ssDNA는 많은 상이한 DNA 올리고뉴클레오타이드 서열들을 포함할 수 있다.
전구체 ssDNA를 정제한 후, 촉매작용은 아연 첨가에 의해 개시될 수 있다("절단").
절단으로 인해, 전구체 ssDNA는 원하는 단일 가닥 DNA 올리고뉴클레오타이드 및 부산물인 촉매적 DNA자임으로 분리되거나 떨어진다. DNA자임은 표적 DNA 올리고뉴클레오타이드, 예를 들어 크로마토그래피를 통해 표적 DNA 올리고뉴클레오타이드로부터 분리될 수 있다.
- 본 발명의 방법의 이점
헬퍼 플라스미드(Marchi et al., 2014) 또는 효소(Schmidt et al, 2015)를 갖는 단일 가닥의 파지미드 DNA의 생명공학적 생산 방법이 당업계에 기술되어 있다. 또한, 사용자 정의된 서열에서 제한 엔도뉴클레아제의 도움으로 긴 원형의 ssDNA를 소화시키는 것을 포함하는 방법이 당업계에 기술되어 있다(Ducani et al., 2013).
문헌(Ducani et al. (2013))에 기술된 방법에서, 단백질 기반의 제한 뉴클레아제가 이용되고, 따라서 추가 시약으로 요구된다. 이러한 효소는 구매하거나 또는 별도의 생명공학적 공정 중에서 생산할 필요가 있다. 또한, Ducani 등이 기술 한 방법에서, 모든 스테이플 올리고뉴클레오타이드는 지루하고 확장되지 않는 예비 겔 전기 영동을 사용하여 정제된다. 본 명세서에 기술된 방법에서는, 표적 DNA 올리고뉴클레오타이드 서열을 생성하기 위해 요구되는 모든 성분이 위유전자와 함께 헬퍼 플라스미드 파지미드의 조합 시스템 중에 이미 함유되어 있기 때문에, 추가의 비용 집약적 또는 노동 집약적인 시약들이 소화를 위해 필요하지 않다.
본 발명의 필수적인 하나의 측면은, 유도된 진화, 즉 DNA자임(Gu et al., 2013)을 통해 생산된 촉매적으로 활성인 DNA 서열의 사용이다. 상기 촉매적으로 활성인 DNA 서열은 연결된 전구체 ssDNA 중에 포함되어 있으며, 표적 DNA 올리고뉴클레오타이드 서열과 함께 생명공학적 공정 중에서 증폭된다. 문헌(Gu et al.)에 의하여 제시된 방법은 상이한 길이의 올리고뉴클레오타이드를 생산하지만, 이는 임의의 서열의 것이 아니다. 이들의 전구체 DNA는 동일한 불필요한(redundant) 서열의 수개의 카피본을 포함하고 있다. 본 명세서에 기술된 방법은, DNA 오리가미의 생산에 필요한 임의의 서열을 갖는 올리고뉴클레오타이드의 생산을 가능하게 한다. 또한, 문헌(Gu et al.)은 동일한 말단 서열을 사용하고, 그리하여 동일한 DNA자임을 사용하는 반면에, 본 발명의 방법은 상이한 표적 올리고뉴클레오타이드 서열들을 생성하기 위한 상이한 DNA자임 서열들을 사용한다.
하기 실시 예 및 도면은 본 발명을 예시하는 것으로서, 본 발명을 한정하는 것은 아니다.
도 1: 연결된 전구체 단일 가닥 DNA의 생명공학적 생산을 보여주는 흐름도.
도 2: 헬퍼 파지미드 단일 가닥 DNA의 헬퍼 플라스미드-보조된 생산의 개략도.
도 3: 표적 분자를 추출하기 위한 5개의 표적 서열(굵은 선) 및 (5+1)x2인 측면에 인접한 촉매적 DNA자임 서열(헤어핀 구조)을 함유하는 파지미드의 개략도.
도 4: 생명공학적으로 생산된 단일 가닥 출발 물질을 이용한 DNA 오리가미 나노로드의 생산.
A, 파지미드의 도식적 표현(왼쪽)과 DNA 나노로드의 내부 가닥 토폴로지.
B, 화학적 고상 합성을 통해 생성된 스테이플 올리고뉴클레오타이드를 사용하는 10-나선 번들의 자가-조립체의 생성물이 있는 아가로스 겔의 뷰(왼쪽)를, 본 발명의 방법에 의해 생산된 스테이플 올리고뉴클레오타이드를 사용한 경우(오른쪽)와 비교한다. 두 샘플 모두 비교 가능한 조립 품질을 의미하는 유사한 이동 특성을 나타낸다.
C, 스테이플 올리고뉴클레오타이드가 본 발명의 방법에 의해 생산된 것인 나노로드의 투과 전자 현미경 사진.
도 5: 포함된 DNA자임 서열에 의해 원하는 표적 DNA 올리고뉴클레오타이드 내로 연결된 전구체 ssDNA의 자동촉매적 소화물의 설명.
보여지는 것은 아가로스 겔의 뷰인데, 그 위에 연결된 전구체 ssDNA의 소화 반응의 생성물이 배양 시간의 함수로서 전기 영동적으로 분리되었다.
도 6: DNA자임 서열의 최적화.
A, 왼쪽 : DNA자임의 도식적 표현 (Type I-R3). 필수 염기쌍(본 발명자들의 실험에서 밝혀진 바와 같이)은 글자로 표시되고, 교환 가능한 염기쌍은 줄로 나타내었다. 삼각형은 절단 위치를 나타낸다. 오른쪽: 하나의 염기쌍에서 다른 DNA자임의 두 변이체의 반응 속도론에 대한 겔-전기 영동 분석. 보다 느린 이동 밴드는 절단되지 않은 올리고뉴클레오타이드에 상응하고, 더 빠른 이동 밴드는 반응 생성물에 상응한다. τ는 생성물 밴드의 강도가 비 절단된 DNA자임의 강도를 초과하는 첫 번째 시점으로 정의된다. 상단 겔은 문헌(Gu et al., 2013)에 설명된 바와 같이, 원래 서열의 절단을 나타내는 반면에, 하부 겔은 G-C-염기쌍이 A-T 염기쌍에 의하여 대체된 변이체의 절단을 나타낸다. 비록 이 염기쌍이 Gu 등에 의한 원조 간행물에서 교환가능한 것으로 분류되었음에도 불구하고, 이러한 교환은 DNA자임의 현저하게 낮은 촉매 활성을 야기한다.
B, 왼쪽: DNA자임에 의해 분리된 나노로드를 위한 스테이플을 포함하는 두 개의 파지미드의 도식적 표현. 오른쪽: 두 개의 파지미드의 소화 속도에 대한 겔 전기영동적 분석. 하단 변이체는 Gu 등에 의한 원조 간행물로부터 필수 염기로의 분류에 기초하여 설계된 반면에, 상단 변이체는 필수 염기에 관한 본 발명자들의 연구결과를 기반으로 설계되었다. 상단의 최적화된 변이체는 신속하고 완전한 절단을 보여 주는 반면에(40분 후에 기본적으로 모든 DNA가 원하는 생성물에 해당하는 밴드중에 있음), 하단 변이체는 심지어 배양 24시간 후에도 불완전한 소화를 나타낸다.
도 7: 본 발명의 DNA자임-기반 방법을 사용하여 제조된 DNA 올리고뉴클레오타이드로부터 조립된 나노구조물.
A, 왼쪽 위: 3200 염기 길이의 스캐폴드와 31개의 스테이플 올리고뉴클레오타이드가 하나의 파지미드에 모두 포함되어 있는 48-나선-튜브의 도식적 표현. 왼쪽 아래: 소화되지 않은 파지미드(왼쪽), 소화된 파지미드(가운데) 및 폴딩된 48-나선-튜브(오른쪽)가 전기 영동되어 있는 아가로오스 겔의 이미지. 오른쪽: 네거티브 얼룩 투과 전자 현미경 사진(하단) 및 48-나선-튜브의 클래스 평균(상단).
B, 왼쪽 위: 7249 염기 길이의 M13-스캐폴드 및 161개의 스테이플 올리고뉴클레오타이드로부터 조립된 포인터 물체의 도식적 표현. 왼쪽 하단: 화학적으로 합성된(중앙) 또는 생명공학적으로 생산된(오른쪽) 스테이플을 사용하여 조립된 스캐폴드(왼쪽) 및 구조가 전기 영동되어 있는 아가로오스 겔의 이미지. 오른쪽: 포인터 물체의 네거티브 얼룩 투과 전자 현미경 및 클래스 평균(삽입).
실시예
실시예 1 DNA -기반 나노로드의 생성
프로토 타입으로서, 본 발명의 방법에 의해 독점적으로 제조된 DNA 물질로부터 DNA-기반 나노로드가 생성되었다. DNA-기반 나노로드는 DNA 오리가미(origami) 설계 방법(다음 문헌 참조: Rothemund, 2006; 및 US 특허출원 2007/117109 A1 및 2012/ 251583 A1; US 특허번호 7,842,793 B2 및 8,501,923 B2)으로 설계되고 개발되었으며, 벌집 격자에서 평행하게 정렬되고 가닥 연결을 통해 가교 결합된 10개의 DNA 이중 나선으로 구성된다(도 4A 참조). 나노로드의 경우, 각각 약 100 염기의 길이를 갖는 21개의 DNA 올리고뉴클레오타이드("스테이플 가닥")뿐만 아니라, 2500 개의 염기 길이의 단일 가닥 DNA 백본 분자("스캐폴드 가닥")가 필요하다. 필요한 모든 구성 요소는 이를 위하여 특별히 제작된 파지미드(phagemid)로부터 유래한다.
1.1 재료 및 방법
- 서열:
DNA자임( DNAzymes ):
P1 - [SEQ ID NO. 1] - P2 - P3 - P2' - [SEQ ID NO. 2] - P1'
P2'- [SEQ ID NO. 2] - P1- P3 - P1'- [SEQ ID NO. 1] - P2
여기서 SEQ ID NO. 1 = TAGTTGAGCTGT이고 SEQ ID NO. 2 = ACGTTGAAG;
그리고 여기서 P3은 임의의 서열의 스페이서이고, 여기서 P1 및 P1'은 DNA 이중 나선을 형성하는 2개의 상보적인 서열이다. P2와 P2'에 대해서도 동일하다.
각각 약 100 염기의 길이를 갖는 21 개의 올리고뉴클 레오타이드 :
SEQ ID NO. 3:
TACTCTTAGAAGTGTCCCAACTACACTAGAAGGACAGTGGCGAGAGGATTACGCGCCTAGATCAACTTTAATGTTGACTCGTGCACCCAACATGCTTTTTAGCTC
SEQ ID NO. 4:
GCACATTGAGGGCTGCTATTAAGACACGACTTATCCCTTTCTCAAAAGGCCAGCAAAGCGATCTGGCCCCAATAGGGGAACAAGAGGCAGAACATATCAAAGCGA
SEQ ID NO. 5:
CTTACCGAGAATAGACACCCGCCTTACAGCGAGGCGAAGGGCTTTAAATCAATCTAGAGCATCATACCAGGCGTTTCGTTCTTGGCGCCGCAACCACCTGTATGC
SEQ ID NO. 6:
TAGACCGCGAAAAATGACGGGGAAAGCCTGGCGAATAACTACGTTGCCTGACTCCCGGGGATATTCTCATAGCTCACTAACTATTGTGCTGTAGAGCTCCGTCTA
SEQ ID NO. 7:
GGCGACCAAACTCTCAGGGTTATTGTCTGATTTATCGCGTCCGGCGGTGCTACAGACCCCTGGTCCGCCCCCCTGACAAGTATAAAACCAGCATTTATCAAGGAT
SEQ ID NO. 8:
TCAGGGCATAAATCGCGTTAATATTTTGCGCGGGGATTAAGTTGCGCCTTATCCGGGCTGTAGTATCCACAGAATCACGCGTATGTTTGTCATTGTAAAAAAGAA
SEQ ID NO. 9:
CTTCGGTGTTTGGTCCATCCAAAAAGGATCTTCACAGAAAAATGTTTGCAAGCAGCAGTATTTCATTCAGAAAGCGGTCTGTGACTGGTGATAACCCAATACTCA
SEQ ID NO. 10:
AAGGAGCGGGAAGGCAATGATGAGGCACCTATCTCAAGGCCACGGATACCTGTCCGGCCACTGGTGCGGGAGGGAAGCACTATTAAAGAACCAGTTTGGTTCCGC
SEQ ID NO. 11:
CTACAGGAAGTTGGCTGCATAATTCTCTTTCACCAAATGCCGCAAAAAAAATTGTTGTGTCACCCAGTTACCTTCGGAAACCACTGATCTTTTCTACGTTAAGGGAGCTAGA
SEQ ID NO. 12:
AGAGGCGTGGGCGCTCTTCCGATACGGTGTATCTCAGTTCGGCGACCGCTGGGTAACCCTAAACACTACGTGAACCACCGAAATTCGCGTT
SEQ ID NO. 13:
TCATGAGGATCCTTCGCTGGTAGCGGTGGCTGAAGGCTCGTCCCTCCGAATGCCATCCGTAAGTGATCTTAGGGCGACACGGAATCCGCCTATGGCTTGGTATCT
SEQ ID NO. 14:
AAACTTGCATAGGCAAGCTCCCTCGTGCGTATGTACATTCGCTGTAGCGTCTTGCCCGGCGTCGGAAAACGGATACATATTTGAGACCCACGCTGCGCATTAGCA
SEQ ID NO. 15:
TTCGTTCATGTGAGCCCTTCGGGAAGCGCCCGGTACGCCAGCGGCGAACCCAACGTCAAAGGGAGATAGGAAAGTGCCACCTAAGTGTAGAAGGGGGACGTCTTG
SEQ ID NO. 16:
TCGACGCATGAAGTCGAGCGCCCTTTTTGATCCAGTTCGATGGTACTCACCATGTTGTGCAAACTCCGGTGTCCTGCCTTTTAAATTAAAATCAAGTCAAACCCG
SEQ ID NO. 17:
TAAAAAGAATCAGTACCGCGTATGTATTAAGTGCTCATCATTAATACGGAGGGCGCTGGCAAGCATTCAGGCTCACCAGTTACCAATGCTTGCCGCGTTGTTCCG
SEQ ID NO. 18:
ACAGGACCTACGGCGATCAAGTGATCCCACCAAGTCATTCTGCTGTTGACAATATTATTGAAGCCAGCCGGCTTAATAAGTGGTGGCCTAATATAAAGACAAAAA
SEQ ID NO. 19:
ACCCTGCTAACAGGAACTGTTGGGCGCTGATAATACCGCGCCACTTTAATAGAAAAATAAACAAGTGCTGGCGATCGGCAGCAGCCACTGGCGCTTACGGAACCG
SEQ ID NO. 20:
GCGCTCTGTTTTTTAAGGATCTCAAGAAATTATCAGTCTATTGGGAATACAGCATCTTTTACTTACTGTCTCGTTGTCAGAAGTCATCGTGGCCGGGAATTTTGG
SEQ ID NO. 21:
GAGCGAGGCTCTCCTGCTGGCGTTTTTCGTCTGACGGCTCCACATGAGCGTTCTTCGGGGCGAGAGTTGCGTCACGCTGCGCGTTACAGGGCAGCAATTATGAGT
SEQ ID NO. 22:
AGTCCAATGGCGCTACGCAGGAAAGAACATCCATAGATACGGTCCCCGAGTTGAGTGTTGTTCGTGGACTGTGGCGAGAAAGGACCTCTTCTACCATCTGTCTAT
SEQ ID NO. 23:
TTCAGCCTGTAGGTACTCAAAGGCGGTACTTCCTCGCCAACGTTAAAATCGGCAAAATCCCTTGATGGCCGGGAGCCCCCGATTCAAGGCG
스캐폴드 역할을 하는 파지미드 백본 : SEQ ID NO. 24
GTTGTcgtctctgaagATGTGAGCtacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtcAcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaaCTTAATTGCagcaagagacgCTTCT
전장 파지미드 ssDNA 서열 (= 전구체 ssDNA ): SEQ ID NO. 25:
ttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaaCTTAATTGCacGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTgtGCAATTAATTTTTTAAGAGTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTACTCTTAGAAGTGTCCCAACTACACTAGAAGGACAGTGGCGAGAGGATTACGCGCCTAGATCAACTTTAATGTTGACTCGTGCACCCAACATGCTTTTTAGCTCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGAGCTAAATTTTTTCAATGTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCACATTGAGGGCTGCTATTAAGACACGACTTATCCCTTTCTCAAAAGGCCAGCAAAGCGATCTGGCCCCAATAGGGGAACAAGAGGCAGAACATATCAAAGCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGCTTTGATTTTTTCGGTAAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTTACCGAGAATAGACACCCGCCTTACAGCGAGGCGAAGGGCTTTAAATCAATCTAGAGCATCATACCAGGCGTTTCGTTCTTGGCGCCGCAACCACCTGTATGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCATACAGTTccTGCGGTCTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAGACCGCGAAAAATGACGGGGAAAGCCTGGCGAATAACTACGTTGCCTGACTCCCGGGGATATTCTCATAGCTCACTAACTATTGTGCTGTAGAGCTCCGTCTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGACGGAGTTTTTTTGGTCGCTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGCGACCAAACTCTCAGGGTTATTGTCTGATTTATCGCGTCCGGCGGTGCTACAGACCCCTGGTCCGCCCCCCTGACAAGTATAAAACCAGCATTTATCAAGGATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATCCTTGATTTTTTGCCCTGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCAGGGCATAAATCGCGTTAATATTTTGCGCGGGGATTAAGTTGCGCCTTATCCGGGCTGTAGTATCCACAGAATCACGCGTATGTTTGTCATTGTAAAAAAGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCTTTTTTTTTTTCACCGAAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTTCGGTGTTTGGTCCATCCAAAAAGGATCTTCACAGAAAAATGTTTGCAAGCAGCAGTATTTCATTCAGAAAGCGGTCTGTGACTGGTGATAACCCAATACTCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGAGTATTGTTTTTCGCTCCTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAGGAGCGGGAAGGCAATGATGAGGCACCTATCTCAAGGCCACGGATACCTGTCCGGCCACTGGTGCGGGAGGGAAGCACTATTAAAGAACCAGTTTGGTTCCGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCGGAACCTTTTTTCCTGTAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTACAGGAAGTTGGCTGCATAATTCTCTTTCACCAAATGCCGCAAAAAAAATTGTTGTGTCACCCAGTTACCTTCGGAAACCACTGATCTTTTCTACGTTAAGGGAGCTAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTAGCTCCTTTTTCCACGCCTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAGGCGTGGGCGCTCTTCCGATACGGTGTATCTCAGTTCGGCGACCGCTGGGTAACCCTAAACACTACGTGAACCACCGAAATTCGCGTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAACGCGAAccTTTCCTCATGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCATGAGGATCCTTCGCTGGTAGCGGTGGCTGAAGGCTCGTCCCTCCGAATGCCATCCGTAAGTGATCTTAGGGCGACACGGAATCCGCCTATGGCTTGGTATCTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGATACCATTTTTGCAAGTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAACTTGCATAGGCAAGCTCCCTCGTGCGTATGTACATTCGCTGTAGCGTCTTGCCCGGCGTCGGAAAACGGATACATATTTGAGACCCACGCTGCGCATTAGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCTAATGCTTTTTTGAACGAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTCGTTCATGTGAGCCCTTCGGGAAGCGCCCGGTACGCCAGCGGCGAACCCAACGTCAAAGGGAGATAGGAAAGTGCCACCTAAGTGTAGAAGGGGGACGTCTTGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCAAGACGTTTTTTTGCGTCGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCGACGCATGAAGTCGAGCGCCCTTTTTGATCCAGTTCGATGGTACTCACCATGTTGTGCAAACTCCGGTGTCCTGCCTTTTAAATTAAAATCAAGTCAAACCCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGGGTTTGTTTTTTCTTTTTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAAAAAGAATCAGTACCGCGTATGTATTAAGTGCTCATCATTAATACGGAGGGCGCTGGCAAGCATTCAGGCTCACCAGTTACCAATGCTTGCCGCGTTGTTCCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGGAACAATTTTTGGTCCTGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACAGGACCTACGGCGATCAAGTGATCCCACCAAGTCATTCTGCTGTTGACAATATTATTGAAGCCAGCCGGCTTAATAAGTGGTGGCCTAATATAAAGACAAAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTTTGTCTccTTcAGCAGGGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACCCTGCTAACAGGAACTGTTGGGCGCTGATAATACCGCGCCACTTTAATAGAAAAATAAACAAGTGCTGGCGATCGGCAGCAGCCACTGGCGCTTACGGAACCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGGTTCCGTTTTTACAGAGCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGCTCTGTTTTTTAAGGATCTCAAGAAATTATCAGTCTATTGGGAATACAGCATCTTTTACTTACTGTCTCGTTGTCAGAAGTCATCGTGGCCGGGAATTTTGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCAAAATTTTTTTGCCTCGCTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAGCGAGGCTCTCCTGCTGGCGTTTTTCGTCTGACGGCTCCACATGAGCGTTCTTCGGGGCGAGAGTTGCGTCACGCTGCGCGTTACAGGGCAGCAATTATGAGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACTCATAAccTTTCCATTGGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCCAATGGCGCTACGCAGGAAAGAACATCCATAGATACGGTCCCCGAGTTGAGTGTTGTTCGTGGACTGTGGCGAGAAAGGACCTCTTCTACCATCTGTCTATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATAGACAGTTTTTAGGCTGAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTCAGCCTGTAGGTACTCAAAGGCGGTACTTCCTCGCCAACGTTAAAATCGGCAAAATCCCTTGATGGCCGGGAGCCCCCGATTCAAGGCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGCCTTGATTTTTGCTCACATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATGTGAGCTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAgcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacg
2. 대장균( E. coli ) 배양 조건
이 실시예에서, 파지미드 단일 가닥 DNA는 대장균 액체 배양물에서 생성되고 이로부터 정제되었다. 이를 위해, 화학적으로 적임인(competent) DH5알파 세포를 파지미드 및 헬퍼 플라스미드와 함께 공동-형질전환시켰다. 이와 같이 수득된 균주를 카나마이신 50 mg/l 및 카르베니실린 100 mg/l을 함유하는 2xYT-배지에서 성장시켰다. 37℃에서 밤새 배양한 후, 배양물을 4000 rcf에서 30분간 원심 분리하였다. 고체 PEG 8000 및 NaCl을 각각 3% (m/v)의 최종 농도로 상등액에 첨가하였다. 이어서 파지-유사 입자를 4000 rcf에서 30분 동안 원심 분리하여 침전시켰다. 문헌(Kick et al., 2015)에 기술된 바와 같이 ssDNA를 이들 입자로부터 추출하였다.
3. 소화 및 타겟 구조의 조립
그 다음, 파지미드 단일 가닥 DNA를 37℃에서 3시간 동안 반응 완충액(50 mM Hepes pH7, 100 mM NaCl, 2 mM ZnCl2)에서 배양하였다. 배양 후, DNA는 원하는 분절로 완전히 소화시킨다(도 5 참조). 그 후 DNA를 에탄올과 초산칼륨으로 침전시키고 오리가미 폴딩 완충액(10 mM Tris, 5 mM NaCl, 1 mM EDTA, 20 mM MgCl2) 중에 용해시킨다. DNA 용액을 65℃까지 15분간 가열한 다음, 60℃ 내지 40℃로 천천히 냉각시킨다(1℃ 당 3시간). 겔 전기 영동 분석(도 4B 참조) 및 투과 전자 현미경(도 4C 참조)은 표적 구조의 성공적인 조립을 확인한다.
실시예 2 DNA자임 염기 서열의 최적화
본 발명의 파지미드를 만들기 위해, 본 발명자들은 제한 효소 결합 부위가 삽입되는 것과 같은 방법으로 표적 올리고뉴클레오타이드 사이에 일정한 DNA자임 서열을 위치시키지 않는다. DNA자임의 서열은 생산될 올리고뉴클레오타이드의 말단 서열에 의존한다. 문헌(Gu et al., Biotechniques 2013)은 단순히 동일한 말단 서열을 사용하고 따라서 동일한 DNA자임을 사용하는 반면에, 본 발명의 방법은 상이한 표적 올리고뉴클레오타이드 서열을 생성하기 위해 상이한 DNA자임 서열을 사용한다.
DNA자임 서열 중의 필수 염기를 밝혀내기 위하여, 본 발명자들은 올리고뉴클레오타이드(두 개의 예시를 위해 도 6A 참조) 및 전장-길이 파지미드의 5개 버전(두 개의 예시를 위해 도 6B 참조)으로서 개별 DNA자임의 약 40개의 상이한 변이체를 스크리닝하였다. 본 발명자들은 DNA자임의 원조 간행물(Gu et al., JACS 2013)에서 필수적인 것으로 분류되지 않은 몇몇 염기가 실제로는 필수적이라는 것을 발견했다. 이는 본 발명자들이 수용가능한 시간에 원하는 생성물로 완전히 절단된 파지미드를 만들 수 있게 하였다(도 6B 참조).
DNA자임 올리고뉴클레오타이드(도 6A에 나타난 바와 같음):
G-C (상부): SEQ ID NO. 26
TTTTTTGCCCTGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCAGGGCATAAAT
A-T (하부): SEQ ID NO. 27
TTTTTTGCCCTGATAGTTGAGCTaTCACAGAATGTGAtGTTGAAGTCAGGGCATAAAT
파지미드 (도 6B에 나타난 바와 같음):
최적화된 버전 ( 상부): SEQ ID NO. 28
ttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaaCTTAATTGCacGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTgtGCAATTAATTTTTTAAGAGTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTACTCTTAGAAGTGTCCCAACTACACTAGAAGGACAGTGGCGAGAGGATTACGCGCCTAGATCAACTTTAATGTTGACTCGTGCACCCAACATGCTTTTTAGCTCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGAGCTAAATTTTTTCAATGTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCACATTGAGGGCTGCTATTAAGACACGACTTATCCCTTTCTCAAAAGGCCAGCAAAGCGATCTGGCCCCAATAGGGGAACAAGAGGCAGAACATATCAAAGCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGCTTTGATTTTTTCGGTAAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTTACCGAGAATAGACACCCGCCTTACAGCGAGGCGAAGGGCTTTAAATCAATCTAGAGCATCATACCAGGCGTTTCGTTCTTGGCGCCGCAACCACCTGTATGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCATACAGTTccTGCGGTCTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAGACCGCGAAAAATGACGGGGAAAGCCTGGCGAATAACTACGTTGCCTGACTCCCGGGGATATTCTCATAGCTCACTAACTATTGTGCTGTAGAGCTCCGTCTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGACGGAGTTTTTTTGGTCGCTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGCGACCAAACTCTCAGGGTTATTGTCTGATTTATCGCGTCCGGCGGTGCTACAGACCCCTGGTCCGCCCCCCTGACAAGTATAAAACCAGCATTTATCAAGGATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATCCTTGATTTTTTGCCCTGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCAGGGCATAAATCGCGTTAATATTTTGCGCGGGGATTAAGTTGCGCCTTATCCGGGCTGTAGTATCCACAGAATCACGCGTATGTTTGTCATTGTAAAAAAGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCTTTTTTTTTTTCACCGAAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTTCGGTGTTTGGTCCATCCAAAAAGGATCTTCACAGAAAAATGTTTGCAAGCAGCAGTATTTCATTCAGAAAGCGGTCTGTGACTGGTGATAACCCAATACTCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGAGTATTGTTTTTCGCTCCTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAGGAGCGGGAAGGCAATGATGAGGCACCTATCTCAAGGCCACGGATACCTGTCCGGCCACTGGTGCGGGAGGGAAGCACTATTAAAGAACCAGTTTGGTTCCGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCGGAACCTTTTTTCCTGTAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTACAGGAAGTTGGCTGCATAATTCTCTTTCACCAAATGCCGCAAAAAAAATTGTTGTGTCACCCAGTTACCTTCGGAAACCACTGATCTTTTCTACGTTAAGGGAGCTAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTAGCTCCTTTTTCCACGCCTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAGGCGTGGGCGCTCTTCCGATACGGTGTATCTCAGTTCGGCGACCGCTGGGTAACCCTAAACACTACGTGAACCACCGAAATTCGCGTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAACGCGAAccTTTCCTCATGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCATGAGGATCCTTCGCTGGTAGCGGTGGCTGAAGGCTCGTCCCTCCGAATGCCATCCGTAAGTGATCTTAGGGCGACACGGAATCCGCCTATGGCTTGGTATCTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGATACCATTTTTGCAAGTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAACTTGCATAGGCAAGCTCCCTCGTGCGTATGTACATTCGCTGTAGCGTCTTGCCCGGCGTCGGAAAACGGATACATATTTGAGACCCACGCTGCGCATTAGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCTAATGCTTTTTTGAACGAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTCGTTCATGTGAGCCCTTCGGGAAGCGCCCGGTACGCCAGCGGCGAACCCAACGTCAAAGGGAGATAGGAAAGTGCCACCTAAGTGTAGAAGGGGGACGTCTTGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCAAGACGTTTTTTTGCGTCGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCGACGCATGAAGTCGAGCGCCCTTTTTGATCCAGTTCGATGGTACTCACCATGTTGTGCAAACTCCGGTGTCCTGCCTTTTAAATTAAAATCAAGTCAAACCCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGGGTTTGTTTTTTCTTTTTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAAAAAGAATCAGTACCGCGTATGTATTAAGTGCTCATCATTAATACGGAGGGCGCTGGCAAGCATTCAGGCTCACCAGTTACCAATGCTTGCCGCGTTGTTCCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGGAACAATTTTTGGTCCTGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACAGGACCTACGGCGATCAAGTGATCCCACCAAGTCATTCTGCTGTTGACAATATTATTGAAGCCAGCCGGCTTAATAAGTGGTGGCCTAATATAAAGACAAAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTTTGTCTccTTcAGCAGGGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACCCTGCTAACAGGAACTGTTGGGCGCTGATAATACCGCGCCACTTTAATAGAAAAATAAACAAGTGCTGGCGATCGGCAGCAGCCACTGGCGCTTACGGAACCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGGTTCCGTTTTTACAGAGCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGCTCTGTTTTTTAAGGATCTCAAGAAATTATCAGTCTATTGGGAATACAGCATCTTTTACTTACTGTCTCGTTGTCAGAAGTCATCGTGGCCGGGAATTTTGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCAAAATTTTTTTGCCTCGCTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAGCGAGGCTCTCCTGCTGGCGTTTTTCGTCTGACGGCTCCACATGAGCGTTCTTCGGGGCGAGAGTTGCGTCACGCTGCGCGTTACAGGGCAGCAATTATGAGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACTCATAAccTTTCCATTGGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCCAATGGCGCTACGCAGGAAAGAACATCCATAGATACGGTCCCCGAGTTGAGTGTTGTTCGTGGACTGTGGCGAGAAAGGACCTCTTCTACCATCTGTCTATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATAGACAGTTTTTAGGCTGAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTCAGCCTGTAGGTACTCAAAGGCGGTACTTCCTCGCCAACGTTAAAATCGGCAAAATCCCTTGATGGCCGGGAGCCCCCGATTCAAGGCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGCCTTGATTTTTGCTCACATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATGTGAGCTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAgcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacg
최적화되지 않은 버전(하부): SEQ ID NO. 29
ttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaaCTTAATTGCgttgAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGCAATTAATTTTTTAAGAGTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTACTCTTAGAAGTGTCCCAACTACACTAGAAGGACAGTGGCGAGAGGATTACGCGCCTAGATCAACTTTAATGTTGACTCGTGCACCCAACATGCTTTTTAGCTCGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGAGCTAAATTTTTTCAATGTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCACATTGAGGGCTGCTATTAAGACACGACTTATCCCTTTCTCAAAAGGCCAGCAAAGCGATCTGGCCCCAATAGGGGAACAAGAGGCAGAACATATCAAAGCGAGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTTCGCTTTGTTTTTTCGGTAAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTTACCGAGAATAGACACCCGCCTTACAGCGAGGCGAAGGGCTTTAAATCAATCTAGAGCATCATACCAGGCGTTTCGTTCTTGGCGCCGCAACCACCTGTATGCGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGCATACAGTTTTTGCGGTCTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAGACCGCGAAAAATGACGGGGAAAGCCTGGCGAATAACTACGTTGCCTGACTCCCGGGGATATTCTCATAGCTCACTAACTATTGTGCTGTAGAGCTCCGTCTAGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTTAGACGGATTTTaTTGGTCGCTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGCGACCAAACTCTCAGGGTTATTGTCTGATTTATCGCGTCCGGCGGTGCTACAGACCCCTGGTCCGCCCCCCTGACAAGTATAAAACCAGCATTTATCAAGGATGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTATCCTTGATTTTTTGCCCTGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCAGGGCATAAATCGCGTTAATATTTTGCGCGGGGATTAAGTTGCGCCTTATCCGGGCTGTAGTATCCACAGAATCACGCGTATGTTTGTCATTGTAAAAAAGAAGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTTTCTTTTTTTTTTCACCGAAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTTCGGTGTTTGGTCCATCCAAAAAGGATCTTCACAGAAAAATGTTTGCAAGCAGCAGTATTTCATTCAGAAAGCGGTCTGTGACTGGTGATAACCCAATACTCAGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTTGAGTATTTTTTTCGCTCCTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAGGAGCGGGAAGGCAATGATGAGGCACCTATCTCAAGGCCACGGATACCTGTCCGGCCACTGGTGCGGGAGGGAAGCACTATTAAAGAACCAGTTTGGTTCCGCGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGCGGAACCTTTTaTCCTGTAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTACAGGAAGTTGGCTGCATAATTCTCTTTCACCAAATGCCGCAAAAAAAATTGTTGTGTCACCCAGTTACCTTCGGAAACCACTGATCTTTTCTACGTTAAGGGAGCTAGAGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTTCTAGCTCTTTTTCCACGCCTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAGGCGTGGGCGCTCTTCCGATACGGTGTATCTCAGTTCGGCGACCGCTGGGTAACCCTAAACACTACGTGAACCACCGAAATTCGCGTTGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTAACGCGAAaaTTaCCTCATGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCATGAGGATCCTTCGCTGGTAGCGGTGGCTGAAGGCTCGTCCCTCCGAATGCCATCCGTAAGTGATCTTAGGGCGACACGGAATCCGCCTATGGCTTGGTATCTGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTAGATACCATTTTaGCAAGTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAACTTGCATAGGCAAGCTCCCTCGTGCGTATGTACATTCGCTGTAGCGTCTTGCCCGGCGTCGGAAAACGGATACATATTTGAGACCCACGCTGCGCATTAGCAGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTTGCTAATGTTTTTTGAACGAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTCGTTCATGTGAGCCCTTCGGGAAGCGCCCGGTACGCCAGCGGCGAACCCAACGTCAAAGGGAGATAGGAAAGTGCCACCTAAGTGTAGAAGGGGGACGTCTTGGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTCAAGACGTTTTTTTGCGTCGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCGACGCATGAAGTCGAGCGCCCTTTTTGATCCAGTTCGATGGTACTCACCATGTTGTGCAAACTCCGGTGTCCTGCCTTTTAAATTAAAATCAAGTCAAACCCGGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTCGGGTTTGTTTTaTCTTTTTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAAAAAGAATCAGTACCGCGTATGTATTAAGTGCTCATCATTAATACGGAGGGCGCTGGCAAGCATTCAGGCTCACCAGTTACCAATGCTTGCCGCGTTGTTCCGGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTCGGAACAATTTTTGGTCCTGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACAGGACCTACGGCGATCAAGTGATCCCACCAAGTCATTCTGCTGTTGACAATATTATTGAAGCCAGCCGGCTTAATAAGTGGTGGCCTAATATAAAGACAAAAAGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTTTTTTGTCaaTTaAGCAGGGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACCCTGCTAACAGGAACTGTTGGGCGCTGATAATACCGCGCCACTTTAATAGAAAAATAAACAAGTGCTGGCGATCGGCAGCAGCCACTGGCGCTTACGGAACCGGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTCGGTTCCGaTTTTACAGAGCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGCTCTGTTTTTTAAGGATCTCAAGAAATTATCAGTCTATTGGGAATACAGCATCTTTTACTTACTGTCTCGTTGTCAGAAGTCATCGTGGCCGGGAATTTTGGGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTCCAAAATTTTTTTGCCTCGCTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAGCGAGGCTCTCCTGCTGGCGTTTTTCGTCTGACGGCTCCACATGAGCGTTCTTCGGGGCGAGAGTTGCGTCACGCTGCGCGTTACAGGGCAGCAATTATGAGTGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTACTCATAAaTTTCCATTGGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCCAATGGCGCTACGCAGGAAAGAACATCCATAGATACGGTCCCCGAGTTGAGTGTTGTTCGTGGACTGTGGCGAGAAAGGACCTCTTCTACCATCTGTCTATGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTATAGACAGTTTTTAGGCTGAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTCAGCCTGTAGGTACTCAAAGGCGGTACTTCCTCGCCAACGTTAAAATCGGCAAAATCCCTTGATGGCCGGGAGCCCCCGATTCAAGGCGGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTCGCCTTGATTTTTGCTCACATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATGTGAGCtacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtcAcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatact
실시예 3 본 발명의 DNA자임-기반 방법을 사용하여 제조된 DNA 올리고뉴클레오타이드로부터 조립된 추가의 나노 구조물.
나노로드에 추가하여, 본 발명자들은 3200 염기 길이 스캐폴드와 31개의 스테이플 올리고뉴클레오타이드(도 7A 참조)로부터 조립되는 48-나선-튜브를 설계하였다. 나노로드의 경우, 이 48-나선-튜브의 모든 스테이플은 하나의 파지미드상에 코드화되고, 파지미드의 백본은 스캐폴드 역할을 한다. 나노로드와 마찬가지로, 이 물체는 본질적으로 완벽한 1:1 화학양론(stoichiometry)으로 인하여, 스캐폴드위에 과도한 스테이플을 사용하지 않고도 원하는 모양으로 효율적으로 조립된다.
기존의 풀-사이즈 DNA 오리가미 물체의 조립에 대한 우리의 방법의 적용가능성을 보이기 위하여, 저온(cryo) 전자 현미경 연구(Bai et al., 2012)에서 종전에 사용되었던 포인터 물체(도 7b 참조)의 조립에 필요한 모든 161개의 스테이플 올리고뉴클레오타이드를 암호화하는 파지미드를 생산하였다. 나노로드 및 48-나선-번들과 달리, 포인터를 위한 스테이플은 4개의 개별 파지미드에 걸쳐 분포시키며, 별도의 스캐폴드(M13mp18, 7249 염기 길이, Bai 등에 의한 원조 연구에서 사용된 것과 동일한 서열)를 사용한다. 상대 농도의 불확실성을 보완하기 위해, 본 발명자들은 스캐폴드 위에 약간 과량의 스테이플을 사용하였다(1.25 대 1). 비교하여, 화학적으로 합성된 스테이플을 사용하는 어셈블리는 2.5 대 1의 더 큰 과량을 필요로 했다.
파지미드 서열
48나선 튜브 (도 7A): SEQ ID NO. 30
gacgtaacggtgctgtctaacatcgagactgcaattaccccgccagacctttgcacttccacactaatttggtcgatctttgcttaaccgggaactatgtagtctatatgagaatattgagcataaggtgtcagccagcctttatccttgaggcagatcaggtctattcgctcagagtaagatgctaacacccagtagatgacgacgtttaattagggccgagagaccaatgtcacgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaaccTTGATCGGGCACGTAAGAGgttccaactttcaccataatgaaataagatcactaccgggcgtattttttgagttatcgagattttcaggagctaaggaagctaaaatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaatttcgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaatttgatatcgagctcgcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatccaagcctcgagctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtTcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcaccttagcaagagccgcacgacgaccagaggccagttatccagagttaggatacctcaatgtgcatccgctcggttctaaggacattatttcagtcctttaagatctcccgtatagaagcctcacgttagggggcgccgtgccttcacgccctcccatttaggaataccttgtctccgccgtctttattcagtagccctatgcattacgatgtggcgcttcccccgcgtgggcgcagaaatttactgaggcggattcgaaacgactgtgagggcaggataggtgagcaggcactggcacgtaatcaaccaacggactcacccgtgtgcaggcctaaaaacagccctcgaagggcacttggatatgaatgaacccacttgttttgactcgtggaggcgtggttttattactgtgctcagttaacgccgcatgaatttagctctgatcaccgtaagggtaactgcactagacatgttgtgggcatttaagtcctgcagtatctttttgttaggtggaacggcctaggggtaccttccgtgagaaactcccagatgatgcatgttcgagtacttgtgaaatggatggtcgcatcccctcctctcacacattacactgtctcgcgcgggttgcgtcttgaccggtacaagttgtgtaaccttacacctctagaaacattttagcagtcgctccaattgataccacgacctcagcgcgcgttgggagaccgttgccaaaCCTAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTAGGTTTTTTTTGCATCTGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCAGATGCACCAATGTATATAAATAGATCCTTTTAAATTAAATAAATCAATCGTAATACTGAACTAGGTTCTCCCAAGGTGTAACCGTAAACCGCCCCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGGGGCGGTTTTTTAATCCAAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTTGGATTCTGAGTTCTGCACGGCTCAATTCGCGTTAAATTTTTGGCGAGGGCGTGAAGGAGGTCATTACTGGTAAGCTCGAGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCTCGAGCTTTTTCCCGTAGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCTACGGGGTCTATAATATTTGAAGTTGTGGCCCAAGTTTTTTGGGGTCAAGGGAGTTTTCGGAAAAAGAGAAACTCTGGCCACACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGTGGCCAGTTTTTAAAAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTTTTTTTTTTTTTTTTAGGATCTATTACGCTTTTTTTTTTTTTTTTTTTTGCCTGCATCGAGGGTTTTTTTTTTTTTTTTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAAAAATTTTTTACCGCTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAGCGGTAAACAAAACAAGTGCGCCTCCTGCAGTTGCCCACACAAAAAGCACGAACTTCTCATAGCTCACTCTCCCTCATCCATCAGTGTACGGTCAATTCTAGACGCGCGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCGCGCGTTTTTTGAGGGTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAACCCTCCCCCGAGCGAACGTGGCGCGCTCCAGATGCGAATATATGTGAAGCCAGTTACCAGGGAAATACACATCTGCGATGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCATCGCATTTTTATACTGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACAGTATACGGCTAGAGTTCTATGTAGCAACCCGAACTATCGCCCGACCGCTGCGCTGTGTGATACTGCGTACCCCTAGGCCGATGCGACTCGGGAAAAGCTCCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGGAGCTTTTTTTTCGCGTGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACACGCGGCCAGGTCTCAATTGAGACGGGGCGAAGCCCATGCCTTGTCGCCTTGCCCTGAATTATCAGGGGACTCCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGGAGTCCCTTTTTAGTTTCTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAGAAACTGCCGTGGCCTAACTTTGGTAATCCGGCGGTTTTTTTGTTAAAACTGGTGTTGGTAAAATCGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCGATTTTTTTTTTTTCCATGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCATGGAAAAACGTTAAAGCCGTTTAGAGCTAATCACTACGTGAACCGTCCGCCAGCACTGTTGTAATTCACCCCGCCATAGGGTAATCCCTTCTCGATGAAGTGCAGCAAAGGTGATCTCCTTAGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCTAAGGATTTTTACACCGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACGGTGTAACAGCCACTGGTGTATTCAGCGTTAAAACCCTAGAGGTGCATTACGTGCCAGGATCCATATACCCAGGGATTGGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCCAATCCTTTTTAAAAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTTTTTTTTTTTTTTTTCACCGTCATCCCATTTTTTTTTTTTTTTTTTTTTGGGCGCTAAGAAAGTTTTTTTTTTTTTTTTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAAAAATTTTTCTCATCCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGGATGAGCATTTTATCCGGTGTAAGACCTTAAATACCCTTACGGTGATCAGTAATGGGAAATCGTCGTGAACAGGAATCGCCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGGCGATTTTTTTCTGGAGCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGCTCCAGTATCTCAGTTCGGTACCGGAAACATGCGATAAAGGCTGGCGGTCAGAGGTGGCACAATCTCGATAACCGTTTTTCCCGCTCAACGCGAGATTCACAAACGGAAGAGGTAACCACCACAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGTGGTGGTTTTTAACAGGTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTACCTGTTGTTCCGACCCTGCCAGAAACCCGACAGGACTATAGCTCTCCCCGCCTTGCTGTAGAGCTGGGCCCATCAGGCGGGCAAGAATGGGTCGTTCCCGCCGTCTGGGAGTTTCTCGTACTCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGAGTACGTTTTTTGCACGAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTCGTGCAAGATACAAATCGAATAGGCTAAGGCCGAAAAGGCGGATAACTGGCCTCTAGAACCTGAGGTCGAAATCTTCAACGTTAAAGACAACGTGAAACAAATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATTTGTTTTTTTTCTTCAGGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCCTGAAGGTGGTACCTCAAGATACTGGGTGTTAGGGTACATTGAGCCTTGAAAGGCCGTAATATCCAGCGGTTATACATCTTAATAGACCTATAGACCCGGTTAAAAGGTCCCACCTAAGCTCATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATGAGCTTTTTTTCCTCCGCTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAGCGGAGGACTGAAATAATGGCCCCCTGGCGGAGACGGGAAGCGCCACATCTAAAAAAAAACGCCCGGCCGTTCTGGGCTTCTTCTTAAATGCACATTAACTCTCAGCAAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTTGCTGATTTTTAACCTTGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACAAGGTTTACGTCGTGGTATCAATTATATATTTTGTTAACTTGGTCGTCGTGCATCAAGGTTAGCGTTATAGTGTGGTTAGACACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGTCTAACCTTTTTCTGACGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACGTCAGACCGAGCTGCCACGCTCGATATCAAATATCTATCGAAATCGTTAAATCAATTGTACCGCGCAACCTCTTACGTGCCCCCGGTAATCGACCGCTCAATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATTGAGCGTTTTTATTAATAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTATTAATGTTCCAACTTGGTCTGACCGTTAAGCATGATGAAGTGACGCTCAGTGGAACGAAACATCACATATCCTGGAGTCCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGGACTCCTTTTTTGATTCGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCGAATCAATTTCTGCGCCCACGCGGAACCCTCACGAGTCCGTCTGCAAGCAGCAGCAAGAAGTTAAGGGATTTTGGATCAAAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTTTGATCTTTTTGCTTTACGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGTAAAGCATCCAAGTGCCCTCACGGGTAGTCGTTAGGATCTTCACCGCACTCACGATCCTTTGATCTTTGTTTAAATTGGTTGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCAACCAATTTTTTGTGCTCAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTGAGCACAGAGCTGTCACGCTGCGCGAACGACTTTTAGCAGAGCGAGGTTGAAGTAAAACCAGGTTCATTCATACTGCTCTTGTCTGCGCTCTGCTGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCAGCAGAGTTTTTATGAATTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAATTCATGAGAAAGGAAGGGAGGGCGCTGCGTGATACGGTCTTTAATATGAATAAGCCATACGAAATTCCTGGCAGTGTAGCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGCTACACTTTTTATTAAGCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGCTTAATGGCAAGCAAGGGTGAACACTTTTCATTAGGCCGGATAAAACTTTTCTTCATTGGTTAATTAAACGTCGTCATCTCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGAGATGATTTTTATGAGAATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATTCTCATGATCTGTATCCAGCCATTGGGATATATCGAACTGACTGAAATGCCTCATACGATGTGATTTTTTAGCTTTATTTCAAGTTGGACATTTCCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGGAAATGTTTTTTGGTTGCGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACGCAACCCTGACACCTTATAAGGAGCGACTGCTGGTCAAAAAATACGCGGGCTCTTGCTAAGGTGAGCCGTTGCTAAAATGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACATTTTATTTTTAAAAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTTTTTTTTTTTTTTTTCTTGTACATGTGTGTTTTTTTTTTTTTTTTTTTTATCACAACAGGCGTTTTTTTTTTTTTTTTTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAAAAATTTTTAAAAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTTTTTTTTTTTTTTTTATTGCAGTATAAATTTTTTTTTTTTTTTTTTTTTATAGGCCAACAGGATTTTTTTTTTTTTTTTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAAAAATTTTTAATCGGTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCACCGATTCCTAAATGGAATGAGTGTAGAACGTGCTGCCTGCTCACCAACGGCATTCTGCCGACATGGTGAGTAAGTTTGGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCCAAACTTTTTTGTCGAAATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATTTC
포인터 파트 1 (도 7B): SEQ ID NO. 31
atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacTGCCCTTTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAAAGGGCGGGTGCCTCGCTCACTGCTTAATGTTGCATGCGGTCACGTTGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCAACGTGATTTTTACAACAATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATTGTTGTTCCGGCCAACCAGGGTGGTGCCAAGCGACAGGAAGCCGGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCCGGCTTTTTTTAAAAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTTTTTTGGGTGAGACGGGCGAGTTTTTTTTTTAAAGGGTCGTGCCGAGGATCCCCGGGTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAACCCTTTTTCGATTTCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGAAATCGCCTGGCCCTTGCCCCAGAGTAAAATAACATCACTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAGTGATGTTTTTCTGATTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAAATCAGCCCCCGGCAGGCGAAAATCCTGACGCTGGTTGAGAGAGGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCCTCTCTTTTTTTTACGGCCTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGGCCGTAAAGCACTAAATTTTTTTTTTTCGGAACCGGAAAGCCGGCGATTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAATCGTTTTTGCTAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTAGCGGTCCTTTTTTTTTTTTTGATGGTGGTTCAATAGCCCTTGGGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCCCAAGGTTTTTCGTAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTACGTGGCAACAGCTGAGAAGTTTTCCTTTTTTTTTTCAGTCACGACGTTGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACAACGTCTTTTTCCCGCGCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGCGCGGGGACGTGCTTCGCCGCTACAGCTTTCAATAGGAATTGCGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCGCAATTTTTTTAAAACCGCTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGCGGTTTTTTTTTTTTTGCGTAGAGTTTTTTTTTTATAGGGTTGAGTAAAGAACTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAGTTCTTTTTTTCTTAGGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACCTAAGGGTTTTTTTTTTAAGAAAGCGAAAGGTCACGCTAGCTCTTTTTTTTTTGAATTCGTAATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATTACGAATTTTTATTCAACGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGTTGAATGAGTGTAAAGTGTAATTGTTAAGGAAGATGATAATCATGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCATGATTATTTTTGGCGGGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACCCGCCGCGCCCGCTTTAATGAATCAGTTTGGCCTTATAAATCAAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTTGATTTTTTTTGAGTTAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTAACTCGCGTCTTTTTTTTTTTGAAATGGATCACCATCAACTGTTTTTTTTTTATAGCAAACATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATGTTTGCTTTTTACGTTTTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAAAACGTTAAAACTAGCTTGAGAGATCTGGAGCTCATTGAATCCCCCTCGAATCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGATTCGATTTTTCTTAGAGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACTCTAAGCTGCATTCCAGTCGCGTGAACCGTCTATCAGGGCGATGTTTTTTTTTTGCCCACTAGGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCCTAGTGTTTTTGTAAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTTACCGGCGCGTAAACAACCCGAAATTTTTTAAAAATTCTGAGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACTCAGAATTTTTCTACACTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAGTGTAGCGGAGCGGGCGCTAGGGCGAAAAACCAACAAGAGTCCACTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGTGGACTTTTTTGATAAAATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATTTTATCCTCGTTCGTGCATCTGGTGTAGCACCAGCACTCAGAGCAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTGCTCTGTTTTTGTTAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTAACCAGAGGAATTTTTTTTTTAAACGCTCCATCACCTCATTAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTAATGAGTTTTTCTTCTACTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAGTAGAAGGTTGGGTAACGCCTTTTTTTTTTAGGGTGTTTTTATCCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGGATAAATTTTTATCAAAGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCTTTGATTAGTAAGAGTCTGTGGGCGATCCCAGGCAACAATAACCCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGGGTTATTTTTTGCTGCCAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTGGCAGCTTTCGCGCGAGCAACACCGCTCCTGATTTAGAACCTGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCAGGTTCTTTTTTTCGTAATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATTACGAATCAGTGCAGAATCCTGATTGCCTTCACCAGGTCGAGGTATCACCCAAATCAAGTTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAAACTTTTTTTGCAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTGCAACGCTAGTTTGACATATCTTTTTTTTTTTGGTCAGTTGGCACCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGGTGCCATTTTTATTAATCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGATTAATAGCTCAACTGGAAGTCTTTTGATCTATTATACCATCCTAGTCCTGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCAGGACTATTTTTATTACCAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTGGTAATATCCGAATTGAGCCAATTCTAGGTCAGGAAAAAGATGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCATCTTTTTTTTCGGCCGATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATCGGCCGGCGGATTCAGTATTGGCAAAGAGATGATGATTAACAATAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTATTGTTTTTTTCCAGAAGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACTTCTGGTCGGTACGCAGGCCACCATTTAGAGCTTGACGGCTAAAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTTTAGCCTTTTTAGAACTGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCAGTTCTGAGAGCTGTTTAGCATTGCATCATTAGAGAAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTTCTCTATTTTTCCTTTCGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCGAAAGGGTACGGTGTCATGTTTTGCGGATGGCTTAGAGCTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAGCTCTATTTTTCGCACCTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAGGTGCGGGGTTGATTCGAAGGTTACTTTAGGAGCACTAACGTTTTAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTAAAACGTTTTTTCCTCCTAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTAGGAGGCCCAAATTAACCGTTTTTTTTTTTTGCAGCCATTTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAAAATTTTTTATCACATTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAATGTGATGGGATACTGCAGGTACGGCCAGTTTTTCTTCTTCACCGGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCCGGTGATTTTTTTATGTTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAACATAATAAAGCGAAGATAATTGCACGTGATGATGGCATCGGAAGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACTTCCGATTTTTCGCCCTGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCAGGGCGCCGGAAGCAAGCTAACTTTTTTTTTTTCACATTAAGTGTTTTTTTTTTGACTCCAACGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACGTTGGATTTTTTGAAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTTCAATCGTTGTACCAACCTAATTTTTTTTTTAACATCGCCATTAAGATTTTCGAGCGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCGCTCGATTTTTATGACCAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTGGTCATAGCTGTTTCCTGTGTGAAAAGCCTGTTTTAACCATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATGGTTAATTTTTTGCTTATATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTATAAGCAAAAGGGGAAGCCTTTTTTTAATATTTCAATCATATGCGGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCCGCATATTTTTTATTTTGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACAAAATAATATACGAGCGTACTATGGTTTTTTTTTTTTGCTTTTGACGCTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAAGCGcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt
포인터 파트 2 (도 7B): SEQ ID NO. 32
atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacGGTGATGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCATCACCAAAAACTCCGCTCACAATTCCACACTTTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAAAAGTTTTTCTGTTGGCTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGCCAACAGATACGTGGTAATGCCGGGGAAGAAGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCTTCTTCTTTTTACTAATCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGATTAGTCTTTAATTTTTTTTTTTGCGCGAATATGATAACGGAACTGACGAGAAACACCAGTCAATAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTATTGACTTTTTTATAAATAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTATTTATGTCAATCCCTTCTGAAACAAGAGAATCGATCCTGAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTCAGGATTTTTTTGATTTATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATAAATCACAGACACCACATTCAACTAATGATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATCATTAGTTTTTACCAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTGGTCATTGGAATTTTTTTTTTCGGTAATCGTAATATTTTGTGCAATGCTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAGCATTGTTTTTTTTGTAGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCTACAAAAAGAACTGTTGTGAATTACCTTATAGAAATTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAATTTTTTTTTTCAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTGAATGGCTACCAGTAAATTGGCAGATTCACCATTTTTTTTTTGTCACAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGTGACAATTTTTTTGTTCTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAGAACAATTACATAACAAACAATCATAATAGTACCGACAAAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTTTGTCGTTTTTTTTCTCATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATGAGAAAGGTAAATTGAAATCTACAAAAGAAGAGCAACACTATCATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATGATAGTTTTTTCTCACATATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTATGTGAGTGAAGTTACAAGCCAACGATTTAACATAAATTGTAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTACAATTTTTTTAAATAATTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAATTATTTTTTTTTTAAATTCGCATTTCGGATTCACAGGCAATAGCATTAGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCTAATGCTTTTTATTAAATTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAATTTAATCAGCTCATTAAATGTTTAATAAATATAAAGGAATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATTCCTTTTTTTTTTAAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTTAAATCATTCCTTTTTTTTTTGTGGGAACAAACTCAGGAAAATAGTAGTGAAAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTTTCACTTTTTTCAGGGTACTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGTACCCTGAAAGAGGTCTAAACCAATTATTTTTTTTTTAATCAAGATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATCTTGATTTTTTCTACCATTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAATGGTAGCGCCATATCGTAACAGAATCAGCACGTATAAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTTATACGTTTTTAAGTCTATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATAGACTTCAACCAGACCACCGCGCCTCCGGTATCTAACGAGCGTCTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGACGCTCTTTTTTCATGTTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAACATGAGGCGGTGACCGTAAGCGAGTACCACCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGGTGGTATTTTTATCTGCTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAGCAGATTATCAAAAACAGATAGGCAGATTATACAAGACCTAAACTATATGTATCAATAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTATTGATTTTTTAATATTTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAAATATTTTTTTTTTTTTCAAACCCTCAATCATGCTGAACACCAGAAGAGGTTTAAATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATTTAAACTTTTTTTCTCTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAAGAGAACAATGTTGGGAACCATCACGGATTAAAGTTGCAGCATTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAATGCTTTTTAAAAAAATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATTTTTTTTTTTTTAAATATGCTTTTTTTTTTAACTAAAGGGATTTTTTTTTTTGTGCTGCAAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTTGCAGCTTTTTAAGAGCAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTGCTCTTCATTCCCATTTGGGCGGCACCGCGACGACAGTAAAACGCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGCGTTTTTTTTTTCTATTGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCAATAGATAATAAAGGCTTACAATAGCAGCGAATAAACAGCTTGATAATAAGTAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTACTTATTTTTTTGACTCAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTGAGTCGAGCTTCAAAGCGAAATATCGCCTGAGGCTACTAAAGAAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTTCTTTATTTTTAAAAAGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACTTTTTTTTTTAATTCGACAACTTTTAAAACATCGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCGATGTTTTTTTGCTTTGATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATCAAAGCGGTATATTTTATATAACACCTCTTCGCTATCGGCCTTGCCTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGGCAAGGTTTTTTCAAAGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACTTTGAGGTGCAGGGATTTCTTAATAATTTTTAAAGTCAGATTTATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATAAATCTTTTTTTTTAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTAAAATCAGGTCTTTGGCATCATTTTTTTTTTATTCTACTGATTTTTTTTTTTCGCACTCCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGGAGTGCTTTTTGTTTCGGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCCGAAACATTTCGGTAGATTTGCGCAACTATTACCGCTAGCAATACTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGTATTGCTTTTTAACGTTCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGAACGTTATTAATCGTATTATAAACAACTGAATTTTGTCGTCTTTCCAGACGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGTCTGGATTTTTAAAAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTTTTTTTTTGAGTAACATTTACCTTTTGAGGCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGCCTCAATTTTTCCGAATAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTATTCGGAAACAGTTAGATTAAGACGCTGTTATATAATTTAATGGGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCCCATTATTTTTACACCCTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAGGGTGTTTGGATATAGATAAATTTACGAGCATGTTTTTTTTTTTAGAAACCAATCAACGGGTATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATACCCGTTTTTTTGGATATGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCATATCCAAAAGAAATTAGCAACGCAAGGAGTTAAATCTAAATTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAATTTAGATTTTTAAAATGATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATCATTTTTTTCCATTACGCATAACGACAATGTAGAAAGGAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTCCTTTCTTTTTAAAAATGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACATTTTTTTTTTTCGGGAGAAACTCATTACCGTAATCTTGACAAGAACTGACCTTGTACAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTGTACAATTTTTATCTCAAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTTGAGATGGTTTAAATTACCTTATTTCAAAATTAAGCTAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTAGCTTAATTTTTCGTAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTACGAGACAAAATTCCTCATATTTTTTTTTTTATTTTAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTAAAATAATTTTTCTCGTTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAAACGAGTAGCCGGAGAGTTCTAGCGAAAAGCCTAAAAGGGACATTCTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGAATGTCattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt
포인터 파트 3 (도 7B): SEQ ID NO. 33
atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacGGGTCATATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTATGACCCTGAAATCGGTCTGGCCTTACCTACATTTGACGAGAGCGGGAGCTATTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAATAGTTTTTGAAGGTTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAACCTTCCCTTAGAATCCTTGCCAATCGCATATTTTAAGTACCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGGTACTTATTTTTGGACGATTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAATCGTCCGGATATAATAACGGACTGACCAGACGGTCAGTTACTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGTAACTGTTTTTAAAAATTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAATTTTTTTTTTATCAACGTAACAAAGCTGCTCATTCAGTAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTACTGAATTTTTTGTTTAAAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTTTAAACTTTTTTTTTTAGTTCGCGATTTTGGCTATCATTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAATGATTTTTGTTGGCGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACGCCAACCTGAAAGCGTAAGAAGATAGAACATATGTACCCCGGTTTGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCAAACCGGTTTTTCCTAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTAGGTAGAAAGATTCATCAGTACCAGACGACGATTTTTTTTTTTAAAAACACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGTTTTTAATTTTTCTATTTTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAAAATAGCGAGAGGCTTTTGCGTTAATAATAGGAATAATAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTATTATTCTTTTTCCAGTCGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCGACTGGATAGCGTCCATTTTTTTTTTATACTGCGAAATGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCATTTCGCTTTTTGCCTCGTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTACGAGGCCAGATACAAGGACGTTGAGAGGGTTAAAGATTCAAATATTAGCTCATTGCTGGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCCAGCAATTTTTAAAACTTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAAGTTTTGCCAGAGGGGGTAATAGTAATACCAGTCTAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTAGACTGGTTTTTCGAGGGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACCCTCGTTTTGAGATTAACGAACTATTCAACCCAGTCAAATTATTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAATAATTTTTTTTTGATAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTATCAAAATCATAGGTCTTTTTTTTTTTGAGAGACTACCTATAAGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCTTATAGTTTTTTACTAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTAGTATTACCTGAGAAAATTATAACAGAGGGTGCCACGTGAGGGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCCCTCACGTTTTTTGTATGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACATACAGTATAAAAATCGCGAATTGCGTAAAATACCATCTAAAGATGGAAATTCGCCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGGCGAATTTTTTCAAAATTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAATTTTGCTTCTGTGAATAAGGCTTGCCCAACATTATTACTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAAGTATTTTTATCGCGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACGCGATAGGCTGGCGCTATTAGGAACCGAATTCGCCTGAATATACAGTAACACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGTTACTGTTTTTTTGCGTTCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGAACGCAATAAGTTACCTTTGGGAATTAGAGCCTTAGCGTTTGCCATTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAATGGTTTTTGGCTTTCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGAAAGCCTGCAATAGTGTTCATTTGATTTCAACTGTGTAGGAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTCCTACATTTTTATATCAGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACTGATATAAGTATATTTTTTTTTTGCCCGGAATAGGTAGGCTGAGTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAACTCTTTTTTTCTTGATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATCAAGAAAAATGATTTTTTTTTTCCATATGAATAATACATCCAATTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAATTGTTTTTTATTATATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATATAATACTAGAATGTGATAATTTTAACCCAAAGACAAAATTTTTTTTTTGGGACCGACTTGAGTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAACTCTTTTTTTAAAATGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCATTTTAAAGTACACAGCGATTCCCATGTATACCGAAGCCCTTTTTGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCAAAAAGTTTTTATTAATTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAATTAATAAAGACAGAGGCGATAAAGCTTAATACTTCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGAAGTATTTTTTAGATGGCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGCCATCTCAACAGTTTCAAATAAGACAAAAAGACACCACGGAATAATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATTATTCCTTTTTCAAAGTTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAACTTTGAAAGAGGTTTTTTTTTTACAGATGAACGGTCATCAAGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCTTGATGTTTTTTACTTTACTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGTAAAGTAATTCGCTAATGCAGAACGCGCCTTTTTTTTTTTGTTTATCAACATATACTTCAATCATTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAATGATTTTTCTTCGTTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAACGAAGGCACAGCCCTCATAGTTTTTTTTTTTTAGCCCCACAAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTTGTGGGTTTTTTACGTATTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAATACGTACAAACAGCAAGAAACAATGAACCCTGAACTCACGTTGTAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTACAACGTTTTTTCGACTTGCTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGCAAGTCGAAATGAGGTTTAGCGGATAATAGCGGGGGAAACGCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGCGTTTCTTTTTAGTTCGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACGAACTTTTTCAAATTGAGACGTCAGATGATTGCTTTGAATACCAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTGGTATTTTTTTAACATGTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAACATGTTCATGTCCAGACTCATTTTAATAACGGTCACCGTCCGACATTCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGAATGTCGTTTTTACAGGGTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCACCCTGTATCGGTAGGCTCCATTAGACGGTGTTTAACGTCAAAAATGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCATTTTTGTTTTTTCGGATCCTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGGATCCGAGGGTATTGACCCCACGGAGATCCCTCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGAGGGATTTTTTCTCTTAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTAAGAGGAATATTCCTAATGAAAAGAACGAACATGGGCGCCCTGTAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTACAGGGCTTTTTATCTATATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATATAGATTTTGCAATCCTTTGATTTAGAAGTATTAGACTTTACATTAGTAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTACTAATGTTTTTCCGTTTGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCAAACGGGTAAAATTTTTTTTTTTACGTAATGCCACTGCCGGAACACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGTTCCGGCTTTTTAACTTCACTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGTGAAGTTAAAGGGAATCATTGGAAGCAAGATTAGAGAATCAACAGTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAACTGTTGTTTTTAAGGTACGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGTACCTTTTGAAATATTCTAAATATAATGCTGAACTCAAACTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGTTTGAGTTTTTTTCAACGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCGTTGAACCTCCCAGCTACAATTTTATCCGATTTTTGAGAATTAACACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGTTAATTCTTTTTTACGGCTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAGCCGTACCGCATTCCAAGAATAATCGGTAAGCAGATAGCCTTTTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAAAAGGCattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt
포인터 파트 4 (도 7B): SEQ ID NO. 34
atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacAAATAAAGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCTTTATTTTCAAAAAAATTATCAGCAGCTATCTCCGTAACACTGAGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACTCAGTGTTTTTTTGAAATTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAATTTCAACAGTTTCTTTTTTTTTTAGCGGAGTGAGAAACAACAACACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGTTGTTGTTTTTTGGCTGCTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTAGCAGCCTTTACAGTTTTTTTTTTAGAGAATAACATAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTATGTTATTTTTTGACGTTGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCAACGTCTAAGAACGCGAGGCAACTAATAACTCCAACGCGAACGACAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGTCGTTCTTTTTATGGCGAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTCGCCATATTAGGGTAATTGAGCGCTTAAGCCCAATACCGATAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTATCGGTTTTTTACTATTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAAATAGTTGCTATCCTTATCACTCATCGAATAATATCGTCAGAAGCAATATAACTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGTTATATTTTTTGGATTGGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCCAATCCAAATAAGAAACTGAATCTAAAATCTCCATCGTAGCCGCTTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAAGCGGCTTTTTTGCTCTGTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCACAGAGCCTAAGGAATTAGCAAATCTTCGGTCGGTTTTAATAAGAAACCCTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAGGGTTTCTTTTTCCTGTTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAAACAGGAAGAAAAGCTGTCTTTATAAACAACAAGAAAATAATAAGAACACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGTTCTTATTTTTTCTTTTGCGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCGCAAAAGGAGCTTTGCACCCGACTTGCGGGAGGTTTTAATTGCAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTGCAATTTTTTTATTGTTCATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTGAACAATAGCAATTTGCTTTCACTCATCTGCAACGGCATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATGCCGTTTTTTTTTACTTATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATAAGTAACTTTTTTTTTTGATCTAAAGTTTTAGTTACAAAATAAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTTATTTTTTTTTACTCAATTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAATTGAGTAATATCAGAACAACTAAATTTGCCCTGTATGGAGATATAGCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGCTATATTTTTTTTCAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTGAACACAGGGATAGCAAGCCCCCACCACCCGACGACAACCGACACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGTCGGTTGTTTTTGCGGTTAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTAACCGCCATTGTATCACATCTTCTATTCTTACGCGATAGCTACATAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTATGTAGCTTTTTAGGCGTTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAACGCCTGTAGCAGTACTCAGCCGCGACCGAAAACTTTAGGGCTTATACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTATAAGCCCTTTTTAGTCTTATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATAAGACTCATAGACGGATATTCATGAGCCGCCGCCAGCATCGCCTCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGAGGCGATTTTTTGTATCATGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCATGATACCGCCACGCACCATTACCATTAGTTTCATCGTAAACAGTGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACACTGTTTTTTTGGCGGTACTAGTTGAGCTGTCACAGAATGTGACGTTGAAGGTACCGCCACCAATGAAACCATCGAAGTTTGCCCTATTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTAATAGGGCTTTTTCTCGACGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCGTCGAGAGGGTCAGGCGCATGCTCCATATCATAAGTGAGGAAGGCGGAACAGCCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGGCTGTTCTTTTTAGTAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTACTCCTCAATTTTTTTTTTTTAAGCAGTAGCGACAGAATCATAGCAGCATTAGGATGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACATCCTATTTTTTGGAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTCCAAGAAGGAATTTTTTTTTTACCGAGGATTAAATAAGAATTTTTTTTTTTAAACACCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGGTGTTTTTTTTATTCACATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATGTGAATTAAATACCCAACCAGCGCTCCGGCTTAGGTTGGGAGAAGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCTTCTCCTTTTTCTGAGGTATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTACCTCAGAGAATAGGAAATACCAAGCTTTAATTCAGCAGCGGAACAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTGTTCCGTTTTTGGCCTTGATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTCAAGGCCGCGTAGAAACTTATTACTTGTCACAAAATGCTGATGCAAATAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTATTTGCTTTTTAGAAAAAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTTTTTCTTTTCATAATCCCTTGATATTTTTTTTTTTTCACAAACAAATAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTATTTGTTTTTTTAATAGGCATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTGCCTATTAGCAAAGAAACGTCACCCTCAGCGTCACCAACTAAAACGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCGTTTTATTTTTACACTCATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATGAGTGTACAGAGCCACTTTTTTTTTTCACCCTCAGAGCCGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCGGCTCTGTTTTTCTCTACTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAGTAGAGAAGGACCGTAATGTATCACCTTCCACAGACCAACCTAGTTGCGCCCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGGGCGCAATTTTTTGACAAGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCTTGTCAGAGCCGCCACCCTCAGAACCGACCAGAACCCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGGGTTCTGTTTTTAATCGTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAACGATTGGAAAATCACGGTTGAGGAACCGATTGAGGGAGGTATGGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACCATACCTTTTTTGCGCTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAAGCGCAGTCCGGGGTCATAATGCCCCACCACCAACATAAAGGTGGCAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTGCCACCTTTTTTCATGTATGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCATACATGGCTTTTTTTTTTTTTTGATGATACAGTCTGAAACATGAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTCATGTTTTTTTTGGGTGGGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCCCACCCTCTGGTAATAAGTTTTAATCTGAATTTCAGACTGTAGCGCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGCGCTACATTTTTCTGACCTGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCAGGTCAGACCGGAACTGACAGGACGGAACCACATTAAAGCACCAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTGGTGCTTTTTTGTTACTGGTAGTTGAGCTGTCACAGAATGTGACGTTGAAGCCAGTAACAGGAGCCACCTCCTCATTGGTCATAGCCCCCTTAAGCAAAACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTTTTGCTTATTTTTTTCTTTAATAGTTGAGCTGTCACAGAATGTGACGTTGAAGTTAAAGAACTTTTGAAATCGCGAAACCGAGCCAGAAAGACAGCAATTCACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTGAATTGCTTTTTTTGAATTTTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGAAAATTCAGAAGGTAAAAATTATTTGCCCGTAGCATTTTCAAAGCCAGAATGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCATTCTGGTTTTTTAAACTGTTAGTTGAGCTGTCACAGAATGTGACGTTGAAGACAGTTTATTGCAGTATGGTTAATTTTCGCCTGAACGCCAACTACAGAGGTTATCATCAGACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTCTGATGATTTTTTAATTTTATTAGTTGAGCTGTCACAGAATGTGACGTTGAAGATAAAATTTTGCTCTTTCGGAACTTTAGCGTACCGTTCCAGTAAGCGTACGTTGAAGCGTTACCTGTTAGGTAACGTAGTTGAGCTGTACGCTTACattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt
상기 상세한 설명 중에, 청구범위에 및/또는 첨부 도면에 개시된 특징들은 개별적으로 또한 이들의 임의의 조합으로, 본 발명을 다양한 형태로 구현하기 위한 재료일 수 있다.
참고문헌
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SEQUENCE LISTING <110> Technische Universit? M?chen <120> Scalable biotechnological production of DNA single strand molecules of defined sequence and length <130> U30718WO <150> EP 16189976 <151> 2016-09-21 <160> 34 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> DNAzyme <400> 1 tagttgagct gt 12 <210> 2 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> DNAzyme <400> 2 acgttgaag 9 <210> 3 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 3 tactcttaga agtgtcccaa ctacactaga aggacagtgg cgagaggatt acgcgcctag 60 atcaacttta atgttgactc gtgcacccaa catgcttttt agctc 105 <210> 4 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 4 gcacattgag ggctgctatt aagacacgac ttatcccttt ctcaaaaggc cagcaaagcg 60 atctggcccc aataggggaa caagaggcag aacatatcaa agcga 105 <210> 5 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 5 cttaccgaga atagacaccc gccttacagc gaggcgaagg gctttaaatc aatctagagc 60 atcataccag gcgtttcgtt cttggcgccg caaccacctg tatgc 105 <210> 6 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 6 tagaccgcga aaaatgacgg ggaaagcctg gcgaataact acgttgcctg actcccgggg 60 atattctcat agctcactaa ctattgtgct gtagagctcc gtcta 105 <210> 7 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 7 ggcgaccaaa ctctcagggt tattgtctga tttatcgcgt ccggcggtgc tacagacccc 60 tggtccgccc ccctgacaag tataaaacca gcatttatca aggat 105 <210> 8 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 8 tcagggcata aatcgcgtta atattttgcg cggggattaa gttgcgcctt atccgggctg 60 tagtatccac agaatcacgc gtatgtttgt cattgtaaaa aagaa 105 <210> 9 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 9 cttcggtgtt tggtccatcc aaaaaggatc ttcacagaaa aatgtttgca agcagcagta 60 tttcattcag aaagcggtct gtgactggtg ataacccaat actca 105 <210> 10 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 10 aaggagcggg aaggcaatga tgaggcacct atctcaaggc cacggatacc tgtccggcca 60 ctggtgcggg agggaagcac tattaaagaa ccagtttggt tccgc 105 <210> 11 <211> 112 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 11 ctacaggaag ttggctgcat aattctcttt caccaaatgc cgcaaaaaaa attgttgtgt 60 cacccagtta ccttcggaaa ccactgatct tttctacgtt aagggagcta ga 112 <210> 12 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 12 agaggcgtgg gcgctcttcc gatacggtgt atctcagttc ggcgaccgct gggtaaccct 60 aaacactacg tgaaccaccg aaattcgcgt t 91 <210> 13 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 13 tcatgaggat ccttcgctgg tagcggtggc tgaaggctcg tccctccgaa tgccatccgt 60 aagtgatctt agggcgacac ggaatccgcc tatggcttgg tatct 105 <210> 14 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 14 aaacttgcat aggcaagctc cctcgtgcgt atgtacattc gctgtagcgt cttgcccggc 60 gtcggaaaac ggatacatat ttgagaccca cgctgcgcat tagca 105 <210> 15 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 15 ttcgttcatg tgagcccttc gggaagcgcc cggtacgcca gcggcgaacc caacgtcaaa 60 gggagatagg aaagtgccac ctaagtgtag aagggggacg tcttg 105 <210> 16 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 16 tcgacgcatg aagtcgagcg ccctttttga tccagttcga tggtactcac catgttgtgc 60 aaactccggt gtcctgcctt ttaaattaaa atcaagtcaa acccg 105 <210> 17 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 17 taaaaagaat cagtaccgcg tatgtattaa gtgctcatca ttaatacgga gggcgctggc 60 aagcattcag gctcaccagt taccaatgct tgccgcgttg ttccg 105 <210> 18 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 18 acaggaccta cggcgatcaa gtgatcccac caagtcattc tgctgttgac aatattattg 60 aagccagccg gcttaataag tggtggccta atataaagac aaaaa 105 <210> 19 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 19 accctgctaa caggaactgt tgggcgctga taataccgcg ccactttaat agaaaaataa 60 acaagtgctg gcgatcggca gcagccactg gcgcttacgg aaccg 105 <210> 20 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 20 gcgctctgtt ttttaaggat ctcaagaaat tatcagtcta ttgggaatac agcatctttt 60 acttactgtc tcgttgtcag aagtcatcgt ggccgggaat tttgg 105 <210> 21 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 21 gagcgaggct ctcctgctgg cgtttttcgt ctgacggctc cacatgagcg ttcttcgggg 60 cgagagttgc gtcacgctgc gcgttacagg gcagcaatta tgagt 105 <210> 22 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 22 agtccaatgg cgctacgcag gaaagaacat ccatagatac ggtccccgag ttgagtgttg 60 ttcgtggact gtggcgagaa aggacctctt ctaccatctg tctat 105 <210> 23 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 23 ttcagcctgt aggtactcaa aggcggtact tcctcgccaa cgttaaaatc ggcaaaatcc 60 cttgatggcc gggagccccc gattcaaggc g 91 <210> 24 <211> 2630 <212> DNA <213> Artificial Sequence <220> <223> Phagemid backbone that serves as scaffold <400> 24 gttgtcgtct ctgaagatgt gagctacaac gtcgtgactg ggaaaaccct ggcgttaccc 60 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 120 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta 180 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 240 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 300 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 360 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 420 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 480 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 540 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 600 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc 660 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 720 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 780 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 840 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 900 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 960 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 1020 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 1080 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 1140 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 1200 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 1260 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 1320 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 1380 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 1440 tgctgataaa tctggagccg gtgagcgtgg gtcacgcggt atcattgcag cactggggcc 1500 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 1560 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 1620 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 1680 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 1740 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 1800 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 1860 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 1920 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 1980 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 2040 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 2100 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 2160 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 2220 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 2280 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 2340 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 2400 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 2460 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 2520 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 2580 ccccgcgcgt tggccgattc attaacttaa ttgcagcaag agacgcttct 2630 <210> 25 <211> 6813 <212> DNA <213> Artificial Sequence <220> <223> Full phagemid ssDNA sequence <400> 25 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 60 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 120 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 180 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 240 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 300 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 360 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 420 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 480 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 540 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 600 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 660 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 720 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 780 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 840 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 900 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 960 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 1020 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 1080 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 1140 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 1200 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 1260 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 1320 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 1380 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 1440 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 1500 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 1560 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 1620 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taacttaatt gcacgttgaa 1680 gcgttacctg ttaggtaacg tagttgagct gtgcaattaa ttttttaaga gtatagttga 1740 gctgtcacag aatgtgacgt tgaagtactc ttagaagtgt cccaactaca ctagaaggac 1800 agtggcgaga ggattacgcg cctagatcaa ctttaatgtt gactcgtgca cccaacatgc 1860 tttttagctc acgttgaagc gttacctgtt aggtaacgta gttgagctgt gagctaaatt 1920 ttttcaatgt gtagttgagc tgtcacagaa tgtgacgttg aagcacattg agggctgcta 1980 ttaagacacg acttatccct ttctcaaaag gccagcaaag cgatctggcc ccaatagggg 2040 aacaagaggc agaacatatc aaagcgacgt tgaagcgtta cctgttaggt aacgtagttg 2100 agctgtcgct ttgatttttt cggtaagtag ttgagctgtc acagaatgtg acgttgaagc 2160 ttaccgagaa tagacacccg ccttacagcg aggcgaaggg ctttaaatca atctagagca 2220 tcataccagg cgtttcgttc ttggcgccgc aaccacctgt atgcacgttg aagcgttacc 2280 tgttaggtaa cgtagttgag ctgtgcatac agttcctgcg gtctatagtt gagctgtcac 2340 agaatgtgac gttgaagtag accgcgaaaa atgacgggga aagcctggcg aataactacg 2400 ttgcctgact cccggggata ttctcatagc tcactaacta ttgtgctgta gagctccgtc 2460 tacgttgaag cgttacctgt taggtaacgt agttgagctg tagacggagt ttttttggtc 2520 gctagttgag ctgtcacaga atgtgacgtt gaaggcgacc aaactctcag ggttattgtc 2580 tgatttatcg cgtccggcgg tgctacagac ccctggtccg cccccctgac aagtataaaa 2640 ccagcattta tcaaggatac gttgaagcgt tacctgttag gtaacgtagt tgagctgtat 2700 ccttgatttt ttgccctgat agttgagctg tcacagaatg tgacgttgaa gtcagggcat 2760 aaatcgcgtt aatattttgc gcggggatta agttgcgcct tatccgggct gtagtatcca 2820 cagaatcacg cgtatgtttg tcattgtaaa aaagaacgtt gaagcgttac ctgttaggta 2880 acgtagttga gctgttcttt ttttttttca ccgaagtagt tgagctgtca cagaatgtga 2940 cgttgaagct tcggtgtttg gtccatccaa aaaggatctt cacagaaaaa tgtttgcaag 3000 cagcagtatt tcattcagaa agcggtctgt gactggtgat aacccaatac tcacgttgaa 3060 gcgttacctg ttaggtaacg tagttgagct gtgagtattg tttttcgctc ctttagttga 3120 gctgtcacag aatgtgacgt tgaagaagga gcgggaaggc aatgatgagg cacctatctc 3180 aaggccacgg atacctgtcc ggccactggt gcgggaggga agcactatta aagaaccagt 3240 ttggttccgc acgttgaagc gttacctgtt aggtaacgta gttgagctgt gcggaacctt 3300 ttttcctgta gtagttgagc tgtcacagaa tgtgacgttg aagctacagg aagttggctg 3360 cataattctc tttcaccaaa tgccgcaaaa aaaattgttg tgtcacccag ttaccttcgg 3420 aaaccactga tcttttctac gttaagggag ctagacgttg aagcgttacc tgttaggtaa 3480 cgtagttgag ctgtctagct cctttttcca cgccttagtt gagctgtcac agaatgtgac 3540 gttgaagagg cgtgggcgct cttccgatac ggtgtatctc agttcggcga ccgctgggta 3600 accctaaaca ctacgtgaac caccgaaatt cgcgttacgt tgaagcgtta cctgttaggt 3660 aacgtagttg agctgtaacg cgaacctttc ctcatgatag ttgagctgtc acagaatgtg 3720 acgttgaagt catgaggatc cttcgctggt agcggtggct gaaggctcgt ccctccgaat 3780 gccatccgta agtgatctta gggcgacacg gaatccgcct atggcttggt atctacgttg 3840 aagcgttacc tgttaggtaa cgtagttgag ctgtagatac catttttgca agttttagtt 3900 gagctgtcac agaatgtgac gttgaagaaa cttgcatagg caagctccct cgtgcgtatg 3960 tacattcgct gtagcgtctt gcccggcgtc ggaaaacgga tacatatttg agacccacgc 4020 tgcgcattag cacgttgaag cgttacctgt taggtaacgt agttgagctg tgctaatgct 4080 tttttgaacg aatagttgag ctgtcacaga atgtgacgtt gaagttcgtt catgtgagcc 4140 cttcgggaag cgcccggtac gccagcggcg aacccaacgt caaagggaga taggaaagtg 4200 ccacctaagt gtagaagggg gacgtcttga cgttgaagcg ttacctgtta ggtaacgtag 4260 ttgagctgtc aagacgtttt tttgcgtcga tagttgagct gtcacagaat gtgacgttga 4320 agtcgacgca tgaagtcgag cgcccttttt gatccagttc gatggtactc accatgttgt 4380 gcaaactccg gtgtcctgcc ttttaaatta aaatcaagtc aaacccgacg ttgaagcgtt 4440 acctgttagg taacgtagtt gagctgtcgg gtttgttttt tctttttata gttgagctgt 4500 cacagaatgt gacgttgaag taaaaagaat cagtaccgcg tatgtattaa gtgctcatca 4560 ttaatacgga gggcgctggc aagcattcag gctcaccagt taccaatgct tgccgcgttg 4620 ttccgacgtt gaagcgttac ctgttaggta acgtagttga gctgtcggaa caatttttgg 4680 tcctgttagt tgagctgtca cagaatgtga cgttgaagac aggacctacg gcgatcaagt 4740 gatcccacca agtcattctg ctgttgacaa tattattgaa gccagccggc ttaataagtg 4800 gtggcctaat ataaagacaa aaacgttgaa gcgttacctg ttaggtaacg tagttgagct 4860 gtttttgtct ccttcagcag ggttagttga gctgtcacag aatgtgacgt tgaagaccct 4920 gctaacagga actgttgggc gctgataata ccgcgccact ttaatagaaa aataaacaag 4980 tgctggcgat cggcagcagc cactggcgct tacggaaccg acgttgaagc gttacctgtt 5040 aggtaacgta gttgagctgt cggttccgtt tttacagagc gtagttgagc tgtcacagaa 5100 tgtgacgttg aagcgctctg ttttttaagg atctcaagaa attatcagtc tattgggaat 5160 acagcatctt ttacttactg tctcgttgtc agaagtcatc gtggccggga attttggacg 5220 ttgaagcgtt acctgttagg taacgtagtt gagctgtcca aaattttttt gcctcgctta 5280 gttgagctgt cacagaatgt gacgttgaag agcgaggctc tcctgctggc gtttttcgtc 5340 tgacggctcc acatgagcgt tcttcggggc gagagttgcg tcacgctgcg cgttacaggg 5400 cagcaattat gagtacgttg aagcgttacc tgttaggtaa cgtagttgag ctgtactcat 5460 aacctttcca ttggatagtt gagctgtcac agaatgtgac gttgaagtcc aatggcgcta 5520 cgcaggaaag aacatccata gatacggtcc ccgagttgag tgttgttcgt ggactgtggc 5580 gagaaaggac ctcttctacc atctgtctat acgttgaagc gttacctgtt aggtaacgta 5640 gttgagctgt atagacagtt tttaggctga atagttgagc tgtcacagaa tgtgacgttg 5700 aagttcagcc tgtaggtact caaaggcggt acttcctcgc caacgttaaa atcggcaaaa 5760 tcccttgatg gccgggagcc cccgattcaa ggcgacgttg aagcgttacc tgttaggtaa 5820 cgtagttgag ctgtcgcctt gatttttgct cacattagtt gagctgtcac agaatgtgac 5880 gttgaagatg tgagctacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 5940 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 6000 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat 6060 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 6120 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 6180 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 6240 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 6300 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 6360 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 6420 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 6480 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 6540 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 6600 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 6660 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 6720 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 6780 taagatcctt gagagttttc gccccgaaga acg 6813 <210> 26 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> DNAzyme oligonucleotide <400> 26 ttttttgccc tgatagttga gctgtcacag aatgtgacgt tgaagtcagg gcataaat 58 <210> 27 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> DNAzyme oligonucleotide <400> 27 ttttttgccc tgatagttga gctatcacag aatgtgatgt tgaagtcagg gcataaat 58 <210> 28 <211> 6813 <212> DNA <213> Artificial Sequence <220> <223> Phagemid <400> 28 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 60 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 120 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 180 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 240 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 300 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 360 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 420 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 480 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 540 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 600 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 660 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 720 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 780 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 840 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 900 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 960 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 1020 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 1080 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 1140 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 1200 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 1260 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 1320 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 1380 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 1440 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 1500 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 1560 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 1620 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taacttaatt gcacgttgaa 1680 gcgttacctg ttaggtaacg tagttgagct gtgcaattaa ttttttaaga gtatagttga 1740 gctgtcacag aatgtgacgt tgaagtactc ttagaagtgt cccaactaca ctagaaggac 1800 agtggcgaga ggattacgcg cctagatcaa ctttaatgtt gactcgtgca cccaacatgc 1860 tttttagctc acgttgaagc gttacctgtt aggtaacgta gttgagctgt gagctaaatt 1920 ttttcaatgt gtagttgagc tgtcacagaa tgtgacgttg aagcacattg agggctgcta 1980 ttaagacacg acttatccct ttctcaaaag gccagcaaag cgatctggcc ccaatagggg 2040 aacaagaggc agaacatatc aaagcgacgt tgaagcgtta cctgttaggt aacgtagttg 2100 agctgtcgct ttgatttttt cggtaagtag ttgagctgtc acagaatgtg acgttgaagc 2160 ttaccgagaa tagacacccg ccttacagcg aggcgaaggg ctttaaatca atctagagca 2220 tcataccagg cgtttcgttc ttggcgccgc aaccacctgt atgcacgttg aagcgttacc 2280 tgttaggtaa cgtagttgag ctgtgcatac agttcctgcg gtctatagtt gagctgtcac 2340 agaatgtgac gttgaagtag accgcgaaaa atgacgggga aagcctggcg aataactacg 2400 ttgcctgact cccggggata ttctcatagc tcactaacta ttgtgctgta gagctccgtc 2460 tacgttgaag cgttacctgt taggtaacgt agttgagctg tagacggagt ttttttggtc 2520 gctagttgag ctgtcacaga atgtgacgtt gaaggcgacc aaactctcag ggttattgtc 2580 tgatttatcg cgtccggcgg tgctacagac ccctggtccg cccccctgac aagtataaaa 2640 ccagcattta tcaaggatac gttgaagcgt tacctgttag gtaacgtagt tgagctgtat 2700 ccttgatttt ttgccctgat agttgagctg tcacagaatg tgacgttgaa gtcagggcat 2760 aaatcgcgtt aatattttgc gcggggatta agttgcgcct tatccgggct gtagtatcca 2820 cagaatcacg cgtatgtttg tcattgtaaa aaagaacgtt gaagcgttac ctgttaggta 2880 acgtagttga gctgttcttt ttttttttca ccgaagtagt tgagctgtca cagaatgtga 2940 cgttgaagct tcggtgtttg gtccatccaa aaaggatctt cacagaaaaa tgtttgcaag 3000 cagcagtatt tcattcagaa agcggtctgt gactggtgat aacccaatac tcacgttgaa 3060 gcgttacctg ttaggtaacg tagttgagct gtgagtattg tttttcgctc ctttagttga 3120 gctgtcacag aatgtgacgt tgaagaagga gcgggaaggc aatgatgagg cacctatctc 3180 aaggccacgg atacctgtcc ggccactggt gcgggaggga agcactatta aagaaccagt 3240 ttggttccgc acgttgaagc gttacctgtt aggtaacgta gttgagctgt gcggaacctt 3300 ttttcctgta gtagttgagc tgtcacagaa tgtgacgttg aagctacagg aagttggctg 3360 cataattctc tttcaccaaa tgccgcaaaa aaaattgttg tgtcacccag ttaccttcgg 3420 aaaccactga tcttttctac gttaagggag ctagacgttg aagcgttacc tgttaggtaa 3480 cgtagttgag ctgtctagct cctttttcca cgccttagtt gagctgtcac agaatgtgac 3540 gttgaagagg cgtgggcgct cttccgatac ggtgtatctc agttcggcga ccgctgggta 3600 accctaaaca ctacgtgaac caccgaaatt cgcgttacgt tgaagcgtta cctgttaggt 3660 aacgtagttg agctgtaacg cgaacctttc ctcatgatag ttgagctgtc acagaatgtg 3720 acgttgaagt catgaggatc cttcgctggt agcggtggct gaaggctcgt ccctccgaat 3780 gccatccgta agtgatctta gggcgacacg gaatccgcct atggcttggt atctacgttg 3840 aagcgttacc tgttaggtaa cgtagttgag ctgtagatac catttttgca agttttagtt 3900 gagctgtcac agaatgtgac gttgaagaaa cttgcatagg caagctccct cgtgcgtatg 3960 tacattcgct gtagcgtctt gcccggcgtc ggaaaacgga tacatatttg agacccacgc 4020 tgcgcattag cacgttgaag cgttacctgt taggtaacgt agttgagctg tgctaatgct 4080 tttttgaacg aatagttgag ctgtcacaga atgtgacgtt gaagttcgtt catgtgagcc 4140 cttcgggaag cgcccggtac gccagcggcg aacccaacgt caaagggaga taggaaagtg 4200 ccacctaagt gtagaagggg gacgtcttga cgttgaagcg ttacctgtta ggtaacgtag 4260 ttgagctgtc aagacgtttt tttgcgtcga tagttgagct gtcacagaat gtgacgttga 4320 agtcgacgca tgaagtcgag cgcccttttt gatccagttc gatggtactc accatgttgt 4380 gcaaactccg gtgtcctgcc ttttaaatta aaatcaagtc aaacccgacg ttgaagcgtt 4440 acctgttagg taacgtagtt gagctgtcgg gtttgttttt tctttttata gttgagctgt 4500 cacagaatgt gacgttgaag taaaaagaat cagtaccgcg tatgtattaa gtgctcatca 4560 ttaatacgga gggcgctggc aagcattcag gctcaccagt taccaatgct tgccgcgttg 4620 ttccgacgtt gaagcgttac ctgttaggta acgtagttga gctgtcggaa caatttttgg 4680 tcctgttagt tgagctgtca cagaatgtga cgttgaagac aggacctacg gcgatcaagt 4740 gatcccacca agtcattctg ctgttgacaa tattattgaa gccagccggc ttaataagtg 4800 gtggcctaat ataaagacaa aaacgttgaa gcgttacctg ttaggtaacg tagttgagct 4860 gtttttgtct ccttcagcag ggttagttga gctgtcacag aatgtgacgt tgaagaccct 4920 gctaacagga actgttgggc gctgataata ccgcgccact ttaatagaaa aataaacaag 4980 tgctggcgat cggcagcagc cactggcgct tacggaaccg acgttgaagc gttacctgtt 5040 aggtaacgta gttgagctgt cggttccgtt tttacagagc gtagttgagc tgtcacagaa 5100 tgtgacgttg aagcgctctg ttttttaagg atctcaagaa attatcagtc tattgggaat 5160 acagcatctt ttacttactg tctcgttgtc agaagtcatc gtggccggga attttggacg 5220 ttgaagcgtt acctgttagg taacgtagtt gagctgtcca aaattttttt gcctcgctta 5280 gttgagctgt cacagaatgt gacgttgaag agcgaggctc tcctgctggc gtttttcgtc 5340 tgacggctcc acatgagcgt tcttcggggc gagagttgcg tcacgctgcg cgttacaggg 5400 cagcaattat gagtacgttg aagcgttacc tgttaggtaa cgtagttgag ctgtactcat 5460 aacctttcca ttggatagtt gagctgtcac agaatgtgac gttgaagtcc aatggcgcta 5520 cgcaggaaag aacatccata gatacggtcc ccgagttgag tgttgttcgt ggactgtggc 5580 gagaaaggac ctcttctacc atctgtctat acgttgaagc gttacctgtt aggtaacgta 5640 gttgagctgt atagacagtt tttaggctga atagttgagc tgtcacagaa tgtgacgttg 5700 aagttcagcc tgtaggtact caaaggcggt acttcctcgc caacgttaaa atcggcaaaa 5760 tcccttgatg gccgggagcc cccgattcaa ggcgacgttg aagcgttacc tgttaggtaa 5820 cgtagttgag ctgtcgcctt gatttttgct cacattagtt gagctgtcac agaatgtgac 5880 gttgaagatg tgagctacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 5940 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 6000 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat 6060 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 6120 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 6180 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 6240 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 6300 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 6360 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 6420 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 6480 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 6540 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 6600 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 6660 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 6720 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 6780 taagatcctt gagagttttc gccccgaaga acg 6813 <210> 29 <211> 6731 <212> DNA <213> Artificial Sequence <220> <223> Phagemid <400> 29 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 60 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 120 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 180 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 240 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 300 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 360 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 420 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 480 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 540 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 600 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 660 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 720 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 780 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 840 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 900 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 960 acttaattgc gttgaagcgt tacctgttag gtaacgtagt tgagctgcaa ttaatttttt 1020 aagagtatag ttgagctgtc acagaatgtg acgttgaagt actcttagaa gtgtcccaac 1080 tacactagaa ggacagtggc gagaggatta cgcgcctaga tcaactttaa tgttgactcg 1140 tgcacccaac atgcttttta gctcgttgaa gcgttacctg ttaggtaacg tagttgagct 1200 gagctaaatt ttttcaatgt gtagttgagc tgtcacagaa tgtgacgttg aagcacattg 1260 agggctgcta ttaagacacg acttatccct ttctcaaaag gccagcaaag cgatctggcc 1320 ccaatagggg aacaagaggc agaacatatc aaagcgagtt gaagcgttac ctgttaggta 1380 acgtagttga gcttcgcttt gttttttcgg taagtagttg agctgtcaca gaatgtgacg 1440 ttgaagctta ccgagaatag acacccgcct tacagcgagg cgaagggctt taaatcaatc 1500 tagagcatca taccaggcgt ttcgttcttg gcgccgcaac cacctgtatg cgttgaagcg 1560 ttacctgtta ggtaacgtag ttgagctgca tacagttttt gcggtctata gttgagctgt 1620 cacagaatgt gacgttgaag tagaccgcga aaaatgacgg ggaaagcctg gcgaataact 1680 acgttgcctg actcccgggg atattctcat agctcactaa ctattgtgct gtagagctcc 1740 gtctagttga agcgttacct gttaggtaac gtagttgagc ttagacggat tttattggtc 1800 gctagttgag ctgtcacaga atgtgacgtt gaaggcgacc aaactctcag ggttattgtc 1860 tgatttatcg cgtccggcgg tgctacagac ccctggtccg cccccctgac aagtataaaa 1920 ccagcattta tcaaggatgt tgaagcgtta cctgttaggt aacgtagttg agctatcctt 1980 gattttttgc cctgatagtt gagctgtcac agaatgtgac gttgaagtca gggcataaat 2040 cgcgttaata ttttgcgcgg ggattaagtt gcgccttatc cgggctgtag tatccacaga 2100 atcacgcgta tgtttgtcat tgtaaaaaag aagttgaagc gttacctgtt aggtaacgta 2160 gttgagcttt cttttttttt tcaccgaagt agttgagctg tcacagaatg tgacgttgaa 2220 gcttcggtgt ttggtccatc caaaaaggat cttcacagaa aaatgtttgc aagcagcagt 2280 atttcattca gaaagcggtc tgtgactggt gataacccaa tactcagttg aagcgttacc 2340 tgttaggtaa cgtagttgag cttgagtatt tttttcgctc ctttagttga gctgtcacag 2400 aatgtgacgt tgaagaagga gcgggaaggc aatgatgagg cacctatctc aaggccacgg 2460 atacctgtcc ggccactggt gcgggaggga agcactatta aagaaccagt ttggttccgc 2520 gttgaagcgt tacctgttag gtaacgtagt tgagctgcgg aaccttttat cctgtagtag 2580 ttgagctgtc acagaatgtg acgttgaagc tacaggaagt tggctgcata attctctttc 2640 accaaatgcc gcaaaaaaaa ttgttgtgtc acccagttac cttcggaaac cactgatctt 2700 ttctacgtta agggagctag agttgaagcg ttacctgtta ggtaacgtag ttgagcttct 2760 agctcttttt ccacgcctta gttgagctgt cacagaatgt gacgttgaag aggcgtgggc 2820 gctcttccga tacggtgtat ctcagttcgg cgaccgctgg gtaaccctaa acactacgtg 2880 aaccaccgaa attcgcgttg ttgaagcgtt acctgttagg taacgtagtt gagctaacgc 2940 gaaaattacc tcatgatagt tgagctgtca cagaatgtga cgttgaagtc atgaggatcc 3000 ttcgctggta gcggtggctg aaggctcgtc cctccgaatg ccatccgtaa gtgatcttag 3060 ggcgacacgg aatccgccta tggcttggta tctgttgaag cgttacctgt taggtaacgt 3120 agttgagcta gataccattt tagcaagttt tagttgagct gtcacagaat gtgacgttga 3180 agaaacttgc ataggcaagc tccctcgtgc gtatgtacat tcgctgtagc gtcttgcccg 3240 gcgtcggaaa acggatacat atttgagacc cacgctgcgc attagcagtt gaagcgttac 3300 ctgttaggta acgtagttga gcttgctaat gttttttgaa cgaatagttg agctgtcaca 3360 gaatgtgacg ttgaagttcg ttcatgtgag cccttcggga agcgcccggt acgccagcgg 3420 cgaacccaac gtcaaaggga gataggaaag tgccacctaa gtgtagaagg gggacgtctt 3480 ggttgaagcg ttacctgtta ggtaacgtag ttgagctcaa gacgtttttt tgcgtcgata 3540 gttgagctgt cacagaatgt gacgttgaag tcgacgcatg aagtcgagcg ccctttttga 3600 tccagttcga tggtactcac catgttgtgc aaactccggt gtcctgcctt ttaaattaaa 3660 atcaagtcaa acccggttga agcgttacct gttaggtaac gtagttgagc tcgggtttgt 3720 tttatctttt tatagttgag ctgtcacaga atgtgacgtt gaagtaaaaa gaatcagtac 3780 cgcgtatgta ttaagtgctc atcattaata cggagggcgc tggcaagcat tcaggctcac 3840 cagttaccaa tgcttgccgc gttgttccgg ttgaagcgtt acctgttagg taacgtagtt 3900 gagctcggaa caatttttgg tcctgttagt tgagctgtca cagaatgtga cgttgaagac 3960 aggacctacg gcgatcaagt gatcccacca agtcattctg ctgttgacaa tattattgaa 4020 gccagccggc ttaataagtg gtggcctaat ataaagacaa aaagttgaag cgttacctgt 4080 taggtaacgt agttgagctt ttttgtcaat taagcagggt tagttgagct gtcacagaat 4140 gtgacgttga agaccctgct aacaggaact gttgggcgct gataataccg cgccacttta 4200 atagaaaaat aaacaagtgc tggcgatcgg cagcagccac tggcgcttac ggaaccggtt 4260 gaagcgttac ctgttaggta acgtagttga gctcggttcc gattttacag agcgtagttg 4320 agctgtcaca gaatgtgacg ttgaagcgct ctgtttttta aggatctcaa gaaattatca 4380 gtctattggg aatacagcat cttttactta ctgtctcgtt gtcagaagtc atcgtggccg 4440 ggaattttgg gttgaagcgt tacctgttag gtaacgtagt tgagctccaa aatttttttg 4500 cctcgcttag ttgagctgtc acagaatgtg acgttgaaga gcgaggctct cctgctggcg 4560 tttttcgtct gacggctcca catgagcgtt cttcggggcg agagttgcgt cacgctgcgc 4620 gttacagggc agcaattatg agtgttgaag cgttacctgt taggtaacgt agttgagcta 4680 ctcataaatt tccattggat agttgagctg tcacagaatg tgacgttgaa gtccaatggc 4740 gctacgcagg aaagaacatc catagatacg gtccccgagt tgagtgttgt tcgtggactg 4800 tggcgagaaa ggacctcttc taccatctgt ctatgttgaa gcgttacctg ttaggtaacg 4860 tagttgagct atagacagtt tttaggctga atagttgagc tgtcacagaa tgtgacgttg 4920 aagttcagcc tgtaggtact caaaggcggt acttcctcgc caacgttaaa atcggcaaaa 4980 tcccttgatg gccgggagcc cccgattcaa ggcggttgaa gcgttacctg ttaggtaacg 5040 tagttgagct cgccttgatt tttgctcaca ttagttgagc tgtcacagaa tgtgacgttg 5100 aagatgtgag ctacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 5160 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 5220 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag 5280 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 5340 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 5400 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 5460 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 5520 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 5580 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 5640 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 5700 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 5760 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 5820 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 5880 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 5940 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 6000 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 6060 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 6120 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 6180 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 6240 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 6300 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 6360 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 6420 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 6480 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 6540 ggagccggtg agcgtgggtc acgcggtatc attgcagcac tggggccaga tggtaagccc 6600 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 6660 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 6720 tcatatatac t 6731 <210> 30 <211> 9059 <212> DNA <213> Artificial Sequence <220> <223> Phagemid 48helix tube <400> 30 gacgtaacgg tgctgtctaa catcgagact gcaattaccc cgccagacct ttgcacttcc 60 acactaattt ggtcgatctt tgcttaaccg ggaactatgt agtctatatg agaatattga 120 gcataaggtg tcagccagcc tttatccttg aggcagatca ggtctattcg ctcagagtaa 180 gatgctaaca cccagtagat gacgacgttt aattagggcc gagagaccaa tgtcacgcat 240 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 300 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 360 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 420 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 480 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 540 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 600 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 660 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaacct tgatcgggca 720 cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg tattttttga 780 gttatcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac 840 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc 900 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa 960 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc 1020 tcatccggaa tttcgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca 1080 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata 1140 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga 1200 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc 1260 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc 1320 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat 1380 tcaggttcat catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca 1440 acagtactgc gatgagtggc agggcggggc gtaatttgat atcgagctcg cttggactcc 1500 tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt 1560 gccgccgggc gttttttatt ggtgagaatc caagcctcga gctgtcagac caagtttact 1620 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1680 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1740 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1800 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1860 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1920 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1980 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 2040 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 2100 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 2160 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 2220 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 2280 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 2340 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 2400 gctggccttt tgctcacctt agcaagagcc gcacgacgac cagaggccag ttatccagag 2460 ttaggatacc tcaatgtgca tccgctcggt tctaaggaca ttatttcagt cctttaagat 2520 ctcccgtata gaagcctcac gttagggggc gccgtgcctt cacgccctcc catttaggaa 2580 taccttgtct ccgccgtctt tattcagtag ccctatgcat tacgatgtgg cgcttccccc 2640 gcgtgggcgc agaaatttac tgaggcggat tcgaaacgac tgtgagggca ggataggtga 2700 gcaggcactg gcacgtaatc aaccaacgga ctcacccgtg tgcaggccta aaaacagccc 2760 tcgaagggca cttggatatg aatgaaccca cttgttttga ctcgtggagg cgtggtttta 2820 ttactgtgct cagttaacgc cgcatgaatt tagctctgat caccgtaagg gtaactgcac 2880 tagacatgtt gtgggcattt aagtcctgca gtatcttttt gttaggtgga acggcctagg 2940 ggtaccttcc gtgagaaact cccagatgat gcatgttcga gtacttgtga aatggatggt 3000 cgcatcccct cctctcacac attacactgt ctcgcgcggg ttgcgtcttg accggtacaa 3060 gttgtgtaac cttacacctc tagaaacatt ttagcagtcg ctccaattga taccacgacc 3120 tcagcgcgcg ttgggagacc gttgccaaac ctagacgttg aagcgttacc tgttaggtaa 3180 cgtagttgag ctgtctaggt tttttttgca tctggtagtt gagctgtcac agaatgtgac 3240 gttgaagcca gatgcaccaa tgtatataaa tagatccttt taaattaaat aaatcaatcg 3300 taatactgaa ctaggttctc ccaaggtgta accgtaaacc gccccacgtt gaagcgttac 3360 ctgttaggta acgtagttga gctgtggggc ggttttttaa tccaagtagt tgagctgtca 3420 cagaatgtga cgttgaagct tggattctga gttctgcacg gctcaattcg cgttaaattt 3480 ttggcgaggg cgtgaaggag gtcattactg gtaagctcga ggacgttgaa gcgttacctg 3540 ttaggtaacg tagttgagct gtcctcgagc tttttcccgt agatagttga gctgtcacag 3600 aatgtgacgt tgaagtctac ggggtctata atatttgaag ttgtggccca agttttttgg 3660 ggtcaaggga gttttcggaa aaagagaaac tctggccaca cgttgaagcg ttacctgtta 3720 ggtaacgtag ttgagctgtg tggccagttt ttaaaaaaaa tagttgagct gtcacagaat 3780 gtgacgttga agtttttttt tttttttttt ttaggatcta ttacgctttt tttttttttt 3840 ttttttgcct gcatcgaggg tttttttttt tttttttttt acgttgaagc gttacctgtt 3900 aggtaacgta gttgagctgt aaaaaaaatt ttttaccgct atagttgagc tgtcacagaa 3960 tgtgacgttg aagtagcggt aaacaaaaca agtgcgcctc ctgcagttgc ccacacaaaa 4020 agcacgaact tctcatagct cactctccct catccatcag tgtacggtca attctagacg 4080 cgcgcacgtt gaagcgttac ctgttaggta acgtagttga gctgtgcgcg cgttttttga 4140 gggttttagt tgagctgtca cagaatgtga cgttgaagaa accctccccc gagcgaacgt 4200 ggcgcgctcc agatgcgaat atatgtgaag ccagttacca gggaaataca catctgcgat 4260 gaacgttgaa gcgttacctg ttaggtaacg tagttgagct gttcatcgca tttttatact 4320 gtttagttga gctgtcacag aatgtgacgt tgaagaacag tatacggcta gagttctatg 4380 tagcaacccg aactatcgcc cgaccgctgc gctgtgtgat actgcgtacc cctaggccga 4440 tgcgactcgg gaaaagctcc acgttgaagc gttacctgtt aggtaacgta gttgagctgt 4500 ggagcttttt tttcgcgtgt ttagttgagc tgtcacagaa tgtgacgttg aagaacacgc 4560 ggccaggtct caattgagac ggggcgaagc ccatgccttg tcgccttgcc ctgaattatc 4620 aggggactcc acgttgaagc gttacctgtt aggtaacgta gttgagctgt ggagtccctt 4680 tttagtttct atagttgagc tgtcacagaa tgtgacgttg aagtagaaac tgccgtggcc 4740 taactttggt aatccggcgg tttttttgtt aaaactggtg ttggtaaaat cggacgttga 4800 agcgttacct gttaggtaac gtagttgagc tgtccgattt tttttttttc catgtagttg 4860 agctgtcaca gaatgtgacg ttgaagcatg gaaaaacgtt aaagccgttt agagctaatc 4920 actacgtgaa ccgtccgcca gcactgttgt aattcacccc gccatagggt aatcccttct 4980 cgatgaagtg cagcaaaggt gatctcctta gcacgttgaa gcgttacctg ttaggtaacg 5040 tagttgagct gtgctaagga tttttacacc gtttagttga gctgtcacag aatgtgacgt 5100 tgaagaacgg tgtaacagcc actggtgtat tcagcgttaa aaccctagag gtgcattacg 5160 tgccaggatc catataccca gggattggca cgttgaagcg ttacctgtta ggtaacgtag 5220 ttgagctgtg ccaatccttt ttaaaaaaaa tagttgagct gtcacagaat gtgacgttga 5280 agtttttttt tttttttttt ttcaccgtca tcccattttt tttttttttt ttttttgggc 5340 gctaagaaag tttttttttt tttttttttt acgttgaagc gttacctgtt aggtaacgta 5400 gttgagctgt aaaaaaaatt tttctcatcc gtagttgagc tgtcacagaa tgtgacgttg 5460 aagcggatga gcattttatc cggtgtaaga ccttaaatac ccttacggtg atcagtaatg 5520 ggaaatcgtc gtgaacagga atcgccaacg ttgaagcgtt acctgttagg taacgtagtt 5580 gagctgttgg cgattttttt ctggagcgta gttgagctgt cacagaatgt gacgttgaag 5640 cgctccagta tctcagttcg gtaccggaaa catgcgataa aggctggcgg tcagaggtgg 5700 cacaatctcg ataaccgttt ttcccgctca acgcgagatt cacaaacgga agaggtaacc 5760 accacaacgt tgaagcgtta cctgttaggt aacgtagttg agctgttgtg gtggttttta 5820 acaggtatag ttgagctgtc acagaatgtg acgttgaagt acctgttgtt ccgaccctgc 5880 cagaaacccg acaggactat agctctcccc gccttgctgt agagctgggc ccatcaggcg 5940 ggcaagaatg ggtcgttccc gccgtctggg agtttctcgt actcgacgtt gaagcgttac 6000 ctgttaggta acgtagttga gctgtcgagt acgttttttg cacgagtagt tgagctgtca 6060 cagaatgtga cgttgaagct cgtgcaagat acaaatcgaa taggctaagg ccgaaaaggc 6120 ggataactgg cctctagaac ctgaggtcga aatcttcaac gttaaagaca acgtgaaaca 6180 aatacgttga agcgttacct gttaggtaac gtagttgagc tgtatttgtt ttttttcttc 6240 aggatagttg agctgtcaca gaatgtgacg ttgaagtcct gaaggtggta cctcaagata 6300 ctgggtgtta gggtacattg agccttgaaa ggccgtaata tccagcggtt atacatctta 6360 atagacctat agacccggtt aaaaggtccc acctaagctc atacgttgaa gcgttacctg 6420 ttaggtaacg tagttgagct gtatgagctt tttttcctcc gcttagttga gctgtcacag 6480 aatgtgacgt tgaagagcgg aggactgaaa taatggcccc ctggcggaga cgggaagcgc 6540 cacatctaaa aaaaaacgcc cggccgttct gggcttcttc ttaaatgcac attaactctc 6600 agcaaaacgt tgaagcgtta cctgttaggt aacgtagttg agctgttttg ctgattttta 6660 accttgttag ttgagctgtc acagaatgtg acgttgaaga caaggtttac gtcgtggtat 6720 caattatata ttttgttaac ttggtcgtcg tgcatcaagg ttagcgttat agtgtggtta 6780 gacacgttga agcgttacct gttaggtaac gtagttgagc tgtgtctaac ctttttctga 6840 cgtttagttg agctgtcaca gaatgtgacg ttgaagaacg tcagaccgag ctgccacgct 6900 cgatatcaaa tatctatcga aatcgttaaa tcaattgtac cgcgcaacct cttacgtgcc 6960 cccggtaatc gaccgctcaa tacgttgaag cgttacctgt taggtaacgt agttgagctg 7020 tattgagcgt ttttattaat agtagttgag ctgtcacaga atgtgacgtt gaagctatta 7080 atgttccaac ttggtctgac cgttaagcat gatgaagtga cgctcagtgg aacgaaacat 7140 cacatatcct ggagtccaac gttgaagcgt tacctgttag gtaacgtagt tgagctgttg 7200 gactcctttt ttgattcgat agttgagctg tcacagaatg tgacgttgaa gtcgaatcaa 7260 tttctgcgcc cacgcggaac cctcacgagt ccgtctgcaa gcagcagcaa gaagttaagg 7320 gattttggat caaaaacgtt gaagcgttac ctgttaggta acgtagttga gctgtttttg 7380 atctttttgc tttacgtagt tgagctgtca cagaatgtga cgttgaagcg taaagcatcc 7440 aagtgccctc acgggtagtc gttaggatct tcaccgcact cacgatcctt tgatctttgt 7500 ttaaattggt tgacgttgaa gcgttacctg ttaggtaacg tagttgagct gtcaaccaat 7560 tttttgtgct cagtagttga gctgtcacag aatgtgacgt tgaagctgag cacagagctg 7620 tcacgctgcg cgaacgactt ttagcagagc gaggttgaag taaaaccagg ttcattcata 7680 ctgctcttgt ctgcgctctg ctgacgttga agcgttacct gttaggtaac gtagttgagc 7740 tgtcagcaga gtttttatga attttagttg agctgtcaca gaatgtgacg ttgaagaaat 7800 tcatgagaaa ggaagggagg gcgctgcgtg atacggtctt taatatgaat aagccatacg 7860 aaattcctgg cagtgtagcg acgttgaagc gttacctgtt aggtaacgta gttgagctgt 7920 cgctacactt tttattaagc gtagttgagc tgtcacagaa tgtgacgttg aagcgcttaa 7980 tggcaagcaa gggtgaacac ttttcattag gccggataaa acttttcttc attggttaat 8040 taaacgtcgt catctcaacg ttgaagcgtt acctgttagg taacgtagtt gagctgttga 8100 gatgattttt atgagaatta gttgagctgt cacagaatgt gacgttgaag attctcatga 8160 tctgtatcca gccattggga tatatcgaac tgactgaaat gcctcatacg atgtgatttt 8220 ttagctttat ttcaagttgg acatttccac gttgaagcgt tacctgttag gtaacgtagt 8280 tgagctgtgg aaatgttttt tggttgcgtt agttgagctg tcacagaatg tgacgttgaa 8340 gacgcaaccc tgacacctta taaggagcga ctgctggtca aaaaatacgc gggctcttgc 8400 taaggtgagc cgttgctaaa atgtacgttg aagcgttacc tgttaggtaa cgtagttgag 8460 ctgtacattt tatttttaaa aaaaatagtt gagctgtcac agaatgtgac gttgaagttt 8520 tttttttttt tttttttctt gtacatgtgt gttttttttt tttttttttt tatcacaaca 8580 ggcgtttttt tttttttttt tttttacgtt gaagcgttac ctgttaggta acgtagttga 8640 gctgtaaaaa aaatttttaa aaaaaatagt tgagctgtca cagaatgtga cgttgaagtt 8700 tttttttttt ttttttttat tgcagtataa attttttttt tttttttttt ttataggcca 8760 acaggatttt tttttttttt ttttttacgt tgaagcgtta cctgttaggt aacgtagttg 8820 agctgtaaaa aaaattttta atcggtgtag ttgagctgtc acagaatgtg acgttgaagc 8880 accgattcct aaatggaatg agtgtagaac gtgctgcctg ctcaccaacg gcattctgcc 8940 gacatggtga gtaagtttgg aacgttgaag cgttacctgt taggtaacgt agttgagctg 9000 ttccaaactt ttttgtcgaa attagttgag ctgtcacaga atgtgacgtt gaagatttc 9059 <210> 31 <211> 8027 <212> DNA <213> Artificial Sequence <220> <223> Phagemid pointer - part 1 <400> 31 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 900 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 960 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 1020 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 1080 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 1140 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 1200 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 1260 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 1320 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 1380 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 1440 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 1500 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 1560 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 1620 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcactgc 1680 cctttgtagt tgagctgtca cagaatgtga cgttgaagca aagggcgggt gcctcgctca 1740 ctgcttaatg ttgcatgcgg tcacgttgac gttgaagcgt tacctgttag gtaacgtagt 1800 tgagctgtca acgtgatttt tacaacaatt agttgagctg tcacagaatg tgacgttgaa 1860 gattgttgtt ccggccaacc agggtggtgc caagcgacag gaagccggaa cgttgaagcg 1920 ttacctgtta ggtaacgtag ttgagctgtt ccggcttttt ttaaaaaaaa tagttgagct 1980 gtcacagaat gtgacgttga agtttttttt ttgggtgaga cgggcgagtt ttttttttaa 2040 agggtcgtgc cgaggatccc cgggttttta cgttgaagcg ttacctgtta ggtaacgtag 2100 ttgagctgta aaaacccttt ttcgatttcg tagttgagct gtcacagaat gtgacgttga 2160 agcgaaatcg cctggccctt gccccagagt aaaataacat cacttacgtt gaagcgttac 2220 ctgttaggta acgtagttga gctgtaagtg atgtttttct gatttttagt tgagctgtca 2280 cagaatgtga cgttgaagaa aatcagcccc cggcaggcga aaatcctgac gctggttgag 2340 agagggacgt tgaagcgtta cctgttaggt aacgtagttg agctgtccct ctcttttttt 2400 tacggcctag ttgagctgtc acagaatgtg acgttgaagg gccgtaaagc actaaatttt 2460 tttttttcgg aaccggaaag ccggcgattt ttacgttgaa gcgttacctg ttaggtaacg 2520 tagttgagct gtaaaaatcg tttttgctaa aaatagttga gctgtcacag aatgtgacgt 2580 tgaagttttt agcggtcctt tttttttttt tgatggtggt tcaatagccc ttgggcacgt 2640 tgaagcgtta cctgttaggt aacgtagttg agctgtgccc aaggtttttc gtaaaaatag 2700 ttgagctgtc acagaatgtg acgttgaagt ttttacgtgg caacagctga gaagttttcc 2760 tttttttttt cagtcacgac gttgtacgtt gaagcgttac ctgttaggta acgtagttga 2820 gctgtacaac gtctttttcc cgcgcgtagt tgagctgtca cagaatgtga cgttgaagcg 2880 cgcggggacg tgcttcgccg ctacagcttt caataggaat tgcggacgtt gaagcgttac 2940 ctgttaggta acgtagttga gctgtccgca atttttttaa aaccgctagt tgagctgtca 3000 cagaatgtga cgttgaaggc ggtttttttt tttttgcgta gagttttttt tttatagggt 3060 tgagtaaaga acttacgttg aagcgttacc tgttaggtaa cgtagttgag ctgtaagttc 3120 tttttttctt aggtttagtt gagctgtcac agaatgtgac gttgaagaac ctaagggttt 3180 tttttttaag aaagcgaaag gtcacgctag ctcttttttt tttgaattcg taatacgttg 3240 aagcgttacc tgttaggtaa cgtagttgag ctgtattacg aatttttatt caacgtagtt 3300 gagctgtcac agaatgtgac gttgaagcgt tgaatgagtg taaagtgtaa ttgttaagga 3360 agatgataat catgacgttg aagcgttacc tgttaggtaa cgtagttgag ctgtcatgat 3420 tatttttggc gggtttagtt gagctgtcac agaatgtgac gttgaagaac ccgccgcgcc 3480 cgctttaatg aatcagtttg gccttataaa tcaaaacgtt gaagcgttac ctgttaggta 3540 acgtagttga gctgttttga ttttttttga gttaaatagt tgagctgtca cagaatgtga 3600 cgttgaagtt taactcgcgt cttttttttt ttgaaatgga tcaccatcaa ctgttttttt 3660 tttatagcaa acatacgttg aagcgttacc tgttaggtaa cgtagttgag ctgtatgttt 3720 gctttttacg ttttgtagtt gagctgtcac agaatgtgac gttgaagcaa aacgttaaaa 3780 ctagcttgag agatctggag ctcattgaat ccccctcgaa tcgacgttga agcgttacct 3840 gttaggtaac gtagttgagc tgtcgattcg atttttctta gagttagttg agctgtcaca 3900 gaatgtgacg ttgaagactc taagctgcat tccagtcgcg tgaaccgtct atcagggcga 3960 tgtttttttt ttgcccacta ggaacgttga agcgttacct gttaggtaac gtagttgagc 4020 tgttcctagt gtttttgtaa aaaatagttg agctgtcaca gaatgtgacg ttgaagtttt 4080 ttaccggcgc gtaaacaacc cgaaattttt taaaaattct gagtacgttg aagcgttacc 4140 tgttaggtaa cgtagttgag ctgtactcag aatttttcta cactgtagtt gagctgtcac 4200 agaatgtgac gttgaagcag tgtagcggag cgggcgctag ggcgaaaaac caacaagagt 4260 ccactacgtt gaagcgttac ctgttaggta acgtagttga gctgtagtgg acttttttga 4320 taaaattagt tgagctgtca cagaatgtga cgttgaagat tttatcctcg ttcgtgcatc 4380 tggtgtagca ccagcactca gagcaaacgt tgaagcgtta cctgttaggt aacgtagttg 4440 agctgtttgc tctgtttttg ttaaaaatag ttgagctgtc acagaatgtg acgttgaagt 4500 ttttaaccag aggaattttt tttttaaacg ctccatcacc tcattagacg ttgaagcgtt 4560 acctgttagg taacgtagtt gagctgtcta atgagttttt cttctactta gttgagctgt 4620 cacagaatgt gacgttgaag agtagaaggt tgggtaacgc cttttttttt tagggtgttt 4680 ttatccaacg ttgaagcgtt acctgttagg taacgtagtt gagctgttgg ataaattttt 4740 atcaaagata gttgagctgt cacagaatgt gacgttgaag tctttgatta gtaagagtct 4800 gtgggcgatc ccaggcaaca ataacccaac gttgaagcgt tacctgttag gtaacgtagt 4860 tgagctgttg ggttattttt tgctgccagt agttgagctg tcacagaatg tgacgttgaa 4920 gctggcagct ttcgcgcgag caacaccgct cctgatttag aacctgaacg ttgaagcgtt 4980 acctgttagg taacgtagtt gagctgttca ggttcttttt ttcgtaatta gttgagctgt 5040 cacagaatgt gacgttgaag attacgaatc agtgcagaat cctgattgcc ttcaccaggt 5100 cgaggtatca cccaaatcaa gttttttacg ttgaagcgtt acctgttagg taacgtagtt 5160 gagctgtaaa aaactttttt tgcaaaaata gttgagctgt cacagaatgt gacgttgaag 5220 tttttgcaac gctagtttga catatctttt tttttttggt cagttggcac cgacgttgaa 5280 gcgttacctg ttaggtaacg tagttgagct gtcggtgcca tttttattaa tcgtagttga 5340 gctgtcacag aatgtgacgt tgaagcgatt aatagctcaa ctggaagtct tttgatctat 5400 tataccatcc tagtcctgac gttgaagcgt tacctgttag gtaacgtagt tgagctgtca 5460 ggactatttt tattaccagt agttgagctg tcacagaatg tgacgttgaa gctggtaata 5520 tccgaattga gccaattcta ggtcaggaaa aagatgcacg ttgaagcgtt acctgttagg 5580 taacgtagtt gagctgtgca tctttttttt cggccgatta gttgagctgt cacagaatgt 5640 gacgttgaag atcggccggc ggattcagta ttggcaaaga gatgatgatt aacaataaac 5700 gttgaagcgt tacctgttag gtaacgtagt tgagctgttt attgtttttt tccagaagtt 5760 agttgagctg tcacagaatg tgacgttgaa gacttctggt cggtacgcag gccaccattt 5820 agagcttgac ggctaaagac gttgaagcgt tacctgttag gtaacgtagt tgagctgtct 5880 ttagcctttt tagaactggt agttgagctg tcacagaatg tgacgttgaa gccagttctg 5940 agagctgttt agcattgcat cattagagaa aacgttgaag cgttacctgt taggtaacgt 6000 agttgagctg ttttctctat ttttcctttc ggtagttgag ctgtcacaga atgtgacgtt 6060 gaagccgaaa gggtacggtg tcatgttttg cggatggctt agagcttacg ttgaagcgtt 6120 acctgttagg taacgtagtt gagctgtaag ctctattttt cgcaccttta gttgagctgt 6180 cacagaatgt gacgttgaag aaggtgcggg gttgattcga aggttacttt aggagcacta 6240 acgttttaac gttgaagcgt tacctgttag gtaacgtagt tgagctgtta aaacgttttt 6300 tcctcctaat agttgagctg tcacagaatg tgacgttgaa gttaggaggc ccaaattaac 6360 cgtttttttt ttttgcagcc atttttttac gttgaagcgt tacctgttag gtaacgtagt 6420 tgagctgtaa aaaaattttt tatcacattt agttgagctg tcacagaatg tgacgttgaa 6480 gaatgtgatg ggatactgca ggtacggcca gtttttcttc ttcaccggca cgttgaagcg 6540 ttacctgtta ggtaacgtag ttgagctgtg ccggtgattt ttttatgttg tagttgagct 6600 gtcacagaat gtgacgttga agcaacataa taaagcgaag ataattgcac gtgatgatgg 6660 catcggaagt acgttgaagc gttacctgtt aggtaacgta gttgagctgt acttccgatt 6720 tttcgccctg gtagttgagc tgtcacagaa tgtgacgttg aagccagggc gccggaagca 6780 agctaacttt ttttttttca cattaagtgt tttttttttg actccaacgt acgttgaagc 6840 gttacctgtt aggtaacgta gttgagctgt acgttggatt ttttgaaaaa atagttgagc 6900 tgtcacagaa tgtgacgttg aagttttttc aatcgttgta ccaacctaat ttttttttta 6960 acatcgccat taagattttc gagcggacgt tgaagcgtta cctgttaggt aacgtagttg 7020 agctgtccgc tcgattttta tgaccaatag ttgagctgtc acagaatgtg acgttgaagt 7080 tggtcatagc tgtttcctgt gtgaaaagcc tgttttaacc atacgttgaa gcgttacctg 7140 ttaggtaacg tagttgagct gtatggttaa ttttttgctt atatagttga gctgtcacag 7200 aatgtgacgt tgaagtataa gcaaaagggg aagccttttt ttaatatttc aatcatatgc 7260 gggacgttga agcgttacct gttaggtaac gtagttgagc tgtcccgcat attttttatt 7320 ttgttagttg agctgtcaca gaatgtgacg ttgaagacaa aataatatac gagcgtacta 7380 tggttttttt tttttgcttt tgacgctttt tacgttgaag cgttacctgt taggtaacgt 7440 agttgagctg taaaaagcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 7500 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 7560 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 7620 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 7680 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 7740 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 7800 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 7860 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg 7920 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 7980 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagt 8027 <210> 32 <211> 7899 <212> DNA <213> Artificial Sequence <220> <223> Phagemid pointer - part 2 <400> 32 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 900 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 960 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 1020 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 1080 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 1140 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 1200 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 1260 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 1320 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 1380 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 1440 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 1500 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 1560 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 1620 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacggt 1680 gatggtagtt gagctgtcac agaatgtgac gttgaagcca tcaccaaaaa ctccgctcac 1740 aattccacac tttttttacg ttgaagcgtt acctgttagg taacgtagtt gagctgtaaa 1800 aaaagttttt ctgttggcta gttgagctgt cacagaatgt gacgttgaag gccaacagat 1860 acgtggtaat gccggggaag aaggacgttg aagcgttacc tgttaggtaa cgtagttgag 1920 ctgtccttct tctttttact aatcgtagtt gagctgtcac agaatgtgac gttgaagcga 1980 ttagtcttta attttttttt ttgcgcgaat atgataacgg aactgacgag aaacaccagt 2040 caataacgtt gaagcgttac ctgttaggta acgtagttga gctgttattg acttttttat 2100 aaatagtagt tgagctgtca cagaatgtga cgttgaagct atttatgtca atcccttctg 2160 aaacaagaga atcgatcctg agacgttgaa gcgttacctg ttaggtaacg tagttgagct 2220 gtctcaggat ttttttgatt tattagttga gctgtcacag aatgtgacgt tgaagataaa 2280 tcacagacac cacattcaac taatgatacg ttgaagcgtt acctgttagg taacgtagtt 2340 gagctgtatc attagttttt accaaaaata gttgagctgt cacagaatgt gacgttgaag 2400 tttttggtca ttggaatttt ttttttcggt aatcgtaata ttttgtgcaa tgcttacgtt 2460 gaagcgttac ctgttaggta acgtagttga gctgtaagca ttgttttttt tgtagatagt 2520 tgagctgtca cagaatgtga cgttgaagtc tacaaaaaga actgttgtga attaccttat 2580 agaaattttt acgttgaagc gttacctgtt aggtaacgta gttgagctgt aaaaattttt 2640 tttttcaaaa atagttgagc tgtcacagaa tgtgacgttg aagtttttga atggctacca 2700 gtaaattggc agattcacca tttttttttt gtcacaacgt tgaagcgtta cctgttaggt 2760 aacgtagttg agctgttgtg acaatttttt tgttctatag ttgagctgtc acagaatgtg 2820 acgttgaagt agaacaatta cataacaaac aatcataata gtaccgacaa aaacgttgaa 2880 gcgttacctg ttaggtaacg tagttgagct gtttttgtcg ttttttttct cattagttga 2940 gctgtcacag aatgtgacgt tgaagatgag aaaggtaaat tgaaatctac aaaagaagag 3000 caacactatc atacgttgaa gcgttacctg ttaggtaacg tagttgagct gtatgatagt 3060 tttttctcac atatagttga gctgtcacag aatgtgacgt tgaagtatgt gagtgaagtt 3120 acaagccaac gatttaacat aaattgtaaa cgttgaagcg ttacctgtta ggtaacgtag 3180 ttgagctgtt tacaattttt ttaaataatt tagttgagct gtcacagaat gtgacgttga 3240 agaattattt tttttttaaa ttcgcatttc ggattcacag gcaatagcat taggacgttg 3300 aagcgttacc tgttaggtaa cgtagttgag ctgtcctaat gctttttatt aaatttagtt 3360 gagctgtcac agaatgtgac gttgaagaat ttaatcagct cattaaatgt ttaataaata 3420 taaaggaata cgttgaagcg ttacctgtta ggtaacgtag ttgagctgta ttcctttttt 3480 ttttaaaaaa tagttgagct gtcacagaat gtgacgttga agttttttaa atcattcctt 3540 ttttttttgt gggaacaaac tcaggaaaat agtagtgaaa aacgttgaag cgttacctgt 3600 taggtaacgt agttgagctg tttttcactt ttttcagggt actagttgag ctgtcacaga 3660 atgtgacgtt gaaggtaccc tgaaagaggt ctaaaccaat tatttttttt ttaatcaaga 3720 tacgttgaag cgttacctgt taggtaacgt agttgagctg tatcttgatt ttttctacca 3780 tttagttgag ctgtcacaga atgtgacgtt gaagaatggt agcgccatat cgtaacagaa 3840 tcagcacgta taagacgttg aagcgttacc tgttaggtaa cgtagttgag ctgtcttata 3900 cgtttttaag tctattagtt gagctgtcac agaatgtgac gttgaagata gacttcaacc 3960 agaccaccgc gcctccggta tctaacgagc gtctacgttg aagcgttacc tgttaggtaa 4020 cgtagttgag ctgtagacgc tcttttttca tgttgtagtt gagctgtcac agaatgtgac 4080 gttgaagcaa catgaggcgg tgaccgtaag cgagtaccac caacgttgaa gcgttacctg 4140 ttaggtaacg tagttgagct gttggtggta tttttatctg ctgtagttga gctgtcacag 4200 aatgtgacgt tgaagcagca gattatcaaa aacagatagg cagattatac aagacctaaa 4260 ctatatgtat caatagacgt tgaagcgtta cctgttaggt aacgtagttg agctgtctat 4320 tgatttttta atatttgtag ttgagctgtc acagaatgtg acgttgaagc aaatattttt 4380 ttttttttca aaccctcaat catgctgaac accagaagag gtttaaatac gttgaagcgt 4440 tacctgttag gtaacgtagt tgagctgtat ttaaactttt tttctctttt agttgagctg 4500 tcacagaatg tgacgttgaa gaaagagaac aatgttggga accatcacgg attaaagttg 4560 cagcattttt acgttgaagc gttacctgtt aggtaacgta gttgagctgt aaaaatgctt 4620 tttaaaaaaa ttagttgagc tgtcacagaa tgtgacgttg aagatttttt tttttttaaa 4680 tatgcttttt tttttaacta aagggatttt tttttttgtg ctgcaagacg ttgaagcgtt 4740 acctgttagg taacgtagtt gagctgtctt gcagcttttt aagagcaata gttgagctgt 4800 cacagaatgt gacgttgaag ttgctcttca ttcccatttg ggcggcaccg cgacgacagt 4860 aaaacgcgac gttgaagcgt tacctgttag gtaacgtagt tgagctgtcg cgtttttttt 4920 ttctattgat agttgagctg tcacagaatg tgacgttgaa gtcaatagat aataaaggct 4980 tacaatagca gcgaataaac agcttgataa taagtaacgt tgaagcgtta cctgttaggt 5040 aacgtagttg agctgttact tatttttttg actcaaatag ttgagctgtc acagaatgtg 5100 acgttgaagt ttgagtcgag cttcaaagcg aaatatcgcc tgaggctact aaagaagacg 5160 ttgaagcgtt acctgttagg taacgtagtt gagctgtctt ctttattttt aaaaagttta 5220 gttgagctgt cacagaatgt gacgttgaag aacttttttt tttaattcga caacttttaa 5280 aacatcgcac gttgaagcgt tacctgttag gtaacgtagt tgagctgtgc gatgtttttt 5340 tgctttgatt agttgagctg tcacagaatg tgacgttgaa gatcaaagcg gtatatttta 5400 tataacacct cttcgctatc ggccttgcct acgttgaagc gttacctgtt aggtaacgta 5460 gttgagctgt aggcaaggtt ttttcaaagt ttagttgagc tgtcacagaa tgtgacgttg 5520 aagaactttg aggtgcaggg atttcttaat aatttttaaa gtcagattta tacgttgaag 5580 cgttacctgt taggtaacgt agttgagctg tataaatctt tttttttaaa aatagttgag 5640 ctgtcacaga atgtgacgtt gaagttttta aaatcaggtc tttggcatca tttttttttt 5700 attctactga tttttttttt tcgcactcca acgttgaagc gttacctgtt aggtaacgta 5760 gttgagctgt tggagtgctt tttgtttcgg gtagttgagc tgtcacagaa tgtgacgttg 5820 aagcccgaaa catttcggta gatttgcgca actattaccg ctagcaatac tacgttgaag 5880 cgttacctgt taggtaacgt agttgagctg tagtattgct ttttaacgtt cgtagttgag 5940 ctgtcacaga atgtgacgtt gaagcgaacg ttattaatcg tattataaac aactgaattt 6000 tgtcgtcttt ccagacgacg ttgaagcgtt acctgttagg taacgtagtt gagctgtcgt 6060 ctggattttt aaaaaaaata gttgagctgt cacagaatgt gacgttgaag tttttttttt 6120 tttgagtaac atttaccttt tgaggcgacg ttgaagcgtt acctgttagg taacgtagtt 6180 gagctgtcgc ctcaattttt ccgaataata gttgagctgt cacagaatgt gacgttgaag 6240 ttattcggaa acagttagat taagacgctg ttatataatt taatggggac gttgaagcgt 6300 tacctgttag gtaacgtagt tgagctgtcc ccattatttt tacacccttt agttgagctg 6360 tcacagaatg tgacgttgaa gaagggtgtt tggatataga taaatttacg agcatgtttt 6420 tttttttaga aaccaatcaa cgggtatacg ttgaagcgtt acctgttagg taacgtagtt 6480 gagctgtata cccgtttttt tggatatgta gttgagctgt cacagaatgt gacgttgaag 6540 catatccaaa agaaattagc aacgcaagga gttaaatcta aattacgttg aagcgttacc 6600 tgttaggtaa cgtagttgag ctgtaattta gatttttaaa atgattagtt gagctgtcac 6660 agaatgtgac gttgaagatc atttttttcc attacgcata acgacaatgt agaaaggaga 6720 cgttgaagcg ttacctgtta ggtaacgtag ttgagctgtc tcctttcttt ttaaaaatgt 6780 tagttgagct gtcacagaat gtgacgttga agacattttt ttttttcggg agaaactcat 6840 taccgtaatc ttgacaagaa ctgaccttgt acagacgttg aagcgttacc tgttaggtaa 6900 cgtagttgag ctgtctgtac aatttttatc tcaagtagtt gagctgtcac agaatgtgac 6960 gttgaagctt gagatggttt aaattacctt atttcaaaat taagctaacg ttgaagcgtt 7020 acctgttagg taacgtagtt gagctgttag cttaattttt cgtaaaaata gttgagctgt 7080 cacagaatgt gacgttgaag tttttacgag acaaaattcc tcatattttt ttttttattt 7140 taacgttgaa gcgttacctg ttaggtaacg tagttgagct gttaaaataa tttttctcgt 7200 ttttagttga gctgtcacag aatgtgacgt tgaagaaaac gagtagccgg agagttctag 7260 cgaaaagcct aaaagggaca ttctacgttg aagcgttacc tgttaggtaa cgtagttgag 7320 ctgtagaatg tcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 7380 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 7440 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 7500 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 7560 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 7620 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 7680 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 7740 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga 7800 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 7860 ccctgataaa tgcttcaata atattgaaaa aggaagagt 7899 <210> 33 <211> 8036 <212> DNA <213> Artificial Sequence <220> <223> Phagemid pointer - part 3 <400> 33 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 900 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 960 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 1020 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 1080 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 1140 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 1200 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 1260 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 1320 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 1380 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 1440 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 1500 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 1560 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 1620 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacggg 1680 tcatatagtt gagctgtcac agaatgtgac gttgaagtat gaccctgaaa tcggtctggc 1740 cttacctaca tttgacgaga gcgggagcta tttttacgtt gaagcgttac ctgttaggta 1800 acgtagttga gctgtaaaaa tagtttttga aggttatagt tgagctgtca cagaatgtga 1860 cgttgaagta accttccctt agaatccttg ccaatcgcat attttaagta ccacgttgaa 1920 gcgttacctg ttaggtaacg tagttgagct gtggtactta tttttggacg atttagttga 1980 gctgtcacag aatgtgacgt tgaagaatcg tccggatata ataacggact gaccagacgg 2040 tcagttacta cgttgaagcg ttacctgtta ggtaacgtag ttgagctgta gtaactgttt 2100 ttaaaaattg tagttgagct gtcacagaat gtgacgttga agcaattttt tttttatcaa 2160 cgtaacaaag ctgctcattc agtaacgttg aagcgttacc tgttaggtaa cgtagttgag 2220 ctgttactga attttttgtt taaagtagtt gagctgtcac agaatgtgac gttgaagctt 2280 taaacttttt tttttagttc gcgattttgg ctatcatttt tacgttgaag cgttacctgt 2340 taggtaacgt agttgagctg taaaaatgat ttttgttggc gttagttgag ctgtcacaga 2400 atgtgacgtt gaagacgcca acctgaaagc gtaagaagat agaacatatg taccccggtt 2460 tgacgttgaa gcgttacctg ttaggtaacg tagttgagct gtcaaaccgg tttttcctaa 2520 aaatagttga gctgtcacag aatgtgacgt tgaagttttt aggtagaaag attcatcagt 2580 accagacgac gatttttttt tttaaaaaca cgttgaagcg ttacctgtta ggtaacgtag 2640 ttgagctgtg tttttaattt ttctattttg tagttgagct gtcacagaat gtgacgttga 2700 agcaaaatag cgagaggctt ttgcgttaat aataggaata ataacgttga agcgttacct 2760 gttaggtaac gtagttgagc tgttattatt ctttttccag tcgatagttg agctgtcaca 2820 gaatgtgacg ttgaagtcga ctggatagcg tccatttttt ttttatactg cgaaatgacg 2880 ttgaagcgtt acctgttagg taacgtagtt gagctgtcat ttcgcttttt gcctcgtata 2940 gttgagctgt cacagaatgt gacgttgaag tacgaggcca gatacaagga cgttgagagg 3000 gttaaagatt caaatattag ctcattgctg gcacgttgaa gcgttacctg ttaggtaacg 3060 tagttgagct gtgccagcaa tttttaaaac ttatagttga gctgtcacag aatgtgacgt 3120 tgaagtaagt tttgccagag ggggtaatag taataccagt ctaacgttga agcgttacct 3180 gttaggtaac gtagttgagc tgttagactg gtttttcgag ggtttagttg agctgtcaca 3240 gaatgtgacg ttgaagaacc ctcgttttga gattaacgaa ctattcaacc cagtcaaatt 3300 atttacgttg aagcgttacc tgttaggtaa cgtagttgag ctgtaaataa tttttttttg 3360 ataaatagtt gagctgtcac agaatgtgac gttgaagttt atcaaaatca taggtctttt 3420 tttttttgag agactaccta taaggacgtt gaagcgttac ctgttaggta acgtagttga 3480 gctgtcctta tagtttttta ctaaaatagt tgagctgtca cagaatgtga cgttgaagtt 3540 ttagtattac ctgagaaaat tataacagag ggtgccacgt gagggacgtt gaagcgttac 3600 ctgttaggta acgtagttga gctgtccctc acgttttttg tatgtttagt tgagctgtca 3660 cagaatgtga cgttgaagaa catacagtat aaaaatcgcg aattgcgtaa aataccatct 3720 aaagatggaa attcgccaac gttgaagcgt tacctgttag gtaacgtagt tgagctgttg 3780 gcgaattttt tcaaaattat agttgagctg tcacagaatg tgacgttgaa gtaattttgc 3840 ttctgtgaat aaggcttgcc caacattatt actttttacg ttgaagcgtt acctgttagg 3900 taacgtagtt gagctgtaaa aagtattttt atcgcgttta gttgagctgt cacagaatgt 3960 gacgttgaag aacgcgatag gctggcgcta ttaggaaccg aattcgcctg aatatacagt 4020 aacacgttga agcgttacct gttaggtaac gtagttgagc tgtgttactg tttttttgcg 4080 ttcgtagttg agctgtcaca gaatgtgacg ttgaagcgaa cgcaataagt tacctttggg 4140 aattagagcc ttagcgtttg ccatttttac gttgaagcgt tacctgttag gtaacgtagt 4200 tgagctgtaa aaatggtttt tggctttcgt agttgagctg tcacagaatg tgacgttgaa 4260 gcgaaagcct gcaatagtgt tcatttgatt tcaactgtgt aggagacgtt gaagcgttac 4320 ctgttaggta acgtagttga gctgtctcct acatttttat atcagttagt tgagctgtca 4380 cagaatgtga cgttgaagac tgatataagt atattttttt tttgcccgga ataggtaggc 4440 tgagttttta cgttgaagcg ttacctgtta ggtaacgtag ttgagctgta aaaactcttt 4500 ttttcttgat tagttgagct gtcacagaat gtgacgttga agatcaagaa aaatgatttt 4560 ttttttccat atgaataata catccaattt ttacgttgaa gcgttacctg ttaggtaacg 4620 tagttgagct gtaaaaattg ttttttatta tattagttga gctgtcacag aatgtgacgt 4680 tgaagatata atactagaat gtgataattt taacccaaag acaaaatttt ttttttggga 4740 ccgacttgag tttttacgtt gaagcgttac ctgttaggta acgtagttga gctgtaaaaa 4800 ctcttttttt aaaatgtagt tgagctgtca cagaatgtga cgttgaagca ttttaaagta 4860 cacagcgatt cccatgtata ccgaagccct ttttgaacgt tgaagcgtta cctgttaggt 4920 aacgtagttg agctgttcaa aaagttttta ttaattatag ttgagctgtc acagaatgtg 4980 acgttgaagt aattaataaa gacagaggcg ataaagctta atacttcgac gttgaagcgt 5040 tacctgttag gtaacgtagt tgagctgtcg aagtattttt tagatggcgt agttgagctg 5100 tcacagaatg tgacgttgaa gcgccatctc aacagtttca aataagacaa aaagacacca 5160 cggaataata cgttgaagcg ttacctgtta ggtaacgtag ttgagctgta ttattccttt 5220 ttcaaagttg tagttgagct gtcacagaat gtgacgttga agcaactttg aaagaggttt 5280 tttttttaca gatgaacggt catcaagaac gttgaagcgt tacctgttag gtaacgtagt 5340 tgagctgttc ttgatgtttt ttactttact agttgagctg tcacagaatg tgacgttgaa 5400 ggtaaagtaa ttcgctaatg cagaacgcgc cttttttttt ttgtttatca acatatactt 5460 caatcatttt tacgttgaag cgttacctgt taggtaacgt agttgagctg taaaaatgat 5520 ttttcttcgt tatagttgag ctgtcacaga atgtgacgtt gaagtaacga aggcacagcc 5580 ctcatagttt ttttttttta gccccacaag acgttgaagc gttacctgtt aggtaacgta 5640 gttgagctgt cttgtgggtt ttttacgtat ttagttgagc tgtcacagaa tgtgacgttg 5700 aagaatacgt acaaacagca agaaacaatg aaccctgaac tcacgttgta acgttgaagc 5760 gttacctgtt aggtaacgta gttgagctgt tacaacgttt tttcgacttg ctagttgagc 5820 tgtcacagaa tgtgacgttg aaggcaagtc gaaatgaggt ttagcggata atagcggggg 5880 aaacgcaacg ttgaagcgtt acctgttagg taacgtagtt gagctgttgc gtttcttttt 5940 agttcgttta gttgagctgt cacagaatgt gacgttgaag aacgaacttt ttcaaattga 6000 gacgtcagat gattgctttg aataccaaac gttgaagcgt tacctgttag gtaacgtagt 6060 tgagctgttt ggtatttttt taacatgttt agttgagctg tcacagaatg tgacgttgaa 6120 gaacatgttc atgtccagac tcattttaat aacggtcacc gtccgacatt cacgttgaag 6180 cgttacctgt taggtaacgt agttgagctg tgaatgtcgt ttttacaggg tgtagttgag 6240 ctgtcacaga atgtgacgtt gaagcaccct gtatcggtag gctccattag acggtgttta 6300 acgtcaaaaa tgacgttgaa gcgttacctg ttaggtaacg tagttgagct gtcatttttg 6360 ttttttcgga tcctagttga gctgtcacag aatgtgacgt tgaagggatc cgagggtatt 6420 gaccccacgg agatccctca acgttgaagc gttacctgtt aggtaacgta gttgagctgt 6480 tgagggattt tttctcttaa atagttgagc tgtcacagaa tgtgacgttg aagtttaaga 6540 ggaatattcc taatgaaaag aacgaacatg ggcgccctgt aacgttgaag cgttacctgt 6600 taggtaacgt agttgagctg ttacagggct ttttatctat attagttgag ctgtcacaga 6660 atgtgacgtt gaagatatag attttgcaat cctttgattt agaagtatta gactttacat 6720 tagtaacgtt gaagcgttac ctgttaggta acgtagttga gctgttacta atgtttttcc 6780 gtttggtagt tgagctgtca cagaatgtga cgttgaagcc aaacgggtaa aatttttttt 6840 tttacgtaat gccactgccg gaacacgttg aagcgttacc tgttaggtaa cgtagttgag 6900 ctgtgttccg gctttttaac ttcactagtt gagctgtcac agaatgtgac gttgaaggtg 6960 aagttaaagg gaatcattgg aagcaagatt agagaatcaa cagttacgtt gaagcgttac 7020 ctgttaggta acgtagttga gctgtaactg ttgtttttaa ggtacgtagt tgagctgtca 7080 cagaatgtga cgttgaagcg taccttttga aatattctaa atataatgct gaactcaaac 7140 tacgttgaag cgttacctgt taggtaacgt agttgagctg tagtttgagt ttttttcaac 7200 ggtagttgag ctgtcacaga atgtgacgtt gaagccgttg aacctcccag ctacaatttt 7260 atccgatttt tgagaattaa cacgttgaag cgttacctgt taggtaacgt agttgagctg 7320 tgttaattct tttttacggc tttagttgag ctgtcacaga atgtgacgtt gaagaagccg 7380 taccgcattc caagaataat cggtaagcag atagcctttt tacgttgaag cgttacctgt 7440 taggtaacgt agttgagctg taaaaaggca ttaagcgcgg cgggtgtggt ggttacgcgc 7500 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc 7560 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg 7620 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca 7680 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc 7740 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct 7800 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa 7860 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc 7920 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 7980 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagt 8036 <210> 34 <211> 7904 <212> DNA <213> Artificial Sequence <220> <223> Phagemid pointer - part 4 <400> 34 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 900 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 960 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 1020 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 1080 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 1140 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 1200 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 1260 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 1320 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 1380 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 1440 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 1500 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 1560 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 1620 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacaaa 1680 taaagtagtt gagctgtcac agaatgtgac gttgaagctt tattttcaaa aaaattatca 1740 gcagctatct ccgtaacact gagtacgttg aagcgttacc tgttaggtaa cgtagttgag 1800 ctgtactcag tgtttttttg aaatttagtt gagctgtcac agaatgtgac gttgaagaat 1860 ttcaacagtt tctttttttt ttagcggagt gagaaacaac aacacgttga agcgttacct 1920 gttaggtaac gtagttgagc tgtgttgttg ttttttggct gctatagttg agctgtcaca 1980 gaatgtgacg ttgaagtagc agcctttaca gttttttttt tagagaataa cataacgttg 2040 aagcgttacc tgttaggtaa cgtagttgag ctgttatgtt attttttgac gttggtagtt 2100 gagctgtcac agaatgtgac gttgaagcca acgtctaaga acgcgaggca actaataact 2160 ccaacgcgaa cgacaacgtt gaagcgttac ctgttaggta acgtagttga gctgttgtcg 2220 ttctttttat ggcgaatagt tgagctgtca cagaatgtga cgttgaagtt cgccatatta 2280 gggtaattga gcgcttaagc ccaataccga taaacgttga agcgttacct gttaggtaac 2340 gtagttgagc tgtttatcgg ttttttacta tttttagttg agctgtcaca gaatgtgacg 2400 ttgaagaaaa tagttgctat ccttatcact catcgaataa tatcgtcaga agcaatataa 2460 ctacgttgaa gcgttacctg ttaggtaacg tagttgagct gtagttatat tttttggatt 2520 gggtagttga gctgtcacag aatgtgacgt tgaagcccaa tccaaataag aaactgaatc 2580 taaaatctcc atcgtagccg cttacgttga agcgttacct gttaggtaac gtagttgagc 2640 tgtaagcggc ttttttgctc tgtgtagttg agctgtcaca gaatgtgacg ttgaagcaca 2700 gagcctaagg aattagcaaa tcttcggtcg gttttaataa gaaaccctac gttgaagcgt 2760 tacctgttag gtaacgtagt tgagctgtag ggtttctttt tcctgttttt agttgagctg 2820 tcacagaatg tgacgttgaa gaaaacagga agaaaagctg tctttataaa caacaagaaa 2880 ataataagaa cacgttgaag cgttacctgt taggtaacgt agttgagctg tgttcttatt 2940 ttttcttttg cgtagttgag ctgtcacaga atgtgacgtt gaagcgcaaa aggagctttg 3000 cacccgactt gcgggaggtt ttaattgcaa acgttgaagc gttacctgtt aggtaacgta 3060 gttgagctgt ttgcaatttt tttattgttc atagttgagc tgtcacagaa tgtgacgttg 3120 aagtgaacaa tagcaatttg ctttcactca tctgcaacgg catacgttga agcgttacct 3180 gttaggtaac gtagttgagc tgtatgccgt ttttttttac ttattagttg agctgtcaca 3240 gaatgtgacg ttgaagataa gtaacttttt tttttgatct aaagttttag ttacaaaata 3300 aaacgttgaa gcgttacctg ttaggtaacg tagttgagct gttttatttt tttttactca 3360 atttagttga gctgtcacag aatgtgacgt tgaagaattg agtaatatca gaacaactaa 3420 atttgccctg tatggagata tagcaacgtt gaagcgttac ctgttaggta acgtagttga 3480 gctgttgcta tatttttttt caaaaatagt tgagctgtca cagaatgtga cgttgaagtt 3540 tttgaacaca gggatagcaa gcccccacca cccgacgaca accgacacgt tgaagcgtta 3600 cctgttaggt aacgtagttg agctgtgtcg gttgtttttg cggttaatag ttgagctgtc 3660 acagaatgtg acgttgaagt taaccgccat tgtatcacat cttctattct tacgcgatag 3720 ctacataacg ttgaagcgtt acctgttagg taacgtagtt gagctgttat gtagcttttt 3780 aggcgttgta gttgagctgt cacagaatgt gacgttgaag caacgcctgt agcagtactc 3840 agccgcgacc gaaaacttta gggcttatac gttgaagcgt tacctgttag gtaacgtagt 3900 tgagctgtat aagccctttt tagtcttatt agttgagctg tcacagaatg tgacgttgaa 3960 gataagactc atagacggat attcatgagc cgccgccagc atcgcctcac gttgaagcgt 4020 tacctgttag gtaacgtagt tgagctgtga ggcgattttt tgtatcatgt agttgagctg 4080 tcacagaatg tgacgttgaa gcatgatacc gccacgcacc attaccatta gtttcatcgt 4140 aaacagtgta cgttgaagcg ttacctgtta ggtaacgtag ttgagctgta cactgttttt 4200 ttggcggtac tagttgagct gtcacagaat gtgacgttga aggtaccgcc accaatgaaa 4260 ccatcgaagt ttgccctatt acgttgaagc gttacctgtt aggtaacgta gttgagctgt 4320 aatagggctt tttctcgacg gtagttgagc tgtcacagaa tgtgacgttg aagccgtcga 4380 gagggtcagg cgcatgctcc atatcataag tgaggaaggc ggaacagcca cgttgaagcg 4440 ttacctgtta ggtaacgtag ttgagctgtg gctgttcttt ttagtaaaaa tagttgagct 4500 gtcacagaat gtgacgttga agtttttact cctcaatttt ttttttttaa gcagtagcga 4560 cagaatcata gcagcattag gatgtacgtt gaagcgttac ctgttaggta acgtagttga 4620 gctgtacatc ctattttttg gaaaaatagt tgagctgtca cagaatgtga cgttgaagtt 4680 tttccaagaa ggaatttttt ttttaccgag gattaaataa gaattttttt ttttaaacac 4740 cgacgttgaa gcgttacctg ttaggtaacg tagttgagct gtcggtgttt tttttattca 4800 cattagttga gctgtcacag aatgtgacgt tgaagatgtg aattaaatac ccaaccagcg 4860 ctccggctta ggttgggaga agaacgttga agcgttacct gttaggtaac gtagttgagc 4920 tgttcttctc ctttttctga ggtatagttg agctgtcaca gaatgtgacg ttgaagtacc 4980 tcagagaata ggaaatacca agctttaatt cagcagcgga acaaacgttg aagcgttacc 5040 tgttaggtaa cgtagttgag ctgtttgttc cgtttttggc cttgatagtt gagctgtcac 5100 agaatgtgac gttgaagtca aggccgcgta gaaacttatt acttgtcaca aaatgctgat 5160 gcaaataaac gttgaagcgt tacctgttag gtaacgtagt tgagctgttt atttgctttt 5220 tagaaaaaat agttgagctg tcacagaatg tgacgttgaa gttttttctt ttcataatcc 5280 cttgatattt tttttttttc acaaacaaat aacgttgaag cgttacctgt taggtaacgt 5340 agttgagctg ttatttgttt ttttaatagg catagttgag ctgtcacaga atgtgacgtt 5400 gaagtgccta ttagcaaaga aacgtcaccc tcagcgtcac caactaaaac gaacgttgaa 5460 gcgttacctg ttaggtaacg tagttgagct gttcgtttta tttttacact cattagttga 5520 gctgtcacag aatgtgacgt tgaagatgag tgtacagagc cacttttttt tttcaccctc 5580 agagccgacg ttgaagcgtt acctgttagg taacgtagtt gagctgtcgg ctctgttttt 5640 ctctacttta gttgagctgt cacagaatgt gacgttgaag aagtagagaa ggaccgtaat 5700 gtatcacctt ccacagacca acctagttgc gcccacgttg aagcgttacc tgttaggtaa 5760 cgtagttgag ctgtgggcgc aattttttga caaggtagtt gagctgtcac agaatgtgac 5820 gttgaagcct tgtcagagcc gccaccctca gaaccgacca gaacccacgt tgaagcgtta 5880 cctgttaggt aacgtagttg agctgtgggt tctgttttta atcgttttag ttgagctgtc 5940 acagaatgtg acgttgaaga aacgattgga aaatcacggt tgaggaaccg attgagggag 6000 gtatggtacg ttgaagcgtt acctgttagg taacgtagtt gagctgtacc ataccttttt 6060 tgcgctttta gttgagctgt cacagaatgt gacgttgaag aaagcgcagt ccggggtcat 6120 aatgccccac caccaacata aaggtggcaa cgttgaagcg ttacctgtta ggtaacgtag 6180 ttgagctgtt gccacctttt ttcatgtatg tagttgagct gtcacagaat gtgacgttga 6240 agcatacatg gctttttttt ttttttgatg atacagtctg aaacatgaac gttgaagcgt 6300 tacctgttag gtaacgtagt tgagctgttc atgttttttt tgggtggggt agttgagctg 6360 tcacagaatg tgacgttgaa gccccaccct ctggtaataa gttttaatct gaatttcaga 6420 ctgtagcgca cgttgaagcg ttacctgtta ggtaacgtag ttgagctgtg cgctacattt 6480 ttctgacctg tagttgagct gtcacagaat gtgacgttga agcaggtcag accggaactg 6540 acaggacgga accacattaa agcaccagac gttgaagcgt tacctgttag gtaacgtagt 6600 tgagctgtct ggtgcttttt tgttactggt agttgagctg tcacagaatg tgacgttgaa 6660 gccagtaaca ggagccacct cctcattggt catagccccc ttaagcaaaa cgttgaagcg 6720 ttacctgtta ggtaacgtag ttgagctgtt ttgcttattt ttttctttaa tagttgagct 6780 gtcacagaat gtgacgttga agttaaagaa cttttgaaat cgcgaaaccg agccagaaag 6840 acagcaattc acgttgaagc gttacctgtt aggtaacgta gttgagctgt gaattgcttt 6900 ttttgaattt ttagttgagc tgtcacagaa tgtgacgttg aagaaaattc agaaggtaaa 6960 aattatttgc ccgtagcatt ttcaaagcca gaatgacgtt gaagcgttac ctgttaggta 7020 acgtagttga gctgtcattc tggtttttta aactgttagt tgagctgtca cagaatgtga 7080 cgttgaagac agtttattgc agtatggtta attttcgcct gaacgccaac tacagaggtt 7140 atcatcagac gttgaagcgt tacctgttag gtaacgtagt tgagctgtct gatgattttt 7200 taattttatt agttgagctg tcacagaatg tgacgttgaa gataaaattt tgctctttcg 7260 gaactttagc gtaccgttcc agtaagcgta cgttgaagcg ttacctgtta ggtaacgtag 7320 ttgagctgta cgcttacatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 7380 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 7440 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 7500 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 7560 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 7620 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 7680 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 7740 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc 7800 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 7860 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagt 7904

Claims (21)

  1. 다음의 단계를 포함하는 DNA 단일 가닥 분자의 재조합적 대량 생산 방법:
    (1) 위유전자(pseudogene) 핵산을 제공하는 단계,
    여기서 상기 위유전자 핵산은 (i) 적어도 하나의 표적 DNA 올리고뉴클레오타이드 또는 폴리뉴클레오타이드 서열 및 (ii) 각각의 표적 DNA 올리고뉴클레오타이드 또는 폴리뉴클레오타이드 서열에 인접하는, 2개 자가-절단 DNA 서열의 적어도 하나의 절단 세트를 포함하는 핵산임,
    여기서 상기 위유전자 핵산은 상기 표적 DNA 올리고뉴클레오티드 또는 폴리뉴클레오티드 서열 하나당 상기 절단 세트 하나를 포함하고, 상기 절단 세트는 2개의 자가-절단 DNA 서열이 서로 역순으로 연결되어 1개의 절단 세트를 형성하는 것임,
    (2) 위유전자 핵산을 벡터 속으로 통합시키고, 박테리아 세포를 상기 벡터로 형질전환시키고, 그리고 박테리아 배양 조건 하에서 상기 벡터로부터 전구체 ssDNA를 생성시키는 단계,
    여기서 상기 전구체 ssDNA는 위유전자 핵산을 포함함;
    (3) 박테리아 배양물로부터 전구체 ssDNA를 분리하는 단계;
    (4) 자가-절단 DNA 서열이 활성화되는 반응 조건 하에서 전구체 ssDNA를 소화시키어, 상기 적어도 하나의 절단 세트를 잘라내는 단계; 및
    (5) 적어도 하나의 표적 단일 가닥 DNA 올리고뉴클레오타이드 또는 폴리뉴클레오타이드를 분리하고, 적어도 하나의 표적 단일 가닥 DNA 올리고뉴클레오타이드 또는 폴리뉴클레오타이드를 수득하는 단계,
    여기서 상기 대량 생산이란 표적 단일 가닥 DNA 올리고뉴클레오티드 또는 폴리뉴클레오티드의 그램 규모 생산을 허용함.
  2. 제1항에 있어서, 자가-절단 DNA 서열은 자가-절단 데옥시리보자임(desoxyribozymes) 또는 DNA자임(DNAzymes)인 것인 방법.
  3. 제2항에 있어서, DNA자임(DNAzymes)은 Zn2+-의존성 DNA자임인 것인 방법.
  4. 제1항에 있어서, 위유전자 핵산은 2개, 3개, 4개 또는 50개를 초과하는 표적 DNA 올리고뉴클레오타이드 서열을 포함하는 것인 방법.
  5. 제1항에 있어서, 표적 DNA 올리고뉴클레오타이드 또는 폴리뉴클레오타이드 서열은 20 뉴클레오타이드 내지 수천 뉴클레오타이드 범위의 길이를 갖는 것인 방법.
  6. 제1항에 있어서, 박테리아는 대장균(E. coli)으로부터 선택되는 것인 방법.
  7. 제1항에 있어서, 단계 (2)의 벡터는 하기와 같은 추가 성분들 중 적어도 하나를 임의로 포함하는 적어도 하나의 파지미드인 것인 방법:
    - 포장 서열(a packaging sequence),
    - 적어도 하나의, 세포 분열 동안 파지미드의 증식을 보장하는 성분, 및
    - 선택 마커(a selection marker).
  8. 제7항에 있어서, 선택 마커는 항생제 내성 유전자인 것인 방법.
  9. 제7항에 있어서, 하기와 같은 추가 성분들 중 적어도 하나를 포함하는 헬퍼 플라스미드 또는 헬퍼 파지가 추가로 사용되는 것인 방법:
    - 박테리오파지의 단백질을 코딩하는 유전자,
    - 적어도 하나의, 세포 분열 동안 헬퍼 플라스미드의 증식을 보장하는 성분, 및
    - 선택 마커.
  10. 제9항에 있어서, 박테리오파지는 M13 박테리오파지인 것인 방법.
  11. 제9항에 있어서, 선택 마커는 항생제 내성 유전자인 것인 방법.
  12. 제1항에 있어서, 단계 (2)에서의 벡터는 적어도 하나의 파지미드이고, 롤링 서클 증폭(RCA)을 통해 박테리아 세포 내에서 증폭되어 파지미드 ssDNA를 생성하는 것인 방법.
  13. 제12항에 있어서, 파지미드 ssDNA는 세포로부터 분비되는 파지-유사 입자 속으로 포장되는 것인 방법.
  14. 제1항에 있어서, 단계 (3)은 세포 배양물로부터 파지-유사 입자를 추출하고, 파지-유사 입자로부터 파지미드 ssDNA를 추출하는 것을 포함하는 것인 방법.
  15. 제1항에 있어서, 단계 (4)의 상기 소화가 Zn2+ 이온의 첨가에 의해 유발되는 것인 방법.
  16. 제1항에 있어서, 단계 (5)에서 적어도 하나의 표적 단일 가닥 DNA 올리고뉴클레오타이드 또는 폴리뉴클레오타이드를 분리하는 것은
    2개의 자가-절단 DNA 서열이 서로 역순으로 연결된 절단 세트로부터 분리하는 것을 포함하는 것인 방법.
  17. 제16항에 있어서, 자가-절단 DNA 서열은 DNA자임인 것인 방법.
  18. 제16항에 있어서, 자가-절단 DNA 서열로부터 분리하는 것은 크로마토그래피를 통하여 수행되는 것인 방법.
  19. 제1항에 있어서, 다음의 추가 단계를 포함하는 것인 방법:
    (6) DNA 오리가미 구조, 타일 기반(tile-based) DNA 나노 구조, 또는 결정형 DNA 나노 물질로 자가-조립 및/또는 폴딩시키는 것; 및 다른 분자를 결합, 검출 또는 가공하기 위한 앱타머 또는 DNA자임으로서 사용하는 것으로부터 선택되는, 적어도 하나의 표적 단일 가닥 DNA 올리고뉴클레오타이드 또는 폴리뉴클레오타이드의 추가 처리.
  20. 제1항에 있어서, 박테리아 배양은 생물 반응기 중에서 수행되는 것인 방법.
  21. 제1항 내지 제20항 중 어느 하나의 항에 따른 방법으로 수득된 적어도 하나의 표적 단일 가닥 DNA 올리고뉴클레오타이드 또는 폴리뉴클레오타이드를 하기에서 사용하는 방법:
    - DNA 나노 기술에서,
    - 연구 도구로서,
    - 진단용 프로브로서,
    - 유전자 합성에서,
    - 유전자 요법에서, 또는
    - 분자 감지에서.
KR1020197008132A 2016-09-21 2017-07-17 정의된 서열 및 길이의 dna 단일 가닥 분자의 확장 가능한 생명공학적 생산 KR102528337B1 (ko)

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CN109563539A (zh) 2016-06-15 2019-04-02 慕尼黑路德维希马克西米利安斯大学 使用dna纳米技术的单分子检测或定量
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