KR20120064656A - Kras G12V RNA를 특이적으로 인지할 수 있는 트랜스-스플라이싱 그룹 I 리보자임 - Google Patents
Kras G12V RNA를 특이적으로 인지할 수 있는 트랜스-스플라이싱 그룹 I 리보자임 Download PDFInfo
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Abstract
본 발명은 단 염기 변이 전사체를 특이적으로 인지할 수 있는 트랜스-스플라이싱 그룹 I 리보자임에 관한 것으로서, 더욱 상세하게는 Kras 유전자의 12번째 코돈의 단 염기 변이 Ras RNA를 특이적으로 인식하여 치환할 수 있는 트랜스-스플라이싱 그룹 I 리보자임에 관한 것이다.
본 발명의 리보자임은 단 염기 변이된 유전자 전사체를 정상 유전자 전사체와 구별하여 특이적으로 인식하므로, 이러한 리보자임을 이용하여 돌연변이 전사체를 발현하는 암세포를 특이적으로 치료 및 진단이 가능하다.
본 발명의 리보자임은 단 염기 변이된 유전자 전사체를 정상 유전자 전사체와 구별하여 특이적으로 인식하므로, 이러한 리보자임을 이용하여 돌연변이 전사체를 발현하는 암세포를 특이적으로 치료 및 진단이 가능하다.
Description
본 발명은 Kras 단 염기 변이 전사체를 특이적으로 인지할 수 있는 트랜스-스플라이싱 그룹 I 리보자임에 관한 것이다.
현재 항암치료로 주로 이용되고 있는 방사선 치료법과 항암화학요법 등은 이런 방법에 저항성을 보이는 환자에게는 비효과적이며 정상세포에도 부작용을 유발할 수 있는 한계점을 가지고 있었다. 따라서, 보다 특이적인 암 치료를 위해 암세포에서만 특이적인 세포사를 유도하는 방법이 필요하다.
라스 단백질(Ras protein)은 세포 성장, 증식과 분화를 조절하는 소 지티파제 단백질(small GTPase protein)으로 진화적으로 매우 보존되어 있고 각 조직마다 서로 다른 양을 발현한다.
이러한 라스의 아이소폼인 H-ras, N-ras, K-ras들은 서로 다른 위치에 의해 같은 활성자(activator)와 작동자(effector)를 가지고도 다른 기능을 한다(John F. Hancock. RAS proteins: different signals from diffrent locations. Nature Molecular Cell Biology 4; 373-384(2003)). RAS 아이소폼은 약 85%의 유사성을 가지고 있으며, H-ras와 N-ras의 하나 혹은 동시의 녹아웃 실험을 통해 정상 발달(normal development)에 필수적이지 않음이 알려졌다.
그러나, K-ras의 경우에는 배아 치사(embryonic lethal)가 되는 필수적인 단백질임이 보고되었다(Johnson, L. et al., K-ras is an essential gene in the mouse with partial functional overlap with N-ras. Genes Dev. 11; 2468-2481(1997)).
정상 세포에서의 RAS 단백질의 활성은 GTP와 GDP의 결합 비율에 의해 조절되어진다(Campbell, S. L. et al., Increasing complexity of Ras signaling. Oncogene 17;1395-1413(1998)). RAS 단백질의 활성 상태는 GTP나 GDP의 결합 여부에 의해 결정이 되는데, RAS-GTP는 활성 상태이고 RAS-GDP는 불활성 상태로 RAF, PI3K, RALGDS, PLCε등의 하위 단계 단백질들의 연속적인 인산화를 유도하여 외부로부터 신호전달을 하는 "분자 스위치(molecular switch)" 역할을 한다(Fabbro, D. et al., Protein tyrosine kinase inhibitors: new treatment modalities Curr . Opin. Pharmacol. 2; 374-381 (2002)).
비활성 상태의 RAS는 GEFs(guanine nucleotide exchange factors)에 의해 GDP를 방출하고 GTP 바인딩을 허용함으로써 활성화될 수 있다. Ras 신호화의 종결은 GAPs(GTPase activating proteins)에 의해 RAS의 내재성의 GTPase를 활성화시켜 GTP를 GDP로 전환함으로써 불활성화된다(John F. Hancock. RAS proteins: different signals from diffrent locations. Nature Molecular Cell Biology 4; 373-384(2003)).
RAS의 발현은 조직 특이적으로 발현되는데 각 조직의 암에서 RAS 돌연변이가 많이 발견되었다. Kras의 경우 대장, 흉선 그리고 폐에서 많이 발현이 되며 결장암, 췌장암, 폐암에서 Kras의 G12V 단 염기 돌연변이는 RAS 단백질의 구조적인 변화에 의해 GAP의 친화도와 활성도가 감소하게 된다. 따라서, RAS의 GTPase 활성이 감소되어 RAS는 계속적으로 단백질의 활성을 유지시킨다(Fabbro, D. et al., P A. Protein tyrosine kinase inhibitors: new treatment modalities, Curr . Opin . Pharmacol. 2; 374-381 (2002); Fahd Al-Mulla et al., Structural differences between valine-12 and aspartate-12 ras protein may modify carcinoma aggression. J. Pathol 18; 433-438(1999)).
Kras 유전자와 인간 암의 관계를 살펴보면 NSCLC(Non small cell carcinoma)의 33%, 결장암의 44% 그리고 췌장암의 90%에서 K-ras 유전자의 돌연변이가 발견된다. 다른 ras 종류의 경우 N-ras는 흑색종(13%), 간암(30%), 그리고 형질구성 백혈병(30%)에서 그 변이가 발견되며, H-ras는 방광(10%)그리고 신장암(10%)에서 발견된다(Mu D.Q. et al., Xualues of mutations of K-ras oncogene at codon 12 in detection of pancreatic cancer. World J Gastroenterol. 10(4); 471-5. (2004)).
이 중 특히 폐암의 경우 통증이 없어 발병의 진단이 늦고 또한 현재 폐암의 85% 이상이 사망하는 것으로 알려져 있다. 실제 대장암, 유방암 그리고 전립선암보다 더 많은 사람이 폐암으로 사망한다.
폐암은 암세포의 현미경적 형태에 따라 크게 비소세포성폐암(Non Small Cell Lung Cancer;NSCLC)와 소세포성폐암(Small Cell Lung Cancer;SCLC)으로 나뉘는데, 비소세포성폐암은 폐암의 65~75%, 소세포성폐암은 15~30%, 기타 폐암이 10%를 차지한다.
상기 비소세포성폐암은 편평상피암(squamous cell carcinoma)과 선암(adenocarcinoma) 그리고 대세포암종(large cell carcinoma)으로 분류한다(Minamoto T., Mai M. and Ronai Z. K-ras mutation: early detection in molecular diagnosis and risk assessment of colorectal, pancreas, and lung cancers a review. Cancer Detect Prev, 24(1); 1-12. (2004)).
상기 편평상피암은 폐암 중 가장 흔한 형태로 폐 중심부에 잘 생기며, 남자 폐암에 가장 맣은 형태로 흡연과 가장 관련이 많은 것으로 알려져 있다. 또한, 선암은 폐의 주변에서 주로 발견되며 간, 뇌, 뼈 등으로 전이가 잘 일어난다. 마지막으로 대세포암종은 전체 폐암의 4~10%를 차지하며, 폐 주변에 주로 발생하며, 암세포가 빠르게 증식하여 전이되는 속도가 빠른 경향이 있다.
상기 소세포성폐암은 주로 기도에서 처음 발생한다. 대개 기관의 표면이나 선을 따라서 생성되며 대부분은(80%) 폐 중앙부에 생기고 나머지(20%)는 주변부에서 발생한다. 전반적으로 악성도가 강해서 림프계통이나 혈액순환을 통해서 조기에 멀리 전이되는 경향이 있다. 소세포성폐암 암세포는 대개는 작고 동그란 형태로 자라는데, 그 모양을 본 따서 연맥세포암(oat cell)이라고도 한다.
나머지 기타 암으로, 세기관지폐포암(bronchioloalveoiar carcinoma)은 천천히 자라는 암세포이며, 나이가 많은 사람에게서 종종 발견되며 진행정도도 다양하다. 또한, 전체 폐암의 1% 미만으로 발생하는 거대세포암(giant cell carcinoma)은 주로 주변부에서 크게 나타나며, 악성도가 심해서 빨리 진행된다.
이 중 편평상피암의 46%와 선암의 44%가 K-ras의 돌연변이로 인해 발병되는데, 그 중 40%가 K-ras의 단 염기 변이로 알려져 있다(Minamoto T., Mai M., and Ronai Z. K-ras mutation: early detection in molecular diagnosis and risk assessment of colorectal, pancreas, and lung cancers. Cancer Detect Prev. 24(1); 1-12. (2000)).
Kras는 세포 내에서 세포의 성장, 분열, 분화의 신호전달에 관여하는 상위단계의 단백질로, Kras의 돌연변이 중 12번, 13번, 61번 코돈이 핫 스팟(hot spot)으로 알려져 있다. 특히 Kras의 12, 13, 61번째 코돈의 돌연변이는 GTP의 가수분해에 관여하는 도메인을 암호화하는 부위에서 구조적 변화를 일으켜 세포의 계속적인 성장을 유도하여 암세포로 발전시키게 된다.
한편, Tetrahmena thermophila로부터의 그룹 I 인트론 리보자임이 실험관 내에서 자기 자신의 3'끝에 부착되어 있는 엑손 RNA를 트랜스하게 존재하는 5'엑손을 표적으로 한 후 부착시킴으로써 별도로 존재하는 전사체를 서로 연결시킬 수 있는 트랜스-스플라이싱 반응을 일으킬 수 있음이 보고되었다(Been, M. and Cech, T. One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity. Cell 47; 207-216. (1986)). 이러한 리보자임은 포유류 세포 내에서도 일어날 수 있음이 밝혀졌다(Jones, J.T., Lee, S.W., and Sullenger, B.A Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells. Nat . Med. 2; 643-648. (1996); Lee, S.W. Effects of substrate RNA structure on the trans-splicing reaction by group I intron of Tetrahymena thermophila. Korean Journal of Microbiology 35; 211-217. (1999)).
이는 곧 그룹 I 리보자임의 트랜스-스플라이싱 반응을 통하여 여러 유전질환과 관련있는 유전자의 전사체도 보정될 수 있음을 시사하며 실제로 최근에 겸상적혈구(sickle cell) 질병을 갖은 환자의 적혈구 전구(erythrocyte precursor) 세포에서 β-글로빈 유전자 전사체가 트랜스-스플라이싱 리보자임에 의해 항-겸상적혈구화(anti-sickling), γ-글로빈 유전자 전사체로 전환될 수 있음이 밝혀졌다(Lan, N., Howrey, R.P., Lee, S.W., Howrey, R.P., Lee, S.W., Howrey, R.P., Smith, C.A., and Sullenger, B.A. Ribozyme-Mediated Repair of Sickle β-Globin mRNAs in Erythrocyte Precursors. Science 280; 1593-1596. (1998); Lan, N., Rooney, B.L., Lee, S.W., Howrey, R.P., Smith, C.A., and Sullenger, B.A. Enhancing RNA repair efficiency by combining trans-splicing ribozymes that recognize different accessible sites on a target RNA. Mol . Ther . 2; 245-255. (2000)).
또한, 여러 다른 부위를 인지하는 리보자임들을 함께 반응시켰을 경우 트랜스-스플라이싱 반응이 더욱 증가한다(Lan, N., Rooney, B.L., Lee, S.W., Howrey, R.P., Smith, C.A., and Sullenger, B.A. Enhancing RNA repair efficiency by combining trans-splicing ribozymes that recognize different accessible sites on a target RNA. Mol . Ther . 2; 245-255. (2000)).
그리고, 트랜스-스플라이싱 리보자임이 포유류 세포 내에 있는 근긴장성이양증 단백질 키나아제(myotonic dystrophy protein kinase) 유전자 전사체도 변형할 수 있다고 보고되었다(Phylactou, L.A., Darrah, C. and Wood, M.A.J.. Ribozyme-mediated trans-splicing of a trinucleotide repeat. Nat . Genet. 18; 378-381. (1998)). 선천성근긴장증(Myotonic congenita)을 유발하는 돌연변이 골격근 클로라이드 채널(mutant skeletal muscle chloride channel) RNA를 기능 면에서도 보정할 수 있었(Rogers, C.S., Vanoye, C.G., Sullenger, B.A., and George, Jr., A.L. Functional repair of a mutant chloride channel using a trans-splicing ribozyme. J. Clin . Invest. 110; 1783-1798. (2002)).
리보자임의 특이도 및 활성도를 증가하기 위해 표적 RNA 인지부위인 내부 가이드 염기서열(internal guide sequence;IGS)를 변형한 IGS를 갖는 리보자임을 식물 병원체인 오이 모자이크 바이러스 RNA를 표적으로 하여 사이토톡신을 트랜스-스플라이싱할 수 있도록 고안하였을 때, 효모 내에서 바이러스 RNA만 발현하는 세포만 특이적으로 그 성장을 방해할 수 있음이 발표되었다(Ayre, B.G., Kohler, U., Goodman, H.M., and Haseloff, J. Design of highly specific cytotoxins by using trans-splicing ribozymes. Proc . Natl . Acad . Sci. 96; 3507-3512. (1999)).
다른 예로 위와 같이 내부 가이드 염기서열이 변형되어 있고 돌연변이 p53 RNA를 표적으로 하여 트랜스-스플라이싱할 수 있는 리보자임을 인체의 결핍성 p53이 관련된 암세포에서 발현시킬 경우 돌연변이 p53 전사체를 효과적으로 보정함으로써 정상적인 p53의 활성을 유도하였다(Watanabe, T., and Sullenger, B.A. Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts. Proc . Natl . Acad . Sci. 97; 8490-8494. (2000)). 이러한 연구는 트랜스-스플라이싱 리보자임이 돌연변이 유전자 RNA를 정상 RNA로 보정 가능함을 시사하고 있다.
또한, 최근에 C형 간염바이러스의 IRES RNA를 특이적으로 인지하는 리보자임을 개발하였으며 이러한 리보자임에 의해 HCV IRES RNA를 세포사 유발 RNA로 치환함으로써 표적 RNA를 발현하는 세포만 선택적으로 세포사가 유도할 수 있었다(Ryu, K.J., Kim, J.H., and Lee, S.W. Ribozyme-mediated selective induction of new gene activity in hepatitis C virus internal ribosome entry site-expressing cells by targeted trans-splicing. Mol . Ther. 7; 386-395. (2003)).
따라서, 이와 같은 결과는 트랜스-스플라이싱 리보자임에 의해 암 특이 유전자 전사체가 표적이 된 후 그 RNA가 다른 유전자 전사체로 치환되도록 재 프로그램될 수 있음을 시사한다.
지금까지 암 치료법에서 ras를 타겟으로 많은 연구들이 진행되어져 왔는데, RAS 단백질의 발현을 막고자 하는 안티센스 올리고뉴클레오타이드 등의 시도나 억제제를 이용하여 RAS의 멤브레인 로컬라이제이션(membrane localization)을 블록하려는 시도들이 보고되었으며, 또한 ras의 기능을 막기 위해 하위단계의 상호작용을 하는 단백질들의 억제제들을 이용한 시도 또한 연구되어왔다.
그러나, 이러한 시도들은 특정 RAS를 표적하거나 변이된 RAS를 특이적으로 제거 또는 억제하지 못하여 정상세포에까지 영향을 미치는 문제점들이 있었으며, 또한 발현되는 RAS의 일부분을 제거 억제하는 것만으로는 암이 치료되지는 않는다는 문제점이 있다. 그리고, 최근 많은 연구가 진행 중인 siRNA를 이용한 Kras G12V의 RNA 간섭은 돌연변이 Kras에 대해 특이적이지만 오프-타겟 효과와 변역반응을 건드리는 점에서 적당하지 않다.
이에, 본 발명자들은 상기와 같은 점을 감안하여 Tetrahymena thermophila의 그룹 I 인트론 리보자임을 이용한 유전자 치료 방법에 대해 예의 연구 노력한 결과, Kras의 12번째 코돈의 단 염기 변이를 선택적으로 인지하는 트랜스-스플라이싱 리보자임을 개발하고, 이러한 리보자임이 in vitro와 in vivo에서 돌연변이 Kras 전사체를 인지, 절단하고 타겟 부위의 하류(down-steam) 부위에 리보자임의 3' 엑손을 연결할 수 있으며, 이때 리보자임의 엑손을 치료 유전자 또는 리포터 유전자로 치환함으로써 암세포를 선택적으로 치료하거나 진단할 수 있음을 확인하고, 이를 토대로 본 발명을 완성하였다.
따라서, 본 발명의 목적은 단 염기 변이 Ras RNA를 특이적으로 인식하여 치환할 수 있는 트랜스-스플라이싱 그룹 I 리보자임 및 이의 용도를 제공하는 것이다.
상기 목적을 달성하기 위하여, 본 발명은
단 염기 변이 Ras RNA를 특이적으로 인식하여 치환할 수 있는 트랜스-스플라이싱 그룹 I 리보자임 및 이의 용도를 제공한다.
또한, 본 발명은 상술한 리보자임을 발현하는 벡터 및 이의 용도를 제공한다.
이때, 상기 리보자임은 Kras G12V RNA를 특이적으로 표적하며, 3' 엑손에는 치료 유전자 또는 리포터 유전자를 함유할 수 있다.
본 발명의 리보자임은 단 염기 변이된 유전자 전사체를 정상 유전자 전사체와 구별하여 특이적으로 인식하므로, 이러한 리보자임을 이용하여 돌연변이 전사체를 발현하는 암세포를 특이적으로 치료 및 진단이 가능하다.
도 1은 트랜스-스플라이싱 리보자임 매개 RNA 리프로그램을 나타낸 모식도이다.
도 2는 Kras RNA의 이차 구조를 나타낸 것이다[화살표: Kras의 12번째 코돈].
도 3은 Kras RNA의 모식도와 특이성 트랜스-스플라이싱 리보자임을 나타낸 것이다.
도 4는 in vitro 돌연변이 형성을 통해 Kras 야생형으로부터 제작된 Kras 12V의 구조를 나타낸 것이다.
도 5는 in vitro에서 KRib1, KRib2의 Kras G12V 트랜스-스플라이싱 산물의 분석을 나타낸 것이다[A: RT-PCR 분석, B-C: 서열분석(B: KRib1, C: KRib2)].
도 6은 P1/P10 헬릭스와 안티센스 부위를 변형함으로써 특이성과 효율성이 증대된 트랜스-스플라이싱 리보자임을 나타낸 것이다[A. 변형된 리보자임의 모식도, 화살표: trans-splicing junction; B. 변형된 KRib1 , KRib2의 구조체].
도 7은 in vitro에서 변형된 리보자임의 트랜스-스플라이싱 효율성 및 특이성 분석 결과를 나타낸 것이다[A-B: 트랜스-접합 산물의 RT-PCR 분석, C-D: 트랜스-접합 RT-PCR 산물의 서열분석(C: AS300KRib1, D: AS100KRib2)].
도 8은 트랜스-스플라이싱 리보자임에 의한 트랜스진의 선별적 유도를 확인하기 위한 리포터 에세이 결과를 나타낸 것이다[A: 293 세포에서 리포터 분석을 위한 구조체, B: Kras (야생형 또는 12V) RNA 및 레닐라 루시퍼라제가 함유된 리보자임(KRib1 세트 / KRib2 세트)의 트랜스펙션].
도 9는 트랜스-스플라이싱 리보자임에 의한 트랜스진의 선별적 발현을 확인하기 위한 RT-PCR 분석 결과를 것이다[A: 293 세포에서 리포터 분석을 위한 구조체, B: Kras (wild or 12V) RNA가 함유된 리보자임(KRib1 세트 / KRib2 세트)의 RT-PCR; W: 야생형 Kras, 12V: Kras G12V, IGS: 내부 가이드 염기서열, D)KRib: 불활성 리보자임].
도 10은 Kras G12V를 내생적으로 발현하는 세포주 SW480에서 TK 유전자 유도에 의한 세포 퇴행을 나타낸 것이다[A: SW480 세포에서 TK 분석을 위한 구조체, B: GFP 유전자 함유 AS100KRib2-TK의 트랜스펙션]
도 11은 박테리아에서 상동 재조합에 의한 재조합 아데노바이러스의 세대를 나타낸 것이다[A: 재조합 아데노바이러스 전달 시스템의 모식도, B: 아데노바이러스 리보자임 재조합의 구조체].
도 12는 재조합 Ad-AS300KRib1-TK의 분석을 나타낸 것이다[A. 바이러스 정제 전 PCR에 의한 바이러스 게놈 확인, B: Kras G12V 변이된 SW480에서 바이러스 정제 전 RT-PCR에 의한 Ad-AS300KRib1-TK 발현 확인; TK : Thymidine Kinase, LITR : Left Inverted Terminal Repeat].
도 13은 재조합 Ad-AS100KRib2-TK의 분석을 나타낸 것이다[A. 바이러스 정제 전 PCR에 의한 바이러스 게놈 확인, B: Kras G12V 변이된 SW480에서 바이러스 정제 전 RT-PCR에 의한 Ad-AS100KRib2-TK 발현 확인; TK : Thymidine Kinase, LITR : Left Inverted Terminal Repeat].
도 14는 재조합 Ad-AS100KRib2-LacZ의 분석을 나타낸 것이다[A. 바이러스 정제 전 PCR에 의한 바이러스 게놈 확인, B: Kras G12V 변이된 SW480에서 바이러스 정제 전 RT-PCR에 의한 Ad-AS100KRib2-LacZ 발현 확인; LITR : Left Inverted Terminal Repeat].
도 15는 Kras G12V 변이된 암 세포 SW480에서 아데노바이러스 전달 시스템을 이용한 트랜스-접합 RNA 산물의 분석을 나타낸 것이다.
도 16은 종양세포주에서 아데노바이러스 전달 시스템에 의한 특이적 트랜스-스플라이싱 리보자임의 세포 생존율을 분석한 것이다[A: SW480(KrasG12V)에서 세포 생존율 MTS 분석([left panel: M.O.I dependent / 100uM GCV , right panel GCV dose dependent / virus 150M.O.I), B: CAPAN-1(KrasG12V)에서 세포 생존율 MTS 분석(M.O.I dependent / 100uM GCV), C: HT-29(wild Kras)에서 세포 생존율 MTS 분석(left panel: M.O.I dependent / 100uM GCV , right panel GCV dose dependent / virus 150M.O.I)].
도 17은 이종이식 마우스 모델에서 아데노바이러스 인코딩 Kras G12V-특이적 리보자임의 분석을 나타낸 것이다[A: 복강 내 CAPAN-1(Kras G12V) 종양 모델(n=6 또는 7)에서 Ad-리보자임에 의한 효율적이고 특이적인 종양 퇴행 보임, B: 피하 내 HT-29(wild type Kras) 종양 모델(n=7)에서 Ad-리보자임에 의한 종양 퇴행 없음].
도 2는 Kras RNA의 이차 구조를 나타낸 것이다[화살표: Kras의 12번째 코돈].
도 3은 Kras RNA의 모식도와 특이성 트랜스-스플라이싱 리보자임을 나타낸 것이다.
도 4는 in vitro 돌연변이 형성을 통해 Kras 야생형으로부터 제작된 Kras 12V의 구조를 나타낸 것이다.
도 5는 in vitro에서 KRib1, KRib2의 Kras G12V 트랜스-스플라이싱 산물의 분석을 나타낸 것이다[A: RT-PCR 분석, B-C: 서열분석(B: KRib1, C: KRib2)].
도 6은 P1/P10 헬릭스와 안티센스 부위를 변형함으로써 특이성과 효율성이 증대된 트랜스-스플라이싱 리보자임을 나타낸 것이다[A. 변형된 리보자임의 모식도, 화살표: trans-splicing junction; B. 변형된 KRib1 , KRib2의 구조체].
도 7은 in vitro에서 변형된 리보자임의 트랜스-스플라이싱 효율성 및 특이성 분석 결과를 나타낸 것이다[A-B: 트랜스-접합 산물의 RT-PCR 분석, C-D: 트랜스-접합 RT-PCR 산물의 서열분석(C: AS300KRib1, D: AS100KRib2)].
도 8은 트랜스-스플라이싱 리보자임에 의한 트랜스진의 선별적 유도를 확인하기 위한 리포터 에세이 결과를 나타낸 것이다[A: 293 세포에서 리포터 분석을 위한 구조체, B: Kras (야생형 또는 12V) RNA 및 레닐라 루시퍼라제가 함유된 리보자임(KRib1 세트 / KRib2 세트)의 트랜스펙션].
도 9는 트랜스-스플라이싱 리보자임에 의한 트랜스진의 선별적 발현을 확인하기 위한 RT-PCR 분석 결과를 것이다[A: 293 세포에서 리포터 분석을 위한 구조체, B: Kras (wild or 12V) RNA가 함유된 리보자임(KRib1 세트 / KRib2 세트)의 RT-PCR; W: 야생형 Kras, 12V: Kras G12V, IGS: 내부 가이드 염기서열, D)KRib: 불활성 리보자임].
도 10은 Kras G12V를 내생적으로 발현하는 세포주 SW480에서 TK 유전자 유도에 의한 세포 퇴행을 나타낸 것이다[A: SW480 세포에서 TK 분석을 위한 구조체, B: GFP 유전자 함유 AS100KRib2-TK의 트랜스펙션]
도 11은 박테리아에서 상동 재조합에 의한 재조합 아데노바이러스의 세대를 나타낸 것이다[A: 재조합 아데노바이러스 전달 시스템의 모식도, B: 아데노바이러스 리보자임 재조합의 구조체].
도 12는 재조합 Ad-AS300KRib1-TK의 분석을 나타낸 것이다[A. 바이러스 정제 전 PCR에 의한 바이러스 게놈 확인, B: Kras G12V 변이된 SW480에서 바이러스 정제 전 RT-PCR에 의한 Ad-AS300KRib1-TK 발현 확인; TK : Thymidine Kinase, LITR : Left Inverted Terminal Repeat].
도 13은 재조합 Ad-AS100KRib2-TK의 분석을 나타낸 것이다[A. 바이러스 정제 전 PCR에 의한 바이러스 게놈 확인, B: Kras G12V 변이된 SW480에서 바이러스 정제 전 RT-PCR에 의한 Ad-AS100KRib2-TK 발현 확인; TK : Thymidine Kinase, LITR : Left Inverted Terminal Repeat].
도 14는 재조합 Ad-AS100KRib2-LacZ의 분석을 나타낸 것이다[A. 바이러스 정제 전 PCR에 의한 바이러스 게놈 확인, B: Kras G12V 변이된 SW480에서 바이러스 정제 전 RT-PCR에 의한 Ad-AS100KRib2-LacZ 발현 확인; LITR : Left Inverted Terminal Repeat].
도 15는 Kras G12V 변이된 암 세포 SW480에서 아데노바이러스 전달 시스템을 이용한 트랜스-접합 RNA 산물의 분석을 나타낸 것이다.
도 16은 종양세포주에서 아데노바이러스 전달 시스템에 의한 특이적 트랜스-스플라이싱 리보자임의 세포 생존율을 분석한 것이다[A: SW480(KrasG12V)에서 세포 생존율 MTS 분석([left panel: M.O.I dependent / 100uM GCV , right panel GCV dose dependent / virus 150M.O.I), B: CAPAN-1(KrasG12V)에서 세포 생존율 MTS 분석(M.O.I dependent / 100uM GCV), C: HT-29(wild Kras)에서 세포 생존율 MTS 분석(left panel: M.O.I dependent / 100uM GCV , right panel GCV dose dependent / virus 150M.O.I)].
도 17은 이종이식 마우스 모델에서 아데노바이러스 인코딩 Kras G12V-특이적 리보자임의 분석을 나타낸 것이다[A: 복강 내 CAPAN-1(Kras G12V) 종양 모델(n=6 또는 7)에서 Ad-리보자임에 의한 효율적이고 특이적인 종양 퇴행 보임, B: 피하 내 HT-29(wild type Kras) 종양 모델(n=7)에서 Ad-리보자임에 의한 종양 퇴행 없음].
이하, 본 발명을 더욱 상세하게 설명하면 다음과 같다.
본 발명의 리보자임은 암 진행 여부를 진단할 수 있는 라스(Ras) 유전자의 단 염기 변이, 특히 Kras 유전자의 12번째 코돈의 단 염기 변이 Ras RNA를 특이적으로 인식하여 치환할 수 있는 트랜스-스플라이싱 그룹 I 리보자임이다.
도 1은 트랜스-스플라이싱 리보자임 매개 RNA 리프로그램을 나타낸 모식도이다.
도 1을 참조하면, 본 발명의 리보자임은 발암의 원인이 되는 온코진(oncogene)인 Kras의 돌연변이 중 12번째 코돈이 GGT에서 GTT로 변이된 KRas 전사체를 인지, 절단하고 타겟 부위의 하류(down-steam) 부위에 리보자임의 3'엑손을 연결한다. 이때 리보자임의 3' 엑손은 치료 유전자 또는 리포터 유전자를 함유함으로써 돌연변이 KRas 전사체를 보정하여 암세포만을 선택적으로 세포사를 유발하거나 진단하여, 치료 또는 진단할 수 있다.
그룹 I 인트론은 6개의 내부 가이드 염기서열(internal guide sequence; IGS)로 타겟을 인지하게 되는데, 이때 5' 끝의 G?U 와블 염기쌍(wobble base pair)은 필수이다. 따라서, 본 발명의 리보자임은 12번째 코돈이 GTT로 변이된 K-ras 12V를 타겟팅하는데 있어서 35번째 nt인 T와 G?U 와블 염기쌍을 이루도록 내부 가이드 염기서열로 'GCAGCU'을 갖거나, 36번째 nt인 T와 G?U 와블 염기쌍을 이루도록 내부 가이드 염기서열로 'GACAGC'를 갖게 하였다.
여기서, 치료 유전자(therapeutic gene)는 암 세포 내에서 발현되어 치료학적 효과를 나타내는 뉴클레오타이드 서열을 말한다. 예를 들면, 약제감수성 유전자, 세포사멸 유전자, 세포증식 억제 유전자, 세포독성 유전자, 종양 억제인자 유전자, 항원성 유전자, 항신생 혈관 생성 유전자, 사이토카인 유전자 등이 포함되며, 이에 한정되는 것은 아니다.
상기 약제감수성 유전자(drug-sensitizing gene)는 독성이 없는 전구체(prodrug)를 독성물질로 전환시키는 효소에 대한 유전자로 유전자가 이입된 세포가 사멸하게 되므로 자살 유전자(suicide gene)로도 불린다. 즉, 정상세포에는 독성이 없는 전구체를 전신적으로 투여했을 때 암세포에만 전구체가 독성 대사체(toxic metabolite)로 전환되어 약제에 대한 감수성을 변화시킴으로써 암세포를 파괴시키는 방법이다. 대표적으로 HSV-tk(Herpes simplex virus-thymidine kinase) 유전자와 갠시클로비르(ganciclovir), 대장균의 사이토신 탈아미노효소 (cytosine deaminase, CD) 유전자와 5-플루오로시토신(5-fluorocytosine, 5-FC)가 가능하다.
상기 세포사멸 유전자(proapoptotic gene)는 발현되어 프로그램된 세포 소멸을 유도하는 뉴클레오타이드 서열을 말한다. 당업자에게 공지된 세포사멸 유전자로, p53, 아데노바이러스 E3-11.6K (Ad2 및 Ad5에서 유래) 또는 아데노바이러스 E3-10.5K (Ad에서 유래), 아데노바이러스 E4 유전자, p53 경로 유전자 및 카스파제를 코딩하는 유전자가 포함된다.
상기 세포증식 억제 유전자(cytostatic gene)는 세포 내에서 발현되어 세포 주기 도중에 세포 주기를 정지시키는 뉴클레오타이드 서열을 의미한다. 대표적으로 p21, 망막아세포종 유전자, E2F-Rb 융합 단백질 유전자, 사이클린-종속성 카이네이즈 억제인자를 코딩하는 유전자(예를 들면, p16, p15, p18 및 p19), 성장 중지 특이성 호메오박스(growth arrest specific homeobox, GAX) 유전자 (국제특허출원공개 WO 97/16459호 및 WO 96/30385호) 등이 있다.
상기 세포독성 유전자(cytotoxic gene)는 세포 내에서 발현되어 독성 효과를 나타내는 뉴클레오타이드 서열을 말한다. 일예로, 슈도모나스 외독소(exotoxin), 리신 독소, 디프테리아 독소 등을 코딩하는 뉴클레오타이드 서열 등이 있다.
상기 종양 억제인자 유전자(tumor suppressor gene)는 표적 세포 내에서 발현되어 종양 표현형을 억제할 수 있거나 세포사멸을 유도할 수 있는 뉴클레오타이드 서열을 의미한다. 대표적으로 종양 괴사 인자(tumor necrosis factor-a, TNF-a), p53 유전자, APC 유전자, DPC-4/Smad4 유전자, BRCA-1 유전자, BRCA-2 유전자, WT-1 유전자, 망막아세포종 유전자(Lee et al., Nature, 329,642, 1987), MMAC-1 유전자, 선종양 폴립증 코일 단백질(adenomatous polyposis coil protein)(Albertsen et al. 미국특허공보 US 5,783,666호), 결손된 결장 종양 (DCC) 유전자, MMSC-2 유전자, NF-1 유전자, 염색체 3p21.3에 위치한 비인후 종양 억제인자 유전자(Cheng et al. Proc.Nat.Acad . Sci., 95,3042-3047, 1998), MTS1 유전자, CDK4 유전자, NF-1 유전자, NF-2 유전자 및 VHL 유전자가 포함된다.
상기 항원성 유전자(antigenic gene)는 표적 세포 내에서 발현되어 면역 시스템에서 인식할 수 있는 세포 표면 항원성 단백질을 생산하는 뉴클레오타이드 서열을 말한다. 당업자에게 공지된 항원성 유전자의 예에는 암태아성 항원 (carcinoembryonic antigen, CEA) 및 p53(Levine, A., 국제특허출원공개 WO 94/02167호)이 포함된다.
상기 사이토카인 유전자(cytokine gene)는 세포 내에서 발현되어 사이토카인을 생성하는 뉴클레오타이드 서열을 의미한다. 대표적으로 GM-CSF, 인터류킨(IL-1, IL-2, IL-4, IL-12, IL-10, IL-19,IL-20), 인터페론 α, β, γ(인터페론 α-2β) 및 인터페론 α-2α-1과 같은 융합체 등이 포함된다.
상기 항신생혈관 생성 유전자(anti-angiogenic gene)는 발현되어 항-신생혈관 생성 인자를 세포밖으로 방출하는 뉴클레오타이드 서열을 말한다. 그 예로 안지오스타틴, 혈관 내피 성장 인자(VEGF)의 억제 인자, 엔도스타틴 등이 포함된다.
또한, 본 발명에서는 상술한 리보자임 활성을 통해 타겟 부위의 하류(down-steam) 부위에 리포터 유전자를 접합할 수 있다. 이렇게 접합된 리포터 유전자는 리포터 단백질로 발현되므로, 발현된 리포터 단백질의 활성 또는 양을 측정함으로써 암 세포를 진단할 수 있다.
이때 리포터 유전자는 본 발명이 속하는 분야에서 공지된 것을 사용할 수 있으며, 바람직하게는 LacZ, 클로람페니콜 아세틸 전이효소(CAT: chloramphenicol acetyl transferase), 레닐라 루시퍼라제(Renila luciferase), 반딧불이(firefly) 루시퍼라아제, 적색형광단백질(RFP), 녹색형광단백질(GFP), 분비성 태반 알칼라인 포스파타아제(secreted placental alkaline phosphatase, SEAP) 또는 HSV-tk(Herpes simplex virus-thymidine kinase)를 암호화하는 유전자를 사용한다. 이 중 활성 분석의 편이성 및 민감도 면에서 LacZ를 사용하는 것이 바람직하다.
이러한 리포터 단백질들의 활성은 하기와 같이 당업자에게 공지되어 있는 방법을 사용하여 측정할 수 있다:
반딧불이 루시퍼라아제(de Wet J. et al., Mol. Cell Biol., 7, 725-737, 1987 참조), 레닐라 루시퍼라아제([Lorenz W.W. et al., PNAS 88, 4438-42, 1991) 참조), 클로람페니콜 아세틸 전이효소(Gorman C. et al.,Mol. Cell Biol., 2, 1044-1051, 1982 참조), LacZ(Hall C.V. et al., J. Mol. Appl . Genet ., 2,101-109, 1983 참조), 인간 성장 호르몬(Selden R. et al.., Mol. Cell Biol., 6, 3173-3179, 1986 참조), 녹색 형광 단백질(Chalfie M. et al., Science, 263, 802-805, 1994 참조) 및 분비성 태반 알칼린 포스파타아제(Berger, J. et al., Gene, 66, 1-10, 1988 참조). 또한, 티미딘 키나제(thymidine kinase)를 리포터 단백질로 한 경우 PET(positron emission tomography) 이미징 법을 이용할 수 있다.
본 발명의 실시예 1에 따르면, 발암의 원인이 되는 온코진(oncogene)인 Kras의 돌연변이 중 12번째 코돈이 GGT에서 GTT로 변이된 Kras를 타겟으로 하여 KRib1, KRib2 트랜스-스플라이싱 리보자임을 제작하였다.
이때 12번째 코돈이 GTT로 변이된 K-ras 12V를 타겟팅하는데 있어서 35번째 nt인 T와 G?U 와블 염기쌍을 이루도록 디자인한 KRib1, 36번째 nt인 T와 G?U 와블 염기쌍을 이루도록 디자인한 KRib2를 제작하였다.
본 발명의 실시예 2에 따르면, 제작된 리보자임을 in vitro에서 트랜스-스플라이싱 반응 실험을 수행하였고, Kras G12V를 타겟팅하는 리보자임 KRib1, KRib2이 특이적으로 Kras G12V를 인지하여 절단하고 리보자임의 3' 엑손을 정확한 타겟 부위에 연결한다는 것을 확인할 수 있었다.
본 발명의 실시예 3에 따르면, IGS만 갖는 리보자임과 효율성을 높이기 위한 P1, P10, AS(안티센스) 구조를 추가한 변형(modification) 리보자임을 제작하고, 이들을 가지고 in vitro 트랜스-스플라이싱 반응을 수행하였다. 그 결과, P1, P10, AS가 있는 리보자임이 특이성과 효율성 면에서 우수하다는 것을 확인하였다.
본 발명의 실시예 4에 따르면, 세포 내에서도 트랜스-접합 산물 즉, 트랜스진의 발현이 유도되는지를 리포터 유전자를 사용하여 리포터 분석을 수행하였고, 그 결과 세포주에서 루시퍼라제의 발현을 확인하여, 세포 내에서도 본 발명의 리보자임이 특이적으로 트랜스-스플라이싱을 일으키며, 트랜스진의 발현이 유도된다는 것을 알 수 있었다.
또한, 트랜스-스플라이싱을 RNA 레벨에서 보기 위해 293 세포에 리보자임들과 Kras(G12V, 야생형) RNA를 코-트랜스펙션시킨 후 RT-PCR을 했다. 그 결과 1 세트에서 AS300KRib1-F.luc과 2 세트에서 AS100KRib2-F.luc이 in vitro 트랜스-스플라이싱 분석을 수행했던 것과 일치하는 결과인 트랜스-스플라이싱 밴드라고 예상 되어지는 크기의 밴드가 나왔다. 이 밴드가 비특이성 밴드가 아닌 트랜스-스플라이싱 산물임을 확인하기 위해 시퀀싱을 하여 Kras G12V의 타겟 부위에서 정확하게 트랜스-스플라이싱이 일어남을 확인하였다.
본 발명의 실시예 5에 따르면, 리보자임의 3'엑손에 TK 유전자(HSV-TK(herpes simplex virus thymidine kinase))를 도입하는 클로닝된 pAVQ CMV AS300KRib1-TK와 pAVQ CMV AS100KRib2-TK들 중 AS100KRib2-TK를 가지고 TK 분석을 통해 세포 생존력을 관찰하였다. TK 분석 결과, HT-29 세포에서는 GCV를 처리하거나 미처리 시에 GFP의 발광에는 어떠한 변화도 없는 것으로 보아 세포의 생존력에 영향을 끼치지 않음을 볼 수가 있었다.
그러나, SW480 세포에서는 AS100KRib2-TK를 트랜스펙션한 세포에서 GCV를 처리하였을 때 GCV를 처리하지 않은 세포에 비해 GFP의 발광 수가 감소한 것을 볼 수가 있었다.
한편, 유전자 치료에서 중요시 되어야 할 점은 재조합 유전자를 세포에 전달시키는 방법이다.
일반적인 방법으로 크게 물리화학적인 방법으로 재조합 DNA를 직접 투여하는 방법과 생물학적인 방법으로 재조합 바이러스 벡터를 들 수가 있다. DNA를 직접 투여하는 방법은 간단하기 때문에 위험성이 낮고 경제적이라는 장점을 가지고 있지만, 재조합 DNA의 전달률이 낮은 단점을 가지고 있다.
이에 본 발명은 상기 리보자임의 세포 전달 방법으로서 위험성도 적고 DNA 전달율도 우수한 아데노 바이러스 전달 시스템을 포함한다.
아데노바이러스는 지름이 약 70~80 nm로 크며 내부는 20~26×106 달톤의 dsDNA를 가진 DNA 바이러스이다. 게놈의 양끝에는 ITR(inverted terminal repeat)이 있고, 이곳에서 바이러스 복제가 시작된다. 아데노바이러스의 게놈은 E1-E4의 초기부위, L1-L5의 후기부위로 구분된다.
아데노바이러스 벡터의 장점은 첫째, 인체 내 종양 발생에 무관한 바이러스로 알려져 있고, 오랜 바이러스 백신의 개발로 위험성이 낮은 바이러스이다. 둘째, 높은 역가의 바이러스를 제조할 수 있다. 셋째, 감수분열이 끝난 세포와 거의 모든 인체의 세포에 감염시킬 수 있다. 넷째, 아데노바이러스 벡터에 10kb에 가까운 DNA를 삽입할 수 있다.
단점은 첫째, 아데노바이러스가 숙주의 게놈에 통합하지 못함으로 영구적인 효과를 보지 못한다. 둘째, 바이러스에 대한 항원?항체반응으로 치료효과가 짧다. 셋째, 많은 적가 투여 시 부작용을 고려하지 않을 수 없다.
본 발명의 실시예 6에 따르면, BJ5183이라는 박테리아에서 리보자임 트랜스퍼 벡터와 pAdenovactor ΔE1/E3 백본 벡터의 코-트랜스포메이션을 하여 상동성 재조합을 시켜 재조합 리보자임을 만들었고, 이를 293 세포에 트랜스펙션함으로써 재조합 리보자임 바이러스를 얻게 되었다.
이렇게 얻은 재조합 리보자임 바이러스는 293 세포에 감염하여 증폭하는데 증폭하는 과정에서 재조합 리보자임 바이러스가 제대로 만들어지고 있는지 확인하기 위해서 바이러스 게놈 DNA를 추출하여 리보자임의 TK 유전자와 바이러스의 LITR을 PCR을 통해 확인하였다.
또한, SW480 세포에 감염하여 RNA 레벨에서도 TK 유전자를 RT-PCR을 통해 확인하였다. 이렇게 확인된 재조합 리보자임 바이러스들은 Vivapure? AdenoPACKTM 100 RT를 사용하여 정제하였다.
이렇게 정제된 Ad-AS300KRib1-TK와 Ad-AS100KRib2-TK 바이러스들은 종양 세포에 감염하여 종양 세포에서 Ad-리보자임의 특이성과 효울성을 보았다. SW480 세포에 감염한 결과 Ad-AS300KRib1-TK와 Ad-AS100KRib2-TK에서 트랜스-스플라이싱 밴드를 보았고, 이것을 시퀀싱을 통해 타겟 부위에서 일어났는지를 확인하였다.
본 발명의 실시예 7에 따르면, Ad-리보자임들이 특이적으로 트랜스-스플라이싱을 일으켜 트랜스진을 유도함을 보기 위해서 MTS 분석을 진행하였다. 그 결과, Kras G12V 변이된 세포주인 SW480 세포와 CAPAN-1 세포에서는 대조군과 비교하였을 때 Ad-리보자임들만이 세포사가 유도됨을 볼 수 있었고, Kras 야생형인 HT-29 세포에서는 대조군과 비교하여 세포사가 일어나지 않음을 관찰하였다. 이는 Ad-리보자임이 Kras G12V를 특이적으로 인지하여 트랜스-스플라이싱하고 트랜스진을 유도하여 특이적으로 세포사를 일으킬 수 있음을 나타낸다.
본 발명의 실시예 8에 따르면, Ad-리보자임의 in vivo에서 특이성과 효율성을 알아보기 위해 이종이식 마우스 모델(xenograft mice model) 실험을 수행하였다.
그 결과, Kras 변이된 종양에서는 대조군과 비교하여 Ad-AS100KRib2-TK의 종양 무게가 감소하였다. 그러나, Kras 야생형 종양의 경우에는 음성 대조군인 Ad-CMV-LacZ와 양성 대조군인 Ad-CMV-TK와 비교했을 때, Ad-리보자임들이 Kras 야생형 종양에서는 작용을 하지 않는 것을 확인할 수 있었다.
이러한 결과는 본 발명에 따른 Ad-리보자임이 Kras G12V만을 발현하는 종양을 특이적으로 퇴행(regression)시킬 수 있다는 것을 보여준다.
이와 같이 본 발명의 리보자임은 암 특이적 유전자를 치료 유전자로 보정하여 유전자 발현을 유도한다. 그 결과 암 세포만을 선택적으로 세포사를 유발할 수 있다.
따라서, 본 발명에서는 상기 리보자임을 유효성분으로 포함하는 항암용 약학적 조성물을 제공한다.
본 발명의 항암용 약학적 조성물은 투여를 위해서 상기 기재한 유효 성분 이외에 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 약제학적 조성물로 바람직하게 제제화할 수 있다.
본 발명의 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 액상 용액으로 제제화되는 조성물에 있어서 허용 가능한 약제학적 담체로는, 멸균 및 생체에 적합한 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 알부민 주사용액, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있으며, 표적 기관에 특이적으로 작용할 수 있도록 표적 기관 특이적 항체 또는 기타 리간드를 상기 담체와 결합시켜 사용할 수 있다.
본 발명의 약제학적 조성물은 비경구 투여가 바람직하고, 예컨대 정맥내 투여, 복강내 투여, 종양내 투여, 근육내 투여, 피하 투여, 또는 국부 투여를 이용하여 투여할 수 있으나, 이에 한정되는 것은 아니다.
본 발명 항암용 약학적 조성물의 투여량은 질환의 종류, 질환의 중증도, 조성물에 함유된 유효 성분 및 다른 성분의 종류 및 함량, 제형의 종류 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다.
본 발명의 약학적 조성물은 단독으로, 또는 외과적 수술요법 등의 보조 치료 방법들과 병행하여 사용할 수 있다. 본 발명의 조성물과 함께 이용될 수 있는 화학 요법제(chemotherapeutic agent)는 시스플라틴(cisplatin), 카르보플라틴 carboplatin), 프로카르바진(procarbazine), 메클로레타민(mechlorethamine), 시클로포스파미드(cyclophosphamide), 이포스파미드 (ifosfamide), 멜팔란(melphalan), 클로라부실(chlorambucil), 비술판(bisulfan), 니트로소우레아 (nitrosourea), 디악티노마이신(dactinomycin), 다우노루비신(daunorubicin), 독소루비신(doxorubicin), 블레오마이신(bleomycin), 플리코마이신(plicomycin), 마이토마이신(mitomycin), 에토포시드(etoposide), 탁목시펜(tamoxifen), 택솔 (taxol), 트랜스플라티눔(transplatinum), 5-플루오로우라실(5-fluorouracil), 빈크리스틴(vincristin), 빈블라스틴(vinblastin) 및 메토트렉세이트(methotrexate) 등을 포함한다. 발명의 조성물과 함께 이용될 수 있는 방사 요법은 X-선 조사 및 -선 조사 등이다.
아울러, 본 발명의 리보자임은 암 특이적 유전자에 리포터 유전자를 접합하여 암세포만을 선택적으로 진단할 수 있다.
따라서, 본 발명에서는 상기 리보자임을 유효성분으로 포함하는 암 진단용 조성물을 제공한다.
[실시예]
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나. 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
실시예 1: Kras G12V RNA 를 특이적으로 인지할 수 있는 트랜스- 스플라이싱 그룹 I 리보자임 제작
1. K-
ras
타겟팅
리보자임의
디자인
리보자임의 내부 가이드 염기서열(Internal guide sequence; IGS)이 타겟 주형 RNA를 인식하여 트랜스-스플라이싱 기작을 일으키는데 있어서 타겟 RNA의 2차 구조가 영향을 미치게 되므로 mfold program(RNA 2차 structure form program)을 이용하여 2차 구조를 예측하였으며, 타겟하려고 하는 12번 코돈 부위가 리보자임이 트랜스-스플라이싱을 일으키는데 용이한 구조인지를 확인하였다.
Kras의 12번째 코돈이 Gly에서 Val으로 치환된 Kras cDNA의 2차 구조를 mfold 프로그램(Web상에서 제공되는 RNA 2차 구조를 분석하는 program-copyright 1996 Dr. M. Zuker)을 이용하여 분석함으로써 변이된 Kras의 12번째 코돈이 리보자임이 타겟팅 하기에 적합한 구조인지를 확인하였다(도 2 참조).
기존 맵핑 방법을 통하여 리보자임이 타겟팅 하기에 적합한 부분을 찾아 분석한 결과와 비교하여 볼 때 Kras의 12번째 코돈이 Gly에서 Val으로 단 염기 변이가 일어난 경우의 이차구조가 타겟팅 가능한 구조라는 것을 확인하고 특이 리보자임을 제작하기로 결정하였다.
Kras의 12번째 코돈이 GGT에서 GTT로 치환된 변이를 타겟팅하는 리보자임의 내부 가이드 염기서열(internal guide sequence; IGS)을 디자인하였다. 이때, IGS가 변이된 단 염기와 G?U 염기쌍을 이루게 한 KRib1(GCAGCT)과 변이된 다음의 염기와 G?U 염기쌍을 이루게 한 KRib2(GACAGC) 두 가지의 리보자임을 디자인하였다(도 3 참조).
2. K-
ras
12V(기질) 구조체 제작
2-1.
In
vitro
돌연변이 형성 및
클로닝
도 4는 in vitro 돌연변이 형성을 통해 Kras 야생형으로부터 제작된 Kras 12V의 구조를 나타낸 것이다.
타겟 구조체를 만들기 위하여 GeneEditor in vitro 부위 유도 돌연변이 형성 시스템(site-Directed mutagenesis System, Promega)를 사용하여 Kras 야생형으로부터 12번째 코돈이 발린으로 치환된 Kras 12V를 제작하였다.
서열번호 1의 프라이머(표 1 참조)를 사용하였으며, 이를 pUC19에 5' BamHI, 3' HindIII로 클로닝하였다.
서열번호 | 프라이머 서열 | |
1 | 5'-GTTGGAGCTGGTGGCGTAGGC-3 | Kras wild |
2 | 5'-TAATACGACTCACTA-3' | Kras 5' BamHI |
3 | 5'-GGGTAATACGACTCACTATAGGAGGTAAAAGTTATCAGGCATGC-3' | kras rib T7-5' |
4 | 5'-GGGTAATACGACTCACTATAGACAGCAAAAGTTATCAGGCATGC-3' | RY-PCR |
5 | 5'-TGTAAAACGACGGCCAGTG-3 | RY-RT |
6 | 5'-CGATGATCACGAAGACGCCAAAAACATA-3' | kras p1p10-rib1-5 |
7 | 5'-CGACATGATCACGAAGACGCCAAAAACATA-3' | kras p1p10-rib2-5 |
8 | 5'-AAGGAAAAAAGCGGCCGCTTATTACAATTTGGACTTT-3' | krib 3' |
9 | 5'-GGGAAGCTTGCAAGAGTGCCTTGACGA-3 | kras as 5' |
10 | 5'-GGGAAGGCTTCCATCAATTACTACTTGC-3 | kras as 3' |
11 | 5'-GGGTAATACGACTCACTATAGGGCTACTAGGACCATAGGTA-3' | kras as300 5' |
12 | 5'-CGCGGATCCACTGAATATAAACTTGTGGTA-3' | kras t/s BamHI5' |
13 | 5'-CCCAAGCTTGCCCAACACCGGCATAAAG-3 | cell t/s luci 3' nest |
14 | 5'-GTGATCATCGCGATGATCACGCTTCGTACCCCTGCCAT-3' | TK 5' NruI 1set |
15 | 5'-GTGATCATCGCGACATGATCACGCTTCGTACCCCTGCCAT-3' | TK 5' NruI 2setI |
16 | 5'-AAGGAAAAAATTCGAATTAGCCTCCCCCAT-3' | 3' TK BstBI |
17 | 5'-TAATACGACTCACTATAGGG-3' | T7-R-Primer |
18 | 5'-CCTTTCCCGCAATTTGACGGTCTT-3' | IGS rev seq |
19 | 5'-CGGGAATTCGTGGTAATGACAAGC-3' | 5' TK stable |
20 | 5'-CGGGGATCCATCCAGGATAAAGACGTGCATGGG-3' | 3' TK stable |
21 | 5'-GGAATTCTGGAGTTTGTGACGTGGCG-3' | 5' LITR EcoRI |
22 | 5'-GCTCTAGATGGCCAAATCTTACTCGGTTACGC-3' | 3' LITR XbaI |
23 | 5'-GGAATTCATGGTCGTTTTACAACGTCGTG-3' | 5' viral LacZ |
24 | 5'-GGGAAGCTTCGGATTGACCGTAATGGGA-3' | cancer.lacZ-3 |
2-1-1. 벡터와 삽입물의 제조
Kras PCR 산물 DNA 1μg을 BamHI(0.3u)/ HindⅢ(0.3u)와 10X B 버퍼 (Roche)와 20 ㎕까지의 dH2O 37 ℃에서 3시간 동안 반응시켜 2.5% TBE 겔에 전기영동을 하여 크기를 확인하였다. 그 다음 3μg의 pUC19 벡터도 동일하게 BamHI(0.3u)/ HindⅢ(0.3u) 처리하여 잘린 것을 확인한 후 페놀 & EtOH 침전 반응을 시킨 후 정량을 하였다.
2-1-2.
DNA
용출(
insert
)
겔 추출(DNA)(Omega bio tec. E.Z.N.A Kit)를 이용하여 DNA 크기에 맞는 농도로 0.5 X TAE 아가로스 겔을 만들어 DNA를 로딩한 다음 롱 웨이브 UV로 밴드를 확인한 후 원하는 밴드 부분만을 정확히 잘라냈다.
각각의 겔 조각을 1.5㎕ 튜브에 50 ~ 300 ㎎이 되도록 넣고 용출 버퍼 0.5 ㎕씩을 넣고 60 ℃에서 10분간 완전히 녹이는데, 200㎎ 이상일 경우 10분 이상 녹였다. 겔을 완전히 녹인 후 검체 튜브에 겔 추출 컬럼을 끼운 후 녹은 용액을 컬럼에 옮겨 13000 rpm으로 60초간 돌린 후 가라앉은 용액을 버리고 워쉬Ⅰ버퍼 0.5 ㎕ 을 각기 컬럼에 넣고 60초간 돌린 후 다시 걸러진 버퍼를 버렸다.
컬럼을 새로운 1.5㎕ 튜브에 옮겨 끼운 후 dH2O 30㎕를 넣고 1분간 보관 후 2분 정도 원심분리를 돌려 DNA를 얻어낸 후 정량 하여 -20 ℃에 보관하였다.
2-1-3. 연결
효소 처리된 pUC19과 삽입물(trans-splicing product)을 각각 벡터 : 삽입물(1:3) 비율로 T₄리가아제(Roche)로 4℃에서 16시간 동안 반응시켰다.
2-1-4. 형질전환
연결 산물을 대장균(JM109)에 형질전환하기 위해 수용성 세포(competent cell)로 제작된 JM109에 연결 산물 1㎕를 넣고 전기형질전환장치(electrophorator)를 25μF, 1.8Kv, 펄스 조절기(pulse controller) 400Ω으로 셋팅하여 형질전환한 후 1ml 후레쉬 배지에 넣어 잘 섞은 후 1시간 동안 배양하였다.
X-gal, IPTG를 넣어 만든 아가 플레이트에 골고루 깐 다음 37℃ 배양기에 16시간 동안 배양시킨 후 컬러 스크리닝을 통하여 백색 콜로니를 선택적으로 5ml 암피실린이 포함된 LB 배지에 접종하여 16시간 동안 37℃ 흔들면서 배양하였다.
2-2. 염기서열 확인
In vitro 돌연변이 형성을 통하여 제작된 Kras 12V의 염기서열을 확인하였다. 종결자 준비 반응 혼합물(terminator ready reaction mixture, PE applied Biosystems) 3㎕, 정량된 DNA 100ng, 서열번호 2의 프라이머(표 1 참조) 3.2pmol을 총 10㎕ 반응으로 (96℃-10', 50℃-5', 60℃-4')× 25 사이클 반응하였다.
이후 정제를 위하여 dH2O 40㎕ 첨가한 다음 3M NaoAC(1/10 volume), 100% EtOH(2 volume)를 넣고 보르텍스 후 13000 rpm, 4℃에서 30분간 원심분리하고 70% EtOH(400㎕)로 세척한 다음 EtOH를 제거하고 speed-vacuum으로 말린 후 주형 억제 시약(template suppression reagent) 15㎕에 녹였다. 이어서 보르텍스 & 스핀 다운 후 시퀀싱 튜브에 옮겨 자동 시퀀서(ABI 310 Genetic Analyzer)로 시퀀싱을 시행하였다.
3. 기질
RNA
(K-
ras
)의 제조
3-1. K-
ras
기질 제조
기질로 이용될 Kras 12V와 대조군으로 사용될 Kras 야생형의 DNA를 Hind III로 잘라서 페놀 추출, EtoH 침전하여 준비하였다.
3-2. 기질 전사
상기 2-1과 같은 방법으로 제조된 DNA를 전사를 통하여 RNA를 만들게 되는데 DNA 주형(1㎍), 1x 전사 버퍼, 10mM DTT (Sigma), 0.5mM ATP, GTP, CTP, UTP (Roche), 80U RNase 억제제(Kosco), T7 RNA 중합효소(Takara), DEPC-H2O를 100㎕까지 넣어서 섞은 후 37℃에서 3시간동안 반응시켰다.
이를 위해 5'프라이머 제작 시에 T7 프로모터 부분을 붙이게 되며, 이후 남아있는 DNA를 제거하기 위해 5U DNase I(promega)를 37℃에서 30분간 처리하여 DNA 주형을 완전히 제거한 후 페놀 추출(PH7.0), 에탄올 침전을 통하여 RNA만을 순수 분리 정제한 후 TE 버퍼(10mM Tris-Hcl PH7.5, 1mM EDTA)에 농축시켜 놓았다.
4.
KRib1
,
KRib2
(
Kras
targeting
ribozyme
) 제조
4-1.
리보자임
기질 제조
Kras의 12번 코돈을 인지하여 트랜스-스플라이싱할 수 있는 내부 가이드 염기서열(Internal guide sequence;IGS)를 가진 리보자임을 제작하기 위해 서열번호 3, 4의 프라이머(표 1 참조)를 사용하여 pt7L21로부터 PCR하여 KRib1, KRib2를 제작하였다. 또한, d)pt7L21로부터 PCR을 통하여 촉매의 코어(catalytic core)가 제거된 d)KRib1, d)KRib2를 제작하여 대조군으로 사용하였다.
4-2.
리보자임
전사
기질과 같은 방법으로 전사를 하는데, 이때 dNTP 양을 1.5mM로 늘려서 자가-스플라이싱을 최대한 방지하며,1x 스플라이싱 버퍼(40Mm Tris (ph7.5), 5mM MgCl2, 10mM DTT, 4mM spermidin)를 사용하였다.
5.
RNA
용출
원하는 RNA만을 얻기 위해, RNA 용액의 1/2 볼륨의 변성 RNA 로딩 염료(10㎕ Formamide, 10mM EDTA, 0.01% Bromophenol blue, 0.01% Xylenecyanol)를 섞은 후 95℃에서 5분간 처리하여 변성시켰다.
변성된 RNA를 우레아가 들어있는 6% 폴리아크릴아마이드 겔에 로딩하여 0.5x TBE 버퍼에서 40분간 230V로 전기영동한 후 원하는 크기의 RNA 밴드 부분의 겔을 잘라서 용출 버퍼(0.6M Ammonium acetate, 1mM EDTA, 0.2% SDS) 400㎕에 넣어서 37 ℃에서 3시간 동안 반응시켰다. 그 다음 젤은 스핀 다운으로 가라앉은 후 그 상층액을 따서 페놀 추출과 EtOH 침전을 통하여 정제하고 TE 버퍼에 최종적으로 농축한 후 UV-분광광도계를 이용하여 RNA를 정량하였다. 이때 리보자임은 비-자가-스플라이싱 산물만을 용출하여 UV-분광광도계를 용출하여 정량하였다.
6. 페놀 추출 및 에탄올 침전
용액과 동량의 페놀 : 클로로포름 : 이소아밀알콜 (25:24:1)을 넣고 보르텍스를 이용하여 완전히 섞어준 뒤 4℃ 10000ⅹg에서 10분간 원심분리하였다. 상층 액만을 조심스럽게 1.5ml 튜브에 옮긴 후 2배 부피의 100% EtOH(-70℃)과 1/10 볼륨의 3M 소듐 아세테이트를 넣어 잘 섞어준 뒤 -70℃에서 40분간 넣어주었다.
4℃에서 30분간 원심분리 후 상층액을 조심스럽게 제거하고 70% EtOH (-70℃) 400㎕를 넣어 골고루 벽면을 씻은 다음 10분간 원심분리를 한 후 EtOH을 모두 제거한 후 10초 정도 말리고 TE 버퍼에 녹였다.
실시예 2: In vitro 에서 트랜스- 스플라이싱 리보자임 KRib1 , KRib2 의 특이성 확인
1. 트랜스-
스플라이싱
반응
상기 실시예 1에서 디자인한 리보자임 KRib1, KRib2의 특이성을 확인하기 위하여, 먼저 시험관 상에서 트랜스-스플라이싱 반응을 하였다. In vitro 돌연변이 형성을 통하여 제작된 Kras G12V RNA(10nM)와 KRib1과 KRib2 리보자임(50nM)의 RNA를 시험관 내에서 트랜스-스플라이싱 반응을 하였다.
특이 IGS를 갖는 리보자임(KRib1, KRib2)과 Kras의 트랜스-스플라이싱 반응을 위하여 기질 RNA와 리보자임의 절대량을 10nΜ : 50nΜ로 정하였으며 기질 RNA(2x reaction buffer, substrate RNA, 0.2mM dGTP, DEPC-H2O)는 37℃에서 미리 5분간 예열시켰다. 리보자임 RNA(2x reaction buffer, Ribozyme RNA, DEPC-H2O)는 50℃에서 5분간 반응시킨 후 37℃에서 2분간 반응시킴으로써 올바른 3차 구조 형성을 유도한 후 두 반응물을 잘 섞어준 다음 37℃에서 3시간 동안 반응시켰다.
2.
역전사
트랜스-스플라이싱된 RNA 전사체의 3' 엑손(Ribozyme) 부분에 결합하는 서열번호 5의 3'프라이머(표 1 참조)와 5x RT 버퍼, 트랜스-스플라이싱 산물(10㎕)를 잘 섞은 다음 65℃에서 5분간 반응시킨 다음 상온에서 10분간 보관하였다.
상온 보관 후 0.5mM dNTP(BMS)와 MMLV 역전사효소 1U(Promega)와 DEPC dH20로 총 50㎕를 만들어 37℃에서 1시간 동안 반응시켰다. 이후 95℃에서 5분간 반응시켜 MMLV-RT의 활성을 완전히 제거 후 4℃에서 보관하였다.
3.
PCR
RT 반응을 마친 반응물(20㎕)을 서열번호 2와 5의 프라이머(표 1 참조)를 사용하여 MgCl2 50mM, 5% DMSO를 넣고(63℃ 20 sec annealing/ 25 cycle) PCR을 수행하였다.
4.
클로닝
(
RT
-
PCR
산물) 및 염기서열 분석
pUC19에 RT-PCR 산물 클로닝(상기 실시예 1의 2-1과 동일)을 하였으며, 서열번호 2의 프라이머(표 1 참조)(3.2 pmol)를 사용하여 시퀀싱(상기 실시예 2의 2-2와 동일)을 수행하였다.
도 5는 in vitro에서 KRib1, KRib2의 Kras G12V 트랜스-스플라이싱 산물의 분석을 나타낸 것이다[A: RT-PCR 분석, B-C: 서열분석(B: KRib1, C: KRib2)].
도 5A를 참조하면, Kras에 결합하는 5' 프라이머와 리보자임 3' 엑손에 결합하는 3' 프라이머를 이용하여 RT-PCR을 수행한 결과, KRib1과 KRib2와 Kras G12V RNA를 반응시킨 샘플에서만 트랜스-스플라이싱이 일어났을 때 나올 수 있는 산물 크기의 밴드를 얻었다.
반면, 촉매성 코어(catalytic core) 부위를 제거한 불활성 리보자임인 (d)KRib1, d)KRib2)과 Kras G12V RNA를 반응시켰을 때 혹은 KRib1과 KRib2와 야생형 Kras RNA만을 각각 반응시켰을 때는 어떠한 산물도 볼 수 없었다.
또한, 도 5B 및 5C를 참조하면, PCR 산물을 클로닝 후 시퀀싱한 결과, 정확하게 타겟 부위에서 트랜스-스플라이싱이 일어나 5' 에는 Kras의 염기서열이 존재하고 3' 쪽으로는 리보자임의 3' 엑손 염기서열이 존재하는 것을 확인할 수 있었다.
이러한 결과를 통해 Kras G12V를 타겟팅하는 리보자임인 KRib1과 KRib2가 in vitro에서 특이적으로 Kras G12V를 인지하여 절단하고 리보자임의 3' 엑손을 정확한 타겟 부위에 연결한다는 것을 확인할 수 있었다.
실시예 3: 변형 정도에 따른 트랜스- 스플라이싱 리보자임의 in vitro 에서 효율성 및 특이성 확인
타겟에 대한 특이적인 6개의 염기서열인 IGS 외에 P1/ P10 헬릭스와 안티센스 부위를 변형(modify)함으로써 트랜스-스플라이싱 리보자임의 특이성과 효율성을 높일 수 있는데, 이러한 강화된 리보자임을 디자인함에 있어서 각 부위를 점차 변형 정도에 따른 특이성과 효율성을 확인하고자 하였다.
도 6은 P1/P10 헬릭스와 안티센스 부위를 변형함으로써 특이성과 효율성이 증대된 트랜스-스플라이싱 리보자임을 나타낸 것이다.
도 6을 참조하면, IGS만을 가진 리보자임, P1/ P10 구조를 더해준 리보자임, 안티센스 100개 또는 300개를 가진 리보자임 이렇게 각 부위를 점차 더해준 변형된 리보자임 RNA 시리즈를 KRib1과 KRib2 두 종류의 리보자임에 대하여 각각 제조하였다.
1. 특이성 증가시킨 특이성
리보자임
제작
리보자임의 IGS에서 3' 엑손 부위를 서열번호 6의 프라이머와 서열번호 8의 프라이머/서열번호 7의 프라이머와 서열번호 8의 프라이머(표 1 참조)를 이용하여 PCR 한 후 Hind III와 Nru I으로 SEAP 프로모터 벡터에 클로닝하였다.
상기 벡터에 클로닝된 리보자임 뒤에 Nru I과 Xba I으로 루시퍼라제를 클로닝하였는데, 이때 3' 엑손인 루시퍼라제가 Kras에 연결되어 트랜스진의 단백질이 올바르게 발현되도록 루시퍼라제 유전자 앞에 여분의 뉴클레오타이드를 더하여 프레임을 맞춰 주었다.
그 후 Kras 서열상에서 IGS에 의해 인지되는 곳으로부터 3' 말단 쪽으로 상보적인 100nt, 300nt의 안티센스 스트랜드를 서열번호 9의 프라이머와 서열번호 10의 프라이머/ 서열번호 9의 프라이머와 서열번호 11의 프라이머(표 1 참조)를 이용하여 PCR을 수행하였으며, 이를 SEAP KRib 1, SEAP KRib 2 구조체의 IGS 앞에 Hind III 효소 부위로 클로닝하였다.
이렇게 완성된 SEAP-KRib 1, SEAP KRib 2, SEAP AS100 KRib1, SEAP AS100 KRib2, SEAP AS300 KRib1, SEAP AS300 KRib2를 pAV 3.0 벡터로 BamH I과 Not I 부위를 이용하여 각각 클로닝하는 작업을 통하여 CMV 프로모터를 가진 리보자임 구조체 셋트를 제작하였다.
2. 트랜스-
스플라이싱
리보자임의
in
vitro
에서 효율성 및 특이성 확인
상기에서 제작된 각각의 리보자임들(50nM)을 in vitro 상에서 Kras 야생형 RNA 또는 Kras G12V RNA(10nM)와 트랜스-스플라이싱 반응하였으며 Kras에 결합하는 5' 프라이머와 리보자임 3' 엑손에 결합하는 3' 프라이머를 이용하여 상기 실시예 2와 같이 RT-PCR을 수행하였다.
도 7은 in vitro에서 변형된 리보자임의 트랜스-스플라이싱 효율성 및 특이성 분석 결과를 나타낸 것이다.
도 7A 및 7B를 참조하면, KRib1 또는 KRib2와 Kras G12V RNA를 반응시킨 샘플에서 트랜스-스플라이싱이 일어났을 때 나올 수 있는 산물 크기의 밴드를 얻었다.
in vitro 상에서의 변형 정도에 따른 KRib1의 2 셋트의 트랜스-스플라이싱 반응 실험의 결과 KRib1 셋트의 경우 안티센스 100개를 갖는 리보자임 보다 300개를 갖는 리보자임의 트랜스-접합(t/s) 산물 양이 많음을 PCR로 확인할 수 있었으며, KRib2의 경우 안티센스 100개를 갖는 리보자임과 300개를 갖는 리보자임의 트랜스-접합 산물 양이 같음을 PCR로 확인할 수 있었다.
또한, 변형된 정도에 따라 Kras 야생형 RNA와 트랜스-스플라이싱한 샘플에서도 트랜스-스플라이싱 산물과 같은 크기의 PCR 밴드가 흐리게 나왔으나, 이를 클로닝 후 시퀀싱한 결과 트랜스-스플라이싱되지 않은 리보자임이 PCR되었거나 기질이 PCR된 비특이적 PCR 산물임을 확인할 수 있었다.
그리고, 촉매성 코어 부위를 제거한 불활성 리보자임인 (d)KRib1, d)KRib2)과 Kras RNA를 반응시켰을 때 혹은 리보자임과 야행형 Kras RNA만을 각각 반응시켰을 때는 어떠한 산물도 볼 수 없었다.
도 7C 및 도 7D를 참조하면, PCR 산물을 클로닝 후 시퀀싱한 결과, 정확하게 타겟 부위에서 트랜스-스플라이싱이 일어나 5' 에는 Kras의 염기서열이 존재하고 3' 쪽으로는 리보자임의 3' 엑손에 루시퍼라제(Luciferase) 염기서열이 존재하는 것을 확인할 수 있었다.
실시예
4: 세포주에서의 트랜스-
스플라이싱
특이성, 효율성 확인
특이성 리보자임이 세포내에서 단 염기 변이된 Kras G12V RNA를 표적으로 리보자임에 부착된 특정유전자를 트랜스-스플라이싱하는지 알아보기 위해서, Kras 야생형을 발현하는 293 세포에서 3' 엑손에 반딧불의 루시퍼라제를 태깅한 리보자임을 디자인하고 리포터 에세이를 수행하였다.
루시퍼라제를 태깅한 리보자임은 12번째 코돈이 Gly(ggt)에서 Val(gtt)로 단 염기 변이된 Kras G12V RNA를 타겟팅하는데, 이때 IGS를 변이된 단 염기와 G?U 염기쌍을 이루게 한 KRib1(GCAGCT) 1 셋트와 변이된 다음의 염기와 G?U 염기쌍을 이루게 한 KRib2(GACAGC) 2 셋트 두 가지의 리보자임을 디자인하였다.
이렇게 디자인된 1 셋트와 2 셋트 리보자임들은 pAV3.0 벡터에 AS100KRib/ AS300KRib의 1 셋트, 2 셋트의 구조체, pAV AS100 KRib1, pAV as300 KRib1의 구조체를 제작하였다. 클로닝된 리보자임들을 이용하여 Kras 야생형을 발현하는 293 세포에서 트랜스-접합 산물 분석을 수행하였다. 기질인 Kras G12V는 RNA 상태로, 리보자임은 DNA 플라스미드 상태로 코-트랜스펙션시켰다.
1. 세포 배양
293 세포는 10% FBS(fetal bovine serum), DMEM(Dulbecco's modified eagle medium; HyClone), 0.5% antibiotic/antimycotic solution이 첨가된 배지로 37℃, 5% CO₂배양기에서 배양하였으며, 3~4일마다 계대배양하였다.
이때, 1X PBS(8g NaCl, 0.2g KCl, 1.14g Na2HPO4, 0.2g KH4PO4/L)로 세포를 씻어준 후, 1x 트립신/EDTA(8.2g NaCl, 0.2g KCl, 1.14g Na2HPO₄, 0.2g KH2PO4, 0.029g Na2EDTA?dH20, 1g trypsin, pH 7.35/L) 1㎖를 처리하여 CO2 배양기에 1~3분간 두었다. 배지 9ml로 트립신을 불활성화시킨 뒤 1500×g로 2분 30초간 원심분리하여 상청액을 제거하고 배지로 재현탁하였다. 새 100mm 디쉬에 이중 1/3을 씨딩하여 배양하였다.
2.
DMRIE
-C에 의한
DNA
,
RNA
코트랜스펙션
클로닝으로 얻은 도 8A 및 도 9A 구조체들의 DNA와 Kras RNA를(상기 실시예 1의 3-2와 동일한 방법으로 제조) 293 세포에 코트랜스펙션시켰다. 환원된 혈청 배지(Reduced serum media) Opti-MEMⅠ 300㎕에 구조체 DNA 1㎍을 넣고 Opti-MEMⅠ300㎕에 DMRIE-C (Invitrogen) 3㎕를 넣은 튜브와 섞어 30분간 상온에서 반응시켰다.
Opti-MEMⅠ 600㎕와 DMRIE-C 3㎕에 Kras 야생형, Kras 12V RNA 4㎍을 넣고 섞어준 뒤 세척한 세포에 오버레이하였다. 4시간 동안 5% CO2 배양기에서 배양한 후 콤플리트 성장 배지(complete growth media)를 교체해 주었다.
3.
듀얼
루시퍼라제
분석
35mm 디쉬에 트랜스펙션한 각각 세포의 배지를 제거하고 1x PBS로 잘 닦아준다. 여기서 1x 수동 용해 버퍼(passive lysis buffer)를 200㎕ 넣고 상온에서 15분간 용해시킨 후 세포를 수확한 뒤 13000rpm으로 30초간 원심분리하여 상층액 만을 새 튜브에 옮겼다.
루미노미터 튜브에 LARII(Luciferase assay reagent II)을 100 ㎕를 넣고 여기에 세포 용해물 20㎕를 넣어 섞은 후 루미노미터로 읽었다. 다시 여기에 Stop & Glo reagent mix(Stop & Glo 20㎕ + Stop & Glo buffer 1㎖)를 100㎕넣고 섞은 후 루미노미터(TD+20/20)로 읽었다. 지연 시간은 3초, 통합 시간(integrate time)은 12초로 하고 감도 단계(sensitivity level)는 각각의 세포에 알맞게 60%로 설정하였다.
4.
RT
-
PCR
을 통한
세포내에서의
트랜스-
스플라이싱
산물의 발현 확인
4-1.
RNA
분리
세포에서 총 RNA를 분리하기 위해, 20mM EDTA가 첨가된 Trizol(Invitrogen)을 사용하였다. 매뉴얼을 기본으로 Trizol 1㎖을 처리한 후 5분간 배양하고 세포를 떼어낸 후 클로로포름을 200㎕ 첨가한 후 보르텍스를 하여 4℃ 원심분리기에서 10분간 원심분리하였다. 상층액을 새로운 튜브에 옮긴 후 EtOH 침전하였다. 그 후 DNase(Promega)를 처리하였다.
4-2.
역전사
상기 2에서 분리한 총 RNA 중 5㎕를 L-아르기닌아마이드가 첨가된 OligodT(Invitrigen)를 사용하여 총 RNA를 잘 섞은 다음 65℃에서 5분간 배양시킨 다음 4℃에서 5분간 보관하였다. 그 후 5x RT buffer, 0.5mM dNTP(BMS)와 MMLV 역전사효소 1U (Promega)와 DEPC dH20로 총 25㎕를 만들어 37℃에서 1시간 동안 반응시켰다. 이후, 95℃에서 5분간 반응 시켜 MMLV-RT의 활성을 완전히 제거 후 4℃에서 보관했다.
4-3.
PCR
RT 반응을 마친 cDNA는 서열번호 12,13의 프라이머(표 1 참조)를 사용하여 PCR 버퍼, dNTP, PCR 태그 중합효소, 5% DMSO를 넣고 (58℃ 30sec annealing/ 30 cycle) PCR하였다. 로딩 대조군으로 사용된 GAPDH는 프라이머를 사용하여 PCR 버퍼, dNTP, PCR 태그 중합효소를 넣고 PCR하였다.
4-4.
클로닝
(
RT
-
PCR
산물) 및 염기서열 분석
pGEM-T 이지 벡터 시스템(Promega)을 이용하여 TA-벡터에 RT-PCR 산물 클로닝을 하였으며, 서열번호 17의 프라이머(표 1 참조) 3.2 pmol을 사용하여 시퀀싱을 수행하였다.
4-4-1.
RT
-
PCR
산물 용출
DNA 겔 추출(Bio basic inc.)을 이용하여 상기 실시예 1의 2-1-2와 같은 방법으로 DNA를 용출을 했다.
4-4-2. 연결
TA-벡터와 RT-PCR 산물 용출한 DNA(trans-splicing product)를 상기 실시예 1의 2-1-3과 같은 방법으로 연결했다.
4-4-3. 형질전환
연결 산물을 대장균(JM109)에 상기 실시예 1의 2-1-4와 같은 방법으로 형질전환했다.
4-4-4. 시퀀싱을 통한 염기 서열 분석
콜로니를 접종하여 미니-준비로 얻어진 DNA를 EcoRI으로 잘라서 삽입물이 들어간 클론을 얻었다. 상기 실시예 1의 2-2와 같은 방법으로 시퀀싱을 통해 서열을 확인하였다.
5. 트랜스-
스플라이싱
특이성, 효율성 확인
도 8B는 트랜스-스플라이싱 리보자임에 의한 트랜스진의 리포터 에세이 결과를 나타낸 것이다.
도 8B를 참조하면, pAV AS100 KRib1을 Kras 야생형 또는 Kras G12V와 코-트랜스펙션한 경우 Kras 야생형을 넣어준 샘플에서는 루시퍼라제의 발현이 없었고 Kras G12V를 넣어준 샘플에서는 루시퍼라제가 적은 양이지만 발현된다는 것을 확인할 수 있었다.
그런데, pAV AS300 KRib1은 Kras 야생형을 넣어준 샘플 보다 Kras G12V를 넣어준 샘플에서 더 높은 루시퍼라제 값을 관찰할 수 있었으나, Kras G12V에 특이적으로 트랜스진이 발현되지 않음을 관찰하였다. 대조군으로 사용한 Mock이나 기질(Kras G12V)만 넣어준 샘플, 벡터 대조군의 샘플에서는 루시퍼라제가 발현되지 않았다.
이러한 결과로부터, AS100KRib1이 실험관 내에서 확인된 결과와 동일하게 세포에서도 Kras G12V에 특이적으로 트랜스진을 발현하는 것을 관찰할 수 있었다.
도 9B는 트랜스-스플라이싱 리보자임에 의한 트랜스진의 선별적 발현을 확인하기 위한 RT-PCR 분석 결과를 것이다
도 9B를 참조하면, Krib 1 셋트에서는 KRAS G12V RNA와 AS300KRib1-F,luc(레인 8)에서만 특이적으로 T/S 산물의 밴드를 볼 수가 있었다. 또한, 2 셋트에서는 KRAS G12V RNA와 AS100KRib2-F.luc(레인 6)에서만 T/S 산물 밴드를 특이적으로 볼 수 있었다.
이러한 특이적인 밴드가 트랜스-접합 산물인지 그리고 정확한 부위에서 스플라이싱이 일어났는지 알아보기 위해 트랜스-스플라이싱 밴드를 시퀀싱하여 확인하였다. 그 결과, 정확한 부위에서 트랜스-스플라이싱이 일어났음을 알 수 있었다. 이로부터 본 발명의 리보자임은 특이적으로 Kras 야생형과 Kras G12V인 단 염기 변이를 구별하여 트랜스-스플라이싱을 일으킨다는 것을 확인할 수 있었다.
실시예 5: In vivo 에서 트랜스- 스플라이싱 트랜스진 발현을 통한 세포 사멸 유도 확인
그룹 I 리보자임은 특이적인 부위를 인지하여 절단하는 것 이외에도 자신의 3' 엑손을 타겟 부위의 5' 엑손에 연결하는 역할을 함으로써 새로운 트래스진의 발현을 유도할 수가 있다. 따라서, Kras 리보자임의 3'엑손에 HSV-TK(thymidine kinase) 유전자를 삽입함으로써 전구 약물인 GCV을 처리하면 특이적으로 트랜스-스플라이싱이 된 새로운 트랜스진을 발현하는 세포에서 세포사를 유도하게 된다.
도 10A는 SW480 세포에서 TK 분석을 위한 구조체를 나타낸 것이다.
도 10A를 참조하면, TK 유전자를 도입한 리보자임이 세포사를 유도할 수 있는지 알아보기 위해 먼저 pAVQ 트랜스퍼 벡터에 TK 유전자가 삽입된 구조체를 제작하였다.
1.
Kras
-
리보자임
-
TK
구조체
클로닝
1-1. 벡터와 삽입물 준비
AS300KRib1-F.luc와 AS100KRib2-F.luc를 가지고 3' 엑손에 F.루시퍼라제 대신 HSV-TK의 TK 유전자를 삽입하기 위해 클로닝을 하였다.
pAVQ 벡터였던 리보자임 구조체들 3μg을 BglII(3U, Rocho)와 BstBI(3U, NEB) 제한효소를 이용하여 37℃ 3시간 자른 후 1% TAE 겔에 걸어 잘린 것을 확인 한 후, 플라스미드를 용출하였다.
TK 유전자를 얻기 위해 pCDNA3.1(+) CMV-TK의 구조체를 이용하여 TK 유전자를 서열번호 14, 15의 프라이머와 서열번호 16의 프라이머(표 1 참조)를 가지고 PCR[96℃-5'→(96℃-30", 58℃-1'30", 72℃-1'30") × 30 cycles → 72℃-7']로 증폭하였다.
이렇게 만들어진 TK DNA를 페놀 추출 및 에탄올 침전 후 3μg에 NruI(3U;Rocho)과 BstBI(3U;NEB) 제한효소를 이용하여 dH2O를 최종 50㎕까지 넣고 섞어 37℃에 3시간 동안 반응시킨 후 페놀 추출 및 에탄올 침전을 통하여 DNA를 정제하고 dH2O에 녹였다.
1-2. 연결
효소 처리된 pAVQ KRib1/KRib2 벡터와 삽입물(TK gene)을 각각 벡터:삽입물(1:3) 비율로 T₄리가아제(Roche)로 4℃에서 16시간 동안 반응시켰다.
1-3. 형질전환
연결한 벡터와 삽입물을 DH5α 수용성 세포에 42℃에서 1분 30초간 열 쇼크를 주어 형질전환을 한 후 5분간 얼음에 보관하고 1㎖의 새로운 LB를 첨가하여 37℃에서 30분간 배양시켰다. 그 후 카나마이신 아가 플레이트에 골고루 깐 다음 37℃ 배양기에 16시간 동안 배양시킨 후 콜로니를 암피실린이 포함된 5㎖ LB 배지에 접종하여 16시간 동안 37℃에서 흔들면서 배양하였다.
1-4. 시퀀싱을 통한
클로닝
확인
콜로니를 접종하여 미니-준비로 얻어진 DNA를 상기 실시예 1의 2-2와 같은 방법으로 서열번호 18의 프라이머(표 1 참조) 시퀀싱을 하여 확인하였다.
2. 자살유전자 활성 분석
실험을 하기 하루 전에 SW480(Kras G12V) 세포과 HT-29(wild type Kras) 세포를 35mm 디쉬에 2×105 세포를 배양하였다. 클로닝된 도 10A의 구조체와 함께 pEGFP-NI(250ng)을 코-트랜스펙션하였다.
2-1.
DNA
트랜스펙션
구조체와 함께 pEGFP-NI(250ng)을 welfec M Gold 트랜스펙션 시약(Welgen)을 사용하여 코-트랜스펙션하였다. PBS로 세포를 세척한 후 환원된 혈청 배지 OPTI-MEM 2㎖을 첨가하여 37℃ 5% CO2 배양기에서 배양하였다. 환원된 혈청 배지 OPTI-MEM 100㎕에 GFP 250ng와 리보자임 1μg을 섞은 후 골드 인핸서 4㎕를 OPTI-MEM 100ul에 희석시킨 후 DNA 혼합물에 섞어 15분간 배양했다.
이 DNA 혼합물과 OPTI-MEM 100㎕에 희석시킨 M gold 6㎕를 반응시킨 후 15분 배양했다. 배양 후 세포 위에 골고루 첨가해주었다. 그 후 37℃ 5% CO2 배양기에서 5시간 배양 후 콤플리트 메디아로 교체해 주었다.
2-2. 세포 생존율 관찰
24시간 후 200 μM GCV(Cymevene?, Roche)을 각 디쉬에 첨가해 주고 3일 동안 배양했다. AxioCam HR4 version 5.05.10. Zeiss 현미경을 이용하여 현광파장을 9 필터(488~509nm)로 하여 트랜스펙션된 세포에서 GFP의 발광 수를 측정하였다. 세포의 생존율(%)은 아래와 같은 공식을 이용하였다.
[수학식 1]
3. 세포 사멸 유도 확인
Kras 야생형을 내생적으로 발현하는 세포주 HT-29와 Kras G12V를 내생적으로 발현하는 세포주 SW480에 AS100KRib2-TK를 GFP 유전자와 함께 플라스미드 상태로 트랜스펙션하였다.
도 10B를 참조하면, HT-29 세포에서는 GCV를 처리하거나 미처리 시에 GFP의 발광에는 어떠한 변화도 않은 것으로 보아 세포의 생존력에 영향을 끼치지 않음을 볼 수가 있었다.
그러나, SW480 세포에서는 AS100KRib2-TK를 트랜스펙션한 세포에서 GCV를 처리하였을 때 GCV를 처리하지 않은 세포에 비해 GFP의 발광 수가 감소한 것을 볼 수가 있었다. 이는 AS100KRib2-TK가 특이적인 트랜스-스플라이싱에 의한 TK 유전자의 발현으로 전구약물인 GCV를 톡신으로 바꾸어 세포 생존율을 감소시켰음을 알 수가 있다.
이러한 결과로 부터, 본 발명의 리보자임은 야생형 Kras와 변이된 Kras를 특이적으로 구별하여 트랜스-스플라이싱하는 것을 확인할 수 있었다.
실시예
6:
아데노바이러스
전달 시스템을 위한 바이러스 구조체 제작
pAVQ 트랜스퍼 벡터에 TK 유전자가 삽입된 구조체인 pAVQ 트랜스퍼 벡터 리보자임을 pAdenovactor ΔE1/E3 백본 벡터와 함께 BJ5183 균주의 박테리아에 코-트랜스펙션시켜 트랜스퍼 벡터 리보자임과 pAdenovactor ΔE1/E3 백본 벡터를 상동 재조합시켰다. 이러한 방법으로 도 11B의 아데노바이러스 재조합 트랜스-스플라이싱 리보자임을 제작하였다.
1. 전달 시스템의 개발을 위한 재조합
아데노바이러스
벡터
클로닝된 도 10A의 pAdenovactor-CMV5-IRES-GFP 트랜스퍼 벡터(Qbiogene)들을 박테리아(BJ5183)에서 pAdenovactor ΔE1/E3 백본 벡터(Qbiogene)와 함께 코-트랜스포메이션시켜서 상동 재조합시켰다(도 11A 참조).
먼저, 트랜스퍼 벡터를 제한효소 PmeI으로 선형화시킨 후 페놀 추출 및 에탄올 침전을 통해 정제한 후 정량하여 1μg과 100ng의 pAdenovactor ΔE1/E3 백본 벡터를 BJ5183에 코-트랜스포메이션했다.
이렇게 얻은 도 11B의 BJ5183에서 상동 재조합된 재조합 벡터들은 PacI으로 선형화하여 페놀 추출 및 에탄올 침전을 통해 정제한 후 정량하여 293 세포에 트랜스펙션하였다. 293 세포에 트랜스펙션된 선형화된 재조합 벡터들은 3번의 플라크 정제를 통해 재조합 아데노바이러스를 만들었다.
293 세포를 이용하여 증폭시킨 재조합 바이러스들은 Vivapure? AdenoPACKTM 100 RT(Sartorius AG.)를 사용하여 정제하고 농축시켰다. 재조합 아데노바이러스들의 적가를 결정하기 위해 TCID50(tissue culture infectious dose for 50% of the cells) 방법을 이용하였다. 바이랄 스톡(Viral stock)들은 10%의 수크로스와 함께 -80℃에 보관하였다.
1-1. 정제 전에
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AS300KRib1
-
TK
의 분석
상동 재조합시킨 바이러스 재조합 리보자임들은 293 세포에 트랜스펙션함으로써 감염 능력을 가진 Ad-리보자임-TK 바이러스를 얻게 되었다. 이러한 바이러스들은 적가가 낮기 때문에 293 세포에 여러 번의 감염을 통해 고 적가를 얻어야한다. 고 적가의 바이러스를 얻게 되면 정제 과정을 통해 순수한 바이러스들을 정제하게 된다.
따라서, 바이러스들을 정제하기 전에 바이러스가 잘 생성되고 있는지, 올바른 Ad-리보자임들이 만들어지고 있는지 확인을 해야 한다. 또한, 여러 클론들 중 효율이 좋은 클론을 선별해야 한다. 이러한 확인을 위해 여러 번의 감염 중 저 적가에서 바이러스의 게놈을 얻어 PCR을 통해 리보자임의 3'엑손의 TK(thymidine kinase) 유전자와 pAdenovactor ΔE1/E3 백본 벡터의 LITR(left inverted terminal repeat)을 PCR을 통해 확인하였다(도 12A).
또한, 저 적가의 Ad-AS300KRib1-TK를 Kras G12V 변이된 세포주인 SW480 세포에 감염하여 Ad-AS300KRib1-TK의 발현을 확인하였다(도 12B). 바이러스는 클론들의 밴드를 비교하여 클론 18번을 가지고 바이러스 정제를 진행하였다.
1-2. 정제 전에
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AS100KRib2
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TK
의 분석
상기 1-1과 같은 방식으로 Ad-AS100KRib2-TK을 분석하였다(도 13). 바이러스의 게놈을 뽑아 PCR을 해본 결과 리보자임의 3'엑손의 TK(thymidine kinase) 유전자와 pAdenovactor ΔE1/E3 백본 벡터의 LITR(left inverted terminal repeat)을 확인하였다(도 13A). 그리고 SW480 세포에 감염하여 Ad-AS100KRib2-TK의 발현을 확인하였다(도 13B). 바이러스는 클론들의 밴드를 비교한 결과 클론 1번을 가지고 바이러스 정제를 진행하였다.
1-3. 정제 전에
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AS100KRib2
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LacZ
의 분석
상기 1-1과 같은 방식으로 Ad-AS100KRib2-LacZ를 분석하였다(도 14). 바이러스의 게놈을 뽑아 PCR을 해본 결과 리보자임의 3'엑손의 LacZ 유전자와 pAdenovactor ΔE1/E3 백본 벡터의 LITR(left inverted terminal repeat)을 확인하였다(도 14A). 그리고 SW480 세포에 감염하여 Ad-AS100KRib2-LacZ의 발현을 확인하였다(도 14B). 바이러스는 클론들의 밴드를 비교한 결과 클론 30번을 가지고 바이러스 정제를 진행하였다.
2. 재조합 바이러스의 확인
3번의 플라크 정제를 통해 재조합 아데노바이러스를 만드는 중간에 바이러스를 감염한 293 세포에서 바이러스가 제대로 만들어지고 있는지 확인하기 위해 바이러스 게놈 DNA를 추출하여 서열번호 19 내지 24의 프라이머(표 1 참조)를 가지고 PCR[96℃-5'→(96℃-30", 58℃-1'30", 72℃-1') × 30 cycles → 72℃-7']을 통해 증폭하였고, 2% TBE 아가로스 겔에 로딩하여 크기를 확인하였다. 또한, SW480 세포에 주입하여 상기 실시예 4의 5, 6과 같은 방법으로 RT-PCR을 통해 재조합 바이러스들을 확인하였다.
3. 바이러스 재조합들의 트랜스-
스플라이싱
확인
상기 1에서 정제된 Ad-리보자임들을 Kras G12V 변이된 세포주인 SW480에 50 M.O.I의 바이러스를 감염한 후, 48시간 후에 11-3,4와 같은 방법으로 서열번호 1과 14의 프라이머(표 1 참조)를 가지고 RT-PCR과 서열번호 17의 프라이머(표 1 참조)를 가지고 시퀀싱을 통해 트랜스-스플라이싱을 확인하였다.
4. 종양 세포에서
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리보자임을
이용한 T/S 산물 확인
이렇게 제작된 Ad-리보자임은 종양세포에서 특이적으로 트랜스-스플라이싱 일어나 T/S 산물을 생성하는지 확인하기 위해 Kras G12V 변이된 세포주인 SW480 세포에 Ad-리보자임을 감염하였다. 대조군으로 Ad-CMV-TK와 혼합하여 사용하였다. Ad-CMV-TK는 리보자임의 3'엑손의 TK 유전자와 같은 유전자로 리보자임이 없는 구조체이다. 혼합(Mix)은 RNA 추출 시 in vitro 상에서 트랜스-스플라이싱이 일어나지 않는다는 것을 보여주는 것으로 SW480 세포의 내생적인 돌연변이 Kras와 내생적인 Kras 야생형인 HT-29 세포에 Ad-AS100KRib2-TK를 감염한 것을 튜브 상에서 반응시킨 것이다.
도 15는 Kras G12V 변이된 SW480 세포에서 아데노바이러스 전달 시스템을 이용한 트랜스-접합 RNA 산물의 분석 결과를 나타낸 것이다.
도 15를 참조하면, 대조군들은 밴드가 없는 반면에 Ad-AS300KRib1-TK와 Ad0AS100KRib2-TK를 감염한 레인에서만 특이적으로 밴드가 보이는 것을 알 수가 있다.
이러한 특이적 밴드가 리보자임에 의한 특이적인 T/S 산물인지를 확인하기 위해서 시퀀싱을 통해 확인하였다. 그 결과, Ad-리보자임들이 특이적이고 올바르게 작용을 하고 있음을 알 수 있었다.
실시예 7: 아데노바이러스 Kras G12V -특이성 리보자임에 의한 Kras G12V 돌열변이된 암 세포에서 특이적인 세포 사멸
Kras G12V 변이된 암 세포에서 Kras G12V를 타겟팅하는 특이성 리보자임이 세포사를 유도하는지를 실험하기 위해 MTS 분석을 진행하였다. Kras G12V 변이된 세포주인 SW480 세포, CAPAN-1 세포와 Kras 야생형인 HT-29 세포에 Ad-리보자임들을 감염시키고 다음날 GCV를 처리함으로써 암 세포의 세포사를 관찰하였다.
1. 세포 배양
HT-29(colorectal adenocarcinoma)는 10% FBS(fetal bovine serum), DMEM(Dulbecco's modified eagle medium; HyClone), 0.5% antibiotic/antimycotic solution이 첨가된 배지로 SW480(colorectal adenocarcinoma)는 10% FBS(fetal bovine serum), RPMI 1640(Roswell Park Memorial Institute 1640; HyClone), 0.5% 항생/항진균 용액이 첨가된 배지로 CAPAN-1(pancreatic adenocarcinoma)은 10% FBS(fetal bovine serum), RPMI 1640(Roswell Park Memorial Institute 1640; HyClone), 0.5% antibiotic/antimycotic solution이 첨가된 배지로 37℃, 5% CO₂배양기에서 배양하였으며, 3~4일마다 계대배양하였다.
이때, 1X PBS(8g NaCl, 0.2g KCl, 1.14g Na2HPO4, 0.2g KH4PO4/L)로 세포를 씻어준 후, 1x 트립신/EDTA(8.2g NaCl, 0.2g KCl, 1.14g Na2HPO₄, 0.2g KH2PO4, 0.029g Na2EDTA?dH20, 1g trypsin, pH 7.35/L) 1㎖를 처리하여 CO2 배양기에 1~3분간 두었다. 배지 9ml로 트립신을 불활성화시킨 뒤 1500×g로 2분 30초간 원심분리하여 상청액을 제거하고 배지로 재현탁하였다. 새 100mm 디쉬에 이중 1/3을 씨딩하여 배양하였다.
2. 세포 증식 분석
세포 증식(MTS) 분석법은 CellTiter 96Aqueous one solution reagent(MTS, Promega)의 매뉴얼을 기초로 하여 수행하였다.
96-웰 플레이트에 세포를 3×103 cells/well(0.1 ㎖)로 씨딩하였다. 37℃ 5% CO2 배양기에서 밤새도록 배양한 후, 각 웰에 다양한 M.O.I의 바이러스를 감염시켰다. 16시간 후에 각 플레이트 100 uM GCV(Cymevene?, Roche)을 처리하여 5일 동안 배양시켰다. Opti-MEM 100ul에 CellTiter 96Aqueous one solution reagent(MTS, Promega)를 20% 추가하여 각 웰에 첨가한 후 1~4시간 동안 37℃ 5% CO2 배양기에서 배양한 후 세포 생존율을 490nm에서 흡수도를 측정하였다.
3. Kras G12V 돌열변이된 암 세포에서 특이적인 세포 사멸 확인
도 16A는 SW480 세포에서 MTS 분석 결과를 나타낸 것이다.
도 16A의 왼쪽 그래프를 참조하면, 동일하게 GCV를 100uM로 처리하였을 때 Ad-리보자임들은 M.O.I 의존적으로 감염하였다. 도 16A의 오른쪽 그래프를 참조하면, 동일하게 Ad-리보자임들을 150 M.O.I로 감염하고 GCV 복용별로 처리를 하였다.
Ad-CMV-TK과 이미 보고된 논문에서 특이성과 효율이 인정된 Ad-CMV-리보자임-TK가 양성 대조군으로 사용되었으며, Ad-CMV-LacZ는 음성 대조군으로 사용되었다.
그 결과, 양성 대조군과 음성 대조군과 비교하였을 때 Ad-리보자임들은 특이적인 트랜스-스플라이싱과 트랜스진이 유도되어 세포사가 일어남을 볼 수 있었다.
도 16B 및 16C는 각각 CAPAN-1 세포, HT-29 세포에서 MTS 분석 결과를 나타낸 것이다.
도 16B의 경우도 동일하게 GCV를 100uM로 처리하고, Ad-리보자임들은 M.O.I 의존적으로 감염한 결과, Ad-리보자임들에서 특이적인 세포사가 일어남을 볼 수 있었다. 그러나, 도 16C 야생형 Kras인 HT-29 세포에서는 양성 바이러스들을 제외하고는 세포사가 일어나지 않음을 볼 수 있었다.
이러한 결과로부터 Ad-리보자임들이 특이적으로 변이된 Kras G12V를 인지하여 트랜스-스플라이싱을 해서 트랜스진을 유도했음을 알 수가 있다.
실시예
8:
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리보자임에
의한 이종이식 모델에서의 종양 퇴행 확인
1. 실험동물
특이적으로 병원체 부재 조건인 4주에서 5주의 수컷 BALB/cAnNCrI 누드 마우스(Orient Bio Inc.)를 실험을 위해 사용하였다. 이종이식 마우스 모델을 만들기 위해, 먼저 마우스 8마리에 CAPAN-1(1×107 cells)을 복강 내 주사한 후 3주 후에 종양 생성시킨 다음 아데노바이러스 리보자임들을 이틀 간격으로 3회(1×109 PFU) 감염시켰다. GCV(50mg/kg)는 바이러스를 처음 감염시킨 날 다음날부터 하루에 2번씩 10일 동안 복강을 통해 처리하였다. 그리고 야생형의 Kras 피하 종양 모델을 만들기 위해서 HT-29을 이용해서 종양 모델을 제작하였다. 수컷 BALB/c 누드 마우스 7마리에 1×107 cells을 피하 주사하여 2주 후에 종양을 생성시켰다. Ad-리보자임은 정맥 내로 이틀 간격으로 3번, 1×109 cell을 처리하였다. GCV(50mg/kg)는 10일 동안 2번씩 복강으로 처리한 후 관찰하였다.
2. 이종이식 모델에서의 종양 퇴행 확인
In vivo에서 Ad-리보자임의 특이성과 효율성을 확인하기 위해서 MTS 분석을 통해 확인된 Ad-리보자임들을 인간 종양이 이식된 이종이식 마우스 모델에 감염했다.
Kras G12V 변이된 CAPAN-1을 i,p로 주사하여 종양을 생성하였고, Ad-리보자임을 처리했다(도 17A 참조). 그 후 GCV를 처리하여 Ad-리보자임에 의해 특이적으로 종양이 퇴행(regression)되는지를 확인했다. 음성 바이러스로는 Ad-CMV-LacZ를 감염했고 양성 바이러스로는 Ad-CMV-TK를 감염했다.
Ad-CMV-LacZ에 비해 Ad-CMV-TK와 Ad-AS100KRib2-TK는 유사하게 Kras G12V 변이된 종양에서 종양 퇴행이 일어남을 알 수가 있었다(P=0.467). 또한, 야생형 Kras를 발현하는 HT-29로 피하의 종양 마우스 모델을 만들었다.
도 17B는 H&E 염색하여 괴사를 제외한 종양을 관찰하여 종양의 중량을 그래프로 나타낸 것이다.
도 17B를 참조하면, 종양의 중량은 음성 대조군과 비슷하게 Ad-리보자임들도 감소하지 않은 것을 알 수 있다(P=0.5137). 그러나, 양성 바이러스인 Ad-CMV-TK인 경우에는 종양의 중량이 감소한 것을 볼 수 있었다. 또한, Kras G12V 변이된 종양과 야생형 Kras 종양의 결과를 비교해 보면 Ad-리보자임은 Kras G12V를 특이적으로 인지하여 작용한다는 것을 알 수가 있다.
<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, DANKOOK UNIVERSITY
<120> TRANS-SPLICING GROUP I RIBOZYME SPECIFIC FOR KRAS G12V RNA
<160> 44
<170> KopatentIn 1.71
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Kras wild primer
<400> 1
gttggagctg gtggcgtagg c 21
<210> 2
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> Kras 5' BamHI primer
<400> 2
taatacgact cacta 15
<210> 3
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> kras rib T7-5' primer
<400> 3
gggtaatacg actcactata ggaggtaaaa gttatcaggc atgc 44
<210> 4
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> RY-PCR primer
<400> 4
gggtaatacg actcactata gacagcaaaa gttatcaggc atgc 44
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> RY-RT primer
<400> 5
tgtaaaacga cggccagtg 19
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> kras p1p10-rib1-5 primer
<400> 6
cgatgatcac gaagacgcca aaaacata 28
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> kras p1p10-rib2-5 primer
<400> 7
cgacatgatc acgaagacgc caaaaacata 30
<210> 8
<211> 37
<212> DNA
<213> Artificial Sequence
<220>
<223> krib 3' primer
<400> 8
aaggaaaaaa gcggccgctt attacaattt ggacttt 37
<210> 9
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> kras as 5' primer
<400> 9
gggaagcttg caagagtgcc ttgacga 27
<210> 10
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> kras as 3' primer
<400> 10
gggaaggctt ccatcaatta ctacttgc 28
<210> 11
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> kras as300 5' priemr
<400> 11
gggtaatacg actcactata gggctactag gaccataggt a 41
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> kras t/s BamHI5' primer
<400> 12
cgcggatcca ctgaatataa acttgtggta 30
<210> 13
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> cell t/s luci 3' nest primer
<400> 13
cccaagcttg cccaacaccg gcataaag 28
<210> 14
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> TK 5' NruI 1set primer
<400> 14
gtgatcatcg cgatgatcac gcttcgtacc cctgccat 38
<210> 15
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> TK 5' NruI 2setI primer
<400> 15
gtgatcatcg cgacatgatc acgcttcgta cccctgccat 40
<210> 16
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' TK BstBI primer
<400> 16
aaggaaaaaa ttcgaattag cctcccccat 30
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> T7-R-Primer
<400> 17
taatacgact cactataggg 20
<210> 18
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> IGS rev seq
<400> 18
cctttcccgc aatttgacgg tctt 24
<210> 19
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> 5' TK stable primer
<400> 19
cgggaattcg tggtaatgac aagc 24
<210> 20
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' TK stable primer
<400> 20
cggggatcca tccaggataa agacgtgcat ggg 33
<210> 21
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> 5' LITR EcoRI primer
<400> 21
ggaattctgg agtttgtgac gtggcg 26
<210> 22
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 3' LITR XbaI primer
<400> 22
gctctagatg gccaaatctt actcggttac gc 32
<210> 23
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> 5' viral LacZ primer
<400> 23
ggaattcatg gtcgttttac aacgtcgtg 29
<210> 24
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> cancer.lacZ-3 primer
<400> 24
gggaagcttc ggattgaccg taatggga 28
<210> 25
<211> 513
<212> RNA
<213> Artificial Sequence
<220>
<223> AS100KRib1
<400> 25
aagcuuccau caauuacuac uugcuuccug uaggaauccu cuauuguugg aucauauucg 60
uccacaaaau gauucugaau uagcuguauc gucaaggcac ucuugcaagc uucauuauca 120
gcagcuaaaa guuaucaggc augcaccugg uagcuagucu uuaaaccaau agauugcauc 180
gguuuaaaag gcaagaccgu caaauugcgg gaaagggguc aacagccguu caguaccaag 240
ucucagggga aacuuugaga uggccuugca aaggguaugg uaauaagcug acggacaugg 300
uccuaaccac gcagccaagu ccuaagucaa cagaucuucu guugauaugg augcaguuca 360
cagacuaaau gucggucggg gaagauguau ucuucucaua agauauaguc ggaccucucc 420
uuaaugggag cuagcggaug aagugaugca acacuggagc cgcugggaac uaauuuguau 480
gcgaaaguau auugauuagu uuuggaguac ucg 513
<210> 26
<211> 713
<212> RNA
<213> Artificial Sequence
<220>
<223> AS300KRib1
<400> 26
aagcuucuac uaggaccaua gguacaucuu cagaguccuu aacucuuuua auuuguucuc 60
uauaauggug aauaucuuca aaugauuuag uauuauuuau ggcaaauaca caaagaaagc 120
ccuccccagu ccucauguac uggucccuca uugcacugua cuccucuuga ccugcugugu 180
cgagaauauc caagagacag guuucuccau caauuacuac uugcuuccug uaggaauccu 240
cuauuguugg aucauauucg uccacaaaau gauucugaau uagcuguauc gucaaggcac 300
ucuugcaagc uucauuauca gcagcuaaaa guuaucaggc augcaccugg uagcuagucu 360
uuaaaccaau agauugcauc gguuuaaaag gcaagaccgu caaauugcgg gaaagggguc 420
aacagccguu caguaccaag ucucagggga aacuuugaga uggccuugca aaggguaugg 480
uaauaagcug acggacaugg uccuaaccac gcagccaagu ccuaagucaa cagaucuucu 540
guugauaugg augcaguuca cagacuaaau gucggucggg gaagauguau ucuucucaua 600
agauauaguc ggaccucucc uuaaugggag cuagcggaug aagugaugca acacuggagc 660
cgcugggaac uaauuuguau gcgaaaguau auugauuagu uuuggaguac ucg 713
<210> 27
<211> 515
<212> RNA
<213> Artificial Sequence
<220>
<223> AS100KRib2
<400> 27
aagcuuccau caauuacuac uugcuuccug uaggaauccu cuauuguugg aucauauucg 60
uccacaaaau gauucugaau uagcuguauc gucaaggcac ucuugcaagc uucaucaugg 120
ccgacagcaa aaguuaucag gcaugcaccu gguagcuagu cuuuaaacca auagauugca 180
ucgguuuaaa aggcaagacc gucaaauugc gggaaagggg ucaacagccg uucaguacca 240
agucucaggg gaaacuuuga gauggccuug caaaggguau gguaauaagc ugacggacau 300
gguccuaacc acgcagccaa guccuaaguc aacagaucuu cuguugauau ggaugcaguu 360
cacagacuaa augucggucg gggaagaugu auucuucuca uaagauauag ucggaccucu 420
ccuuaauggg agcuagcgga ugaagugaug caacacugga gccgcuggga acuaauuugu 480
augcgaaagu auauugauua guuuuggagu acucg 515
<210> 28
<211> 715
<212> RNA
<213> Artificial Sequence
<220>
<223> AS300KRib2
<400> 28
aagcuucuac uaggaccaua gguacaucuu cagaguccuu aacucuuuua auuuguucuc 60
uauaauggug aauaucuuca aaugauuuag uauuauuuau ggcaaauaca caaagaaagc 120
ccuccccagu ccucauguac uggucccuca uugcacugua cuccucuuga ccugcugugu 180
cgagaauauc caagagacag guuucuccau caauuacuac uugcuuccug uaggaauccu 240
cuauuguugg aucauauucg uccacaaaau gauucugaau uagcuguauc gucaaggcac 300
ucuugcaagc uucaucaugg ccgacagcaa aaguuaucag gcaugcaccu gguagcuagu 360
cuuuaaacca auagauugca ucgguuuaaa aggcaagacc gucaaauugc gggaaagggg 420
ucaacagccg uucaguacca agucucaggg gaaacuuuga gauggccuug caaaggguau 480
gguaauaagc ugacggacau gguccuaacc acgcagccaa guccuaaguc aacagaucuu 540
cuguugauau ggaugcaguu cacagacuaa augucggucg gggaagaugu auucuucuca 600
uaagauauag ucggaccucu ccuuaauggg agcuagcgga ugaagugaug caacacugga 660
gccgcuggga acuaauuugu augcgaaagu auauugauua guuuuggagu acucg 715
<210> 29
<211> 2184
<212> DNA
<213> Artificial Sequence
<220>
<223> AS100KRib1-F.luci
<400> 29
aagcttccat caattactac ttgcttcctg taggaatcct ctattgttgg atcatattcg 60
tccacaaaat gattctgaat tagctgtatc gtcaaggcac tcttgcaagc ttcattatca 120
gcagctaaaa gttatcaggc atgcacctgg tagctagtct ttaaaccaat agattgcatc 180
ggtttaaaag gcaagaccgt caaattgcgg gaaaggggtc aacagccgtt cagtaccaag 240
tctcagggga aactttgaga tggccttgca aagggtatgg taataagctg acggacatgg 300
tcctaaccac gcagccaagt cctaagtcaa cagatcttct gttgatatgg atgcagttca 360
cagactaaat gtcggtcggg gaagatgtat tcttctcata agatatagtc ggacctctcc 420
ttaatgggag ctagcggatg aagtgatgca acactggagc cgctgggaac taatttgtat 480
gcgaaagtat attgattagt tttggagtac tcgcgatgat cacgaagacg ccaaaaacat 540
aaagaaaggc ccggcgccat tctatcctct agaggatgga accgctggag agcaactgca 600
taaggctatg aagagatacg ccctggttcc tggaacaatt gcttttacag atgcacatat 660
cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc gttcggttgg cagaagctat 720
gaaacgatat gggctgaata caaatcacag aatcgtcgta tgcagtgaaa actctcttca 780
attctttatg ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga 840
catttataat gaacgtgaat tgctcaacag tatgaacatt tcgcagccta ccgtagtgtt 900
tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa aaaaaattac caataatcca 960
gaaaattatt atcatggatt ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt 1020
cgtcacatct catctacctc ccggttttaa tgaatacgat tttgtaccag agtcctttga 1080
tcgtgacaaa acaattgcac tgataatgaa ttcctctgga tctactgggt tacctaaggg 1140
tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg catgccagag atcctatttt 1200
tggcaatcaa atcattccgg atactgcgat tttaagtgtt gttccattcc atcacggttt 1260
tggaatgttt actacactcg gatatttgat atgtggattt cgagtcgtct taatgtatag 1320
atttgaagaa gagctgtttt tacgatccct tcaggattac aaaattcaaa gtgcgttgct 1380
agtaccaacc ctattttcat tcttcgccaa aagcactctg attgacaaat acgatttatc 1440
taatttacac gaaattgctt ctgggggcgc acctctttcg aaagaagtcg gggaagcggt 1500
tgcaaaacgc ttccatcttc cagggatacg acaaggatat gggctcactg agactacatc 1560
agctattctg attacacccg agggggatga taaaccgggc gcggtcggta aagttgttcc 1620
attttttgaa gcgaaggttg tggatctgga taccgggaaa acgctgggcg ttaatcagag 1680
aggcgaatta tgtgtcagag gacctatgat tatgtccggt tatgtaaaca atccggaagc 1740
gaccaacgcc ttgattgaca aggatggatg gctacattct ggagacatag cttactggga 1800
cgaagacgaa cacttcttca tagttgaccg cttgaagtct ttaattaaat acaaaggata 1860
tcaggtggcc cccgctgaat tggaatcgat attgttacaa caccccaaca tcttcgacgc 1920
gggcgtggca ggtcttcccg acgatgacgc cggtgaactt cccgccgccg ttgttgtttt 1980
ggagcacgga aagacgatga cggaaaaaga gatcgtggat tacgtggcca gtcaagtaac 2040
aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac gaagtaccga aaggtcttac 2100
cggaaaactc gacgcaagaa aaatcagaga gatcctcata aaggccaaga agggcggaaa 2160
gtccaaattg taataagcgg ccgc 2184
<210> 30
<211> 2384
<212> DNA
<213> Artificial Sequence
<220>
<223> AS300KRib1-F.luci
<400> 30
aagcttctac taggaccata ggtacatctt cagagtcctt aactctttta atttgttctc 60
tataatggtg aatatcttca aatgatttag tattatttat ggcaaataca caaagaaagc 120
cctccccagt cctcatgtac tggtccctca ttgcactgta ctcctcttga cctgctgtgt 180
cgagaatatc caagagacag gtttctccat caattactac ttgcttcctg taggaatcct 240
ctattgttgg atcatattcg tccacaaaat gattctgaat tagctgtatc gtcaaggcac 300
tcttgcaagc ttcattatca gcagctaaaa gttatcaggc atgcacctgg tagctagtct 360
ttaaaccaat agattgcatc ggtttaaaag gcaagaccgt caaattgcgg gaaaggggtc 420
aacagccgtt cagtaccaag tctcagggga aactttgaga tggccttgca aagggtatgg 480
taataagctg acggacatgg tcctaaccac gcagccaagt cctaagtcaa cagatcttct 540
gttgatatgg atgcagttca cagactaaat gtcggtcggg gaagatgtat tcttctcata 600
agatatagtc ggacctctcc ttaatgggag ctagcggatg aagtgatgca acactggagc 660
cgctgggaac taatttgtat gcgaaagtat attgattagt tttggagtac tcgcgatgat 720
cacgaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatcctct agaggatgga 780
accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 840
gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 900
gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 960
tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 1020
gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgaacatt 1080
tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 1140
aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga ttaccaggga 1200
tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 1260
tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa ttcctctgga 1320
tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg 1380
catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 1440
gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 1500
cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac 1560
aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg 1620
attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc acctctttcg 1680
aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg acaaggatat 1740
gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1800
gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1860
acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 1920
tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1980
ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg cttgaagtct 2040
ttaattaaat acaaaggata tcaggtggcc cccgctgaat tggaatcgat attgttacaa 2100
caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt 2160
cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 2220
tacgtggcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 2280
gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 2340
aaggccaaga agggcggaaa gtccaaattg taataagcgg ccgc 2384
<210> 31
<211> 2188
<212> DNA
<213> Artificial Sequence
<220>
<223> AS100KRib2-F.luci
<400> 31
aagcttccat caattactac ttgcttcctg taggaatcct ctattgttgg atcatattcg 60
tccacaaaat gattctgaat tagctgtatc gtcaaggcac tcttgcaagc ttcatcatgg 120
ccgacagcaa aagttatcag gcatgcacct ggtagctagt ctttaaacca atagattgca 180
tcggtttaaa aggcaagacc gtcaaattgc gggaaagggg tcaacagccg ttcagtacca 240
agtctcaggg gaaactttga gatggccttg caaagggtat ggtaataagc tgacggacat 300
ggtcctaacc acgcagccaa gtcctaagtc aacagatctt ctgttgatat ggatgcagtt 360
cacagactaa atgtcggtcg gggaagatgt attcttctca taagatatag tcggacctct 420
ccttaatggg agctagcgga tgaagtgatg caacactgga gccgctggga actaatttgt 480
atgcgaaagt atattgatta gttttggagt actcgcgaca tgatcacgaa gacgccaaaa 540
acataaagaa aggcccggcg ccattctatc ctctagagga tggaaccgct ggagagcaac 600
tgcataaggc tatgaagaga tacgccctgg ttcctggaac aattgctttt acagatgcac 660
atatcgaggt gaacatcacg tacgcggaat acttcgaaat gtccgttcgg ttggcagaag 720
ctatgaaacg atatgggctg aatacaaatc acagaatcgt cgtatgcagt gaaaactctc 780
ttcaattctt tatgccggtg ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga 840
acgacattta taatgaacgt gaattgctca acagtatgaa catttcgcag cctaccgtag 900
tgtttgtttc caaaaagggg ttgcaaaaaa ttttgaacgt gcaaaaaaaa ttaccaataa 960
tccagaaaat tattatcatg gattctaaaa cggattacca gggatttcag tcgatgtaca 1020
cgttcgtcac atctcatcta cctcccggtt ttaatgaata cgattttgta ccagagtcct 1080
ttgatcgtga caaaacaatt gcactgataa tgaattcctc tggatctact gggttaccta 1140
agggtgtggc ccttccgcat agaactgcct gcgtcagatt ctcgcatgcc agagatccta 1200
tttttggcaa tcaaatcatt ccggatactg cgattttaag tgttgttcca ttccatcacg 1260
gttttggaat gtttactaca ctcggatatt tgatatgtgg atttcgagtc gtcttaatgt 1320
atagatttga agaagagctg tttttacgat cccttcagga ttacaaaatt caaagtgcgt 1380
tgctagtacc aaccctattt tcattcttcg ccaaaagcac tctgattgac aaatacgatt 1440
tatctaattt acacgaaatt gcttctgggg gcgcacctct ttcgaaagaa gtcggggaag 1500
cggttgcaaa acgcttccat cttccaggga tacgacaagg atatgggctc actgagacta 1560
catcagctat tctgattaca cccgaggggg atgataaacc gggcgcggtc ggtaaagttg 1620
ttccattttt tgaagcgaag gttgtggatc tggataccgg gaaaacgctg ggcgttaatc 1680
agagaggcga attatgtgtc agaggaccta tgattatgtc cggttatgta aacaatccgg 1740
aagcgaccaa cgccttgatt gacaaggatg gatggctaca ttctggagac atagcttact 1800
gggacgaaga cgaacacttc ttcatagttg accgcttgaa gtctttaatt aaatacaaag 1860
gatatcaggt ggcccccgct gaattggaat cgatattgtt acaacacccc aacatcttcg 1920
acgcgggcgt ggcaggtctt cccgacgatg acgccggtga acttcccgcc gccgttgttg 1980
ttttggagca cggaaagacg atgacggaaa aagagatcgt ggattacgtg gccagtcaag 2040
taacaaccgc gaaaaagttg cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc 2100
ttaccggaaa actcgacgca agaaaaatca gagagatcct cataaaggcc aagaagggcg 2160
gaaagtccaa attgtaataa gcggccgc 2188
<210> 32
<211> 2377
<212> DNA
<213> Artificial Sequence
<220>
<223> AS300KRib2-F.luci
<400> 32
aagcttctac taggaccata ggtacatctt cagagtcctt aactctttta atttgttctc 60
tataatggtg aatatcttca aatgatttag tattatttat ggcaaataca caaagaaagc 120
cctccccagt cctcatgtac tggtccctca ttgcactgta ctcctcttga cctgctgtgt 180
cgagaatatc caagagacag gtttctccat caattactac ttgcttcctg taggaatcct 240
ctattgttgg atcatattcg tccacaaaat gattctgaat tagctgtatc gtcaaggcac 300
tcttgcaagc ttcatcatgg ccgacagcaa aagttatcag gcatgcacct ggtagctagt 360
ctttaaacca atagattgca tcggtttaaa aggcaagacc gtcaaattgc gggaaagggg 420
tcaacagccg ttcagtacca agtctcaggg gaaactttga gatggccttg caaagggtat 480
ggtaataagc tgacggacat ggtcctaacc acgcagccaa gtcctaagtc aacagatctt 540
ctgttgatat ggatgcagtt cacagactaa atgtcggtcg gggaagatgt attcttctca 600
taagatatag tcggacctct ccttaatggg agctagcgga tgaagtgatg caacactgga 660
gccgctggga actaatttgt atgcgaaagt atattgatta gttttggagt actcgcgaca 720
tgatcacgaa gacgccaaaa acataaagaa aggcccggcg ccattctatc ctctagagga 780
tggaaccgct ggagagcaac tgcataaggc tatgaagaga tacgccctgg ttcctggaac 840
aattgctttt acagatgcac atatcgaggt gaacatcacg tacgcggaat acttcgaaat 900
gtccgttcgg ttggcagaag ctatgaaacg atatgggctg aatacaaatc acagaatcgt 960
cgtatgcagt gaaaactctc ttcaattctt tatgccggtg ttgggcgcgt tatttatcgg 1020
agttgcagtt gcgcccgcga acgacattta taatgaacgt gaattgctca acagtatgaa 1080
catttcgcag cctaccgtag tgtttgtttc caaaaagggg ttgcaaaaaa ttttgaacgt 1140
gcaaaaaaaa ttaccaataa tccagaaaat tattatcatg gattctaaaa cggattacca 1200
gggatttcag tcgatgtaca cgttcgtcac atctcatcta cctcccggtt ttaatgaata 1260
cgattttgta ccagagtcct ttgatcgtga caaaacaatt gcactgataa tgaattcctc 1320
tggatctact gggttaccta agggtgtggc ccttccgcat agaactgcct gcgtcagatt 1380
ctcgcatgcc agagatccta tttttggcaa tcaaatcatt ccggatactg cgattttaag 1440
tgttgttcca ttccatcacg gttttggaat gtttactaca ctcggatatt tgatatgtgg 1500
atttcgagtc gtcttaatgt atagatttga agaagagctg tttttacgat cccttcagga 1560
ttacaaaatt caaagtgcgt tgctagtacc aaccctattt tcattcttcg ccaaaagcac 1620
tctgattgac aaatacgatt tatctaattt acacgaaatt gcttctgggg gcgcacctct 1680
ttcgaaagaa gtcggggaag cggttgcaaa acgcttccat cttccaggga tacgacaagg 1740
atatgggctc actgagacta catcagctat tctgattaca cccgaggggg atgataaacc 1800
gggcgcggtc ggtaaagttg ttccattttt tgaagcgaag gttgtggatc tggataccgg 1860
gaaaacgctg ggcgttaatc agagaggcga attatgtgtc agaggaccta tgattatgtc 1920
cggttatgta aacaatccgg aagcgaccaa cgccttgatt gacaaggatg gatggctaca 1980
ttctggagac atagcttact gggacgaaga cgaacacttc ttcatagttg accgcttgaa 2040
gtctttaatt aaatacaaag gatatcaggt ggcccccgct gaattggaat cgatattgtt 2100
acaacacccc aacatcttcg acgcgggcgt ggcaggtctt cccgacgatg acgccggtga 2160
acttcccgcc gccgttgttg ttttggagca cggaaagacg atgacggaaa aagagatcgt 2220
ggattacgtg gccagtcaag taacaaccgc gaaaaagttg cgcggaggag ttgtgtttgt 2280
ggacgaagta ccgaaaggtc ttaccggaaa actcgacgca agaaaaatca gagagatcct 2340
cataaaggcc aagaagggcg gaaagtccaa attgtaa 2377
<210> 33
<211> 1650
<212> DNA
<213> Artificial Sequence
<220>
<223> AS100KRib1-TK
<400> 33
aagcttccat caattactac ttgcttcctg taggaatcct ctattgttgg atcatattcg 60
tccacaaaat gattctgaat tagctgtatc gtcaaggcac tcttgcaagc ttcattatca 120
gcagctaaaa gttatcaggc atgcacctgg tagctagtct ttaaaccaat agattgcatc 180
ggtttaaaag gcaagaccgt caaattgcgg gaaaggggtc aacagccgtt cagtaccaag 240
tctcagggga aactttgaga tggccttgca aagggtatgg taataagctg acggacatgg 300
tcctaaccac gcagccaagt cctaagtcaa cagatcttct gttgatatgg atgcagttca 360
cagactaaat gtcggtcggg gaagatgtat tcttctcata agatatagtc ggacctctcc 420
ttaatgggag ctagcggatg aagtgatgca acactggagc cgctgggaac taatttgtat 480
gcgaaagtat attgattagt tttggagtac tcgcgatgat cacgcttcgt acccctgcca 540
tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc ggccatagca accgacgtac 600
ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc cgcctggagc agaaaatgcc 660
cacgctactg cgggtttata tagacggtcc tcacgggatg gggaaaacca ccaccacgca 720
actgctggtg gccctgggtt cgcgcgacga tatcgtctac gtacccgagc cgatgactta 780
ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc tacaccacac aacaccgcct 840
cgaccagggt gagatatcgg ccggggacgc ggcggtggta atgacaagcg cccagataac 900
aatgggcatg ccttatgccg tgaccgacgc cgttctggct cctcatgtcg ggggggaggc 960
tgggagttca catgccccgc ccccggccct caccctcatc ttcgaccgcc atcccatcgc 1020
cgccctcctg tgctacccgg ccgcgcgata ccttatgggc agcatgaccc cccaggccgt 1080
gctggcgttc gtggccctca tcccgccgac cttgcccggc acaaacatcg tgttgggggc 1140
ccttccggag gacagacaca tcgaccgcct ggccaaacgc cagcgccccg gcgagcggct 1200
tgacctggct atgctggccg cgattcgccg cgtttacggg ctgcttgcca atacggtgcg 1260
gtatctgcag ggcggcgggt cgtggtggga ggattgggga cagctttcgg ggacggccgt 1320
gccgccccag ggtgccgagc cccagagcaa cgcgggccca cgaccccata tcggggacac 1380
gttatttacc ctgtttcggg cccccgagtt gctggccccc aacggcgacc tgtataacgt 1440
gtttgcctgg gccttggacg tcttggccaa acgcctccgt cccatgcacg tctttatcct 1500
ggattacgac caatcgcccg ccggctgccg ggacgccctg ctgcaactta cctccgggat 1560
ggtccagacc cacgtcacca ccccaggctc cataccgacg atctgcgacc tggcgcgcac 1620
gtttgcccgg gagatggggg aggctaataa 1650
<210> 34
<211> 1850
<212> DNA
<213> Artificial Sequence
<220>
<223> AS300KRib1-TK
<400> 34
aagcttctac taggaccata ggtacatctt cagagtcctt aactctttta atttgttctc 60
tataatggtg aatatcttca aatgatttag tattatttat ggcaaataca caaagaaagc 120
cctccccagt cctcatgtac tggtccctca ttgcactgta ctcctcttga cctgctgtgt 180
cgagaatatc caagagacag gtttctccat caattactac ttgcttcctg taggaatcct 240
ctattgttgg atcatattcg tccacaaaat gattctgaat tagctgtatc gtcaaggcac 300
tcttgcaagc ttcattatca gcagctaaaa gttatcaggc atgcacctgg tagctagtct 360
ttaaaccaat agattgcatc ggtttaaaag gcaagaccgt caaattgcgg gaaaggggtc 420
aacagccgtt cagtaccaag tctcagggga aactttgaga tggccttgca aagggtatgg 480
taataagctg acggacatgg tcctaaccac gcagccaagt cctaagtcaa cagatcttct 540
gttgatatgg atgcagttca cagactaaat gtcggtcggg gaagatgtat tcttctcata 600
agatatagtc ggacctctcc ttaatgggag ctagcggatg aagtgatgca acactggagc 660
cgctgggaac taatttgtat gcgaaagtat attgattagt tttggagtac tcgcgatgat 720
cacgcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 780
ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 840
cgcctggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc tcacgggatg 900
gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 960
gtacccgagc cgatgactta ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc 1020
tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 1080
atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 1140
cctcatgtcg ggggggaggc tgggagttca catgccccgc ccccggccct caccctcatc 1200
ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata ccttatgggc 1260
agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 1320
acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 1380
cagcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 1440
ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggtggga ggattgggga 1500
cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 1560
cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggccccc 1620
aacggcgacc tgtataacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 1680
cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 1740
ctgcaactta cctccgggat ggtccagacc cacgtcacca ccccaggctc cataccgacg 1800
atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaataa 1850
<210> 35
<211> 1654
<212> DNA
<213> Artificial Sequence
<220>
<223> AS100KRib2-TK
<400> 35
aagcttccat caattactac ttgcttcctg taggaatcct ctattgttgg atcatattcg 60
tccacaaaat gattctgaat tagctgtatc gtcaaggcac tcttgcaagc ttcatcatgg 120
ccgacagcaa aagttatcag gcatgcacct ggtagctagt ctttaaacca atagattgca 180
tcggtttaaa aggcaagacc gtcaaattgc gggaaagggg tcaacagccg ttcagtacca 240
agtctcaggg gaaactttga gatggccttg caaagggtat ggtaataagc tgacggacat 300
ggtcctaacc acgcagccaa gtcctaagtc aacagatctt ctgttgatat ggatgcagtt 360
cacagactaa atgtcggtcg gggaagatgt attcttctca taagatatag tcggacctct 420
ccttaatggg agctagcgga tgaagtgatg caacactgga gccgctggga actaatttgt 480
atgcgaaagt atattgatta gttttggagt actcgcgaca tgatcacgct tcgtacccct 540
gccatcaaca cgcgtctgcg ttcgaccagg ctgcgcgttc tcgcggccat agcaaccgac 600
gtacggcgtt gcgccctcgc cggcagcaag aagccacgga agtccgcctg gagcagaaaa 660
tgcccacgct actgcgggtt tatatagacg gtcctcacgg gatggggaaa accaccacca 720
cgcaactgct ggtggccctg ggttcgcgcg acgatatcgt ctacgtaccc gagccgatga 780
cttactggca ggtgctgggg gcttccgaga caatcgcgaa catctacacc acacaacacc 840
gcctcgacca gggtgagata tcggccgggg acgcggcggt ggtaatgaca agcgcccaga 900
taacaatggg catgccttat gccgtgaccg acgccgttct ggctcctcat gtcggggggg 960
aggctgggag ttcacatgcc ccgcccccgg ccctcaccct catcttcgac cgccatccca 1020
tcgccgccct cctgtgctac ccggccgcgc gataccttat gggcagcatg accccccagg 1080
ccgtgctggc gttcgtggcc ctcatcccgc cgaccttgcc cggcacaaac atcgtgttgg 1140
gggcccttcc ggaggacaga cacatcgacc gcctggccaa acgccagcgc cccggcgagc 1200
ggcttgacct ggctatgctg gccgcgattc gccgcgttta cgggctgctt gccaatacgg 1260
tgcggtatct gcagggcggc gggtcgtggt gggaggattg gggacagctt tcggggacgg 1320
ccgtgccgcc ccagggtgcc gagccccaga gcaacgcggg cccacgaccc catatcgggg 1380
acacgttatt taccctgttt cgggcccccg agttgctggc ccccaacggc gacctgtata 1440
acgtgtttgc ctgggccttg gacgtcttgg ccaaacgcct ccgtcccatg cacgtcttta 1500
tcctggatta cgaccaatcg cccgccggct gccgggacgc cctgctgcaa cttacctccg 1560
ggatggtcca gacccacgtc accaccccag gctccatacc gacgatctgc gacctggcgc 1620
gcacgtttgc ccgggagatg ggggaggcta ataa 1654
<210> 36
<211> 1854
<212> DNA
<213> Artificial Sequence
<220>
<223> AS300KRib2-TK
<400> 36
aagcttctac taggaccata ggtacatctt cagagtcctt aactctttta atttgttctc 60
tataatggtg aatatcttca aatgatttag tattatttat ggcaaataca caaagaaagc 120
cctccccagt cctcatgtac tggtccctca ttgcactgta ctcctcttga cctgctgtgt 180
cgagaatatc caagagacag gtttctccat caattactac ttgcttcctg taggaatcct 240
ctattgttgg atcatattcg tccacaaaat gattctgaat tagctgtatc gtcaaggcac 300
tcttgcaagc ttcatcatgg ccgacagcaa aagttatcag gcatgcacct ggtagctagt 360
ctttaaacca atagattgca tcggtttaaa aggcaagacc gtcaaattgc gggaaagggg 420
tcaacagccg ttcagtacca agtctcaggg gaaactttga gatggccttg caaagggtat 480
ggtaataagc tgacggacat ggtcctaacc acgcagccaa gtcctaagtc aacagatctt 540
ctgttgatat ggatgcagtt cacagactaa atgtcggtcg gggaagatgt attcttctca 600
taagatatag tcggacctct ccttaatggg agctagcgga tgaagtgatg caacactgga 660
gccgctggga actaatttgt atgcgaaagt atattgatta gttttggagt actcgcgaca 720
tgatcacgct tcgtacccct gccatcaaca cgcgtctgcg ttcgaccagg ctgcgcgttc 780
tcgcggccat agcaaccgac gtacggcgtt gcgccctcgc cggcagcaag aagccacgga 840
agtccgcctg gagcagaaaa tgcccacgct actgcgggtt tatatagacg gtcctcacgg 900
gatggggaaa accaccacca cgcaactgct ggtggccctg ggttcgcgcg acgatatcgt 960
ctacgtaccc gagccgatga cttactggca ggtgctgggg gcttccgaga caatcgcgaa 1020
catctacacc acacaacacc gcctcgacca gggtgagata tcggccgggg acgcggcggt 1080
ggtaatgaca agcgcccaga taacaatggg catgccttat gccgtgaccg acgccgttct 1140
ggctcctcat gtcggggggg aggctgggag ttcacatgcc ccgcccccgg ccctcaccct 1200
catcttcgac cgccatccca tcgccgccct cctgtgctac ccggccgcgc gataccttat 1260
gggcagcatg accccccagg ccgtgctggc gttcgtggcc ctcatcccgc cgaccttgcc 1320
cggcacaaac atcgtgttgg gggcccttcc ggaggacaga cacatcgacc gcctggccaa 1380
acgccagcgc cccggcgagc ggcttgacct ggctatgctg gccgcgattc gccgcgttta 1440
cgggctgctt gccaatacgg tgcggtatct gcagggcggc gggtcgtggt gggaggattg 1500
gggacagctt tcggggacgg ccgtgccgcc ccagggtgcc gagccccaga gcaacgcggg 1560
cccacgaccc catatcgggg acacgttatt taccctgttt cgggcccccg agttgctggc 1620
ccccaacggc gacctgtata acgtgtttgc ctgggccttg gacgtcttgg ccaaacgcct 1680
ccgtcccatg cacgtcttta tcctggatta cgaccaatcg cccgccggct gccgggacgc 1740
cctgctgcaa cttacctccg ggatggtcca gacccacgtc accaccccag gctccatacc 1800
gacgatctgc gacctggcgc gcacgtttgc ccgggagatg ggggaggcta ataa 1854
<210> 37
<211> 8793
<212> DNA
<213> Artificial Sequence
<220>
<223> pAVQ-AS300KRib1
<400> 37
taacatcatc aataatatac cttattttgg attgaagcca atatgataat gagggggtgg 60
agtttgtgac gtggcgcggg gcgtgggaac ggggcgggtg acgtagtagt gtggcggaag 120
tgtgatgttg caagtgtggc ggaacacatg taagcgacgg atgtggcaaa agtgacgttt 180
ttggtgtgcg ccggtgtaca caggaagtga caattttcgc gcggttttag gcggatgttg 240
tagtaaattt gggcgtaacc gagtaagatt tggccatttt cgcgggaaaa ctgaataaga 300
ggaagtgaaa tctgaataat tttgtgttac tcatagcgcg taatactgcg atctatacat 360
tgaatcaata ttggcaatta gccatattag tcattggtta tatagcataa atcaatattg 420
gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat attggctcat 480
gtccaatatg accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 540
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 600
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 660
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 720
ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca 780
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta 840
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 900
acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 960
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata 1020
accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1080
gagctcgttt agtgaaccgt cagatcctca ctctcttccg catcgctgtc tgcgagggcc 1140
agctgttggg ctcgcggttg aggacaaact cttcgcggtc tttccagtac tcttggatcg 1200
gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg acctgagcca gtccgcatcg 1260
accggatcgg aaaacctctc gagaaaggcg tctaaccagt cacagtcgca aggtaggctg 1320
agcaccgtgg cgggcggcag cgggtggcgg tcggggttgt ttctggcgga ggtgctgctg 1380
atgatgtaat taaagtaggc ggtcttgagc cggcggatgg tcgaggtgag gtgtggcagg 1440
cttgagatcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga cttctgcgct 1500
aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg atctggccat 1560
acacttgagt gacaatgaca tccactttgc ctttctctcc acaggtgtcc actcccaggt 1620
ccaagtttgg aagatcccta ctaggaccat aggtacatct tcagagtcct taactctttt 1680
aatttgttct ctataatggt gaatatcttc aaatgattta gtattattta tggcaaatac 1740
acaaagaaag ccctccccag tcctcatgta ctggtccctc attgcactgt actcctcttg 1800
acctgctgtg tcgagaatat ccaagagaca ggtttctcca tcaattacta cttgcttcct 1860
gtaggaatcc tctattgttg gatcatattc gtccacaaaa tgattctgaa ttagctgtat 1920
cgtcaaggca ctcttgcaag cttcattatc agcagctaaa agttatcagg catgcacctg 1980
gtagctagtc tttaaaccaa tagattgcat cggtttaaaa ggcaagaccg tcaaattgcg 2040
ggaaaggggt caacagccgt tcagtaccaa gtctcagggg aaactttgag atggccttgc 2100
aaagggtatg gtaataagct gacggacatg gtcctaacca cgcagccaag tcctaagtca 2160
acagatcttc tgttgatatg gatgcagttc acagactaaa tgtcggtcgg ggaagatgta 2220
ttcttctcat aagatatagt cggacctctc cttaatggga gctagcggat gaagtgatgc 2280
aacactggag ccgctgggaa ctaatttgta tgcgaaagta tattgattag ttttggagta 2340
ctccgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg 2400
ctgggattac acatggcatg gatgaactgt acaactgagg atcccccgac ctcgacctct 2460
ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca 2520
ctcggaagga catatgggag ggcaaatcat ttggtcgaga tccctcggag atcggatctg 2580
ggcgtggtta agggtgggaa agaatatata aggtgggggt cttatgtagt tttgtatctg 2640
ttttgcagca gccgccgccg ccatgagcac caactcgttt gatggaagca ttgtgagctc 2700
atatttgaca acgcgcatgc ccccatgggc cggggtgcgt cagaatgtga tgggctccag 2760
cattgatggt cgccccgtcc tgcccgcaaa ctctactacc ttgacctacg agaccgtgtc 2820
tggaacgccg ttggagactg cagcctccgc cgccgcttca gccgctgcag ccaccgcccg 2880
cgggattgtg actgactttg ctttcctgag cccgcttgca agcagtgcag cttcccgttc 2940
atccgcccgc gatgacaagt tgacggctct tttggcacaa ttggattctt tgacccggga 3000
acttaatgtc gtttctcagc agctgttgga tctgcgccag caggtttctg ccctgaaggc 3060
ttcctcccct cccaatgcgg tttaaaacat aaataaaaaa ccagactctg tttggatttg 3120
gatcaagcaa gtgtcttgct gtctttattt aggggttttg cgcgcgcggt aggcccggga 3180
ccagcggtct cggtcgttga gggtcctgtg tattttttcc aggacgtggt aaaggtgact 3240
ctggatgttc agatacatgg gcataagccc gtctctgggg tggaggtagc accactgcag 3300
agcttcatgc tgcggggtgg tgttgtagat gatccagtcg tagcaggagc gctgggcgtg 3360
gtgcctaaaa atgtctttca gtagcaagct gattgccagg ggcaggccct tggtgtaagt 3420
gtttacaaag cggttaagct gggatgggtg catacgtggg gatatgagat gcatcttgga 3480
ctgtattttt aggttggcta tgttcccagc catatccctc cggggattca tgttgtgcag 3540
aaccaccagc acagtgtatc cggtgcactt gggaaatttg tcatgtagct tagaaggaaa 3600
tgcgtggaag aacttggaga cgcccttgtg acctccaaga ttttccatgc attcgtccat 3660
aatgatggca atgggcccac gggcggcggc ctgggcgaag atatttctgg gatcactaac 3720
gtcatagttg tgttccagga tgagatcgtc ataggccatt tttacaaagc gcgggcggag 3780
ggtgccagac tgcggtataa tggttccatc cggcccaggg gcgtagttac cctcacagat 3840
ttgcatttcc cacgctttga gttcagatgg ggggatcatg tctacctgcg gggcgatgaa 3900
gaaaacggtt tccggggtag gggagatcag ctgggaagaa agcaggttcc tgagcagctg 3960
cgacttaccg cagccggtgg gcccgtaaat cacacctatt accgggtgca actggtagtt 4020
aagagagctg cagctgccgt catccctgag caggggggcc acttcgttaa gcatgtccct 4080
gactcgcatg ttttccctga ccaaatccgc cagaaggcgc tcgccgccca gcgatagcag 4140
ttcttgcaag gaagcaaagt ttttcaacgg tttgagaccg tccgccgtag gcatgctttt 4200
gagcgtttga ccaagcagtt ccaggcggtc ccacagctcg gtcacctgct ctacggcatc 4260
tcgatccagc atatctcctc gtttcgcggg ttggggcggc tttcgctgta cggcagtagt 4320
cggtgctcgt ccagacgggc cagggtcatg tctttccacg ggcgcagggt cctcgtcagc 4380
gtagtctggg tcacggtgaa ggggtgcgct ccgggctgcg cgctggccag ggtgcgcttg 4440
aggctggtcc tgctggtgct gaagcgctgc cggtcttcgc cctgcgcgtc ggccaggtag 4500
catttgacca tggtgtcata gtccagcccc tccgcggcgt ggcccttggc gcgcagcttg 4560
cccttggagg aggcgccgca cgaggggcag tgcagacttt tgagggcgta gagcttgggc 4620
gcgagaaata ccgattccgg ggagtaggca tccgcgccgc aggccccgca gacggtctcg 4680
cattccacga gccaggtgag ctctggccgt tcggggtcaa aaaccaggtt tcccccatgc 4740
tttttgatgc gtttcttacc tctggtttcc atgagccggt gtccacgctc ggtgacgaaa 4800
aggctgtccg tgtccccgta tacagacttg agagggagtt taaacgaatt caatagcttg 4860
ttgcatgggc ggcgatataa aatgcaaggt gctgctcaaa aaatcaggca aagcctcgcg 4920
caaaaaagaa agcacatcgt agtcatgctc atgcagataa aggcaggtaa gctccggaac 4980
caccacagaa aaagacacca tttttctctc aaacatgtct gcgggtttct gcataaacac 5040
aaaataaaat aacaaaaaaa catttaaaca ttagaagcct gtcttacaac aggaaaaaca 5100
acccttataa gcataagacg gactacggcc atgccggcgt gaccgtaaaa aaactggtca 5160
ccgtgattaa aaagcaccac cgacagctcc tcggtcatgt ccggagtcat aatgtaagac 5220
tcggtaaaca catcaggttg attcatcggt cagtgctaaa aagcgaccga aatagcccgg 5280
gggaatacat acccgcaggc gtagagacaa cattacagcc cccataggag gtataacaaa 5340
attaatagga gagaaaaaca cataaacacc tgaaaaaccc tcctgcctag gcaaaatagc 5400
accctcccgc tccagaacaa catacagcgc ttcacagcgg cagcctaaca gtcagcctta 5460
ccagtaaaaa agaaaaccta ttaaaaaaac accactcgac acggcaccag ctcaatcagt 5520
cacagtgtaa aaaagggcca agtgcagagc gagtatatat aggactaaaa aatgacgtaa 5580
cggttaaagt ccacaaaaaa cacccagaaa accgcacgcg aacctacgcc cagaaacgaa 5640
agccaaaaaa cccacaactt cctcaaatcg tcacttccgt tttcccacgt tacgtaactt 5700
cccattttaa gaaaactaca attcccaaca catacaagtt actccgccct aaaacctacg 5760
tcacccgccc cgttcccacg ccccgcgcca cgtcacaaac tccaccccct cattatcata 5820
ttggcttcaa tccaaaataa ggtatattat tgatgatgtt aattaacatg catggatcca 5880
tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc 5940
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 6000
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 6060
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 6120
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 6180
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 6240
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 6300
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 6360
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 6420
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 6480
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 6540
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 6600
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 6660
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 6720
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 6780
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 6840
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 6900
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 6960
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 7020
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 7080
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 7140
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcagccat 7200
gagattatca aaaaggatct tcacctagat ccttttcacg tagaaagcca gtccgcagaa 7260
acggtgctga ccccggatga atgtcagcta ctgggctatc tggacaaggg aaaacgcaag 7320
cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg cgatagctag actgggcggt 7380
tttatggaca gcaagcgaac cggaattgcc agctggggcg ccctctggta aggttgggaa 7440
gccctgcaaa gtaaactgga tggctttctc gccgccaagg atctgatggc gcaggggatc 7500
aagctctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca 7560
cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac 7620
aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt 7680
tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc 7740
gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg 7800
aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc 7860
tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc 7920
ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat 7980
ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc 8040
cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca 8100
tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga 8160
ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat 8220
tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc 8280
tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gaattttgtt 8340
aaaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aacatccctt 8400
ataaatcaaa agaatagacc gcgatagggt tgagtgttgt tccagtttgg aacaagagtc 8460
cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg 8520
gcccactacg tgaaccatca cccaaatcaa gttttttgcg gtcgaggtgc cgtaaagctc 8580
taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg 8640
tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag 8700
cggtcacgct gcgcgtaacc accacacccg cgcgcttaat gcgccgctac agggcgcgtc 8760
cattcgccat tcaggatcga attaattctt aat 8793
<210> 38
<211> 9939
<212> DNA
<213> Artificial Sequence
<220>
<223> pAVQ-AS300KRib1-TK
<400> 38
taacatcatc aataatatac cttattttgg attgaagcca atatgataat gagggggtgg 60
agtttgtgac gtggcgcggg gcgtgggaac ggggcgggtg acgtagtagt gtggcggaag 120
tgtgatgttg caagtgtggc ggaacacatg taagcgacgg atgtggcaaa agtgacgttt 180
ttggtgtgcg ccggtgtaca caggaagtga caattttcgc gcggttttag gcggatgttg 240
tagtaaattt gggcgtaacc gagtaagatt tggccatttt cgcgggaaaa ctgaataaga 300
ggaagtgaaa tctgaataat tttgtgttac tcatagcgcg taatactgcg atctatacat 360
tgaatcaata ttggcaatta gccatattag tcattggtta tatagcataa atcaatattg 420
gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat attggctcat 480
gtccaatatg accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 540
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 600
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 660
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 720
ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca 780
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta 840
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 900
acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 960
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata 1020
accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1080
gagctcgttt agtgaaccgt cagatcctca ctctcttccg catcgctgtc tgcgagggcc 1140
agctgttggg ctcgcggttg aggacaaact cttcgcggtc tttccagtac tcttggatcg 1200
gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg acctgagcca gtccgcatcg 1260
accggatcgg aaaacctctc gagaaaggcg tctaaccagt cacagtcgca aggtaggctg 1320
agcaccgtgg cgggcggcag cgggtggcgg tcggggttgt ttctggcgga ggtgctgctg 1380
atgatgtaat taaagtaggc ggtcttgagc cggcggatgg tcgaggtgag gtgtggcagg 1440
cttgagatcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga cttctgcgct 1500
aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg atctggccat 1560
acacttgagt gacaatgaca tccactttgc ctttctctcc acaggtgtcc actcccaggt 1620
ccaagtttgg aagatcccta ctaggaccat aggtacatct tcagagtcct taactctttt 1680
aatttgttct ctataatggt gaatatcttc aaatgattta gtattattta tggcaaatac 1740
acaaagaaag ccctccccag tcctcatgta ctggtccctc attgcactgt actcctcttg 1800
acctgctgtg tcgagaatat ccaagagaca ggtttctcca tcaattacta cttgcttcct 1860
gtaggaatcc tctattgttg gatcatattc gtccacaaaa tgattctgaa ttagctgtat 1920
cgtcaaggca ctcttgcaag cttcattatc agcagctaaa agttatcagg catgcacctg 1980
gtagctagtc tttaaaccaa tagattgcat cggtttaaaa ggcaagaccg tcaaattgcg 2040
ggaaaggggt caacagccgt tcagtaccaa gtctcagggg aaactttgag atggccttgc 2100
aaagggtatg gtaataagct gacggacatg gtcctaacca cgcagccaag tcctaagtca 2160
acagatcttc tgttgatatg gatgcagttc acagactaaa tgtcggtcgg ggaagatgta 2220
ttcttctcat aagatatagt cggacctctc cttaatggga gctagcggat gaagtgatgc 2280
aacactggag ccgctgggaa ctaatttgta tgcgaaagta tattgattag ttttggagta 2340
ctcgcgatga tcacgcttcg tacccctgcc atcaacacgc gtctgcgttc gaccaggctg 2400
cgcgttctcg cggccatagc aaccgacgta cggcgttgcg ccctcgccgg cagcaagaag 2460
ccacggaagt ccgcctggag cagaaaatgc ccacgctact gcgggtttat atagacggtc 2520
ctcacgggat ggggaaaacc accaccacgc aactgctggt ggccctgggt tcgcgcgacg 2580
atatcgtcta cgtacccgag ccgatgactt actggcaggt gctgggggct tccgagacaa 2640
tcgcgaacat ctacaccaca caacaccgcc tcgaccaggg tgagatatcg gccggggacg 2700
cggcggtggt aatgacaagc gcccagataa caatgggcat gccttatgcc gtgaccgacg 2760
ccgttctggc tcctcatgtc gggggggagg ctgggagttc acatgccccg cccccggccc 2820
tcaccctcat cttcgaccgc catcccatcg ccgccctcct gtgctacccg gccgcgcgat 2880
accttatggg cagcatgacc ccccaggccg tgctggcgtt cgtggccctc atcccgccga 2940
ccttgcccgg cacaaacatc gtgttggggg cccttccgga ggacagacac atcgaccgcc 3000
tggccaaacg ccagcgcccc ggcgagcggc ttgacctggc tatgctggcc gcgattcgcc 3060
gcgtttacgg gctgcttgcc aatacggtgc ggtatctgca gggcggcggg tcgtggtggg 3120
aggattgggg acagctttcg gggacggccg tgccgcccca gggtgccgag ccccagagca 3180
acgcgggccc acgaccccat atcggggaca cgttatttac cctgtttcgg gcccccgagt 3240
tgctggcccc caacggcgac ctgtataacg tgtttgcctg ggccttggac gtcttggcca 3300
aacgcctccg tcccatgcac gtctttatcc tggattacga ccaatcgccc gccggctgcc 3360
gggacgccct gctgcaactt acctccggga tggtccagac ccacgtcacc accccaggct 3420
ccataccgac gatctgcgac ctggcgcgca cgtttgcccg ggagatgggg gaggctaact 3480
gaagcttttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa 3540
cagctgctgg gattacacat ggcatggatg aactgtacaa ctgaggatcc cccgacctcg 3600
acctctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt 3660
ctctcactcg gaaggacata tgggagggca aatcatttgg tcgagatccc tcggagatcg 3720
gatctgggcg tggttaaggg tgggaaagaa tatataaggt gggggtctta tgtagttttg 3780
tatctgtttt gcagcagccg ccgccgccat gagcaccaac tcgtttgatg gaagcattgt 3840
gagctcatat ttgacaacgc gcatgccccc atgggccggg gtgcgtcaga atgtgatggg 3900
ctccagcatt gatggtcgcc ccgtcctgcc cgcaaactct actaccttga cctacgagac 3960
cgtgtctgga acgccgttgg agactgcagc ctccgccgcc gcttcagccg ctgcagccac 4020
cgcccgcggg attgtgactg actttgcttt cctgagcccg cttgcaagca gtgcagcttc 4080
ccgttcatcc gcccgcgatg acaagttgac ggctcttttg gcacaattgg attctttgac 4140
ccgggaactt aatgtcgttt ctcagcagct gttggatctg cgccagcagg tttctgccct 4200
gaaggcttcc tcccctccca atgcggttta aaacataaat aaaaaaccag actctgtttg 4260
gatttggatc aagcaagtgt cttgctgtct ttatttaggg gttttgcgcg cgcggtaggc 4320
ccgggaccag cggtctcggt cgttgagggt cctgtgtatt ttttccagga cgtggtaaag 4380
gtgactctgg atgttcagat acatgggcat aagcccgtct ctggggtgga ggtagcacca 4440
ctgcagagct tcatgctgcg gggtggtgtt gtagatgatc cagtcgtagc aggagcgctg 4500
ggcgtggtgc ctaaaaatgt ctttcagtag caagctgatt gccaggggca ggcccttggt 4560
gtaagtgttt acaaagcggt taagctggga tgggtgcata cgtggggata tgagatgcat 4620
cttggactgt atttttaggt tggctatgtt cccagccata tccctccggg gattcatgtt 4680
gtgcagaacc accagcacag tgtatccggt gcacttggga aatttgtcat gtagcttaga 4740
aggaaatgcg tggaagaact tggagacgcc cttgtgacct ccaagatttt ccatgcattc 4800
gtccataatg atggcaatgg gcccacgggc ggcggcctgg gcgaagatat ttctgggatc 4860
actaacgtca tagttgtgtt ccaggatgag atcgtcatag gccattttta caaagcgcgg 4920
gcggagggtg ccagactgcg gtataatggt tccatccggc ccaggggcgt agttaccctc 4980
acagatttgc atttcccacg ctttgagttc agatgggggg atcatgtcta cctgcggggc 5040
gatgaagaaa acggtttccg gggtagggga gatcagctgg gaagaaagca ggttcctgag 5100
cagctgcgac ttaccgcagc cggtgggccc gtaaatcaca cctattaccg ggtgcaactg 5160
gtagttaaga gagctgcagc tgccgtcatc cctgagcagg ggggccactt cgttaagcat 5220
gtccctgact cgcatgtttt ccctgaccaa atccgccaga aggcgctcgc cgcccagcga 5280
tagcagttct tgcaaggaag caaagttttt caacggtttg agaccgtccg ccgtaggcat 5340
gcttttgagc gtttgaccaa gcagttccag gcggtcccac agctcggtca cctgctctac 5400
ggcatctcga tccagcatat ctcctcgttt cgcgggttgg ggcggctttc gctgtacggc 5460
agtagtcggt gctcgtccag acgggccagg gtcatgtctt tccacgggcg cagggtcctc 5520
gtcagcgtag tctgggtcac ggtgaagggg tgcgctccgg gctgcgcgct ggccagggtg 5580
cgcttgaggc tggtcctgct ggtgctgaag cgctgccggt cttcgccctg cgcgtcggcc 5640
aggtagcatt tgaccatggt gtcatagtcc agcccctccg cggcgtggcc cttggcgcgc 5700
agcttgccct tggaggaggc gccgcacgag gggcagtgca gacttttgag ggcgtagagc 5760
ttgggcgcga gaaataccga ttccggggag taggcatccg cgccgcaggc cccgcagacg 5820
gtctcgcatt ccacgagcca ggtgagctct ggccgttcgg ggtcaaaaac caggtttccc 5880
ccatgctttt tgatgcgttt cttacctctg gtttccatga gccggtgtcc acgctcggtg 5940
acgaaaaggc tgtccgtgtc cccgtataca gacttgagag ggagtttaaa cgaattcaat 6000
agcttgttgc atgggcggcg atataaaatg caaggtgctg ctcaaaaaat caggcaaagc 6060
ctcgcgcaaa aaagaaagca catcgtagtc atgctcatgc agataaaggc aggtaagctc 6120
cggaaccacc acagaaaaag acaccatttt tctctcaaac atgtctgcgg gtttctgcat 6180
aaacacaaaa taaaataaca aaaaaacatt taaacattag aagcctgtct tacaacagga 6240
aaaacaaccc ttataagcat aagacggact acggccatgc cggcgtgacc gtaaaaaaac 6300
tggtcaccgt gattaaaaag caccaccgac agctcctcgg tcatgtccgg agtcataatg 6360
taagactcgg taaacacatc aggttgattc atcggtcagt gctaaaaagc gaccgaaata 6420
gcccggggga atacataccc gcaggcgtag agacaacatt acagccccca taggaggtat 6480
aacaaaatta ataggagaga aaaacacata aacacctgaa aaaccctcct gcctaggcaa 6540
aatagcaccc tcccgctcca gaacaacata cagcgcttca cagcggcagc ctaacagtca 6600
gccttaccag taaaaaagaa aacctattaa aaaaacacca ctcgacacgg caccagctca 6660
atcagtcaca gtgtaaaaaa gggccaagtg cagagcgagt atatatagga ctaaaaaatg 6720
acgtaacggt taaagtccac aaaaaacacc cagaaaaccg cacgcgaacc tacgcccaga 6780
aacgaaagcc aaaaaaccca caacttcctc aaatcgtcac ttccgttttc ccacgttacg 6840
taacttccca ttttaagaaa actacaattc ccaacacata caagttactc cgccctaaaa 6900
cctacgtcac ccgccccgtt cccacgcccc gcgccacgtc acaaactcca ccccctcatt 6960
atcatattgg cttcaatcca aaataaggta tattattgat gatgttaatt aacatgcatg 7020
gatccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg 7080
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 7140
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 7200
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 7260
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 7320
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 7380
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 7440
aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 7500
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 7560
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 7620
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 7680
gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 7740
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 7800
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 7860
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 7920
ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 7980
taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 8040
tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 8100
cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 8160
gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 8220
cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 8280
ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc 8340
agccatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga aagccagtcc 8400
gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga caagggaaaa 8460
cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg 8520
ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt 8580
tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct gatggcgcag 8640
gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg 8700
attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 8760
acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 8820
tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg 8880
gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 8940
agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 9000
ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 9060
tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 9120
tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 9180
gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt 9240
gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 9300
catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 9360
tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 9420
cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat 9480
tttgttaaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaca 9540
tcccttataa atcaaaagaa tagaccgcga tagggttgag tgttgttcca gtttggaaca 9600
agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 9660
gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttgcggtcg aggtgccgta 9720
aagctctaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 9780
cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 9840
gtgtagcggt cacgctgcgc gtaaccacca cacccgcgcg cttaatgcgc cgctacaggg 9900
cgcgtccatt cgccattcag gatcgaatta attcttaat 9939
<210> 39
<211> 8607
<212> DNA
<213> Artificial Sequence
<220>
<223> pAVQ-AS100KRib2
<400> 39
taacatcatc aataatatac cttattttgg attgaagcca atatgataat gagggggtgg 60
agtttgtgac gtggcgcggg gcgtgggaac ggggcgggtg acgtagtagt gtggcggaag 120
tgtgatgttg caagtgtggc ggaacacatg taagcgacgg atgtggcaaa agtgacgttt 180
ttggtgtgcg ccggtgtaca caggaagtga caattttcgc gcggttttag gcggatgttg 240
tagtaaattt gggcgtaacc gagtaagatt tggccatttt cgcgggaaaa ctgaataaga 300
ggaagtgaaa tctgaataat tttgtgttac tcatagcgcg taatactgcg atctatacat 360
tgaatcaata ttggcaatta gccatattag tcattggtta tatagcataa atcaatattg 420
gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat attggctcat 480
gtccaatatg accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 540
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 600
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 660
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 720
ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca 780
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta 840
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 900
acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 960
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata 1020
accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1080
gagctcgttt agtgaaccgt cagatcctca ctctcttccg catcgctgtc tgcgagggcc 1140
agctgttggg ctcgcggttg aggacaaact cttcgcggtc tttccagtac tcttggatcg 1200
gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg acctgagcca gtccgcatcg 1260
accggatcgg aaaacctctc gagaaaggcg tctaaccagt cacagtcgca aggtaggctg 1320
agcaccgtgg cgggcggcag cgggtggcgg tcggggttgt ttctggcgga ggtgctgctg 1380
atgatgtaat taaagtaggc ggtcttgagc cggcggatgg tcgaggtgag gtgtggcagg 1440
cttgagatcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga cttctgcgct 1500
aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg atctggccat 1560
acacttgagt gacaatgaca tccactttgc ctttctctcc acaggtgtcc actcccaggt 1620
ccaagtttgg aagatcccca tcaattacta cttgcttcct gtaggaatcc tctattgttg 1680
gatcatattc gtccacaaaa tgattctgaa ttagctgtat cgtcaaggca ctcttgcaag 1740
cttcatcatg gccgacagca aaagttatca ggcatgcacc tggtagctag tctttaaacc 1800
aatagattgc atcggtttaa aaggcaagac cgtcaaattg cgggaaaggg gtcaacagcc 1860
gttcagtacc aagtctcagg ggaaactttg agatggcctt gcaaagggta tggtaataag 1920
ctgacggaca tggtcctaac cacgcagcca agtcctaagt caacagatct tctgttgata 1980
tggatgcagt tcacagacta aatgtcggtc ggggaagatg tattcttctc ataagatata 2040
gtcggacctc tccttaatgg gagctagcgg atgaagtgat gcaacactgg agccgctggg 2100
aactaatttg tatgcgaaag tatattgatt agttttggag tactcgcgac atgatcacga 2160
aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga 2220
ttacacatgg catggatgaa ctgtacaact gaggatcccc cgacctcgac ctctggctaa 2280
taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga 2340
aggacatatg ggagggcaaa tcatttggtc gagatccctc ggagatcgga tctgggcgtg 2400
gttaagggtg ggaaagaata tataaggtgg gggtcttatg tagttttgta tctgttttgc 2460
agcagccgcc gccgccatga gcaccaactc gtttgatgga agcattgtga gctcatattt 2520
gacaacgcgc atgcccccat gggccggggt gcgtcagaat gtgatgggct ccagcattga 2580
tggtcgcccc gtcctgcccg caaactctac taccttgacc tacgagaccg tgtctggaac 2640
gccgttggag actgcagcct ccgccgccgc ttcagccgct gcagccaccg cccgcgggat 2700
tgtgactgac tttgctttcc tgagcccgct tgcaagcagt gcagcttccc gttcatccgc 2760
ccgcgatgac aagttgacgg ctcttttggc acaattggat tctttgaccc gggaacttaa 2820
tgtcgtttct cagcagctgt tggatctgcg ccagcaggtt tctgccctga aggcttcctc 2880
ccctcccaat gcggtttaaa acataaataa aaaaccagac tctgtttgga tttggatcaa 2940
gcaagtgtct tgctgtcttt atttaggggt tttgcgcgcg cggtaggccc gggaccagcg 3000
gtctcggtcg ttgagggtcc tgtgtatttt ttccaggacg tggtaaaggt gactctggat 3060
gttcagatac atgggcataa gcccgtctct ggggtggagg tagcaccact gcagagcttc 3120
atgctgcggg gtggtgttgt agatgatcca gtcgtagcag gagcgctggg cgtggtgcct 3180
aaaaatgtct ttcagtagca agctgattgc caggggcagg cccttggtgt aagtgtttac 3240
aaagcggtta agctgggatg ggtgcatacg tggggatatg agatgcatct tggactgtat 3300
ttttaggttg gctatgttcc cagccatatc cctccgggga ttcatgttgt gcagaaccac 3360
cagcacagtg tatccggtgc acttgggaaa tttgtcatgt agcttagaag gaaatgcgtg 3420
gaagaacttg gagacgccct tgtgacctcc aagattttcc atgcattcgt ccataatgat 3480
ggcaatgggc ccacgggcgg cggcctgggc gaagatattt ctgggatcac taacgtcata 3540
gttgtgttcc aggatgagat cgtcataggc catttttaca aagcgcgggc ggagggtgcc 3600
agactgcggt ataatggttc catccggccc aggggcgtag ttaccctcac agatttgcat 3660
ttcccacgct ttgagttcag atggggggat catgtctacc tgcggggcga tgaagaaaac 3720
ggtttccggg gtaggggaga tcagctggga agaaagcagg ttcctgagca gctgcgactt 3780
accgcagccg gtgggcccgt aaatcacacc tattaccggg tgcaactggt agttaagaga 3840
gctgcagctg ccgtcatccc tgagcagggg ggccacttcg ttaagcatgt ccctgactcg 3900
catgttttcc ctgaccaaat ccgccagaag gcgctcgccg cccagcgata gcagttcttg 3960
caaggaagca aagtttttca acggtttgag accgtccgcc gtaggcatgc ttttgagcgt 4020
ttgaccaagc agttccaggc ggtcccacag ctcggtcacc tgctctacgg catctcgatc 4080
cagcatatct cctcgtttcg cgggttgggg cggctttcgc tgtacggcag tagtcggtgc 4140
tcgtccagac gggccagggt catgtctttc cacgggcgca gggtcctcgt cagcgtagtc 4200
tgggtcacgg tgaaggggtg cgctccgggc tgcgcgctgg ccagggtgcg cttgaggctg 4260
gtcctgctgg tgctgaagcg ctgccggtct tcgccctgcg cgtcggccag gtagcatttg 4320
accatggtgt catagtccag cccctccgcg gcgtggccct tggcgcgcag cttgcccttg 4380
gaggaggcgc cgcacgaggg gcagtgcaga cttttgaggg cgtagagctt gggcgcgaga 4440
aataccgatt ccggggagta ggcatccgcg ccgcaggccc cgcagacggt ctcgcattcc 4500
acgagccagg tgagctctgg ccgttcgggg tcaaaaacca ggtttccccc atgctttttg 4560
atgcgtttct tacctctggt ttccatgagc cggtgtccac gctcggtgac gaaaaggctg 4620
tccgtgtccc cgtatacaga cttgagaggg agtttaaacg aattcaatag cttgttgcat 4680
gggcggcgat ataaaatgca aggtgctgct caaaaaatca ggcaaagcct cgcgcaaaaa 4740
agaaagcaca tcgtagtcat gctcatgcag ataaaggcag gtaagctccg gaaccaccac 4800
agaaaaagac accatttttc tctcaaacat gtctgcgggt ttctgcataa acacaaaata 4860
aaataacaaa aaaacattta aacattagaa gcctgtctta caacaggaaa aacaaccctt 4920
ataagcataa gacggactac ggccatgccg gcgtgaccgt aaaaaaactg gtcaccgtga 4980
ttaaaaagca ccaccgacag ctcctcggtc atgtccggag tcataatgta agactcggta 5040
aacacatcag gttgattcat cggtcagtgc taaaaagcga ccgaaatagc ccgggggaat 5100
acatacccgc aggcgtagag acaacattac agcccccata ggaggtataa caaaattaat 5160
aggagagaaa aacacataaa cacctgaaaa accctcctgc ctaggcaaaa tagcaccctc 5220
ccgctccaga acaacataca gcgcttcaca gcggcagcct aacagtcagc cttaccagta 5280
aaaaagaaaa cctattaaaa aaacaccact cgacacggca ccagctcaat cagtcacagt 5340
gtaaaaaagg gccaagtgca gagcgagtat atataggact aaaaaatgac gtaacggtta 5400
aagtccacaa aaaacaccca gaaaaccgca cgcgaaccta cgcccagaaa cgaaagccaa 5460
aaaacccaca acttcctcaa atcgtcactt ccgttttccc acgttacgta acttcccatt 5520
ttaagaaaac tacaattccc aacacataca agttactccg ccctaaaacc tacgtcaccc 5580
gccccgttcc cacgccccgc gccacgtcac aaactccacc ccctcattat catattggct 5640
tcaatccaaa ataaggtata ttattgatga tgttaattaa catgcatgga tccatatgcg 5700
gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc 5760
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 5820
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 5880
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 5940
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 6000
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 6060
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 6120
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 6180
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 6240
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 6300
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 6360
ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 6420
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 6480
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 6540
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 6600
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 6660
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 6720
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 6780
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 6840
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 6900
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 6960
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag ccatgagatt 7020
atcaaaaagg atcttcacct agatcctttt cacgtagaaa gccagtccgc agaaacggtg 7080
ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa 7140
gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg 7200
gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg 7260
caaagtaaac tggatggctt tctcgccgcc aaggatctga tggcgcaggg gatcaagctc 7320
tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg 7380
ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg 7440
ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa 7500
gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct 7560
ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga 7620
ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc 7680
cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac 7740
ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc 7800
cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact 7860
gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga 7920
tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg 7980
ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga 8040
agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga 8100
ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgaattt tgttaaaatt 8160
tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaacatc ccttataaat 8220
caaaagaata gaccgcgata gggttgagtg ttgttccagt ttggaacaag agtccactat 8280
taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac 8340
tacgtgaacc atcacccaaa tcaagttttt tgcggtcgag gtgccgtaaa gctctaaatc 8400
ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga 8460
gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca 8520
cgctgcgcgt aaccaccaca cccgcgcgct taatgcgccg ctacagggcg cgtccattcg 8580
ccattcagga tcgaattaat tcttaat 8607
<210> 40
<211> 9737
<212> DNA
<213> Artificial Sequence
<220>
<223> pAVQ-AS100KRib2-TK
<400> 40
taacatcatc aataatatac cttattttgg attgaagcca atatgataat gagggggtgg 60
agtttgtgac gtggcgcggg gcgtgggaac ggggcgggtg acgtagtagt gtggcggaag 120
tgtgatgttg caagtgtggc ggaacacatg taagcgacgg atgtggcaaa agtgacgttt 180
ttggtgtgcg ccggtgtaca caggaagtga caattttcgc gcggttttag gcggatgttg 240
tagtaaattt gggcgtaacc gagtaagatt tggccatttt cgcgggaaaa ctgaataaga 300
ggaagtgaaa tctgaataat tttgtgttac tcatagcgcg taatactgcg atctatacat 360
tgaatcaata ttggcaatta gccatattag tcattggtta tatagcataa atcaatattg 420
gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat attggctcat 480
gtccaatatg accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 540
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 600
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 660
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 720
ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca 780
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta 840
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 900
acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 960
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata 1020
accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1080
gagctcgttt agtgaaccgt cagatcctca ctctcttccg catcgctgtc tgcgagggcc 1140
agctgttggg ctcgcggttg aggacaaact cttcgcggtc tttccagtac tcttggatcg 1200
gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg acctgagcca gtccgcatcg 1260
accggatcgg aaaacctctc gagaaaggcg tctaaccagt cacagtcgca aggtaggctg 1320
agcaccgtgg cgggcggcag cgggtggcgg tcggggttgt ttctggcgga ggtgctgctg 1380
atgatgtaat taaagtaggc ggtcttgagc cggcggatgg tcgaggtgag gtgtggcagg 1440
cttgagatcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga cttctgcgct 1500
aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg atctggccat 1560
acacttgagt gacaatgaca tccactttgc ctttctctcc acaggtgtcc actcccaggt 1620
ccaagtttgg aagatcccca tcaattacta cttgcttcct gtaggaatcc tctattgttg 1680
gatcatattc gtccacaaaa tgattctgaa ttagctgtat cgtcaaggca ctcttgcaag 1740
cttcatcatg gccgacagca aaagttatca ggcatgcacc tggtagctag tctttaaacc 1800
aatagattgc atcggtttaa aaggcaagac cgtcaaattg cgggaaaggg gtcaacagcc 1860
gttcagtacc aagtctcagg ggaaactttg agatggcctt gcaaagggta tggtaataag 1920
ctgacggaca tggtcctaac cacgcagcca agtcctaagt caacagatct tctgttgata 1980
tggatgcagt tcacagacta aatgtcggtc ggggaagatg tattcttctc ataagatata 2040
gtcggacctc tccttaatgg gagctagcgg atgaagtgat gcaacactgg agccgctggg 2100
aactaatttg tatgcgaaag tatattgatt agttttggag tactcgcgac atgatcacgc 2160
ttcgtacccc tgccatcaac acgcgtctgc gttcgaccag gctgcgcgtt ctcgcggcca 2220
tagcaaccga cgtacggcgt tgcgccctcg ccggcagcaa gaagccacgg aagtccgcct 2280
ggagcagaaa atgcccacgc tactgcgggt ttatatagac ggtcctcacg ggatggggaa 2340
aaccaccacc acgcaactgc tggtggccct gggttcgcgc gacgatatcg tctacgtacc 2400
cgagccgatg acttactggc aggtgctggg ggcttccgag acaatcgcga acatctacac 2460
cacacaacac cgcctcgacc agggtgagat atcggccggg gacgcggcgg tggtaatgac 2520
aagcgcccag ataacaatgg gcatgcctta tgccgtgacc gacgccgttc tggctcctca 2580
tgtcgggggg gaggctggga gttcacatgc cccgcccccg gccctcaccc tcatcttcga 2640
ccgccatccc atcgccgccc tcctgtgcta cccggccgcg cgatacctta tgggcagcat 2700
gaccccccag gccgtgctgg cgttcgtggc cctcatcccg ccgaccttgc ccggcacaaa 2760
catcgtgttg ggggcccttc cggaggacag acacatcgac cgcctggcca aacgccagcg 2820
ccccggcgag cggcttgacc tggctatgct ggccgcgatt cgccgcgttt acgggctgct 2880
tgccaatacg gtgcggtatc tgcagggcgg cgggtcgtgg tgggaggatt ggggacagct 2940
ttcggggacg gccgtgccgc cccagggtgc cgagccccag agcaacgcgg gcccacgacc 3000
ccatatcggg gacacgttat ttaccctgtt tcgggccccc gagttgctgg cccccaacgg 3060
cgacctgtat aacgtgtttg cctgggcctt ggacgtcttg gccaaacgcc tccgtcccat 3120
gcacgtcttt atcctggatt acgaccaatc gcccgccggc tgccgggacg ccctgctgca 3180
acttacctcc gggatggtcc agacccacgt caccacccca ggctccatac cgacgatctg 3240
cgacctggcg cgcacgtttg cccgggagat gggggaggct aataattcga aagatcccaa 3300
cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg 3360
catggatgaa ctgtacaact gaggatcccc cgacctcgac ctctggctaa taaaggaaat 3420
ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg 3480
ggagggcaaa tcatttggtc gagatccctc ggagatcgga tctgggcgtg gttaagggtg 3540
ggaaagaata tataaggtgg gggtcttatg tagttttgta tctgttttgc agcagccgcc 3600
gccgccatga gcaccaactc gtttgatgga agcattgtga gctcatattt gacaacgcgc 3660
atgcccccat gggccggggt gcgtcagaat gtgatgggct ccagcattga tggtcgcccc 3720
gtcctgcccg caaactctac taccttgacc tacgagaccg tgtctggaac gccgttggag 3780
actgcagcct ccgccgccgc ttcagccgct gcagccaccg cccgcgggat tgtgactgac 3840
tttgctttcc tgagcccgct tgcaagcagt gcagcttccc gttcatccgc ccgcgatgac 3900
aagttgacgg ctcttttggc acaattggat tctttgaccc gggaacttaa tgtcgtttct 3960
cagcagctgt tggatctgcg ccagcaggtt tctgccctga aggcttcctc ccctcccaat 4020
gcggtttaaa acataaataa aaaaccagac tctgtttgga tttggatcaa gcaagtgtct 4080
tgctgtcttt atttaggggt tttgcgcgcg cggtaggccc gggaccagcg gtctcggtcg 4140
ttgagggtcc tgtgtatttt ttccaggacg tggtaaaggt gactctggat gttcagatac 4200
atgggcataa gcccgtctct ggggtggagg tagcaccact gcagagcttc atgctgcggg 4260
gtggtgttgt agatgatcca gtcgtagcag gagcgctggg cgtggtgcct aaaaatgtct 4320
ttcagtagca agctgattgc caggggcagg cccttggtgt aagtgtttac aaagcggtta 4380
agctgggatg ggtgcatacg tggggatatg agatgcatct tggactgtat ttttaggttg 4440
gctatgttcc cagccatatc cctccgggga ttcatgttgt gcagaaccac cagcacagtg 4500
tatccggtgc acttgggaaa tttgtcatgt agcttagaag gaaatgcgtg gaagaacttg 4560
gagacgccct tgtgacctcc aagattttcc atgcattcgt ccataatgat ggcaatgggc 4620
ccacgggcgg cggcctgggc gaagatattt ctgggatcac taacgtcata gttgtgttcc 4680
aggatgagat cgtcataggc catttttaca aagcgcgggc ggagggtgcc agactgcggt 4740
ataatggttc catccggccc aggggcgtag ttaccctcac agatttgcat ttcccacgct 4800
ttgagttcag atggggggat catgtctacc tgcggggcga tgaagaaaac ggtttccggg 4860
gtaggggaga tcagctggga agaaagcagg ttcctgagca gctgcgactt accgcagccg 4920
gtgggcccgt aaatcacacc tattaccggg tgcaactggt agttaagaga gctgcagctg 4980
ccgtcatccc tgagcagggg ggccacttcg ttaagcatgt ccctgactcg catgttttcc 5040
ctgaccaaat ccgccagaag gcgctcgccg cccagcgata gcagttcttg caaggaagca 5100
aagtttttca acggtttgag accgtccgcc gtaggcatgc ttttgagcgt ttgaccaagc 5160
agttccaggc ggtcccacag ctcggtcacc tgctctacgg catctcgatc cagcatatct 5220
cctcgtttcg cgggttgggg cggctttcgc tgtacggcag tagtcggtgc tcgtccagac 5280
gggccagggt catgtctttc cacgggcgca gggtcctcgt cagcgtagtc tgggtcacgg 5340
tgaaggggtg cgctccgggc tgcgcgctgg ccagggtgcg cttgaggctg gtcctgctgg 5400
tgctgaagcg ctgccggtct tcgccctgcg cgtcggccag gtagcatttg accatggtgt 5460
catagtccag cccctccgcg gcgtggccct tggcgcgcag cttgcccttg gaggaggcgc 5520
cgcacgaggg gcagtgcaga cttttgaggg cgtagagctt gggcgcgaga aataccgatt 5580
ccggggagta ggcatccgcg ccgcaggccc cgcagacggt ctcgcattcc acgagccagg 5640
tgagctctgg ccgttcgggg tcaaaaacca ggtttccccc atgctttttg atgcgtttct 5700
tacctctggt ttccatgagc cggtgtccac gctcggtgac gaaaaggctg tccgtgtccc 5760
cgtatacaga cttgagaggg agtttaaacg aattcaatag cttgttgcat gggcggcgat 5820
ataaaatgca aggtgctgct caaaaaatca ggcaaagcct cgcgcaaaaa agaaagcaca 5880
tcgtagtcat gctcatgcag ataaaggcag gtaagctccg gaaccaccac agaaaaagac 5940
accatttttc tctcaaacat gtctgcgggt ttctgcataa acacaaaata aaataacaaa 6000
aaaacattta aacattagaa gcctgtctta caacaggaaa aacaaccctt ataagcataa 6060
gacggactac ggccatgccg gcgtgaccgt aaaaaaactg gtcaccgtga ttaaaaagca 6120
ccaccgacag ctcctcggtc atgtccggag tcataatgta agactcggta aacacatcag 6180
gttgattcat cggtcagtgc taaaaagcga ccgaaatagc ccgggggaat acatacccgc 6240
aggcgtagag acaacattac agcccccata ggaggtataa caaaattaat aggagagaaa 6300
aacacataaa cacctgaaaa accctcctgc ctaggcaaaa tagcaccctc ccgctccaga 6360
acaacataca gcgcttcaca gcggcagcct aacagtcagc cttaccagta aaaaagaaaa 6420
cctattaaaa aaacaccact cgacacggca ccagctcaat cagtcacagt gtaaaaaagg 6480
gccaagtgca gagcgagtat atataggact aaaaaatgac gtaacggtta aagtccacaa 6540
aaaacaccca gaaaaccgca cgcgaaccta cgcccagaaa cgaaagccaa aaaacccaca 6600
acttcctcaa atcgtcactt ccgttttccc acgttacgta acttcccatt ttaagaaaac 6660
tacaattccc aacacataca agttactccg ccctaaaacc tacgtcaccc gccccgttcc 6720
cacgccccgc gccacgtcac aaactccacc ccctcattat catattggct tcaatccaaa 6780
ataaggtata ttattgatga tgttaattaa catgcatgga tccatatgcg gtgtgaaata 6840
ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact 6900
gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 6960
atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 7020
caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 7080
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 7140
taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 7200
ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 7260
tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 7320
gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 7380
ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 7440
aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 7500
aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 7560
agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 7620
cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 7680
gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 7740
atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 7800
gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 7860
tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 7920
gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 7980
ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 8040
actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 8100
ccagttaata gtttgcgcaa cgttgttgcc attgctgcag ccatgagatt atcaaaaagg 8160
atcttcacct agatcctttt cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg 8220
atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag 8280
gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc 8340
gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac 8400
tggatggctt tctcgccgcc aaggatctga tggcgcaggg gatcaagctc tgatcaagag 8460
acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc 8520
gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 8580
gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 8640
tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg 8700
ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta 8760
ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta 8820
tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc 8880
gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc 8940
gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg 9000
ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg 9060
ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt 9120
gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc 9180
ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 9240
atcgccttct atcgccttct tgacgagttc ttctgaattt tgttaaaatt tttgttaaat 9300
cagctcattt tttaaccaat aggccgaaat cggcaacatc ccttataaat caaaagaata 9360
gaccgcgata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt 9420
ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc 9480
atcacccaaa tcaagttttt tgcggtcgag gtgccgtaaa gctctaaatc ggaaccctaa 9540
agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg 9600
gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt 9660
aaccaccaca cccgcgcgct taatgcgccg ctacagggcg cgtccattcg ccattcagga 9720
tcgaattaat tcttaat 9737
<210> 41
<211> 11671
<212> DNA
<213> Artificial Sequence
<220>
<223> pAVQ-AS100KRib2-LacZ
<400> 41
taacatcatc aataatatac cttattttgg attgaagcca atatgataat gagggggtgg 60
agtttgtgac gtggcgcggg gcgtgggaac ggggcgggtg acgtagtagt gtggcggaag 120
tgtgatgttg caagtgtggc ggaacacatg taagcgacgg atgtggcaaa agtgacgttt 180
ttggtgtgcg ccggtgtaca caggaagtga caattttcgc gcggttttag gcggatgttg 240
tagtaaattt gggcgtaacc gagtaagatt tggccatttt cgcgggaaaa ctgaataaga 300
ggaagtgaaa tctgaataat tttgtgttac tcatagcgcg taatactgcg atctatacat 360
tgaatcaata ttggcaatta gccatattag tcattggtta tatagcataa atcaatattg 420
gctattggcc attgcatacg ttgtatctat atcataatat gtacatttat attggctcat 480
gtccaatatg accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 540
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 600
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 660
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 720
ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca 780
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta 840
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 900
acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 960
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata 1020
accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1080
gagctcgttt agtgaaccgt cagatcctca ctctcttccg catcgctgtc tgcgagggcc 1140
agctgttggg ctcgcggttg aggacaaact cttcgcggtc tttccagtac tcttggatcg 1200
gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg acctgagcca gtccgcatcg 1260
accggatcgg aaaacctctc gagaaaggcg tctaaccagt cacagtcgca aggtaggctg 1320
agcaccgtgg cgggcggcag cgggtggcgg tcggggttgt ttctggcgga ggtgctgctg 1380
atgatgtaat taaagtaggc ggtcttgagc cggcggatgg tcgaggtgag gtgtggcagg 1440
cttgagatcc agctgttggg gtgagtactc cctctcaaaa gcgggcatga cttctgcgct 1500
aagattgtca gtttccaaaa acgaggagga tttgatattc acctggcccg atctggccat 1560
acacttgagt gacaatgaca tccactttgc ctttctctcc acaggtgtcc actcccaggt 1620
ccaagtttgg aagatcccca tcaattacta cttgcttcct gtaggaatcc tctattgttg 1680
gatcatattc gtccacaaaa tgattctgaa ttagctgtat cgtcaaggca ctcttgcaag 1740
cttcatcatg gccgacagca aaagttatca ggcatgcacc tggtagctag tctttaaacc 1800
aatagattgc atcggtttaa aaggcaagac cgtcaaattg cgggaaaggg gtcaacagcc 1860
gttcagtacc aagtctcagg ggaaactttg agatggcctt gcaaagggta tggtaataag 1920
ctgacggaca tggtcctaac cacgcagcca agtcctaagt caacagatct tctgttgata 1980
tggatgcagt tcacagacta aatgtcggtc ggggaagatg tattcttctc ataagatata 2040
gtcggacctc tccttaatgg gagctagcgg atgaagtgat gcaacactgg agccgctggg 2100
aactaatttg tatgcgaaag tatattgatt agttttggag tactcgcgac atcatcacgt 2160
cgttttacaa cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 2220
atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 2280
atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgctttgcc tggtttccgg 2340
caccagaagc ggtgccggaa agctggctgg agtgcgatct tcctgaggcc gatactgtcg 2400
tcgtcccctc aaactggcag atgcacggtt acgatgcgcc catctacacc aacgtgacct 2460
atcccattac ggtcaatccg ccgtttgttc ccacggagaa tccgacgggt tgttactcgc 2520
tcacatttaa tgttgatgaa agctggctac aggaaggcca gacgcgaatt atttttgatg 2580
gcgttaactc ggcgtttcat ctgtggtgca acgggcgctg ggtcggttac ggccaggaca 2640
gtcgtttgcc gtctgaattt gacctgagcg catttttacg cgccggagaa aaccgcctcg 2700
cggtgatggt gctgcgttgg agtgacggca gttatctgga agatcaggat atgtggcgga 2760
tgagcggcat tttccgtgac gtctcgttgc tgcataaacc gactacacaa atcagcgatt 2820
tccatgttgc cactcgcttt aatgatgatt tcagccgcgc tgtactggag gctgaagttc 2880
agatgtgcgg cgagttgcgt gactacctac gggtaacagt ttctttatgg cagggtgaaa 2940
cgcaggtcgc cagcggcacc gcgcctttcg gcggtgaaat tatcgatgag cgtggtggtt 3000
atgccgatcg cgtcacacta cgtctgaacg tcgaaaaccc gaaactgtgg agcgccgaaa 3060
tcccgaatct ctatcgtgcg gtggttgaac tgcacaccgc cgacggcacg ctgattgaag 3120
cagaagcctg cgatgtcggt ttccgcgagg tgcggattga aaatggtctg ctgctgctga 3180
acggcaagcc gttgctgatt cgaggcgtta accgtcacga gcatcatcct ctgcatggtc 3240
aggtcatgga tgagcagacg atggtgcagg atatcctgct gatgaagcag aacaacttta 3300
acgccgtgcg ctgttcgcat tatccgaacc atccgctgtg gtacacgctg tgcgaccgct 3360
acggcctgta tgtggtggat gaagccaata ttgaaaccca cggcatggtg ccaatgaatc 3420
gtctgaccga tgatccgcgc tggctaccgg cgatgagcga acgcgtaacg cgaatggtgc 3480
agcgcgatcg taatcacccg agtgtgatca tctggtcgct ggggaatgaa tcaggccacg 3540
gcgctaatca cgacgcgctg tatcgctgga tcaaatctgt cgatccttcc cgcccggtgc 3600
agtatgaagg cggcggagcc gacaccacgg ccaccgatat tatttgcccg atgtacgcgc 3660
gcgtggatga agaccagccc ttcccggctg tgccgaaatg gtccatcaaa aaatggcttt 3720
cgctacctgg agagacgcgc ccgctgatcc tttgcgaata cgcccacgcg atgggtaaca 3780
gtcttggcgg tttcgctaaa tactggcagg cgtttcgtca gtatccccgt ttacagggcg 3840
gcttcgtctg ggactgggtg gatcagtcgc tgattaaata tgatgaaaac ggcaacccgt 3900
ggtcggctta cggcggtgat tttggcgata cgccgaacga tcgccagttc tgtatgaacg 3960
gtctggtctt tgccgaccgc acgccgcatc cagcgctgac ggaagcaaaa caccagcagc 4020
agtttttcca gttccgttta tccgggcaaa ccatcgaagt gaccagcgaa tacctgttcc 4080
gtcatagcga taacgagctc ctgcactgga tggtggcgct ggatggtaag ccgctggcaa 4140
gcggtgaagt gcctctggat gtcgctccac aaggtaaaca gttgattgaa ctgcctgaac 4200
taccgcagcc ggagagcgcc gggcaactct ggctcacagt acgcgtagtg caaccgaacg 4260
cgaccgcatg gtcagaagcc gggcacatca gcgcctggca gcagtggcgt ctggcggaaa 4320
acctcagtgt gacgctcccc gccgcgtccc acgccatccc gcatctgacc accagcgaaa 4380
tggatttttg catcgagctg ggtaataagc gttggcaatt taaccgccag tcaggctttc 4440
tttcacagat gtggattggc gataaaaaac aactgctgac gccgctgcgc gatcagttca 4500
cccgtgcacc gctggataac gacattggcg taagtgaagc gacccgcatt gaccctaacg 4560
cctgggtcga acgctggaag gcggcgggcc attaccaggc cgaagcagcg ttgttgcagt 4620
gcacggcaga tacacttgct gatgcggtgc tgattacgac cgctcacgcg tggcagcatc 4680
aggggaaaac cttatttatc agccggaaaa cctaccggat tgatggtagt ggtcaaatgg 4740
cgattaccgt tgatgttgaa gtggcgagcg atacaccgca tccggcgcgg attggcctga 4800
actgccagct ggcgcaggta gcagagcggg taaactggct cggattaggg ccgcaagaaa 4860
actatcccga ccgccttact gccgcctgtt ttgaccgctg ggatctgcca ttgtcagaca 4920
tgtatacccc gtacgtcttc ccgagcgaaa acggtctgcg ctgcgggacg cgcgaattga 4980
attatggccc acaccagtgg cgcggcgact tccagttcaa catcagccgc tacagtcaac 5040
agcaactgat ggaaaccagc catcgccatc tgctgcacgc ggaagaaggc acatggctga 5100
atatcgacgg tttccatatg gggattggtg gcgacgactc ctggagcccg tcagtatcgg 5160
cggaattcca gctgagcgcc ggtcgctacc attaccagtt ggtctggtgt caaaaataat 5220
tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct 5280
gggattacac atggcatgga tgaactgtac aactgaggat cccccgacct cgacctctgg 5340
ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt gtctctcact 5400
cggaaggaca tatgggaggg caaatcattt ggtcgagatc cctcggagat cggatctggg 5460
cgtggttaag ggtgggaaag aatatataag gtgggggtct tatgtagttt tgtatctgtt 5520
ttgcagcagc cgccgccgcc atgagcacca actcgtttga tggaagcatt gtgagctcat 5580
atttgacaac gcgcatgccc ccatgggccg gggtgcgtca gaatgtgatg ggctccagca 5640
ttgatggtcg ccccgtcctg cccgcaaact ctactacctt gacctacgag accgtgtctg 5700
gaacgccgtt ggagactgca gcctccgccg ccgcttcagc cgctgcagcc accgcccgcg 5760
ggattgtgac tgactttgct ttcctgagcc cgcttgcaag cagtgcagct tcccgttcat 5820
ccgcccgcga tgacaagttg acggctcttt tggcacaatt ggattctttg acccgggaac 5880
ttaatgtcgt ttctcagcag ctgttggatc tgcgccagca ggtttctgcc ctgaaggctt 5940
cctcccctcc caatgcggtt taaaacataa ataaaaaacc agactctgtt tggatttgga 6000
tcaagcaagt gtcttgctgt ctttatttag gggttttgcg cgcgcggtag gcccgggacc 6060
agcggtctcg gtcgttgagg gtcctgtgta ttttttccag gacgtggtaa aggtgactct 6120
ggatgttcag atacatgggc ataagcccgt ctctggggtg gaggtagcac cactgcagag 6180
cttcatgctg cggggtggtg ttgtagatga tccagtcgta gcaggagcgc tgggcgtggt 6240
gcctaaaaat gtctttcagt agcaagctga ttgccagggg caggcccttg gtgtaagtgt 6300
ttacaaagcg gttaagctgg gatgggtgca tacgtgggga tatgagatgc atcttggact 6360
gtatttttag gttggctatg ttcccagcca tatccctccg gggattcatg ttgtgcagaa 6420
ccaccagcac agtgtatccg gtgcacttgg gaaatttgtc atgtagctta gaaggaaatg 6480
cgtggaagaa cttggagacg cccttgtgac ctccaagatt ttccatgcat tcgtccataa 6540
tgatggcaat gggcccacgg gcggcggcct gggcgaagat atttctggga tcactaacgt 6600
catagttgtg ttccaggatg agatcgtcat aggccatttt tacaaagcgc gggcggaggg 6660
tgccagactg cggtataatg gttccatccg gcccaggggc gtagttaccc tcacagattt 6720
gcatttccca cgctttgagt tcagatgggg ggatcatgtc tacctgcggg gcgatgaaga 6780
aaacggtttc cggggtaggg gagatcagct gggaagaaag caggttcctg agcagctgcg 6840
acttaccgca gccggtgggc ccgtaaatca cacctattac cgggtgcaac tggtagttaa 6900
gagagctgca gctgccgtca tccctgagca ggggggccac ttcgttaagc atgtccctga 6960
ctcgcatgtt ttccctgacc aaatccgcca gaaggcgctc gccgcccagc gatagcagtt 7020
cttgcaagga agcaaagttt ttcaacggtt tgagaccgtc cgccgtaggc atgcttttga 7080
gcgtttgacc aagcagttcc aggcggtccc acagctcggt cacctgctct acggcatctc 7140
gatccagcat atctcctcgt ttcgcgggtt ggggcggctt tcgctgtacg gcagtagtcg 7200
gtgctcgtcc agacgggcca gggtcatgtc tttccacggg cgcagggtcc tcgtcagcgt 7260
agtctgggtc acggtgaagg ggtgcgctcc gggctgcgcg ctggccaggg tgcgcttgag 7320
gctggtcctg ctggtgctga agcgctgccg gtcttcgccc tgcgcgtcgg ccaggtagca 7380
tttgaccatg gtgtcatagt ccagcccctc cgcggcgtgg cccttggcgc gcagcttgcc 7440
cttggaggag gcgccgcacg aggggcagtg cagacttttg agggcgtaga gcttgggcgc 7500
gagaaatacc gattccgggg agtaggcatc cgcgccgcag gccccgcaga cggtctcgca 7560
ttccacgagc caggtgagct ctggccgttc ggggtcaaaa accaggtttc ccccatgctt 7620
tttgatgcgt ttcttacctc tggtttccat gagccggtgt ccacgctcgg tgacgaaaag 7680
gctgtccgtg tccccgtata cagacttgag agggagttta aacgaattca atagcttgtt 7740
gcatgggcgg cgatataaaa tgcaaggtgc tgctcaaaaa atcaggcaaa gcctcgcgca 7800
aaaaagaaag cacatcgtag tcatgctcat gcagataaag gcaggtaagc tccggaacca 7860
ccacagaaaa agacaccatt tttctctcaa acatgtctgc gggtttctgc ataaacacaa 7920
aataaaataa caaaaaaaca tttaaacatt agaagcctgt cttacaacag gaaaaacaac 7980
ccttataagc ataagacgga ctacggccat gccggcgtga ccgtaaaaaa actggtcacc 8040
gtgattaaaa agcaccaccg acagctcctc ggtcatgtcc ggagtcataa tgtaagactc 8100
ggtaaacaca tcaggttgat tcatcggtca gtgctaaaaa gcgaccgaaa tagcccgggg 8160
gaatacatac ccgcaggcgt agagacaaca ttacagcccc cataggaggt ataacaaaat 8220
taataggaga gaaaaacaca taaacacctg aaaaaccctc ctgcctaggc aaaatagcac 8280
cctcccgctc cagaacaaca tacagcgctt cacagcggca gcctaacagt cagccttacc 8340
agtaaaaaag aaaacctatt aaaaaaacac cactcgacac ggcaccagct caatcagtca 8400
cagtgtaaaa aagggccaag tgcagagcga gtatatatag gactaaaaaa tgacgtaacg 8460
gttaaagtcc acaaaaaaca cccagaaaac cgcacgcgaa cctacgccca gaaacgaaag 8520
ccaaaaaacc cacaacttcc tcaaatcgtc acttccgttt tcccacgtta cgtaacttcc 8580
cattttaaga aaactacaat tcccaacaca tacaagttac tccgccctaa aacctacgtc 8640
acccgccccg ttcccacgcc ccgcgccacg tcacaaactc caccccctca ttatcatatt 8700
ggcttcaatc caaaataagg tatattattg atgatgttaa ttaacatgca tggatccata 8760
tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg 8820
cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 8880
actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 8940
gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 9000
ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 9060
acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 9120
ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 9180
cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 9240
tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 9300
gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 9360
ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 9420
acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 9480
gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 9540
ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 9600
tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 9660
gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 9720
tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 9780
ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 9840
taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 9900
cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 9960
gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 10020
gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct gcagccatga 10080
gattatcaaa aaggatcttc acctagatcc ttttcacgta gaaagccagt ccgcagaaac 10140
ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg 10200
caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt 10260
tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc 10320
cctgcaaagt aaactggatg gctttctcgc cgccaaggat ctgatggcgc aggggatcaa 10380
gctctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 10440
caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 10500
tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 10560
tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt 10620
ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 10680
gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 10740
ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 10800
ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 10860
aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 10920
aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg 10980
gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 11040
gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 11100
ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 11160
ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attttgttaa 11220
aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa catcccttat 11280
aaatcaaaag aatagaccgc gatagggttg agtgttgttc cagtttggaa caagagtcca 11340
ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 11400
ccactacgtg aaccatcacc caaatcaagt tttttgcggt cgaggtgccg taaagctcta 11460
aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 11520
gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 11580
gtcacgctgc gcgtaaccac cacacccgcg cgcttaatgc gccgctacag ggcgcgtcca 11640
ttcgccattc aggatcgaat taattcttaa t 11671
<210> 42
<211> 36428
<212> DNA
<213> Artificial Sequence
<220>
<223> Ad-AS300KRib1-TK
<400> 42
ttaagggtgg gaaagaatat ataaggtggg ggtcttatgt agttttgtat ctgttttgca 60
gcagccgccg ccgccatgag caccaactcg tttgatggaa gcattgtgag ctcatatttg 120
acaacgcgca tgcccccatg ggccggggtg cgtcagaatg tgatgggctc cagcattgat 180
ggtcgccccg tcctgcccgc aaactctact accttgacct acgagaccgt gtctggaacg 240
ccgttggaga ctgcagcctc cgccgccgct tcagccgctg cagccaccgc ccgcgggatt 300
gtgactgact ttgctttcct gagcccgctt gcaagcagtg cagcttcccg ttcatccgcc 360
cgcgatgaca agttgacggc tcttttggca caattggatt ctttgacccg ggaacttaat 420
gtcgtttctc agcagctgtt ggatctgcgc cagcaggttt ctgccctgaa ggcttcctcc 480
cctcccaatg cggtttaaaa cataaataaa aaaccagact ctgtttggat ttggatcaag 540
caagtgtctt gctgtcttta tttaggggtt ttgcgcgcgc ggtaggcccg ggaccagcgg 600
tctcggtcgt tgagggtcct gtgtattttt tccaggacgt ggtaaaggtg actctggatg 660
ttcagataca tgggcataag cccgtctctg gggtggaggt agcaccactg cagagcttca 720
tgctgcgggg tggtgttgta gatgatccag tcgtagcagg agcgctgggc gtggtgccta 780
aaaatgtctt tcagtagcaa gctgattgcc aggggcaggc ccttggtgta agtgtttaca 840
aagcggttaa gctgggatgg gtgcatacgt ggggatatga gatgcatctt ggactgtatt 900
tttaggttgg ctatgttccc agccatatcc ctccggggat tcatgttgtg cagaaccacc 960
agcacagtgt atccggtgca cttgggaaat ttgtcatgta gcttagaagg aaatgcgtgg 1020
aagaacttgg agacgccctt gtgacctcca agattttcca tgcattcgtc cataatgatg 1080
gcaatgggcc cacgggcggc ggcctgggcg aagatatttc tgggatcact aacgtcatag 1140
ttgtgttcca ggatgagatc gtcataggcc atttttacaa agcgcgggcg gagggtgcca 1200
gactgcggta taatggttcc atccggccca ggggcgtagt taccctcaca gatttgcatt 1260
tcccacgctt tgagttcaga tggggggatc atgtctacct gcggggcgat gaagaaaacg 1320
gtttccgggg taggggagat cagctgggaa gaaagcaggt tcctgagcag ctgcgactta 1380
ccgcagccgg tgggcccgta aatcacacct attaccgggt gcaactggta gttaagagag 1440
ctgcagctgc cgtcatccct gagcaggggg gccacttcgt taagcatgtc cctgactcgc 1500
atgttttccc tgaccaaatc cgccagaagg cgctcgccgc ccagcgatag cagttcttgc 1560
aaggaagcaa agtttttcaa cggtttgaga ccgtccgccg taggcatgct tttgagcgtt 1620
tgaccaagca gttccaggcg gtcccacagc tcggtcacct gctctacggc atctcgatcc 1680
agcatatctc ctcgtttcgc gggttggggc ggctttcgct gtacggcagt agtcggtgct 1740
cgtccagacg ggccagggtc atgtctttcc acgggcgcag ggtcctcgtc agcgtagtct 1800
gggtcacggt gaaggggtgc gctccgggct gcgcgctggc cagggtgcgc ttgaggctgg 1860
tcctgctggt gctgaagcgc tgccggtctt cgccctgcgc gtcggccagg tagcatttga 1920
ccatggtgtc atagtccagc ccctccgcgg cgtggccctt ggcgcgcagc ttgcccttgg 1980
aggaggcgcc gcacgagggg cagtgcagac ttttgagggc gtagagcttg ggcgcgagaa 2040
ataccgattc cggggagtag gcatccgcgc cgcaggcccc gcagacggtc tcgcattcca 2100
cgagccaggt gagctctggc cgttcggggt caaaaaccag gtttccccca tgctttttga 2160
tgcgtttctt acctctggtt tccatgagcc ggtgtccacg ctcggtgacg aaaaggctgt 2220
ccgtgtcccc gtatacagac ttgagaggcc tgtcctcgag cggtgttccg cggtcctcct 2280
cgtatagaaa ctcggaccac tctgagacaa aggctcgcgt ccaggccagc acgaaggagg 2340
ctaagtggga ggggtagcgg tcgttgtcca ctagggggtc cactcgctcc agggtgtgaa 2400
gacacatgtc gccctcttcg gcatcaagga aggtgattgg tttgtaggtg taggccacgt 2460
gaccgggtgt tcctgaaggg gggctataaa agggggtggg ggcgcgttcg tcctcactct 2520
cttccgcatc gctgtctgcg agggccagct gttggggtga gtactccctc tgaaaagcgg 2580
gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcacct 2640
ggcccgcggt gatgcctttg agggtggccg catccatctg gtcagaaaag acaatctttt 2700
tgttgtcaag cttggtggca aacgacccgt agagggcgtt ggacagcaac ttggcgatgg 2760
agcgcagggt ttggtttttg tcgcgatcgg cgcgctcctt ggccgcgatg tttagctgca 2820
cgtattcgcg cgcaacgcac cgccattcgg gaaagacggt ggtgcgctcg tcgggcacca 2880
ggtgcacgcg ccaaccgcgg ttgtgcaggg tgacaaggtc aacgctggtg gctacctctc 2940
cgcgtaggcg ctcgttggtc cagcagaggc ggccgccctt gcgcgagcag aatggcggta 3000
gggggtctag ctgcgtctcg tccggggggt ctgcgtccac ggtaaagacc ccgggcagca 3060
ggcgcgcgtc gaagtagtct atcttgcatc cttgcaagtc tagcgcctgc tgccatgcgc 3120
gggcggcaag cgcgcgctcg tatgggttga gtgggggacc ccatggcatg gggtgggtga 3180
gcgcggaggc gtacatgccg caaatgtcgt aaacgtagag gggctctctg agtattccaa 3240
gatatgtagg gtagcatctt ccaccgcgga tgctggcgcg cacgtaatcg tatagttcgt 3300
gcgagggagc gaggaggtcg ggaccgaggt tgctacgggc gggctgctct gctcggaaga 3360
ctatctgcct gaagatggca tgtgagttgg atgatatggt tggacgctgg aagacgttga 3420
agctggcgtc tgtgagacct accgcgtcac gcacgaagga ggcgtaggag tcgcgcagct 3480
tgttgaccag ctcggcggtg acctgcacgt ctagggcgca gtagtccagg gtttccttga 3540
tgatgtcata cttatcctgt cccttttttt tccacagctc gcggttgagg acaaactctt 3600
cgcggtcttt ccagtactct tggatcggaa acccgtcggc ctccgaacgg taagagccta 3660
gcatgtagaa ctggttgacg gcctggtagg cgcagcatcc cttttctacg ggtagcgcgt 3720
atgcctgcgc ggccttccgg agcgaggtgt gggtgagcgc aaaggtgtcc ctgaccatga 3780
ctttgaggta ctggtatttg aagtcagtgt cgtcgcatcc gccctgctcc cagagcaaaa 3840
agtccgtgcg ctttttggaa cgcggatttg gcagggcgaa ggtgacatcg ttgaagagta 3900
tctttcccgc gcgaggcata aagttgcgtg tgatgcggaa gggtcccggc acctcggaac 3960
ggttgttaat tacctgggcg gcgagcacga tctcgtcaaa gccgttgatg ttgtggccca 4020
caatgtaaag ttccaagaag cgcgggatgc ccttgatgga aggcaatttt ttaagttcct 4080
cgtaggtgag ctcttcaggg gagctgagcc cgtgctctga aagggcccag tctgcaagat 4140
gagggttgga agcgacgaat gagctccaca ggtcacgggc cattagcatt tgcaggtggt 4200
cgcgaaaggt cctaaactgg cgacctatgg ccattttttc tggggtgatg cagtagaagg 4260
taagcgggtc ttgttcccag cggtcccatc caaggttcgc ggctaggtct cgcgcggcag 4320
tcactagagg ctcatctccg ccgaacttca tgaccagcat gaagggcacg agctgcttcc 4380
caaaggcccc catccaagta taggtctcta catcgtaggt gacaaagaga cgctcggtgc 4440
gaggatgcga gccgatcggg aagaactgga tctcccgcca ccaattggag gagtggctat 4500
tgatgtggtg aaagtagaag tccctgcgac gggccgaaca ctcgtgctgg cttttgtaaa 4560
aacgtgcgca gtactggcag cggtgcacgg gctgtacatc ctgcacgagg ttgacctgac 4620
gaccgcgcac aaggaagcag agtgggaatt tgagcccctc gcctggcggg tttggctggt 4680
ggtcttctac ttcggctgct tgtccttgac cgtctggctg ctcgagggga gttacggtgg 4740
atcggaccac cacgccgcgc gagcccaaag tccagatgtc cgcgcgcggc ggtcggagct 4800
tgatgacaac atcgcgcaga tgggagctgt ccatggtctg gagctcccgc ggcgtcaggt 4860
caggcgggag ctcctgcagg tttacctcgc atagacgggt cagggcgcgg gctagatcca 4920
ggtgatacct aatttccagg ggctggttgg tggcggcgtc gatggcttgc aagaggccgc 4980
atccccgcgg cgcgactacg gtaccgcgcg gcgggcggtg ggccgcgggg gtgtccttgg 5040
atgatgcatc taaaagcggt gacgcgggcg agcccccgga ggtagggggg gctccggacc 5100
cgccgggaga gggggcaggg gcacgtcggc gccgcgcgcg ggcaggagct ggtgctgcgc 5160
gcgtaggttg ctggcgaacg cgacgacgcg gcggttgatc tcctgaatct ggcgcctctg 5220
cgtgaagacg acgggcccgg tgagcttgag cctgaaagag agttcgacag aatcaatttc 5280
ggtgtcgttg acggcggcct ggcgcaaaat ctcctgcacg tctcctgagt tgtcttgata 5340
ggcgatctcg gccatgaact gctcgatctc ttcctcctgg agatctccgc gtccggctcg 5400
ctccacggtg gcggcgaggt cgttggaaat gcgggccatg agctgcgaga aggcgttgag 5460
gcctccctcg ttccagacgc ggctgtagac cacgccccct tcggcatcgc gggcgcgcat 5520
gaccacctgc gcgagattga gctccacgtg ccgggcgaag acggcgtagt ttcgcaggcg 5580
ctgaaagagg tagttgaggg tggtggcggt gtgttctgcc acgaagaagt acataaccca 5640
gcgtcgcaac gtggattcgt tgatatcccc caaggcctca aggcgctcca tggcctcgta 5700
gaagtccacg gcgaagttga aaaactggga gttgcgcgcc gacacggtta actcctcctc 5760
cagaagacgg atgagctcgg cgacagtgtc gcgcacctcg cgctcaaagg ctacaggggc 5820
ctcttcttct tcttcaatct cctcttccat aagggcctcc ccttcttctt cttctggcgg 5880
cggtggggga ggggggacac ggcggcgacg acggcgcacc gggaggcggt cgacaaagcg 5940
ctcgatcatc tccccgcggc gacggcgcat ggtctcggtg acggcgcggc cgttctcgcg 6000
ggggcgcagt tggaagacgc cgcccgtcat gtcccggtta tgggttggcg gggggctgcc 6060
atgcggcagg gatacggcgc taacgatgca tctcaacaat tgttgtgtag gtactccgcc 6120
gccgagggac ctgagcgagt ccgcatcgac cggatcggaa aacctctcga gaaaggcgtc 6180
taaccagtca cagtcgcaag gtaggctgag caccgtggcg ggcggcagcg ggcggcggtc 6240
ggggttgttt ctggcggagg tgctgctgat gatgtaatta aagtaggcgg tcttgagacg 6300
gcggatggtc gacagaagca ccatgtcctt gggtccggcc tgctgaatgc gcaggcggtc 6360
ggccatgccc caggcttcgt tttgacatcg gcgcaggtct ttgtagtagt cttgcatgag 6420
cctttctacc ggcacttctt cttctccttc ctcttgtcct gcatctcttg catctatcgc 6480
tgcggcggcg gcggagtttg gccgtaggtg gcgccctctt cctcccatgc gtgtgacccc 6540
gaagcccctc atcggctgaa gcagggctag gtcggcgaca acgcgctcgg ctaatatggc 6600
ctgctgcacc tgcgtgaggg tagactggaa gtcatccatg tccacaaagc ggtggtatgc 6660
gcccgtgttg atggtgtaag tgcagttggc cataacggac cagttaacgg tctggtgacc 6720
cggctgcgag agctcggtgt acctgagacg cgagtaagcc ctcgagtcaa atacgtagtc 6780
gttgcaagtc cgcaccaggt actggtatcc caccaaaaag tgcggcggcg gctggcggta 6840
gaggggccag cgtagggtgg ccggggctcc gggggcgaga tcttccaaca taaggcgatg 6900
atatccgtag atgtacctgg acatccaggt gatgccggcg gcggtggtgg aggcgcgcgg 6960
aaagtcgcgg acgcggttcc agatgttgcg cagcggcaaa aagtgctcca tggtcgggac 7020
gctctggccg gtcaggcgcg cgcaatcgtt gacgctctac cgtgcaaaag gagagcctgt 7080
aagcgggcac tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc 7140
ggggttcgag ccccgtatcc ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc 7200
gaacccaggt gtgcgacgtc agacaacggg ggagtgctcc ttttggcttc cttccaggcg 7260
cggcggctgc tgcgctagct tttttggcca ctggccgcgc gcagcgtaag cggttaggct 7320
ggaaagcgaa agcattaagt ggctcgctcc ctgtagccgg agggttattt tccaagggtt 7380
gagtcgcggg acccccggtt cgagtctcgg accggccgga ctgcggcgaa cgggggtttg 7440
cctccccgtc atgcaagacc ccgcttgcaa attcctccgg aaacagggac gagccccttt 7500
tttgcttttc ccagatgcat ccggtgctgc ggcagatgcg cccccctcct cagcagcggc 7560
aagagcaaga gcagcggcag acatgcaggg caccctcccc tcctcctacc gcgtcaggag 7620
gggcgacatc cgcggttgac gcggcagcag atggtgatta cgaacccccg cggcgccggg 7680
cccggcacta cctggacttg gaggagggcg agggcctggc gcggctagga gcgccctctc 7740
ctgagcggta cccaagggtg cagctgaagc gtgatacgcg tgaggcgtac gtgccgcggc 7800
agaacctgtt tcgcgaccgc gagggagagg agcccgagga gatgcgggat cgaaagttcc 7860
acgcagggcg cgagctgcgg catggcctga atcgcgagcg gttgctgcgc gaggaggact 7920
ttgagcccga cgcgcgaacc gggattagtc ccgcgcgcgc acacgtggcg gccgccgacc 7980
tggtaaccgc atacgagcag acggtgaacc aggagattaa ctttcaaaaa agctttaaca 8040
accacgtgcg tacgcttgtg gcgcgcgagg aggtggctat aggactgatg catctgtggg 8100
actttgtaag cgcgctggag caaaacccaa atagcaagcc gctcatggcg cagctgttcc 8160
ttatagtgca gcacagcagg gacaacgagg cattcaggga tgcgctgcta aacatagtag 8220
agcccgaggg ccgctggctg ctcgatttga taaacatcct gcagagcata gtggtgcagg 8280
agcgcagctt gagcctggct gacaaggtgg ccgccatcaa ctattccatg cttagcctgg 8340
gcaagtttta cgcccgcaag atataccata ccccttacgt tcccatagac aaggaggtaa 8400
agatcgaggg gttctacatg cgcatggcgc tgaaggtgct taccttgagc gacgacctgg 8460
gcgtttatcg caacgagcgc atccacaagg ccgtgagcgt gagccggcgg cgcgagctca 8520
gcgaccgcga gctgatgcac agcctgcaaa gggccctggc tggcacgggc agcggcgata 8580
gagaggccga gtcctacttt gacgcgggcg ctgacctgcg ctgggcccca agccgacgcg 8640
ccctggaggc agctggggcc ggacctgggc tggcggtggc acccgcgcgc gctggcaacg 8700
tcggcggcgt ggaggaatat gacgaggacg atgagtacga gccagaggac ggcgagtact 8760
aagcggtgat gtttctgatc agatgatgca agacgcaacg gacccggcgg tgcgggcggc 8820
gctgcagagc cagccgtccg gccttaactc cacggacgac tggcgccagg tcatggaccg 8880
catcatgtcg ctgactgcgc gcaatcctga cgcgttccgg cagcagccgc aggccaaccg 8940
gctctccgca attctggaag cggtggtccc ggcgcgcgca aaccccacgc acgagaaggt 9000
gctggcgatc gtaaacgcgc tggccgaaaa cagggccatc cggcccgacg aggccggcct 9060
ggtctacgac gcgctgcttc agcgcgtggc tcgttacaac agcggcaacg tgcagaccaa 9120
cctggaccgg ctggtggggg atgtgcgcga ggccgtggcg cagcgtgagc gcgcgcagca 9180
gcagggcaac ctgggctcca tggttgcact aaacgccttc ctgagtacac agcccgccaa 9240
cgtgccgcgg ggacaggagg actacaccaa ctttgtgagc gcactgcggc taatggtgac 9300
tgagacaccg caaagtgagg tgtaccagtc tgggccagac tattttttcc agaccagtag 9360
acaaggcctg cagaccgtaa acctgagcca ggctttcaaa aacttgcagg ggctgtgggg 9420
ggtgcgggct cccacaggcg accgcgcgac cgtgtctagc ttgctgacgc ccaactcgcg 9480
cctgttgctg ctgctaatag cgcccttcac ggacagtggc agcgtgtccc gggacacata 9540
cctaggtcac ttgctgacac tgtaccgcga ggccataggt caggcgcatg tggacgagca 9600
tactttccag gagattacaa gtgtcagccg cgcgctgggg caggaggaca cgggcagcct 9660
ggaggcaacc ctaaactacc tgctgaccaa ccggcggcag aagatcccct cgttgcacag 9720
tttaaacagc gaggaggagc gcattttgcg ctacgtgcag cagagcgtga gccttaacct 9780
gatgcgcgac ggggtaacgc ccagcgtggc gctggacatg accgcgcgca acatggaacc 9840
gggcatgtat gcctcaaacc ggccgtttat caaccgccta atggactact tgcatcgcgc 9900
ggccgccgtg aaccccgagt atttcaccaa tgccatcttg aacccgcact ggctaccgcc 9960
ccctggtttc tacaccgggg gattcgaggt gcccgagggt aacgatggat tcctctggga 10020
cgacatagac gacagcgtgt tttccccgca accgcagacc ctgctagagt tgcaacagcg 10080
cgagcaggca gaggcggcgc tgcgaaagga aagcttccgc aggccaagca gcttgtccga 10140
tctaggcgct gcggccccgc ggtcagatgc tagtagccca tttccaagct tgatagggtc 10200
tcttaccagc actcgcacca cccgcccgcg cctgctgggc gaggaggagt acctaaacaa 10260
ctcgctgctg cagccgcagc gcgaaaaaaa cctgcctccg gcatttccca acaacgggat 10320
agagagccta gtggacaaga tgagtagatg gaagacgtac gcgcaggagc acagggacgt 10380
gccaggcccg cgcccgccca cccgtcgtca aaggcacgac cgtcagcggg gtctggtgtg 10440
ggaggacgat gactcggcag acgacagcag cgtcctggat ttgggaggga gtggcaaccc 10500
gtttgcgcac cttcgcccca ggctggggag aatgttttaa aaaaaaaaaa gcatgatgca 10560
aaataaaaaa ctcaccaagg ccatggcacc gagcgttggt tttcttgtat tccccttagt 10620
atgcggcgcg cggcgatgta tgaggaaggt cctcctccct cctacgagag tgtggtgagc 10680
gcggcgccag tggcggcggc gctgggttct cccttcgatg ctcccctgga cccgccgttt 10740
gtgcctccgc ggtacctgcg gcctaccggg gggagaaaca gcatccgtta ctctgagttg 10800
gcacccctat tcgacaccac ccgtgtgtac ctggtggaca acaagtcaac ggatgtggca 10860
tccctgaact accagaacga ccacagcaac tttctgacca cggtcattca aaacaatgac 10920
tacagcccgg gggaggcaag cacacagacc atcaatcttg acgaccggtc gcactggggc 10980
ggcgacctga aaaccatcct gcataccaac atgccaaatg tgaacgagtt catgtttacc 11040
aataagttta aggcgcgggt gatggtgtcg cgcttgccta ctaaggacaa tcaggtggag 11100
ctgaaatacg agtgggtgga gttcacgctg cccgagggca actactccga gaccatgacc 11160
atagacctta tgaacaacgc gatcgtggag cactacttga aagtgggcag acagaacggg 11220
gttctggaaa gcgacatcgg ggtaaagttt gacacccgca acttcagact ggggtttgac 11280
cccgtcactg gtcttgtcat gcctggggta tatacaaacg aagccttcca tccagacatc 11340
attttgctgc caggatgcgg ggtggacttc acccacagcc gcctgagcaa cttgttgggc 11400
atccgcaagc ggcaaccctt ccaggagggc tttaggatca cctacgatga tctggagggt 11460
ggtaacattc ccgcactgtt ggatgtggac gcctaccagg cgagcttgaa agatgacacc 11520
gaacagggcg ggggtggcgc aggcggcagc aacagcagtg gcagcggcgc ggaagagaac 11580
tccaacgcgg cagccgcggc aatgcagccg gtggaggaca tgaacgatca tgccattcgc 11640
ggcgacacct ttgccacacg ggctgaggag aagcgcgctg aggccgaagc agcggccgaa 11700
gctgccgccc ccgctgcgca acccgaggtc gagaagcctc agaagaaacc ggtgatcaaa 11760
cccctgacag aggacagcaa gaaacgcagt tacaacctaa taagcaatga cagcaccttc 11820
acccagtacc gcagctggta ccttgcatac aactacggcg accctcagac cggaatccgc 11880
tcatggaccc tgctttgcac tcctgacgta acctgcggct cggagcaggt ctactggtcg 11940
ttgccagaca tgatgcaaga ccccgtgacc ttccgctcca cgcgccagat cagcaacttt 12000
ccggtggtgg gcgccgagct gttgcccgtg cactccaaga gcttctacaa cgaccaggcc 12060
gtctactccc aactcatccg ccagtttacc tctctgaccc acgtgttcaa tcgctttccc 12120
gagaaccaga ttttggcgcg cccgccagcc cccaccatca ccaccgtcag tgaaaacgtt 12180
cctgctctca cagatcacgg gacgctaccg ctgcgcaaca gcatcggagg agtccagcga 12240
gtgaccatta ctgacgccag acgccgcacc tgcccctacg tttacaaggc cctgggcata 12300
gtctcgccgc gcgtcctatc gagccgcact ttttgagcaa gcatgtccat ccttatatcg 12360
cccagcaata acacaggctg gggcctgcgc ttcccaagca agatgtttgg cggggccaag 12420
aagcgctccg accaacaccc agtgcgcgtg cgcgggcact accgcgcgcc ctggggcgcg 12480
cacaaacgcg gccgcactgg gcgcaccacc gtcgatgacg ccatcgacgc ggtggtggag 12540
gaggcgcgca actacacgcc cacgccgcca ccagtgtcca cagtggacgc ggccattcag 12600
accgtggtgc gcggagcccg gcgctatgct aaaatgaaga gacggcggag gcgcgtagca 12660
cgtcgccacc gccgccgacc cggcactgcc gcccaacgcg cggcggcggc cctgcttaac 12720
cgcgcacgtc gcaccggccg acgggcggcc atgcgggccg ctcgaaggct ggccgcgggt 12780
attgtcactg tgccccccag gtccaggcga cgagcggccg ccgcagcagc cgcggccatt 12840
agtgctatga ctcagggtcg caggggcaac gtgtattggg tgcgcgactc ggttagcggc 12900
ctgcgcgtgc ccgtgcgcac ccgccccccg cgcaactaga ttgcaagaaa aaactactta 12960
gactcgtact gttgtatgta tccagcggcg gcggcgcgca acgaagctat gtccaagcgc 13020
aaaatcaaag aagagatgct ccaggtcatc gcgccggaga tctatggccc cccgaagaag 13080
gaagagcagg attacaagcc ccgaaagcta aagcgggtca aaaagaaaaa gaaagatgat 13140
gatgatgaac ttgacgacga ggtggaactg ctgcacgcta ccgcgcccag gcgacgggta 13200
cagtggaaag gtcgacgcgt aaaacgtgtt ttgcgacccg gcaccaccgt agtctttacg 13260
cccggtgagc gctccacccg cacctacaag cgcgtgtatg atgaggtgta cggcgacgag 13320
gacctgcttg agcaggccaa cgagcgcctc ggggagtttg cctacggaaa gcggcataag 13380
gacatgctgg cgttgccgct ggacgagggc aacccaacac ctagcctaaa gcccgtaaca 13440
ctgcagcagg tgctgcccgc gcttgcaccg tccgaagaaa agcgcggcct aaagcgcgag 13500
tctggtgact tggcacccac cgtgcagctg atggtaccca agcgccagcg actggaagat 13560
gtcttggaaa aaatgaccgt ggaacctggg ctggagcccg aggtccgcgt gcggccaatc 13620
aagcaggtgg cgccgggact gggcgtgcag accgtggacg ttcagatacc cactaccagt 13680
agcaccagta ttgccaccgc cacagagggc atggagacac aaacgtcccc ggttgcctca 13740
gcggtggcgg atgccgcggt gcaggcggtc gctgcggccg cgtccaagac ctctacggag 13800
gtgcaaacgg acccgtggat gtttcgcgtt tcagcccccc ggcgcccgcg cggttcgagg 13860
aagtacggcg ccgccagcgc gctactgccc gaatatgccc tacatccttc cattgcgcct 13920
acccccggct atcgtggcta cacctaccgc cccagaagac gagcaactac ccgacgccga 13980
accaccactg gaacccgccg ccgccgtcgc cgtcgccagc ccgtgctggc cccgatttcc 14040
gtgcgcaggg tggctcgcga aggaggcagg accctggtgc tgccaacagc gcgctaccac 14100
cccagcatcg tttaaaagcc ggtctttgtg gttcttgcag atatggccct cacctgccgc 14160
ctccgtttcc cggtgccggg attccgagga agaatgcacc gtaggagggg catggccggc 14220
cacggcctga cgggcggcat gcgtcgtgcg caccaccggc ggcggcgcgc gtcgcaccgt 14280
cgcatgcgcg gcggtatcct gcccctcctt attccactga tcgccgcggc gattggcgcc 14340
gtgcccggaa ttgcatccgt ggccttgcag gcgcagagac actgattaaa aacaagttgc 14400
atgtggaaaa atcaaaataa aaagtctgga ctctcacgct cgcttggtcc tgtaactatt 14460
ttgtagaatg gaagacatca actttgcgtc tctggccccg cgacacggct cgcgcccgtt 14520
catgggaaac tggcaagata tcggcaccag caatatgagc ggtggcgcct tcagctgggg 14580
ctcgctgtgg agcggcatta aaaatttcgg ttccaccgtt aagaactatg gcagcaaggc 14640
ctggaacagc agcacaggcc agatgctgag ggataagttg aaagagcaaa atttccaaca 14700
aaaggtggta gatggcctgg cctctggcat tagcggggtg gtggacctgg ccaaccaggc 14760
agtgcaaaat aagattaaca gtaagcttga tccccgccct cccgtagagg agcctccacc 14820
ggccgtggag acagtgtctc cagaggggcg tggcgaaaag cgtccgcgcc ccgacaggga 14880
agaaactctg gtgacgcaaa tagacgagcc tccctcgtac gaggaggcac taaagcaagg 14940
cctgcccacc acccgtccca tcgcgcccat ggctaccgga gtgctgggcc agcacacacc 15000
cgtaacgctg gacctgcctc cccccgccga cacccagcag aaacctgtgc tgccaggccc 15060
gaccgccgtt gttgtaaccc gtcctagccg cgcgtccctg cgccgcgccg ccagcggtcc 15120
gcgatcgttg cggcccgtag ccagtggcaa ctggcaaagc acactgaaca gcatcgtggg 15180
tctgggggtg caatccctga agcgccgacg atgcttctga atagctaacg tgtcgtatgt 15240
gtgtcatgta tgcgtccatg tcgccgccag aggagctgct gagccgccgc gcgcccgctt 15300
tccaagatgg ctaccccttc gatgatgccg cagtggtctt acatgcacat ctcgggccag 15360
gacgcctcgg agtacctgag ccccgggctg gtgcagtttg cccgcgccac cgagacgtac 15420
ttcagcctga ataacaagtt tagaaacccc acggtggcgc ctacgcacga cgtgaccaca 15480
gaccggtccc agcgtttgac gctgcggttc atccctgtgg accgtgagga tactgcgtac 15540
tcgtacaagg cgcggttcac cctagctgtg ggtgataacc gtgtgctgga catggcttcc 15600
acgtactttg acatccgcgg cgtgctggac aggggcccta cttttaagcc ctactctggc 15660
actgcctaca acgccctggc tcccaagggt gccccaaatc cttgcgaatg ggatgaagct 15720
gctactgctc ttgaaataaa cctagaagaa gaggacgatg acaacgaaga cgaagtagac 15780
gagcaagctg agcagcaaaa aactcacgta tttgggcagg cgccttattc tggtataaat 15840
attacaaagg agggtattca aataggtgtc gaaggtcaaa cacctaaata tgccgataaa 15900
acatttcaac ctgaacctca aataggagaa tctcagtggt acgaaactga aattaatcat 15960
gcagctggga gagtccttaa aaagactacc ccaatgaaac catgttacgg ttcatatgca 16020
aaacccacaa atgaaaatgg agggcaaggc attcttgtaa agcaacaaaa tggaaagcta 16080
gaaagtcaag tggaaatgca atttttctca actactgagg cgaccgcagg caatggtgat 16140
aacttgactc ctaaagtggt attgtacagt gaagatgtag atatagaaac cccagacact 16200
catatttctt acatgcccac tattaaggaa ggtaactcac gagaactaat gggccaacaa 16260
tctatgccca acaggcctaa ttacattgct tttagggaca attttattgg tctaatgtat 16320
tacaacagca cgggtaatat gggtgttctg gcgggccaag catcgcagtt gaatgctgtt 16380
gtagatttgc aagacagaaa cacagagctt tcataccagc ttttgcttga ttccattggt 16440
gatagaacca ggtacttttc tatgtggaat caggctgttg acagctatga tccagatgtt 16500
agaattattg aaaatcatgg aactgaagat gaacttccaa attactgctt tccactggga 16560
ggtgtgatta atacagagac tcttaccaag gtaaaaccta aaacaggtca ggaaaatgga 16620
tgggaaaaag atgctacaga attttcagat aaaaatgaaa taagagttgg aaataatttt 16680
gccatggaaa tcaatctaaa tgccaacctg tggagaaatt tcctgtactc caacatagcg 16740
ctgtatttgc ccgacaagct aaagtacagt ccttccaacg taaaaatttc tgataaccca 16800
aacacctacg actacatgaa caagcgagtg gtggctcccg ggttagtgga ctgctacatt 16860
aaccttggag cacgctggtc ccttgactat atggacaacg tcaacccatt taaccaccac 16920
cgcaatgctg gcctgcgcta ccgctcaatg ttgctgggca atggtcgcta tgtgcccttc 16980
cacatccagg tgcctcagaa gttctttgcc attaaaaacc tccttctcct gccgggctca 17040
tacacctacg agtggaactt caggaaggat gttaacatgg ttctgcagag ctccctagga 17100
aatgacctaa gggttgacgg agccagcatt aagtttgata gcatttgcct ttacgccacc 17160
ttcttcccca tggcccacaa caccgcctcc acgcttgagg ccatgcttag aaacgacacc 17220
aacgaccagt cctttaacga ctatctctcc gccgccaaca tgctctaccc tatacccgcc 17280
aacgctacca acgtgcccat atccatcccc tcccgcaact gggcggcttt ccgcggctgg 17340
gccttcacgc gccttaagac taaggaaacc ccatcactgg gctcgggcta cgacccttat 17400
tacacctact ctggctctat accctaccta gatggaacct tttacctcaa ccacaccttt 17460
aagaaggtgg ccattacctt tgactcttct gtcagctggc ctggcaatga ccgcctgctt 17520
acccccaacg agtttgaaat taagcgctca gttgacgggg agggttacaa cgttgcccag 17580
tgtaacatga ccaaagactg gttcctggta caaatgctag ctaactacaa cattggctac 17640
cagggcttct atatcccaga gagctacaag gaccgcatgt actccttctt tagaaacttc 17700
cagcccatga gccgtcaggt ggtggatgat actaaataca aggactacca acaggtgggc 17760
atcctacacc aacacaacaa ctctggattt gttggctacc ttgcccccac catgcgcgaa 17820
ggacaggcct accctgctaa cttcccctat ccgcttatag gcaagaccgc agttgacagc 17880
attacccaga aaaagtttct ttgcgatcgc accctttggc gcatcccatt ctccagtaac 17940
tttatgtcca tgggcgcact cacagacctg ggccaaaacc ttctctacgc caactccgcc 18000
cacgcgctag acatgacttt tgaggtggat cccatggacg agcccaccct tctttatgtt 18060
ttgtttgaag tctttgacgt ggtccgtgtg caccggccgc accgcggcgt catcgaaacc 18120
gtgtacctgc gcacgccctt ctcggccggc aacgccacaa cataaagaag caagcaacat 18180
caacaacagc tgccgccatg ggctccagtg agcaggaact gaaagccatt gtcaaagatc 18240
ttggttgtgg gccatatttt ttgggcacct atgacaagcg ctttccaggc tttgtttctc 18300
cacacaagct cgcctgcgcc atagtcaata cggccggtcg cgagactggg ggcgtacact 18360
ggatggcctt tgcctggaac ccgcactcaa aaacatgcta cctctttgag ccctttggct 18420
tttctgacca gcgactcaag caggtttacc agtttgagta cgagtcactc ctgcgccgta 18480
gcgccattgc ttcttccccc gaccgctgta taacgctgga aaagtccacc caaagcgtac 18540
aggggcccaa ctcggccgcc tgtggactat tctgctgcat gtttctccac gcctttgcca 18600
actggcccca aactcccatg gatcacaacc ccaccatgaa ccttattacc ggggtaccca 18660
actccatgct caacagtccc caggtacagc ccaccctgcg tcgcaaccag gaacagctct 18720
acagcttcct ggagcgccac tcgccctact tccgcagcca cagtgcgcag attaggagcg 18780
ccacttcttt ttgtcacttg aaaaacatgt aaaaataatg tactagagac actttcaata 18840
aaggcaaatg cttttatttg tacactctcg ggtgattatt tacccccacc cttgccgtct 18900
gcgccgttta aaaatcaaag gggttctgcc gcgcatcgct atgcgccact ggcagggaca 18960
cgttgcgata ctggtgttta gtgctccact taaactcagg cacaaccatc cgcggcagct 19020
cggtgaagtt ttcactccac aggctgcgca ccatcaccaa cgcgtttagc aggtcgggcg 19080
ccgatatctt gaagtcgcag ttggggcctc cgccctgcgc gcgcgagttg cgatacacag 19140
ggttgcagca ctggaacact atcagcgccg ggtggtgcac gctggccagc acgctcttgt 19200
cggagatcag atccgcgtcc aggtcctccg cgttgctcag ggcgaacgga gtcaactttg 19260
gtagctgcct tcccaaaaag ggcgcgtgcc caggctttga gttgcactcg caccgtagtg 19320
gcatcaaaag gtgaccgtgc ccggtctggg cgttaggata cagcgcctgc ataaaagcct 19380
tgatctgctt aaaagccacc tgagcctttg cgccttcaga gaagaacatg ccgcaagact 19440
tgccggaaaa ctgattggcc ggacaggccg cgtcgtgcac gcagcacctt gcgtcggtgt 19500
tggagatctg caccacattt cggccccacc ggttcttcac gatcttggcc ttgctagact 19560
gctccttcag cgcgcgctgc ccgttttcgc tcgtcacatc catttcaatc acgtgctcct 19620
tatttatcat aatgcttccg tgtagacact taagctcgcc ttcgatctca gcgcagcggt 19680
gcagccacaa cgcgcagccc gtgggctcgt gatgcttgta ggtcacctct gcaaacgact 19740
gcaggtacgc ctgcaggaat cgccccatca tcgtcacaaa ggtcttgttg ctggtgaagg 19800
tcagctgcaa cccgcggtgc tcctcgttca gccaggtctt gcatacggcc gccagagctt 19860
ccacttggtc aggcagtagt ttgaagttcg cctttagatc gttatccacg tggtacttgt 19920
ccatcagcgc gcgcgcagcc tccatgccct tctcccacgc agacacgatc ggcacactca 19980
gcgggttcat caccgtaatt tcactttccg cttcgctggg ctcttcctct tcctcttgcg 20040
tccgcatacc acgcgccact gggtcgtctt cattcagccg ccgcactgtg cgcttacctc 20100
ctttgccatg cttgattagc accggtgggt tgctgaaacc caccatttgt agcgccacat 20160
cttctctttc ttcctcgctg tccacgatta cctctggtga tggcgggcgc tcgggcttgg 20220
gagaagggcg cttctttttc ttcttgggcg caatggccaa atccgccgcc gaggtcgatg 20280
gccgcgggct gggtgtgcgc ggcaccagcg cgtcttgtga tgagtcttcc tcgtcctcgg 20340
actcgatacg ccgcctcatc cgcttttttg ggggcgcccg gggaggcggc ggcgacgggg 20400
acggggacga cacgtcctcc atggttgggg gacgtcgcgc cgcaccgcgt ccgcgctcgg 20460
gggtggtttc gcgctgctcc tcttcccgac tggccatttc cttctcctat aggcagaaaa 20520
agatcatgga gtcagtcgag aagaaggaca gcctaaccgc cccctctgag ttcgccacca 20580
ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca cccccgcttg 20640
aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac gacgaggacc 20700
gctcagtacc aacagaggat aaaaagcaag accaggacaa cgcagaggca aacgaggaac 20760
aagtcgggcg gggggacgaa aggcatggcg actacctaga tgtgggagac gacgtgctgt 20820
tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag cgcagcgatg 20880
tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctattc tcaccgcgcg 20940
taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc aacttctacc 21000
ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa aactgcaaga 21060
tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc ttgcggcagg 21120
gcgctgtcat acctgatatc gcctcgctca acgaagtgcc aaaaatcttt gagggtcttg 21180
gacgcgacga gaagcgcgcg gcaaacgctc tgcaacagga aaacagcgaa aatgaaagtc 21240
actctggagt gttggtggaa ctcgagggtg acaacgcgcg cctagccgta ctaaaacgca 21300
gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag gtcatgagca 21360
cagtcatgag tgagctgatc gtgcgccgtg cgcagcccct ggagagggat gcaaatttgc 21420
aagaacaaac agaggagggc ctacccgcag ttggcgacga gcagctagcg cgctggcttc 21480
aaacgcgcga gcctgccgac ttggaggagc gacgcaaact aatgatggcc gcagtgctcg 21540
ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg cagcgcaagc 21600
tagaggaaac attgcactac acctttcgac agggctacgt acgccaggcc tgcaagatct 21660
ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa aaccgccttg 21720
ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac gtccgcgact 21780
gcgtttactt atttctatgc tacacctggc agacggccat gggcgtttgg cagcagtgct 21840
tggaggagtg caacctcaag gagctgcaga aactgctaaa gcaaaacttg aaggacctat 21900
ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacatcatt ttccccgaac 21960
gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc atgttgcaga 22020
actttaggaa ctttatccta gagcgctcag gaatcttgcc cgccacctgc tgtgcacttc 22080
ctagcgactt tgtgcccatt aagtaccgcg aatgccctcc gccgctttgg ggccactgct 22140
accttctgca gctagccaac taccttgcct accactctga cataatggaa gacgtgagcg 22200
gtgacggtct actggagtgt cactgtcgct gcaacctatg caccccgcac cgctccctgg 22260
tttgcaattc gcagctgctt aacgaaagtc aaattatcgg tacctttgag ctgcagggtc 22320
cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg ctgtggacgt 22380
cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt aggttctacg 22440
aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc cagggccaca 22500
ttcttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta cgaaagggac 22560
ggggggttta cttggacccc cagtccggcg aggagctcaa cccaatcccc ccgccgccgc 22620
agccctatca gcagcagccg cgggcccttg cttcccagga tggcacccaa aaagaagctg 22680
cagctgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc agaggaggtt 22740
ttggacgagg aggaggagga catgatggaa gactgggaga gcctagacga ggaagcttcc 22800
gaggtcgaag aggtgtcaga cgaaacaccg tcaccctcgg tcgcattccc ctcgccggcg 22860
ccccagaaat cggcaaccgg ttccagcatg gctacaacct ccgctcctca ggcgccgccg 22920
gcactgcccg ttcgccgacc caaccgtaga tgggacacca ctggaaccag ggccggtaag 22980
tccaagcagc cgccgccgtt agcccaagag caacaacagc gccaaggcta ccgctcatgg 23040
cgcgggcaca agaacgccat agttgcttgc ttgcaagact gtgggggcaa catctccttc 23100
gcccgccgct ttcttctcta ccatcacggc gtggccttcc cccgtaacat cctgcattac 23160
taccgtcatc tctacagccc atactgcacc ggcggcagcg gcagcggcag caacagcagc 23220
ggccacacag aagcaaaggc gaccggatag caagactctg acaaagccca agaaatccac 23280
agcggcggca gcagcaggag gaggagcgct gcgtctggcg cccaacgaac ccgtatcgac 23340
ccgcgagctt agaaacagga tttttcccac tctgtatgct atatttcaac agagcagggg 23400
ccaagaacaa gagctgaaaa taaaaaacag gtctctgcga tccctcaccc gcagctgcct 23460
gtatcacaaa agcgaagatc agcttcggcg cacgctggaa gacgcggagg ctctcttcag 23520
taaatactgc gcgctgactc ttaaggacta gtttcgcgcc ctttctcaaa tttaagcgcg 23580
aaaactacgt catctccagc ggccacaccc ggcgccagca cctgtcgtca gcgccattat 23640
gagcaaggaa attcccacgc cctacatgtg gagttaccag ccacaaatgg gacttgcggc 23700
tggagctgcc caagactact caacccgaat aaactacatg agcgcgggac cccacatgat 23760
atcccgggtc aacggaatcc gcgcccaccg aaaccgaatt ctcttggaac aggcggctat 23820
taccaccaca cctcgtaata accttaatcc ccgtagttgg cccgctgccc tggtgtacca 23880
ggaaagtccc gctcccacca ctgtggtact tcccagagac gcccaggccg aagttcagat 23940
gactaactca ggggcgcagc ttgcgggcgg ctttcgtcac agggtgcggt cgcccgggca 24000
gggtataact cacctgacaa tcagagggcg aggtattcag ctcaacgacg agtcggtgag 24060
ctcctcgctt ggtctccgtc cggacgggac atttcagatc ggcggcgccg gccgtccttc 24120
attcacgcct cgtcaggcaa tcctaactct gcagacctcg tcctctgagc cgcgctctgg 24180
aggcattgga actctgcaat ttattgagga gtttgtgcca tcggtctact ttaacccctt 24240
ctcgggacct cccggccact atccggatca atttattcct aactttgacg cggtaaagga 24300
ctcggcggac ggctacgact gaatgttaag tggagaggca gagcaactgc gcctgaaaca 24360
cctggtccac tgtcgccgcc acaagtgctt tgcccgcgac tccggtgagt tttgctactt 24420
tgaattgccc gaggatcata tcgagggccc ggcgcacggc gtccggctta ccgcccaggg 24480
agagcttgcc cgtagcctga ttcgggagtt tacccagcgc cccctgctag ttgagcggga 24540
caggggaccc tgtgttctca ctgtgatttg caactgtcct aaccttggat tacatcaaga 24600
tcctctagtt ataactagag tacccgggga tcttattccc tttaactaat aaaaaaaaat 24660
aataaagcat cacttactta aaatcagtta gcaaatttct gtccagttta ttcagcagca 24720
cctccttgcc ctcctcccag ctctggtatt gcagcttcct cctggctgca aactttctcc 24780
acaatctaaa tggaatgtca gtttcctcct gttcctgtcc atccgcaccc actatcttca 24840
tgttgttgca gatgaagcgc gcaagaccgt ctgaagatac cttcaacccc gtgtatccat 24900
atgacacgga aaccggtcct ccaactgtgc cttttcttac tcctcccttt gtatccccca 24960
atgggtttca agagagtccc cctggggtac tctctttgcg cctatccgaa cctctagtta 25020
cctccaatgg catgcttgcg ctcaaaatgg gcaacggcct ctctctggac gaggccggca 25080
accttacctc ccaaaatgta accactgtga gcccacctct caaaaaaacc aagtcaaaca 25140
taaacctgga aatatctgca cccctcacag ttacctcaga agccctaact gtggctgccg 25200
ccgcacctct aatggtcgcg ggcaacacac tcaccatgca atcacaggcc ccgctaaccg 25260
tgcacgactc caaacttagc attgccaccc aaggacccct cacagtgtca gaaggaaagc 25320
tagccctgca aacatcaggc cccctcacca ccaccgatag cagtaccctt actatcactg 25380
cctcaccccc tctaactact gccactggta gcttgggcat tgacttgaaa gagcccattt 25440
atacacaaaa tggaaaacta ggactaaagt acggggctcc tttgcatgta acagacgacc 25500
taaacacttt gaccgtagca actggtccag gtgtgactat taataatact tccttgcaaa 25560
ctaaagttac tggagccttg ggttttgatt cacaaggcaa tatgcaactt aatgtagcag 25620
gaggactaag gattgattct caaaacagac gccttatact tgatgttagt tatccgtttg 25680
atgctcaaaa ccaactaaat ctaagactag gacagggccc tctttttata aactcagccc 25740
acaacttgga tattaactac aacaaaggcc tttacttgtt tacagcttca aacaattcca 25800
aaaagcttga ggttaaccta agcactgcca aggggttgat gtttgacgct acagccatag 25860
ccattaatgc aggagatggg cttgaatttg gttcacctaa tgcaccaaac acaaatcccc 25920
tcaaaacaaa aattggccat ggcctagaat ttgattcaaa caaggctatg gttcctaaac 25980
taggaactgg ccttagtttt gacagcacag gtgccattac agtaggaaac aaaaataatg 26040
ataagctaac tttgtggacc acaccagctc catctcctaa ctgtagacta aatgcagaga 26100
aagatgctaa actcactttg gtcttaacaa aatgtggcag tcaaatactt gctacagttt 26160
cagttttggc tgttaaaggc agtttggctc caatatctgg aacagttcaa agtgctcatc 26220
ttattataag atttgacgaa aatggagtgc tactaaacaa ttccttcctg gacccagaat 26280
attggaactt tagaaatgga gatcttactg aaggcacagc ctatacaaac gctgttggat 26340
ttatgcctaa cctatcagct tatccaaaat ctcacggtaa aactgccaaa agtaacattg 26400
tcagtcaagt ttacttaaac ggagacaaaa ctaaacctgt aacactaacc attacactaa 26460
acggtacaca ggaaacagga gacacaactc caagtgcata ctctatgtca ttttcatggg 26520
actggtctgg ccacaactac attaatgaaa tatttgccac atcctcttac actttttcat 26580
acattgccca agaataaaga atcgtttgtg ttatgtttca acgtgtttat ttttcaattg 26640
cagaaaattt caagtcattt ttcattcagt agtatagccc caccaccaca tagcttatac 26700
agatcaccgt accttaatca aactcacaga accctagtat tcaacctgcc acctccctcc 26760
caacacacag agtacacagt cctttctccc cggctggcct taaaaagcat catatcatgg 26820
gtaacagaca tattcttagg tgttatattc cacacggttt cctgtcgagc caaacgctca 26880
tcagtgatat taataaactc cccgggcagc tcacttaagt tcatgtcgct gtccagctgc 26940
tgagccacag gctgctgtcc aacttgcggt tgcttaacgg gcggcgaagg agaagtccac 27000
gcctacatgg gggtagagtc ataatcgtgc atcaggatag ggcggtggtg ctgcagcagc 27060
gcgcgaataa actgctgccg ccgccgctcc gtcctgcagg aatacaacat ggcagtggtc 27120
tcctcagcga tgattcgcac cgcccgcagc ataaggcgcc ttgtcctccg ggcacagcag 27180
cgcaccctga tctcacttaa atcagcacag taactgcagc acagcaccac aatattgttc 27240
aaaatcccac agtgcaaggc gctgtatcca aagctcatgg cggggaccac agaacccacg 27300
tggccatcat accacaagcg caggtagatt aagtggcgac ccctcataaa cacgctggac 27360
ataaacatta cctcttttgg catgttgtaa ttcaccacct cccggtacca tataaacctc 27420
tgattaaaca tggcgccatc caccaccatc ctaaaccagc tggccaaaac ctgcccgccg 27480
gctatacact gcagggaacc gggactggaa caatgacagt ggagagccca ggactcgtaa 27540
ccatggatca tcatgctcgt catgatatca atgttggcac aacacaggca cacgtgcata 27600
cacttcctca ggattacaag ctcctcccgc gttagaacca tatcccaggg aacaacccat 27660
tcctgaatca gcgtaaatcc cacactgcag ggaagacctc gcacgtaact cacgttgtgc 27720
attgtcaaag tgttacattc gggcagcagc ggatgatcct ccagtatggt agcgcgggtt 27780
tctgtctcaa aaggaggtag acgatcccta ctgtacggag tgcgccgaga caaccgagat 27840
cgtgttggtc gtagtgtcat gccaaatgga acgccggacg tagtcatatt tcctgaagca 27900
aaaccaggtg cgggcgtgac aaacagatct gcgtctccgg tctcgccgct tagatcgctc 27960
tgtgtagtag ttgtagtata tccactctct caaagcatcc aggcgccccc tggcttcggg 28020
ttctatgtaa actccttcat gcgccgctgc cctgataaca tccaccaccg cagaataagc 28080
cacacccagc caacctacac attcgttctg cgagtcacac acgggaggag cgggaagagc 28140
tggaagaacc atgttttttt ttttattcca aaagattatc caaaacctca aaatgaagat 28200
ctattaagtg aacgcgctcc cctccggtgg cgtggtcaaa ctctacagcc aaagaacaga 28260
taatggcatt tgtaagatgt tgcacaatgg cttccaaaag gcaaacggcc ctcacgtcca 28320
agtggacgta aaggctaaac ccttcagggt gaatctcctc tataaacatt ccagcacctt 28380
caaccatgcc caaataattc tcatctcgcc accttctcaa tatatctcta agcaaatccc 28440
gaatattaag tccggccatt gtaaaaatct gctccagagc gccctccacc ttcagcctca 28500
agcagcgaat catgattgca aaaattcagg ttcctcacag acctgtataa gattcaaaag 28560
cggaacatta acaaaaatac cgcgatcccg taggtccctt cgcagggcca gctgaacata 28620
atcgtgcagg tctgcacgga ccagcgcggc cacttccccg ccaggaacct tgacaaaaga 28680
acccacactg attatgacac gcatactcgg agctatgcta accagcgtag ccccgatgta 28740
agctttcaat agcttgttgc atgggcggcg atataaaatg caaggtgctg ctcaaaaaat 28800
caggcaaagc ctcgcgcaaa aaagaaagca catcgtagtc atgctcatgc agataaaggc 28860
aggtaagctc cggaaccacc acagaaaaag acaccatttt tctctcaaac atgtctgcgg 28920
gtttctgcat aaacacaaaa taaaataaca aaaaaacatt taaacattag aagcctgtct 28980
tacaacagga aaaacaaccc ttataagcat aagacggact acggccatgc cggcgtgacc 29040
gtaaaaaaac tggtcaccgt gattaaaaag caccaccgac agctcctcgg tcatgtccgg 29100
agtcataatg taagactcgg taaacacatc aggttgattc atcggtcagt gctaaaaagc 29160
gaccgaaata gcccggggga atacataccc gcaggcgtag agacaacatt acagccccca 29220
taggaggtat aacaaaatta ataggagaga aaaacacata aacacctgaa aaaccctcct 29280
gcctaggcaa aatagcaccc tcccgctcca gaacaacata cagcgcttca cagcggcagc 29340
ctaacagtca gccttaccag taaaaaagaa aacctattaa aaaaacacca ctcgacacgg 29400
caccagctca atcagtcaca gtgtaaaaaa gggccaagtg cagagcgagt atatatagga 29460
ctaaaaaatg acgtaacggt taaagtccac aaaaaacacc cagaaaaccg cacgcgaacc 29520
tacgcccaga aacgaaagcc aaaaaaccca caacttcctc aaatcgtcac ttccgttttc 29580
ccacgttacg taacttccca ttttaagaaa actacaattc ccaacacata caagttactc 29640
cgccctaaaa cctacgtcac ccgccccgtt cccacgcccc gcgccacgtc acaaactcca 29700
ccccctcatt atcatattgg cttcaatcca aaataaggta tattattgat gatgttaatt 29760
aacatgcatg gatccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 29820
gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 29880
ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 29940
acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 30000
cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 30060
caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 30120
gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 30180
tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 30240
aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 30300
ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 30360
cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 30420
tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 30480
tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 30540
ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 30600
aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 30660
aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 30720
aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 30780
gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 30840
gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 30900
caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 30960
ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 31020
attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 31080
ccattgctgc agccatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga 31140
aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga 31200
caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat 31260
agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct 31320
ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct 31380
gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg 31440
aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 31500
actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 31560
ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg 31620
aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 31680
ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 31740
tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 31800
tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 31860
gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 31920
aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg 31980
atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 32040
tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 32100
tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 32160
tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 32220
tcttctgaat tttgttaaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 32280
atcggcaaca tcccttataa atcaaaagaa tagaccgcga tagggttgag tgttgttcca 32340
gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 32400
gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttgcggtcg 32460
aggtgccgta aagctctaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 32520
ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 32580
gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgcgcg cttaatgcgc 32640
cgctacaggg cgcgtccatt cgccattcag gatcgaatta attcttaatt aacatcatca 32700
ataatatacc ttattttgga ttgaagccaa tatgataatg agggggtgga gtttgtgacg 32760
tggcgcgggg cgtgggaacg gggcgggtga cgtagtagtg tggcggaagt gtgatgttgc 32820
aagtgtggcg gaacacatgt aagcgacgga tgtggcaaaa gtgacgtttt tggtgtgcgc 32880
cggtgtacac aggaagtgac aattttcgcg cggttttagg cggatgttgt agtaaatttg 32940
ggcgtaaccg agtaagattt ggccattttc gcgggaaaac tgaataagag gaagtgaaat 33000
ctgaataatt ttgtgttact catagcgcgt aatactgcga tctatacatt gaatcaatat 33060
tggcaattag ccatattagt cattggttat atagcataaa tcaatattgg ctattggcca 33120
ttgcatacgt tgtatctata tcataatatg tacatttata ttggctcatg tccaatatga 33180
ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta 33240
gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 33300
tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 33360
ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 33420
gcagtacatc aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa 33480
tggcccgcct ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac 33540
atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg 33600
cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 33660
agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg 33720
ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta 33780
gtgaaccgtc agatcctcac tctcttccgc atcgctgtct gcgagggcca gctgttgggc 33840
tcgcggttga ggacaaactc ttcgcggtct ttccagtact cttggatcgg aaacccgtcg 33900
gcctccgaac ggtactccgc caccgaggga cctgagccag tccgcatcga ccggatcgga 33960
aaacctctcg agaaaggcgt ctaaccagtc acagtcgcaa ggtaggctga gcaccgtggc 34020
gggcggcagc gggtggcggt cggggttgtt tctggcggag gtgctgctga tgatgtaatt 34080
aaagtaggcg gtcttgagcc ggcggatggt cgaggtgagg tgtggcaggc ttgagatcca 34140
gctgttgggg tgagtactcc ctctcaaaag cgggcatgac ttctgcgcta agattgtcag 34200
tttccaaaaa cgaggaggat ttgatattca cctggcccga tctggccata cacttgagtg 34260
acaatgacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caagtttgga 34320
agatccctac taggaccata ggtacatctt cagagtcctt aactctttta atttgttctc 34380
tataatggtg aatatcttca aatgatttag tattatttat ggcaaataca caaagaaagc 34440
cctccccagt cctcatgtac tggtccctca ttgcactgta ctcctcttga cctgctgtgt 34500
cgagaatatc caagagacag gtttctccat caattactac ttgcttcctg taggaatcct 34560
ctattgttgg atcatattcg tccacaaaat gattctgaat tagctgtatc gtcaaggcac 34620
tcttgcaagc ttcattatca gcagctaaaa gttatcaggc atgcacctgg tagctagtct 34680
ttaaaccaat agattgcatc ggtttaaaag gcaagaccgt caaattgcgg gaaaggggtc 34740
aacagccgtt cagtaccaag tctcagggga aactttgaga tggccttgca aagggtatgg 34800
taataagctg acggacatgg tcctaaccac gcagccaagt cctaagtcaa cagatcttct 34860
gttgatatgg atgcagttca cagactaaat gtcggtcggg gaagatgtat tcttctcata 34920
agatatagtc ggacctctcc ttaatgggag ctagcggatg aagtgatgca acactggagc 34980
cgctgggaac taatttgtat gcgaaagtat attgattagt tttggagtac tcgcgatgat 35040
cacgcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 35100
ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 35160
cgcctggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc tcacgggatg 35220
gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 35280
gtacccgagc cgatgactta ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc 35340
tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 35400
atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 35460
cctcatgtcg ggggggaggc tgggagttca catgccccgc ccccggccct caccctcatc 35520
ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata ccttatgggc 35580
agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 35640
acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 35700
cagcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 35760
ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggtggga ggattgggga 35820
cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 35880
cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggccccc 35940
aacggcgacc tgtataacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 36000
cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 36060
ctgcaactta cctccgggat ggtccagacc cacgtcacca ccccaggctc cataccgacg 36120
atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaactg aagcttttcg 36180
aaggcgaaag atcccaacga aaagagagac cacatggtcc ttcttgagtt tgtaacagct 36240
gctgggatta cacatggcat ggatgaactg tacaactgag gatcccccga cctcgacctc 36300
tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc 36360
actcggaagg acatatggga gggcaaatca tttggtcgag atccctcgga gatcggatct 36420
gggcgtgg 36428
<210> 43
<211> 36226
<212> DNA
<213> Artificial Sequence
<220>
<223> Ad-AS100KRib2-TK
<400> 43
ttaagggtgg gaaagaatat ataaggtggg ggtcttatgt agttttgtat ctgttttgca 60
gcagccgccg ccgccatgag caccaactcg tttgatggaa gcattgtgag ctcatatttg 120
acaacgcgca tgcccccatg ggccggggtg cgtcagaatg tgatgggctc cagcattgat 180
ggtcgccccg tcctgcccgc aaactctact accttgacct acgagaccgt gtctggaacg 240
ccgttggaga ctgcagcctc cgccgccgct tcagccgctg cagccaccgc ccgcgggatt 300
gtgactgact ttgctttcct gagcccgctt gcaagcagtg cagcttcccg ttcatccgcc 360
cgcgatgaca agttgacggc tcttttggca caattggatt ctttgacccg ggaacttaat 420
gtcgtttctc agcagctgtt ggatctgcgc cagcaggttt ctgccctgaa ggcttcctcc 480
cctcccaatg cggtttaaaa cataaataaa aaaccagact ctgtttggat ttggatcaag 540
caagtgtctt gctgtcttta tttaggggtt ttgcgcgcgc ggtaggcccg ggaccagcgg 600
tctcggtcgt tgagggtcct gtgtattttt tccaggacgt ggtaaaggtg actctggatg 660
ttcagataca tgggcataag cccgtctctg gggtggaggt agcaccactg cagagcttca 720
tgctgcgggg tggtgttgta gatgatccag tcgtagcagg agcgctgggc gtggtgccta 780
aaaatgtctt tcagtagcaa gctgattgcc aggggcaggc ccttggtgta agtgtttaca 840
aagcggttaa gctgggatgg gtgcatacgt ggggatatga gatgcatctt ggactgtatt 900
tttaggttgg ctatgttccc agccatatcc ctccggggat tcatgttgtg cagaaccacc 960
agcacagtgt atccggtgca cttgggaaat ttgtcatgta gcttagaagg aaatgcgtgg 1020
aagaacttgg agacgccctt gtgacctcca agattttcca tgcattcgtc cataatgatg 1080
gcaatgggcc cacgggcggc ggcctgggcg aagatatttc tgggatcact aacgtcatag 1140
ttgtgttcca ggatgagatc gtcataggcc atttttacaa agcgcgggcg gagggtgcca 1200
gactgcggta taatggttcc atccggccca ggggcgtagt taccctcaca gatttgcatt 1260
tcccacgctt tgagttcaga tggggggatc atgtctacct gcggggcgat gaagaaaacg 1320
gtttccgggg taggggagat cagctgggaa gaaagcaggt tcctgagcag ctgcgactta 1380
ccgcagccgg tgggcccgta aatcacacct attaccgggt gcaactggta gttaagagag 1440
ctgcagctgc cgtcatccct gagcaggggg gccacttcgt taagcatgtc cctgactcgc 1500
atgttttccc tgaccaaatc cgccagaagg cgctcgccgc ccagcgatag cagttcttgc 1560
aaggaagcaa agtttttcaa cggtttgaga ccgtccgccg taggcatgct tttgagcgtt 1620
tgaccaagca gttccaggcg gtcccacagc tcggtcacct gctctacggc atctcgatcc 1680
agcatatctc ctcgtttcgc gggttggggc ggctttcgct gtacggcagt agtcggtgct 1740
cgtccagacg ggccagggtc atgtctttcc acgggcgcag ggtcctcgtc agcgtagtct 1800
gggtcacggt gaaggggtgc gctccgggct gcgcgctggc cagggtgcgc ttgaggctgg 1860
tcctgctggt gctgaagcgc tgccggtctt cgccctgcgc gtcggccagg tagcatttga 1920
ccatggtgtc atagtccagc ccctccgcgg cgtggccctt ggcgcgcagc ttgcccttgg 1980
aggaggcgcc gcacgagggg cagtgcagac ttttgagggc gtagagcttg ggcgcgagaa 2040
ataccgattc cggggagtag gcatccgcgc cgcaggcccc gcagacggtc tcgcattcca 2100
cgagccaggt gagctctggc cgttcggggt caaaaaccag gtttccccca tgctttttga 2160
tgcgtttctt acctctggtt tccatgagcc ggtgtccacg ctcggtgacg aaaaggctgt 2220
ccgtgtcccc gtatacagac ttgagaggcc tgtcctcgag cggtgttccg cggtcctcct 2280
cgtatagaaa ctcggaccac tctgagacaa aggctcgcgt ccaggccagc acgaaggagg 2340
ctaagtggga ggggtagcgg tcgttgtcca ctagggggtc cactcgctcc agggtgtgaa 2400
gacacatgtc gccctcttcg gcatcaagga aggtgattgg tttgtaggtg taggccacgt 2460
gaccgggtgt tcctgaaggg gggctataaa agggggtggg ggcgcgttcg tcctcactct 2520
cttccgcatc gctgtctgcg agggccagct gttggggtga gtactccctc tgaaaagcgg 2580
gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcacct 2640
ggcccgcggt gatgcctttg agggtggccg catccatctg gtcagaaaag acaatctttt 2700
tgttgtcaag cttggtggca aacgacccgt agagggcgtt ggacagcaac ttggcgatgg 2760
agcgcagggt ttggtttttg tcgcgatcgg cgcgctcctt ggccgcgatg tttagctgca 2820
cgtattcgcg cgcaacgcac cgccattcgg gaaagacggt ggtgcgctcg tcgggcacca 2880
ggtgcacgcg ccaaccgcgg ttgtgcaggg tgacaaggtc aacgctggtg gctacctctc 2940
cgcgtaggcg ctcgttggtc cagcagaggc ggccgccctt gcgcgagcag aatggcggta 3000
gggggtctag ctgcgtctcg tccggggggt ctgcgtccac ggtaaagacc ccgggcagca 3060
ggcgcgcgtc gaagtagtct atcttgcatc cttgcaagtc tagcgcctgc tgccatgcgc 3120
gggcggcaag cgcgcgctcg tatgggttga gtgggggacc ccatggcatg gggtgggtga 3180
gcgcggaggc gtacatgccg caaatgtcgt aaacgtagag gggctctctg agtattccaa 3240
gatatgtagg gtagcatctt ccaccgcgga tgctggcgcg cacgtaatcg tatagttcgt 3300
gcgagggagc gaggaggtcg ggaccgaggt tgctacgggc gggctgctct gctcggaaga 3360
ctatctgcct gaagatggca tgtgagttgg atgatatggt tggacgctgg aagacgttga 3420
agctggcgtc tgtgagacct accgcgtcac gcacgaagga ggcgtaggag tcgcgcagct 3480
tgttgaccag ctcggcggtg acctgcacgt ctagggcgca gtagtccagg gtttccttga 3540
tgatgtcata cttatcctgt cccttttttt tccacagctc gcggttgagg acaaactctt 3600
cgcggtcttt ccagtactct tggatcggaa acccgtcggc ctccgaacgg taagagccta 3660
gcatgtagaa ctggttgacg gcctggtagg cgcagcatcc cttttctacg ggtagcgcgt 3720
atgcctgcgc ggccttccgg agcgaggtgt gggtgagcgc aaaggtgtcc ctgaccatga 3780
ctttgaggta ctggtatttg aagtcagtgt cgtcgcatcc gccctgctcc cagagcaaaa 3840
agtccgtgcg ctttttggaa cgcggatttg gcagggcgaa ggtgacatcg ttgaagagta 3900
tctttcccgc gcgaggcata aagttgcgtg tgatgcggaa gggtcccggc acctcggaac 3960
ggttgttaat tacctgggcg gcgagcacga tctcgtcaaa gccgttgatg ttgtggccca 4020
caatgtaaag ttccaagaag cgcgggatgc ccttgatgga aggcaatttt ttaagttcct 4080
cgtaggtgag ctcttcaggg gagctgagcc cgtgctctga aagggcccag tctgcaagat 4140
gagggttgga agcgacgaat gagctccaca ggtcacgggc cattagcatt tgcaggtggt 4200
cgcgaaaggt cctaaactgg cgacctatgg ccattttttc tggggtgatg cagtagaagg 4260
taagcgggtc ttgttcccag cggtcccatc caaggttcgc ggctaggtct cgcgcggcag 4320
tcactagagg ctcatctccg ccgaacttca tgaccagcat gaagggcacg agctgcttcc 4380
caaaggcccc catccaagta taggtctcta catcgtaggt gacaaagaga cgctcggtgc 4440
gaggatgcga gccgatcggg aagaactgga tctcccgcca ccaattggag gagtggctat 4500
tgatgtggtg aaagtagaag tccctgcgac gggccgaaca ctcgtgctgg cttttgtaaa 4560
aacgtgcgca gtactggcag cggtgcacgg gctgtacatc ctgcacgagg ttgacctgac 4620
gaccgcgcac aaggaagcag agtgggaatt tgagcccctc gcctggcggg tttggctggt 4680
ggtcttctac ttcggctgct tgtccttgac cgtctggctg ctcgagggga gttacggtgg 4740
atcggaccac cacgccgcgc gagcccaaag tccagatgtc cgcgcgcggc ggtcggagct 4800
tgatgacaac atcgcgcaga tgggagctgt ccatggtctg gagctcccgc ggcgtcaggt 4860
caggcgggag ctcctgcagg tttacctcgc atagacgggt cagggcgcgg gctagatcca 4920
ggtgatacct aatttccagg ggctggttgg tggcggcgtc gatggcttgc aagaggccgc 4980
atccccgcgg cgcgactacg gtaccgcgcg gcgggcggtg ggccgcgggg gtgtccttgg 5040
atgatgcatc taaaagcggt gacgcgggcg agcccccgga ggtagggggg gctccggacc 5100
cgccgggaga gggggcaggg gcacgtcggc gccgcgcgcg ggcaggagct ggtgctgcgc 5160
gcgtaggttg ctggcgaacg cgacgacgcg gcggttgatc tcctgaatct ggcgcctctg 5220
cgtgaagacg acgggcccgg tgagcttgag cctgaaagag agttcgacag aatcaatttc 5280
ggtgtcgttg acggcggcct ggcgcaaaat ctcctgcacg tctcctgagt tgtcttgata 5340
ggcgatctcg gccatgaact gctcgatctc ttcctcctgg agatctccgc gtccggctcg 5400
ctccacggtg gcggcgaggt cgttggaaat gcgggccatg agctgcgaga aggcgttgag 5460
gcctccctcg ttccagacgc ggctgtagac cacgccccct tcggcatcgc gggcgcgcat 5520
gaccacctgc gcgagattga gctccacgtg ccgggcgaag acggcgtagt ttcgcaggcg 5580
ctgaaagagg tagttgaggg tggtggcggt gtgttctgcc acgaagaagt acataaccca 5640
gcgtcgcaac gtggattcgt tgatatcccc caaggcctca aggcgctcca tggcctcgta 5700
gaagtccacg gcgaagttga aaaactggga gttgcgcgcc gacacggtta actcctcctc 5760
cagaagacgg atgagctcgg cgacagtgtc gcgcacctcg cgctcaaagg ctacaggggc 5820
ctcttcttct tcttcaatct cctcttccat aagggcctcc ccttcttctt cttctggcgg 5880
cggtggggga ggggggacac ggcggcgacg acggcgcacc gggaggcggt cgacaaagcg 5940
ctcgatcatc tccccgcggc gacggcgcat ggtctcggtg acggcgcggc cgttctcgcg 6000
ggggcgcagt tggaagacgc cgcccgtcat gtcccggtta tgggttggcg gggggctgcc 6060
atgcggcagg gatacggcgc taacgatgca tctcaacaat tgttgtgtag gtactccgcc 6120
gccgagggac ctgagcgagt ccgcatcgac cggatcggaa aacctctcga gaaaggcgtc 6180
taaccagtca cagtcgcaag gtaggctgag caccgtggcg ggcggcagcg ggcggcggtc 6240
ggggttgttt ctggcggagg tgctgctgat gatgtaatta aagtaggcgg tcttgagacg 6300
gcggatggtc gacagaagca ccatgtcctt gggtccggcc tgctgaatgc gcaggcggtc 6360
ggccatgccc caggcttcgt tttgacatcg gcgcaggtct ttgtagtagt cttgcatgag 6420
cctttctacc ggcacttctt cttctccttc ctcttgtcct gcatctcttg catctatcgc 6480
tgcggcggcg gcggagtttg gccgtaggtg gcgccctctt cctcccatgc gtgtgacccc 6540
gaagcccctc atcggctgaa gcagggctag gtcggcgaca acgcgctcgg ctaatatggc 6600
ctgctgcacc tgcgtgaggg tagactggaa gtcatccatg tccacaaagc ggtggtatgc 6660
gcccgtgttg atggtgtaag tgcagttggc cataacggac cagttaacgg tctggtgacc 6720
cggctgcgag agctcggtgt acctgagacg cgagtaagcc ctcgagtcaa atacgtagtc 6780
gttgcaagtc cgcaccaggt actggtatcc caccaaaaag tgcggcggcg gctggcggta 6840
gaggggccag cgtagggtgg ccggggctcc gggggcgaga tcttccaaca taaggcgatg 6900
atatccgtag atgtacctgg acatccaggt gatgccggcg gcggtggtgg aggcgcgcgg 6960
aaagtcgcgg acgcggttcc agatgttgcg cagcggcaaa aagtgctcca tggtcgggac 7020
gctctggccg gtcaggcgcg cgcaatcgtt gacgctctac cgtgcaaaag gagagcctgt 7080
aagcgggcac tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc 7140
ggggttcgag ccccgtatcc ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc 7200
gaacccaggt gtgcgacgtc agacaacggg ggagtgctcc ttttggcttc cttccaggcg 7260
cggcggctgc tgcgctagct tttttggcca ctggccgcgc gcagcgtaag cggttaggct 7320
ggaaagcgaa agcattaagt ggctcgctcc ctgtagccgg agggttattt tccaagggtt 7380
gagtcgcggg acccccggtt cgagtctcgg accggccgga ctgcggcgaa cgggggtttg 7440
cctccccgtc atgcaagacc ccgcttgcaa attcctccgg aaacagggac gagccccttt 7500
tttgcttttc ccagatgcat ccggtgctgc ggcagatgcg cccccctcct cagcagcggc 7560
aagagcaaga gcagcggcag acatgcaggg caccctcccc tcctcctacc gcgtcaggag 7620
gggcgacatc cgcggttgac gcggcagcag atggtgatta cgaacccccg cggcgccggg 7680
cccggcacta cctggacttg gaggagggcg agggcctggc gcggctagga gcgccctctc 7740
ctgagcggta cccaagggtg cagctgaagc gtgatacgcg tgaggcgtac gtgccgcggc 7800
agaacctgtt tcgcgaccgc gagggagagg agcccgagga gatgcgggat cgaaagttcc 7860
acgcagggcg cgagctgcgg catggcctga atcgcgagcg gttgctgcgc gaggaggact 7920
ttgagcccga cgcgcgaacc gggattagtc ccgcgcgcgc acacgtggcg gccgccgacc 7980
tggtaaccgc atacgagcag acggtgaacc aggagattaa ctttcaaaaa agctttaaca 8040
accacgtgcg tacgcttgtg gcgcgcgagg aggtggctat aggactgatg catctgtggg 8100
actttgtaag cgcgctggag caaaacccaa atagcaagcc gctcatggcg cagctgttcc 8160
ttatagtgca gcacagcagg gacaacgagg cattcaggga tgcgctgcta aacatagtag 8220
agcccgaggg ccgctggctg ctcgatttga taaacatcct gcagagcata gtggtgcagg 8280
agcgcagctt gagcctggct gacaaggtgg ccgccatcaa ctattccatg cttagcctgg 8340
gcaagtttta cgcccgcaag atataccata ccccttacgt tcccatagac aaggaggtaa 8400
agatcgaggg gttctacatg cgcatggcgc tgaaggtgct taccttgagc gacgacctgg 8460
gcgtttatcg caacgagcgc atccacaagg ccgtgagcgt gagccggcgg cgcgagctca 8520
gcgaccgcga gctgatgcac agcctgcaaa gggccctggc tggcacgggc agcggcgata 8580
gagaggccga gtcctacttt gacgcgggcg ctgacctgcg ctgggcccca agccgacgcg 8640
ccctggaggc agctggggcc ggacctgggc tggcggtggc acccgcgcgc gctggcaacg 8700
tcggcggcgt ggaggaatat gacgaggacg atgagtacga gccagaggac ggcgagtact 8760
aagcggtgat gtttctgatc agatgatgca agacgcaacg gacccggcgg tgcgggcggc 8820
gctgcagagc cagccgtccg gccttaactc cacggacgac tggcgccagg tcatggaccg 8880
catcatgtcg ctgactgcgc gcaatcctga cgcgttccgg cagcagccgc aggccaaccg 8940
gctctccgca attctggaag cggtggtccc ggcgcgcgca aaccccacgc acgagaaggt 9000
gctggcgatc gtaaacgcgc tggccgaaaa cagggccatc cggcccgacg aggccggcct 9060
ggtctacgac gcgctgcttc agcgcgtggc tcgttacaac agcggcaacg tgcagaccaa 9120
cctggaccgg ctggtggggg atgtgcgcga ggccgtggcg cagcgtgagc gcgcgcagca 9180
gcagggcaac ctgggctcca tggttgcact aaacgccttc ctgagtacac agcccgccaa 9240
cgtgccgcgg ggacaggagg actacaccaa ctttgtgagc gcactgcggc taatggtgac 9300
tgagacaccg caaagtgagg tgtaccagtc tgggccagac tattttttcc agaccagtag 9360
acaaggcctg cagaccgtaa acctgagcca ggctttcaaa aacttgcagg ggctgtgggg 9420
ggtgcgggct cccacaggcg accgcgcgac cgtgtctagc ttgctgacgc ccaactcgcg 9480
cctgttgctg ctgctaatag cgcccttcac ggacagtggc agcgtgtccc gggacacata 9540
cctaggtcac ttgctgacac tgtaccgcga ggccataggt caggcgcatg tggacgagca 9600
tactttccag gagattacaa gtgtcagccg cgcgctgggg caggaggaca cgggcagcct 9660
ggaggcaacc ctaaactacc tgctgaccaa ccggcggcag aagatcccct cgttgcacag 9720
tttaaacagc gaggaggagc gcattttgcg ctacgtgcag cagagcgtga gccttaacct 9780
gatgcgcgac ggggtaacgc ccagcgtggc gctggacatg accgcgcgca acatggaacc 9840
gggcatgtat gcctcaaacc ggccgtttat caaccgccta atggactact tgcatcgcgc 9900
ggccgccgtg aaccccgagt atttcaccaa tgccatcttg aacccgcact ggctaccgcc 9960
ccctggtttc tacaccgggg gattcgaggt gcccgagggt aacgatggat tcctctggga 10020
cgacatagac gacagcgtgt tttccccgca accgcagacc ctgctagagt tgcaacagcg 10080
cgagcaggca gaggcggcgc tgcgaaagga aagcttccgc aggccaagca gcttgtccga 10140
tctaggcgct gcggccccgc ggtcagatgc tagtagccca tttccaagct tgatagggtc 10200
tcttaccagc actcgcacca cccgcccgcg cctgctgggc gaggaggagt acctaaacaa 10260
ctcgctgctg cagccgcagc gcgaaaaaaa cctgcctccg gcatttccca acaacgggat 10320
agagagccta gtggacaaga tgagtagatg gaagacgtac gcgcaggagc acagggacgt 10380
gccaggcccg cgcccgccca cccgtcgtca aaggcacgac cgtcagcggg gtctggtgtg 10440
ggaggacgat gactcggcag acgacagcag cgtcctggat ttgggaggga gtggcaaccc 10500
gtttgcgcac cttcgcccca ggctggggag aatgttttaa aaaaaaaaaa gcatgatgca 10560
aaataaaaaa ctcaccaagg ccatggcacc gagcgttggt tttcttgtat tccccttagt 10620
atgcggcgcg cggcgatgta tgaggaaggt cctcctccct cctacgagag tgtggtgagc 10680
gcggcgccag tggcggcggc gctgggttct cccttcgatg ctcccctgga cccgccgttt 10740
gtgcctccgc ggtacctgcg gcctaccggg gggagaaaca gcatccgtta ctctgagttg 10800
gcacccctat tcgacaccac ccgtgtgtac ctggtggaca acaagtcaac ggatgtggca 10860
tccctgaact accagaacga ccacagcaac tttctgacca cggtcattca aaacaatgac 10920
tacagcccgg gggaggcaag cacacagacc atcaatcttg acgaccggtc gcactggggc 10980
ggcgacctga aaaccatcct gcataccaac atgccaaatg tgaacgagtt catgtttacc 11040
aataagttta aggcgcgggt gatggtgtcg cgcttgccta ctaaggacaa tcaggtggag 11100
ctgaaatacg agtgggtgga gttcacgctg cccgagggca actactccga gaccatgacc 11160
atagacctta tgaacaacgc gatcgtggag cactacttga aagtgggcag acagaacggg 11220
gttctggaaa gcgacatcgg ggtaaagttt gacacccgca acttcagact ggggtttgac 11280
cccgtcactg gtcttgtcat gcctggggta tatacaaacg aagccttcca tccagacatc 11340
attttgctgc caggatgcgg ggtggacttc acccacagcc gcctgagcaa cttgttgggc 11400
atccgcaagc ggcaaccctt ccaggagggc tttaggatca cctacgatga tctggagggt 11460
ggtaacattc ccgcactgtt ggatgtggac gcctaccagg cgagcttgaa agatgacacc 11520
gaacagggcg ggggtggcgc aggcggcagc aacagcagtg gcagcggcgc ggaagagaac 11580
tccaacgcgg cagccgcggc aatgcagccg gtggaggaca tgaacgatca tgccattcgc 11640
ggcgacacct ttgccacacg ggctgaggag aagcgcgctg aggccgaagc agcggccgaa 11700
gctgccgccc ccgctgcgca acccgaggtc gagaagcctc agaagaaacc ggtgatcaaa 11760
cccctgacag aggacagcaa gaaacgcagt tacaacctaa taagcaatga cagcaccttc 11820
acccagtacc gcagctggta ccttgcatac aactacggcg accctcagac cggaatccgc 11880
tcatggaccc tgctttgcac tcctgacgta acctgcggct cggagcaggt ctactggtcg 11940
ttgccagaca tgatgcaaga ccccgtgacc ttccgctcca cgcgccagat cagcaacttt 12000
ccggtggtgg gcgccgagct gttgcccgtg cactccaaga gcttctacaa cgaccaggcc 12060
gtctactccc aactcatccg ccagtttacc tctctgaccc acgtgttcaa tcgctttccc 12120
gagaaccaga ttttggcgcg cccgccagcc cccaccatca ccaccgtcag tgaaaacgtt 12180
cctgctctca cagatcacgg gacgctaccg ctgcgcaaca gcatcggagg agtccagcga 12240
gtgaccatta ctgacgccag acgccgcacc tgcccctacg tttacaaggc cctgggcata 12300
gtctcgccgc gcgtcctatc gagccgcact ttttgagcaa gcatgtccat ccttatatcg 12360
cccagcaata acacaggctg gggcctgcgc ttcccaagca agatgtttgg cggggccaag 12420
aagcgctccg accaacaccc agtgcgcgtg cgcgggcact accgcgcgcc ctggggcgcg 12480
cacaaacgcg gccgcactgg gcgcaccacc gtcgatgacg ccatcgacgc ggtggtggag 12540
gaggcgcgca actacacgcc cacgccgcca ccagtgtcca cagtggacgc ggccattcag 12600
accgtggtgc gcggagcccg gcgctatgct aaaatgaaga gacggcggag gcgcgtagca 12660
cgtcgccacc gccgccgacc cggcactgcc gcccaacgcg cggcggcggc cctgcttaac 12720
cgcgcacgtc gcaccggccg acgggcggcc atgcgggccg ctcgaaggct ggccgcgggt 12780
attgtcactg tgccccccag gtccaggcga cgagcggccg ccgcagcagc cgcggccatt 12840
agtgctatga ctcagggtcg caggggcaac gtgtattggg tgcgcgactc ggttagcggc 12900
ctgcgcgtgc ccgtgcgcac ccgccccccg cgcaactaga ttgcaagaaa aaactactta 12960
gactcgtact gttgtatgta tccagcggcg gcggcgcgca acgaagctat gtccaagcgc 13020
aaaatcaaag aagagatgct ccaggtcatc gcgccggaga tctatggccc cccgaagaag 13080
gaagagcagg attacaagcc ccgaaagcta aagcgggtca aaaagaaaaa gaaagatgat 13140
gatgatgaac ttgacgacga ggtggaactg ctgcacgcta ccgcgcccag gcgacgggta 13200
cagtggaaag gtcgacgcgt aaaacgtgtt ttgcgacccg gcaccaccgt agtctttacg 13260
cccggtgagc gctccacccg cacctacaag cgcgtgtatg atgaggtgta cggcgacgag 13320
gacctgcttg agcaggccaa cgagcgcctc ggggagtttg cctacggaaa gcggcataag 13380
gacatgctgg cgttgccgct ggacgagggc aacccaacac ctagcctaaa gcccgtaaca 13440
ctgcagcagg tgctgcccgc gcttgcaccg tccgaagaaa agcgcggcct aaagcgcgag 13500
tctggtgact tggcacccac cgtgcagctg atggtaccca agcgccagcg actggaagat 13560
gtcttggaaa aaatgaccgt ggaacctggg ctggagcccg aggtccgcgt gcggccaatc 13620
aagcaggtgg cgccgggact gggcgtgcag accgtggacg ttcagatacc cactaccagt 13680
agcaccagta ttgccaccgc cacagagggc atggagacac aaacgtcccc ggttgcctca 13740
gcggtggcgg atgccgcggt gcaggcggtc gctgcggccg cgtccaagac ctctacggag 13800
gtgcaaacgg acccgtggat gtttcgcgtt tcagcccccc ggcgcccgcg cggttcgagg 13860
aagtacggcg ccgccagcgc gctactgccc gaatatgccc tacatccttc cattgcgcct 13920
acccccggct atcgtggcta cacctaccgc cccagaagac gagcaactac ccgacgccga 13980
accaccactg gaacccgccg ccgccgtcgc cgtcgccagc ccgtgctggc cccgatttcc 14040
gtgcgcaggg tggctcgcga aggaggcagg accctggtgc tgccaacagc gcgctaccac 14100
cccagcatcg tttaaaagcc ggtctttgtg gttcttgcag atatggccct cacctgccgc 14160
ctccgtttcc cggtgccggg attccgagga agaatgcacc gtaggagggg catggccggc 14220
cacggcctga cgggcggcat gcgtcgtgcg caccaccggc ggcggcgcgc gtcgcaccgt 14280
cgcatgcgcg gcggtatcct gcccctcctt attccactga tcgccgcggc gattggcgcc 14340
gtgcccggaa ttgcatccgt ggccttgcag gcgcagagac actgattaaa aacaagttgc 14400
atgtggaaaa atcaaaataa aaagtctgga ctctcacgct cgcttggtcc tgtaactatt 14460
ttgtagaatg gaagacatca actttgcgtc tctggccccg cgacacggct cgcgcccgtt 14520
catgggaaac tggcaagata tcggcaccag caatatgagc ggtggcgcct tcagctgggg 14580
ctcgctgtgg agcggcatta aaaatttcgg ttccaccgtt aagaactatg gcagcaaggc 14640
ctggaacagc agcacaggcc agatgctgag ggataagttg aaagagcaaa atttccaaca 14700
aaaggtggta gatggcctgg cctctggcat tagcggggtg gtggacctgg ccaaccaggc 14760
agtgcaaaat aagattaaca gtaagcttga tccccgccct cccgtagagg agcctccacc 14820
ggccgtggag acagtgtctc cagaggggcg tggcgaaaag cgtccgcgcc ccgacaggga 14880
agaaactctg gtgacgcaaa tagacgagcc tccctcgtac gaggaggcac taaagcaagg 14940
cctgcccacc acccgtccca tcgcgcccat ggctaccgga gtgctgggcc agcacacacc 15000
cgtaacgctg gacctgcctc cccccgccga cacccagcag aaacctgtgc tgccaggccc 15060
gaccgccgtt gttgtaaccc gtcctagccg cgcgtccctg cgccgcgccg ccagcggtcc 15120
gcgatcgttg cggcccgtag ccagtggcaa ctggcaaagc acactgaaca gcatcgtggg 15180
tctgggggtg caatccctga agcgccgacg atgcttctga atagctaacg tgtcgtatgt 15240
gtgtcatgta tgcgtccatg tcgccgccag aggagctgct gagccgccgc gcgcccgctt 15300
tccaagatgg ctaccccttc gatgatgccg cagtggtctt acatgcacat ctcgggccag 15360
gacgcctcgg agtacctgag ccccgggctg gtgcagtttg cccgcgccac cgagacgtac 15420
ttcagcctga ataacaagtt tagaaacccc acggtggcgc ctacgcacga cgtgaccaca 15480
gaccggtccc agcgtttgac gctgcggttc atccctgtgg accgtgagga tactgcgtac 15540
tcgtacaagg cgcggttcac cctagctgtg ggtgataacc gtgtgctgga catggcttcc 15600
acgtactttg acatccgcgg cgtgctggac aggggcccta cttttaagcc ctactctggc 15660
actgcctaca acgccctggc tcccaagggt gccccaaatc cttgcgaatg ggatgaagct 15720
gctactgctc ttgaaataaa cctagaagaa gaggacgatg acaacgaaga cgaagtagac 15780
gagcaagctg agcagcaaaa aactcacgta tttgggcagg cgccttattc tggtataaat 15840
attacaaagg agggtattca aataggtgtc gaaggtcaaa cacctaaata tgccgataaa 15900
acatttcaac ctgaacctca aataggagaa tctcagtggt acgaaactga aattaatcat 15960
gcagctggga gagtccttaa aaagactacc ccaatgaaac catgttacgg ttcatatgca 16020
aaacccacaa atgaaaatgg agggcaaggc attcttgtaa agcaacaaaa tggaaagcta 16080
gaaagtcaag tggaaatgca atttttctca actactgagg cgaccgcagg caatggtgat 16140
aacttgactc ctaaagtggt attgtacagt gaagatgtag atatagaaac cccagacact 16200
catatttctt acatgcccac tattaaggaa ggtaactcac gagaactaat gggccaacaa 16260
tctatgccca acaggcctaa ttacattgct tttagggaca attttattgg tctaatgtat 16320
tacaacagca cgggtaatat gggtgttctg gcgggccaag catcgcagtt gaatgctgtt 16380
gtagatttgc aagacagaaa cacagagctt tcataccagc ttttgcttga ttccattggt 16440
gatagaacca ggtacttttc tatgtggaat caggctgttg acagctatga tccagatgtt 16500
agaattattg aaaatcatgg aactgaagat gaacttccaa attactgctt tccactggga 16560
ggtgtgatta atacagagac tcttaccaag gtaaaaccta aaacaggtca ggaaaatgga 16620
tgggaaaaag atgctacaga attttcagat aaaaatgaaa taagagttgg aaataatttt 16680
gccatggaaa tcaatctaaa tgccaacctg tggagaaatt tcctgtactc caacatagcg 16740
ctgtatttgc ccgacaagct aaagtacagt ccttccaacg taaaaatttc tgataaccca 16800
aacacctacg actacatgaa caagcgagtg gtggctcccg ggttagtgga ctgctacatt 16860
aaccttggag cacgctggtc ccttgactat atggacaacg tcaacccatt taaccaccac 16920
cgcaatgctg gcctgcgcta ccgctcaatg ttgctgggca atggtcgcta tgtgcccttc 16980
cacatccagg tgcctcagaa gttctttgcc attaaaaacc tccttctcct gccgggctca 17040
tacacctacg agtggaactt caggaaggat gttaacatgg ttctgcagag ctccctagga 17100
aatgacctaa gggttgacgg agccagcatt aagtttgata gcatttgcct ttacgccacc 17160
ttcttcccca tggcccacaa caccgcctcc acgcttgagg ccatgcttag aaacgacacc 17220
aacgaccagt cctttaacga ctatctctcc gccgccaaca tgctctaccc tatacccgcc 17280
aacgctacca acgtgcccat atccatcccc tcccgcaact gggcggcttt ccgcggctgg 17340
gccttcacgc gccttaagac taaggaaacc ccatcactgg gctcgggcta cgacccttat 17400
tacacctact ctggctctat accctaccta gatggaacct tttacctcaa ccacaccttt 17460
aagaaggtgg ccattacctt tgactcttct gtcagctggc ctggcaatga ccgcctgctt 17520
acccccaacg agtttgaaat taagcgctca gttgacgggg agggttacaa cgttgcccag 17580
tgtaacatga ccaaagactg gttcctggta caaatgctag ctaactacaa cattggctac 17640
cagggcttct atatcccaga gagctacaag gaccgcatgt actccttctt tagaaacttc 17700
cagcccatga gccgtcaggt ggtggatgat actaaataca aggactacca acaggtgggc 17760
atcctacacc aacacaacaa ctctggattt gttggctacc ttgcccccac catgcgcgaa 17820
ggacaggcct accctgctaa cttcccctat ccgcttatag gcaagaccgc agttgacagc 17880
attacccaga aaaagtttct ttgcgatcgc accctttggc gcatcccatt ctccagtaac 17940
tttatgtcca tgggcgcact cacagacctg ggccaaaacc ttctctacgc caactccgcc 18000
cacgcgctag acatgacttt tgaggtggat cccatggacg agcccaccct tctttatgtt 18060
ttgtttgaag tctttgacgt ggtccgtgtg caccggccgc accgcggcgt catcgaaacc 18120
gtgtacctgc gcacgccctt ctcggccggc aacgccacaa cataaagaag caagcaacat 18180
caacaacagc tgccgccatg ggctccagtg agcaggaact gaaagccatt gtcaaagatc 18240
ttggttgtgg gccatatttt ttgggcacct atgacaagcg ctttccaggc tttgtttctc 18300
cacacaagct cgcctgcgcc atagtcaata cggccggtcg cgagactggg ggcgtacact 18360
ggatggcctt tgcctggaac ccgcactcaa aaacatgcta cctctttgag ccctttggct 18420
tttctgacca gcgactcaag caggtttacc agtttgagta cgagtcactc ctgcgccgta 18480
gcgccattgc ttcttccccc gaccgctgta taacgctgga aaagtccacc caaagcgtac 18540
aggggcccaa ctcggccgcc tgtggactat tctgctgcat gtttctccac gcctttgcca 18600
actggcccca aactcccatg gatcacaacc ccaccatgaa ccttattacc ggggtaccca 18660
actccatgct caacagtccc caggtacagc ccaccctgcg tcgcaaccag gaacagctct 18720
acagcttcct ggagcgccac tcgccctact tccgcagcca cagtgcgcag attaggagcg 18780
ccacttcttt ttgtcacttg aaaaacatgt aaaaataatg tactagagac actttcaata 18840
aaggcaaatg cttttatttg tacactctcg ggtgattatt tacccccacc cttgccgtct 18900
gcgccgttta aaaatcaaag gggttctgcc gcgcatcgct atgcgccact ggcagggaca 18960
cgttgcgata ctggtgttta gtgctccact taaactcagg cacaaccatc cgcggcagct 19020
cggtgaagtt ttcactccac aggctgcgca ccatcaccaa cgcgtttagc aggtcgggcg 19080
ccgatatctt gaagtcgcag ttggggcctc cgccctgcgc gcgcgagttg cgatacacag 19140
ggttgcagca ctggaacact atcagcgccg ggtggtgcac gctggccagc acgctcttgt 19200
cggagatcag atccgcgtcc aggtcctccg cgttgctcag ggcgaacgga gtcaactttg 19260
gtagctgcct tcccaaaaag ggcgcgtgcc caggctttga gttgcactcg caccgtagtg 19320
gcatcaaaag gtgaccgtgc ccggtctggg cgttaggata cagcgcctgc ataaaagcct 19380
tgatctgctt aaaagccacc tgagcctttg cgccttcaga gaagaacatg ccgcaagact 19440
tgccggaaaa ctgattggcc ggacaggccg cgtcgtgcac gcagcacctt gcgtcggtgt 19500
tggagatctg caccacattt cggccccacc ggttcttcac gatcttggcc ttgctagact 19560
gctccttcag cgcgcgctgc ccgttttcgc tcgtcacatc catttcaatc acgtgctcct 19620
tatttatcat aatgcttccg tgtagacact taagctcgcc ttcgatctca gcgcagcggt 19680
gcagccacaa cgcgcagccc gtgggctcgt gatgcttgta ggtcacctct gcaaacgact 19740
gcaggtacgc ctgcaggaat cgccccatca tcgtcacaaa ggtcttgttg ctggtgaagg 19800
tcagctgcaa cccgcggtgc tcctcgttca gccaggtctt gcatacggcc gccagagctt 19860
ccacttggtc aggcagtagt ttgaagttcg cctttagatc gttatccacg tggtacttgt 19920
ccatcagcgc gcgcgcagcc tccatgccct tctcccacgc agacacgatc ggcacactca 19980
gcgggttcat caccgtaatt tcactttccg cttcgctggg ctcttcctct tcctcttgcg 20040
tccgcatacc acgcgccact gggtcgtctt cattcagccg ccgcactgtg cgcttacctc 20100
ctttgccatg cttgattagc accggtgggt tgctgaaacc caccatttgt agcgccacat 20160
cttctctttc ttcctcgctg tccacgatta cctctggtga tggcgggcgc tcgggcttgg 20220
gagaagggcg cttctttttc ttcttgggcg caatggccaa atccgccgcc gaggtcgatg 20280
gccgcgggct gggtgtgcgc ggcaccagcg cgtcttgtga tgagtcttcc tcgtcctcgg 20340
actcgatacg ccgcctcatc cgcttttttg ggggcgcccg gggaggcggc ggcgacgggg 20400
acggggacga cacgtcctcc atggttgggg gacgtcgcgc cgcaccgcgt ccgcgctcgg 20460
gggtggtttc gcgctgctcc tcttcccgac tggccatttc cttctcctat aggcagaaaa 20520
agatcatgga gtcagtcgag aagaaggaca gcctaaccgc cccctctgag ttcgccacca 20580
ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca cccccgcttg 20640
aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac gacgaggacc 20700
gctcagtacc aacagaggat aaaaagcaag accaggacaa cgcagaggca aacgaggaac 20760
aagtcgggcg gggggacgaa aggcatggcg actacctaga tgtgggagac gacgtgctgt 20820
tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag cgcagcgatg 20880
tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctattc tcaccgcgcg 20940
taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc aacttctacc 21000
ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa aactgcaaga 21060
tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc ttgcggcagg 21120
gcgctgtcat acctgatatc gcctcgctca acgaagtgcc aaaaatcttt gagggtcttg 21180
gacgcgacga gaagcgcgcg gcaaacgctc tgcaacagga aaacagcgaa aatgaaagtc 21240
actctggagt gttggtggaa ctcgagggtg acaacgcgcg cctagccgta ctaaaacgca 21300
gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag gtcatgagca 21360
cagtcatgag tgagctgatc gtgcgccgtg cgcagcccct ggagagggat gcaaatttgc 21420
aagaacaaac agaggagggc ctacccgcag ttggcgacga gcagctagcg cgctggcttc 21480
aaacgcgcga gcctgccgac ttggaggagc gacgcaaact aatgatggcc gcagtgctcg 21540
ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg cagcgcaagc 21600
tagaggaaac attgcactac acctttcgac agggctacgt acgccaggcc tgcaagatct 21660
ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa aaccgccttg 21720
ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac gtccgcgact 21780
gcgtttactt atttctatgc tacacctggc agacggccat gggcgtttgg cagcagtgct 21840
tggaggagtg caacctcaag gagctgcaga aactgctaaa gcaaaacttg aaggacctat 21900
ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacatcatt ttccccgaac 21960
gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc atgttgcaga 22020
actttaggaa ctttatccta gagcgctcag gaatcttgcc cgccacctgc tgtgcacttc 22080
ctagcgactt tgtgcccatt aagtaccgcg aatgccctcc gccgctttgg ggccactgct 22140
accttctgca gctagccaac taccttgcct accactctga cataatggaa gacgtgagcg 22200
gtgacggtct actggagtgt cactgtcgct gcaacctatg caccccgcac cgctccctgg 22260
tttgcaattc gcagctgctt aacgaaagtc aaattatcgg tacctttgag ctgcagggtc 22320
cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg ctgtggacgt 22380
cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt aggttctacg 22440
aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc cagggccaca 22500
ttcttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta cgaaagggac 22560
ggggggttta cttggacccc cagtccggcg aggagctcaa cccaatcccc ccgccgccgc 22620
agccctatca gcagcagccg cgggcccttg cttcccagga tggcacccaa aaagaagctg 22680
cagctgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc agaggaggtt 22740
ttggacgagg aggaggagga catgatggaa gactgggaga gcctagacga ggaagcttcc 22800
gaggtcgaag aggtgtcaga cgaaacaccg tcaccctcgg tcgcattccc ctcgccggcg 22860
ccccagaaat cggcaaccgg ttccagcatg gctacaacct ccgctcctca ggcgccgccg 22920
gcactgcccg ttcgccgacc caaccgtaga tgggacacca ctggaaccag ggccggtaag 22980
tccaagcagc cgccgccgtt agcccaagag caacaacagc gccaaggcta ccgctcatgg 23040
cgcgggcaca agaacgccat agttgcttgc ttgcaagact gtgggggcaa catctccttc 23100
gcccgccgct ttcttctcta ccatcacggc gtggccttcc cccgtaacat cctgcattac 23160
taccgtcatc tctacagccc atactgcacc ggcggcagcg gcagcggcag caacagcagc 23220
ggccacacag aagcaaaggc gaccggatag caagactctg acaaagccca agaaatccac 23280
agcggcggca gcagcaggag gaggagcgct gcgtctggcg cccaacgaac ccgtatcgac 23340
ccgcgagctt agaaacagga tttttcccac tctgtatgct atatttcaac agagcagggg 23400
ccaagaacaa gagctgaaaa taaaaaacag gtctctgcga tccctcaccc gcagctgcct 23460
gtatcacaaa agcgaagatc agcttcggcg cacgctggaa gacgcggagg ctctcttcag 23520
taaatactgc gcgctgactc ttaaggacta gtttcgcgcc ctttctcaaa tttaagcgcg 23580
aaaactacgt catctccagc ggccacaccc ggcgccagca cctgtcgtca gcgccattat 23640
gagcaaggaa attcccacgc cctacatgtg gagttaccag ccacaaatgg gacttgcggc 23700
tggagctgcc caagactact caacccgaat aaactacatg agcgcgggac cccacatgat 23760
atcccgggtc aacggaatcc gcgcccaccg aaaccgaatt ctcttggaac aggcggctat 23820
taccaccaca cctcgtaata accttaatcc ccgtagttgg cccgctgccc tggtgtacca 23880
ggaaagtccc gctcccacca ctgtggtact tcccagagac gcccaggccg aagttcagat 23940
gactaactca ggggcgcagc ttgcgggcgg ctttcgtcac agggtgcggt cgcccgggca 24000
gggtataact cacctgacaa tcagagggcg aggtattcag ctcaacgacg agtcggtgag 24060
ctcctcgctt ggtctccgtc cggacgggac atttcagatc ggcggcgccg gccgtccttc 24120
attcacgcct cgtcaggcaa tcctaactct gcagacctcg tcctctgagc cgcgctctgg 24180
aggcattgga actctgcaat ttattgagga gtttgtgcca tcggtctact ttaacccctt 24240
ctcgggacct cccggccact atccggatca atttattcct aactttgacg cggtaaagga 24300
ctcggcggac ggctacgact gaatgttaag tggagaggca gagcaactgc gcctgaaaca 24360
cctggtccac tgtcgccgcc acaagtgctt tgcccgcgac tccggtgagt tttgctactt 24420
tgaattgccc gaggatcata tcgagggccc ggcgcacggc gtccggctta ccgcccaggg 24480
agagcttgcc cgtagcctga ttcgggagtt tacccagcgc cccctgctag ttgagcggga 24540
caggggaccc tgtgttctca ctgtgatttg caactgtcct aaccttggat tacatcaaga 24600
tcctctagtt ataactagag tacccgggga tcttattccc tttaactaat aaaaaaaaat 24660
aataaagcat cacttactta aaatcagtta gcaaatttct gtccagttta ttcagcagca 24720
cctccttgcc ctcctcccag ctctggtatt gcagcttcct cctggctgca aactttctcc 24780
acaatctaaa tggaatgtca gtttcctcct gttcctgtcc atccgcaccc actatcttca 24840
tgttgttgca gatgaagcgc gcaagaccgt ctgaagatac cttcaacccc gtgtatccat 24900
atgacacgga aaccggtcct ccaactgtgc cttttcttac tcctcccttt gtatccccca 24960
atgggtttca agagagtccc cctggggtac tctctttgcg cctatccgaa cctctagtta 25020
cctccaatgg catgcttgcg ctcaaaatgg gcaacggcct ctctctggac gaggccggca 25080
accttacctc ccaaaatgta accactgtga gcccacctct caaaaaaacc aagtcaaaca 25140
taaacctgga aatatctgca cccctcacag ttacctcaga agccctaact gtggctgccg 25200
ccgcacctct aatggtcgcg ggcaacacac tcaccatgca atcacaggcc ccgctaaccg 25260
tgcacgactc caaacttagc attgccaccc aaggacccct cacagtgtca gaaggaaagc 25320
tagccctgca aacatcaggc cccctcacca ccaccgatag cagtaccctt actatcactg 25380
cctcaccccc tctaactact gccactggta gcttgggcat tgacttgaaa gagcccattt 25440
atacacaaaa tggaaaacta ggactaaagt acggggctcc tttgcatgta acagacgacc 25500
taaacacttt gaccgtagca actggtccag gtgtgactat taataatact tccttgcaaa 25560
ctaaagttac tggagccttg ggttttgatt cacaaggcaa tatgcaactt aatgtagcag 25620
gaggactaag gattgattct caaaacagac gccttatact tgatgttagt tatccgtttg 25680
atgctcaaaa ccaactaaat ctaagactag gacagggccc tctttttata aactcagccc 25740
acaacttgga tattaactac aacaaaggcc tttacttgtt tacagcttca aacaattcca 25800
aaaagcttga ggttaaccta agcactgcca aggggttgat gtttgacgct acagccatag 25860
ccattaatgc aggagatggg cttgaatttg gttcacctaa tgcaccaaac acaaatcccc 25920
tcaaaacaaa aattggccat ggcctagaat ttgattcaaa caaggctatg gttcctaaac 25980
taggaactgg ccttagtttt gacagcacag gtgccattac agtaggaaac aaaaataatg 26040
ataagctaac tttgtggacc acaccagctc catctcctaa ctgtagacta aatgcagaga 26100
aagatgctaa actcactttg gtcttaacaa aatgtggcag tcaaatactt gctacagttt 26160
cagttttggc tgttaaaggc agtttggctc caatatctgg aacagttcaa agtgctcatc 26220
ttattataag atttgacgaa aatggagtgc tactaaacaa ttccttcctg gacccagaat 26280
attggaactt tagaaatgga gatcttactg aaggcacagc ctatacaaac gctgttggat 26340
ttatgcctaa cctatcagct tatccaaaat ctcacggtaa aactgccaaa agtaacattg 26400
tcagtcaagt ttacttaaac ggagacaaaa ctaaacctgt aacactaacc attacactaa 26460
acggtacaca ggaaacagga gacacaactc caagtgcata ctctatgtca ttttcatggg 26520
actggtctgg ccacaactac attaatgaaa tatttgccac atcctcttac actttttcat 26580
acattgccca agaataaaga atcgtttgtg ttatgtttca acgtgtttat ttttcaattg 26640
cagaaaattt caagtcattt ttcattcagt agtatagccc caccaccaca tagcttatac 26700
agatcaccgt accttaatca aactcacaga accctagtat tcaacctgcc acctccctcc 26760
caacacacag agtacacagt cctttctccc cggctggcct taaaaagcat catatcatgg 26820
gtaacagaca tattcttagg tgttatattc cacacggttt cctgtcgagc caaacgctca 26880
tcagtgatat taataaactc cccgggcagc tcacttaagt tcatgtcgct gtccagctgc 26940
tgagccacag gctgctgtcc aacttgcggt tgcttaacgg gcggcgaagg agaagtccac 27000
gcctacatgg gggtagagtc ataatcgtgc atcaggatag ggcggtggtg ctgcagcagc 27060
gcgcgaataa actgctgccg ccgccgctcc gtcctgcagg aatacaacat ggcagtggtc 27120
tcctcagcga tgattcgcac cgcccgcagc ataaggcgcc ttgtcctccg ggcacagcag 27180
cgcaccctga tctcacttaa atcagcacag taactgcagc acagcaccac aatattgttc 27240
aaaatcccac agtgcaaggc gctgtatcca aagctcatgg cggggaccac agaacccacg 27300
tggccatcat accacaagcg caggtagatt aagtggcgac ccctcataaa cacgctggac 27360
ataaacatta cctcttttgg catgttgtaa ttcaccacct cccggtacca tataaacctc 27420
tgattaaaca tggcgccatc caccaccatc ctaaaccagc tggccaaaac ctgcccgccg 27480
gctatacact gcagggaacc gggactggaa caatgacagt ggagagccca ggactcgtaa 27540
ccatggatca tcatgctcgt catgatatca atgttggcac aacacaggca cacgtgcata 27600
cacttcctca ggattacaag ctcctcccgc gttagaacca tatcccaggg aacaacccat 27660
tcctgaatca gcgtaaatcc cacactgcag ggaagacctc gcacgtaact cacgttgtgc 27720
attgtcaaag tgttacattc gggcagcagc ggatgatcct ccagtatggt agcgcgggtt 27780
tctgtctcaa aaggaggtag acgatcccta ctgtacggag tgcgccgaga caaccgagat 27840
cgtgttggtc gtagtgtcat gccaaatgga acgccggacg tagtcatatt tcctgaagca 27900
aaaccaggtg cgggcgtgac aaacagatct gcgtctccgg tctcgccgct tagatcgctc 27960
tgtgtagtag ttgtagtata tccactctct caaagcatcc aggcgccccc tggcttcggg 28020
ttctatgtaa actccttcat gcgccgctgc cctgataaca tccaccaccg cagaataagc 28080
cacacccagc caacctacac attcgttctg cgagtcacac acgggaggag cgggaagagc 28140
tggaagaacc atgttttttt ttttattcca aaagattatc caaaacctca aaatgaagat 28200
ctattaagtg aacgcgctcc cctccggtgg cgtggtcaaa ctctacagcc aaagaacaga 28260
taatggcatt tgtaagatgt tgcacaatgg cttccaaaag gcaaacggcc ctcacgtcca 28320
agtggacgta aaggctaaac ccttcagggt gaatctcctc tataaacatt ccagcacctt 28380
caaccatgcc caaataattc tcatctcgcc accttctcaa tatatctcta agcaaatccc 28440
gaatattaag tccggccatt gtaaaaatct gctccagagc gccctccacc ttcagcctca 28500
agcagcgaat catgattgca aaaattcagg ttcctcacag acctgtataa gattcaaaag 28560
cggaacatta acaaaaatac cgcgatcccg taggtccctt cgcagggcca gctgaacata 28620
atcgtgcagg tctgcacgga ccagcgcggc cacttccccg ccaggaacct tgacaaaaga 28680
acccacactg attatgacac gcatactcgg agctatgcta accagcgtag ccccgatgta 28740
agctttcaat agcttgttgc atgggcggcg atataaaatg caaggtgctg ctcaaaaaat 28800
caggcaaagc ctcgcgcaaa aaagaaagca catcgtagtc atgctcatgc agataaaggc 28860
aggtaagctc cggaaccacc acagaaaaag acaccatttt tctctcaaac atgtctgcgg 28920
gtttctgcat aaacacaaaa taaaataaca aaaaaacatt taaacattag aagcctgtct 28980
tacaacagga aaaacaaccc ttataagcat aagacggact acggccatgc cggcgtgacc 29040
gtaaaaaaac tggtcaccgt gattaaaaag caccaccgac agctcctcgg tcatgtccgg 29100
agtcataatg taagactcgg taaacacatc aggttgattc atcggtcagt gctaaaaagc 29160
gaccgaaata gcccggggga atacataccc gcaggcgtag agacaacatt acagccccca 29220
taggaggtat aacaaaatta ataggagaga aaaacacata aacacctgaa aaaccctcct 29280
gcctaggcaa aatagcaccc tcccgctcca gaacaacata cagcgcttca cagcggcagc 29340
ctaacagtca gccttaccag taaaaaagaa aacctattaa aaaaacacca ctcgacacgg 29400
caccagctca atcagtcaca gtgtaaaaaa gggccaagtg cagagcgagt atatatagga 29460
ctaaaaaatg acgtaacggt taaagtccac aaaaaacacc cagaaaaccg cacgcgaacc 29520
tacgcccaga aacgaaagcc aaaaaaccca caacttcctc aaatcgtcac ttccgttttc 29580
ccacgttacg taacttccca ttttaagaaa actacaattc ccaacacata caagttactc 29640
cgccctaaaa cctacgtcac ccgccccgtt cccacgcccc gcgccacgtc acaaactcca 29700
ccccctcatt atcatattgg cttcaatcca aaataaggta tattattgat gatgttaatt 29760
aacatgcatg gatccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 29820
gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 29880
ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 29940
acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 30000
cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 30060
caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 30120
gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 30180
tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 30240
aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 30300
ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 30360
cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 30420
tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 30480
tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 30540
ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 30600
aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 30660
aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 30720
aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 30780
gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 30840
gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 30900
caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 30960
ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 31020
attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 31080
ccattgctgc agccatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga 31140
aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga 31200
caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat 31260
agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct 31320
ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct 31380
gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg 31440
aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 31500
actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 31560
ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg 31620
aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 31680
ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 31740
tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 31800
tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 31860
gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 31920
aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg 31980
atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 32040
tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 32100
tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 32160
tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 32220
tcttctgaat tttgttaaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 32280
atcggcaaca tcccttataa atcaaaagaa tagaccgcga tagggttgag tgttgttcca 32340
gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 32400
gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttgcggtcg 32460
aggtgccgta aagctctaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 32520
ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 32580
gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgcgcg cttaatgcgc 32640
cgctacaggg cgcgtccatt cgccattcag gatcgaatta attcttaatt aacatcatca 32700
ataatatacc ttattttgga ttgaagccaa tatgataatg agggggtgga gtttgtgacg 32760
tggcgcgggg cgtgggaacg gggcgggtga cgtagtagtg tggcggaagt gtgatgttgc 32820
aagtgtggcg gaacacatgt aagcgacgga tgtggcaaaa gtgacgtttt tggtgtgcgc 32880
cggtgtacac aggaagtgac aattttcgcg cggttttagg cggatgttgt agtaaatttg 32940
ggcgtaaccg agtaagattt ggccattttc gcgggaaaac tgaataagag gaagtgaaat 33000
ctgaataatt ttgtgttact catagcgcgt aatactgcga tctatacatt gaatcaatat 33060
tggcaattag ccatattagt cattggttat atagcataaa tcaatattgg ctattggcca 33120
ttgcatacgt tgtatctata tcataatatg tacatttata ttggctcatg tccaatatga 33180
ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta 33240
gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 33300
tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 33360
ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 33420
gcagtacatc aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa 33480
tggcccgcct ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac 33540
atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg 33600
cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 33660
agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg 33720
ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta 33780
gtgaaccgtc agatcctcac tctcttccgc atcgctgtct gcgagggcca gctgttgggc 33840
tcgcggttga ggacaaactc ttcgcggtct ttccagtact cttggatcgg aaacccgtcg 33900
gcctccgaac ggtactccgc caccgaggga cctgagccag tccgcatcga ccggatcgga 33960
aaacctctcg agaaaggcgt ctaaccagtc acagtcgcaa ggtaggctga gcaccgtggc 34020
gggcggcagc gggtggcggt cggggttgtt tctggcggag gtgctgctga tgatgtaatt 34080
aaagtaggcg gtcttgagcc ggcggatggt cgaggtgagg tgtggcaggc ttgagatcca 34140
gctgttgggg tgagtactcc ctctcaaaag cgggcatgac ttctgcgcta agattgtcag 34200
tttccaaaaa cgaggaggat ttgatattca cctggcccga tctggccata cacttgagtg 34260
acaatgacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caagtttgga 34320
agatccccat caattactac ttgcttcctg taggaatcct ctattgttgg atcatattcg 34380
tccacaaaat gattctgaat tagctgtatc gtcaaggcac tcttgcaagc ttcatcatgg 34440
ccgacagcaa aagttatcag gcatgcacct ggtagctagt ctttaaacca atagattgca 34500
tcggtttaaa aggcaagacc gtcaaattgc gggaaagggg tcaacagccg ttcagtacca 34560
agtctcaggg gaaactttga gatggccttg caaagggtat ggtaataagc tgacggacat 34620
ggtcctaacc acgcagccaa gtcctaagtc aacagatctt ctgttgatat ggatgcagtt 34680
cacagactaa atgtcggtcg gggaagatgt attcttctca taagatatag tcggacctct 34740
ccttaatggg agctagcgga tgaagtgatg caacactgga gccgctggga actaatttgt 34800
atgcgaaagt atattgatta gttttggagt actcgcgaca tgatcacgct tcgtacccct 34860
gccatcaaca cgcgtctgcg ttcgaccagg ctgcgcgttc tcgcggccat agcaaccgac 34920
gtacggcgtt gcgccctcgc cggcagcaag aagccacgga agtccgcctg gagcagaaaa 34980
tgcccacgct actgcgggtt tatatagacg gtcctcacgg gatggggaaa accaccacca 35040
cgcaactgct ggtggccctg ggttcgcgcg acgatatcgt ctacgtaccc gagccgatga 35100
cttactggca ggtgctgggg gcttccgaga caatcgcgaa catctacacc acacaacacc 35160
gcctcgacca gggtgagata tcggccgggg acgcggcggt ggtaatgaca agcgcccaga 35220
taacaatggg catgccttat gccgtgaccg acgccgttct ggctcctcat gtcggggggg 35280
aggctgggag ttcacatgcc ccgcccccgg ccctcaccct catcttcgac cgccatccca 35340
tcgccgccct cctgtgctac ccggccgcgc gataccttat gggcagcatg accccccagg 35400
ccgtgctggc gttcgtggcc ctcatcccgc cgaccttgcc cggcacaaac atcgtgttgg 35460
gggcccttcc ggaggacaga cacatcgacc gcctggccaa acgccagcgc cccggcgagc 35520
ggcttgacct ggctatgctg gccgcgattc gccgcgttta cgggctgctt gccaatacgg 35580
tgcggtatct gcagggcggc gggtcgtggt gggaggattg gggacagctt tcggggacgg 35640
ccgtgccgcc ccagggtgcc gagccccaga gcaacgcggg cccacgaccc catatcgggg 35700
acacgttatt taccctgttt cgggcccccg agttgctggc ccccaacggc gacctgtata 35760
acgtgtttgc ctgggccttg gacgtcttgg ccaaacgcct ccgtcccatg cacgtcttta 35820
tcctggatta cgaccaatcg cccgccggct gccgggacgc cctgctgcaa cttacctccg 35880
ggatggtcca gacccacgtc accaccccag gctccatacc gacgatctgc gacctggcgc 35940
gcacgtttgc ccgggagatg ggggaggcta ataattcgaa ggcgaaagat cccaacgaaa 36000
agagagacca catggtcctt cttgagtttg taacagctgc tgggattaca catggcatgg 36060
atgaactgta caactgagga tcccccgacc tcgacctctg gctaataaag gaaatttatt 36120
ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg 36180
gcaaatcatt tggtcgagat ccctcggaga tcggatctgg gcgtgg 36226
<210> 44
<211> 38160
<212> DNA
<213> Artificial Sequence
<220>
<223> Ad-AS100Rib2-LacZ
<400> 44
ttaagggtgg gaaagaatat ataaggtggg ggtcttatgt agttttgtat ctgttttgca 60
gcagccgccg ccgccatgag caccaactcg tttgatggaa gcattgtgag ctcatatttg 120
acaacgcgca tgcccccatg ggccggggtg cgtcagaatg tgatgggctc cagcattgat 180
ggtcgccccg tcctgcccgc aaactctact accttgacct acgagaccgt gtctggaacg 240
ccgttggaga ctgcagcctc cgccgccgct tcagccgctg cagccaccgc ccgcgggatt 300
gtgactgact ttgctttcct gagcccgctt gcaagcagtg cagcttcccg ttcatccgcc 360
cgcgatgaca agttgacggc tcttttggca caattggatt ctttgacccg ggaacttaat 420
gtcgtttctc agcagctgtt ggatctgcgc cagcaggttt ctgccctgaa ggcttcctcc 480
cctcccaatg cggtttaaaa cataaataaa aaaccagact ctgtttggat ttggatcaag 540
caagtgtctt gctgtcttta tttaggggtt ttgcgcgcgc ggtaggcccg ggaccagcgg 600
tctcggtcgt tgagggtcct gtgtattttt tccaggacgt ggtaaaggtg actctggatg 660
ttcagataca tgggcataag cccgtctctg gggtggaggt agcaccactg cagagcttca 720
tgctgcgggg tggtgttgta gatgatccag tcgtagcagg agcgctgggc gtggtgccta 780
aaaatgtctt tcagtagcaa gctgattgcc aggggcaggc ccttggtgta agtgtttaca 840
aagcggttaa gctgggatgg gtgcatacgt ggggatatga gatgcatctt ggactgtatt 900
tttaggttgg ctatgttccc agccatatcc ctccggggat tcatgttgtg cagaaccacc 960
agcacagtgt atccggtgca cttgggaaat ttgtcatgta gcttagaagg aaatgcgtgg 1020
aagaacttgg agacgccctt gtgacctcca agattttcca tgcattcgtc cataatgatg 1080
gcaatgggcc cacgggcggc ggcctgggcg aagatatttc tgggatcact aacgtcatag 1140
ttgtgttcca ggatgagatc gtcataggcc atttttacaa agcgcgggcg gagggtgcca 1200
gactgcggta taatggttcc atccggccca ggggcgtagt taccctcaca gatttgcatt 1260
tcccacgctt tgagttcaga tggggggatc atgtctacct gcggggcgat gaagaaaacg 1320
gtttccgggg taggggagat cagctgggaa gaaagcaggt tcctgagcag ctgcgactta 1380
ccgcagccgg tgggcccgta aatcacacct attaccgggt gcaactggta gttaagagag 1440
ctgcagctgc cgtcatccct gagcaggggg gccacttcgt taagcatgtc cctgactcgc 1500
atgttttccc tgaccaaatc cgccagaagg cgctcgccgc ccagcgatag cagttcttgc 1560
aaggaagcaa agtttttcaa cggtttgaga ccgtccgccg taggcatgct tttgagcgtt 1620
tgaccaagca gttccaggcg gtcccacagc tcggtcacct gctctacggc atctcgatcc 1680
agcatatctc ctcgtttcgc gggttggggc ggctttcgct gtacggcagt agtcggtgct 1740
cgtccagacg ggccagggtc atgtctttcc acgggcgcag ggtcctcgtc agcgtagtct 1800
gggtcacggt gaaggggtgc gctccgggct gcgcgctggc cagggtgcgc ttgaggctgg 1860
tcctgctggt gctgaagcgc tgccggtctt cgccctgcgc gtcggccagg tagcatttga 1920
ccatggtgtc atagtccagc ccctccgcgg cgtggccctt ggcgcgcagc ttgcccttgg 1980
aggaggcgcc gcacgagggg cagtgcagac ttttgagggc gtagagcttg ggcgcgagaa 2040
ataccgattc cggggagtag gcatccgcgc cgcaggcccc gcagacggtc tcgcattcca 2100
cgagccaggt gagctctggc cgttcggggt caaaaaccag gtttccccca tgctttttga 2160
tgcgtttctt acctctggtt tccatgagcc ggtgtccacg ctcggtgacg aaaaggctgt 2220
ccgtgtcccc gtatacagac ttgagaggcc tgtcctcgag cggtgttccg cggtcctcct 2280
cgtatagaaa ctcggaccac tctgagacaa aggctcgcgt ccaggccagc acgaaggagg 2340
ctaagtggga ggggtagcgg tcgttgtcca ctagggggtc cactcgctcc agggtgtgaa 2400
gacacatgtc gccctcttcg gcatcaagga aggtgattgg tttgtaggtg taggccacgt 2460
gaccgggtgt tcctgaaggg gggctataaa agggggtggg ggcgcgttcg tcctcactct 2520
cttccgcatc gctgtctgcg agggccagct gttggggtga gtactccctc tgaaaagcgg 2580
gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga ggaggatttg atattcacct 2640
ggcccgcggt gatgcctttg agggtggccg catccatctg gtcagaaaag acaatctttt 2700
tgttgtcaag cttggtggca aacgacccgt agagggcgtt ggacagcaac ttggcgatgg 2760
agcgcagggt ttggtttttg tcgcgatcgg cgcgctcctt ggccgcgatg tttagctgca 2820
cgtattcgcg cgcaacgcac cgccattcgg gaaagacggt ggtgcgctcg tcgggcacca 2880
ggtgcacgcg ccaaccgcgg ttgtgcaggg tgacaaggtc aacgctggtg gctacctctc 2940
cgcgtaggcg ctcgttggtc cagcagaggc ggccgccctt gcgcgagcag aatggcggta 3000
gggggtctag ctgcgtctcg tccggggggt ctgcgtccac ggtaaagacc ccgggcagca 3060
ggcgcgcgtc gaagtagtct atcttgcatc cttgcaagtc tagcgcctgc tgccatgcgc 3120
gggcggcaag cgcgcgctcg tatgggttga gtgggggacc ccatggcatg gggtgggtga 3180
gcgcggaggc gtacatgccg caaatgtcgt aaacgtagag gggctctctg agtattccaa 3240
gatatgtagg gtagcatctt ccaccgcgga tgctggcgcg cacgtaatcg tatagttcgt 3300
gcgagggagc gaggaggtcg ggaccgaggt tgctacgggc gggctgctct gctcggaaga 3360
ctatctgcct gaagatggca tgtgagttgg atgatatggt tggacgctgg aagacgttga 3420
agctggcgtc tgtgagacct accgcgtcac gcacgaagga ggcgtaggag tcgcgcagct 3480
tgttgaccag ctcggcggtg acctgcacgt ctagggcgca gtagtccagg gtttccttga 3540
tgatgtcata cttatcctgt cccttttttt tccacagctc gcggttgagg acaaactctt 3600
cgcggtcttt ccagtactct tggatcggaa acccgtcggc ctccgaacgg taagagccta 3660
gcatgtagaa ctggttgacg gcctggtagg cgcagcatcc cttttctacg ggtagcgcgt 3720
atgcctgcgc ggccttccgg agcgaggtgt gggtgagcgc aaaggtgtcc ctgaccatga 3780
ctttgaggta ctggtatttg aagtcagtgt cgtcgcatcc gccctgctcc cagagcaaaa 3840
agtccgtgcg ctttttggaa cgcggatttg gcagggcgaa ggtgacatcg ttgaagagta 3900
tctttcccgc gcgaggcata aagttgcgtg tgatgcggaa gggtcccggc acctcggaac 3960
ggttgttaat tacctgggcg gcgagcacga tctcgtcaaa gccgttgatg ttgtggccca 4020
caatgtaaag ttccaagaag cgcgggatgc ccttgatgga aggcaatttt ttaagttcct 4080
cgtaggtgag ctcttcaggg gagctgagcc cgtgctctga aagggcccag tctgcaagat 4140
gagggttgga agcgacgaat gagctccaca ggtcacgggc cattagcatt tgcaggtggt 4200
cgcgaaaggt cctaaactgg cgacctatgg ccattttttc tggggtgatg cagtagaagg 4260
taagcgggtc ttgttcccag cggtcccatc caaggttcgc ggctaggtct cgcgcggcag 4320
tcactagagg ctcatctccg ccgaacttca tgaccagcat gaagggcacg agctgcttcc 4380
caaaggcccc catccaagta taggtctcta catcgtaggt gacaaagaga cgctcggtgc 4440
gaggatgcga gccgatcggg aagaactgga tctcccgcca ccaattggag gagtggctat 4500
tgatgtggtg aaagtagaag tccctgcgac gggccgaaca ctcgtgctgg cttttgtaaa 4560
aacgtgcgca gtactggcag cggtgcacgg gctgtacatc ctgcacgagg ttgacctgac 4620
gaccgcgcac aaggaagcag agtgggaatt tgagcccctc gcctggcggg tttggctggt 4680
ggtcttctac ttcggctgct tgtccttgac cgtctggctg ctcgagggga gttacggtgg 4740
atcggaccac cacgccgcgc gagcccaaag tccagatgtc cgcgcgcggc ggtcggagct 4800
tgatgacaac atcgcgcaga tgggagctgt ccatggtctg gagctcccgc ggcgtcaggt 4860
caggcgggag ctcctgcagg tttacctcgc atagacgggt cagggcgcgg gctagatcca 4920
ggtgatacct aatttccagg ggctggttgg tggcggcgtc gatggcttgc aagaggccgc 4980
atccccgcgg cgcgactacg gtaccgcgcg gcgggcggtg ggccgcgggg gtgtccttgg 5040
atgatgcatc taaaagcggt gacgcgggcg agcccccgga ggtagggggg gctccggacc 5100
cgccgggaga gggggcaggg gcacgtcggc gccgcgcgcg ggcaggagct ggtgctgcgc 5160
gcgtaggttg ctggcgaacg cgacgacgcg gcggttgatc tcctgaatct ggcgcctctg 5220
cgtgaagacg acgggcccgg tgagcttgag cctgaaagag agttcgacag aatcaatttc 5280
ggtgtcgttg acggcggcct ggcgcaaaat ctcctgcacg tctcctgagt tgtcttgata 5340
ggcgatctcg gccatgaact gctcgatctc ttcctcctgg agatctccgc gtccggctcg 5400
ctccacggtg gcggcgaggt cgttggaaat gcgggccatg agctgcgaga aggcgttgag 5460
gcctccctcg ttccagacgc ggctgtagac cacgccccct tcggcatcgc gggcgcgcat 5520
gaccacctgc gcgagattga gctccacgtg ccgggcgaag acggcgtagt ttcgcaggcg 5580
ctgaaagagg tagttgaggg tggtggcggt gtgttctgcc acgaagaagt acataaccca 5640
gcgtcgcaac gtggattcgt tgatatcccc caaggcctca aggcgctcca tggcctcgta 5700
gaagtccacg gcgaagttga aaaactggga gttgcgcgcc gacacggtta actcctcctc 5760
cagaagacgg atgagctcgg cgacagtgtc gcgcacctcg cgctcaaagg ctacaggggc 5820
ctcttcttct tcttcaatct cctcttccat aagggcctcc ccttcttctt cttctggcgg 5880
cggtggggga ggggggacac ggcggcgacg acggcgcacc gggaggcggt cgacaaagcg 5940
ctcgatcatc tccccgcggc gacggcgcat ggtctcggtg acggcgcggc cgttctcgcg 6000
ggggcgcagt tggaagacgc cgcccgtcat gtcccggtta tgggttggcg gggggctgcc 6060
atgcggcagg gatacggcgc taacgatgca tctcaacaat tgttgtgtag gtactccgcc 6120
gccgagggac ctgagcgagt ccgcatcgac cggatcggaa aacctctcga gaaaggcgtc 6180
taaccagtca cagtcgcaag gtaggctgag caccgtggcg ggcggcagcg ggcggcggtc 6240
ggggttgttt ctggcggagg tgctgctgat gatgtaatta aagtaggcgg tcttgagacg 6300
gcggatggtc gacagaagca ccatgtcctt gggtccggcc tgctgaatgc gcaggcggtc 6360
ggccatgccc caggcttcgt tttgacatcg gcgcaggtct ttgtagtagt cttgcatgag 6420
cctttctacc ggcacttctt cttctccttc ctcttgtcct gcatctcttg catctatcgc 6480
tgcggcggcg gcggagtttg gccgtaggtg gcgccctctt cctcccatgc gtgtgacccc 6540
gaagcccctc atcggctgaa gcagggctag gtcggcgaca acgcgctcgg ctaatatggc 6600
ctgctgcacc tgcgtgaggg tagactggaa gtcatccatg tccacaaagc ggtggtatgc 6660
gcccgtgttg atggtgtaag tgcagttggc cataacggac cagttaacgg tctggtgacc 6720
cggctgcgag agctcggtgt acctgagacg cgagtaagcc ctcgagtcaa atacgtagtc 6780
gttgcaagtc cgcaccaggt actggtatcc caccaaaaag tgcggcggcg gctggcggta 6840
gaggggccag cgtagggtgg ccggggctcc gggggcgaga tcttccaaca taaggcgatg 6900
atatccgtag atgtacctgg acatccaggt gatgccggcg gcggtggtgg aggcgcgcgg 6960
aaagtcgcgg acgcggttcc agatgttgcg cagcggcaaa aagtgctcca tggtcgggac 7020
gctctggccg gtcaggcgcg cgcaatcgtt gacgctctac cgtgcaaaag gagagcctgt 7080
aagcgggcac tcttccgtgg tctggtggat aaattcgcaa gggtatcatg gcggacgacc 7140
ggggttcgag ccccgtatcc ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc 7200
gaacccaggt gtgcgacgtc agacaacggg ggagtgctcc ttttggcttc cttccaggcg 7260
cggcggctgc tgcgctagct tttttggcca ctggccgcgc gcagcgtaag cggttaggct 7320
ggaaagcgaa agcattaagt ggctcgctcc ctgtagccgg agggttattt tccaagggtt 7380
gagtcgcggg acccccggtt cgagtctcgg accggccgga ctgcggcgaa cgggggtttg 7440
cctccccgtc atgcaagacc ccgcttgcaa attcctccgg aaacagggac gagccccttt 7500
tttgcttttc ccagatgcat ccggtgctgc ggcagatgcg cccccctcct cagcagcggc 7560
aagagcaaga gcagcggcag acatgcaggg caccctcccc tcctcctacc gcgtcaggag 7620
gggcgacatc cgcggttgac gcggcagcag atggtgatta cgaacccccg cggcgccggg 7680
cccggcacta cctggacttg gaggagggcg agggcctggc gcggctagga gcgccctctc 7740
ctgagcggta cccaagggtg cagctgaagc gtgatacgcg tgaggcgtac gtgccgcggc 7800
agaacctgtt tcgcgaccgc gagggagagg agcccgagga gatgcgggat cgaaagttcc 7860
acgcagggcg cgagctgcgg catggcctga atcgcgagcg gttgctgcgc gaggaggact 7920
ttgagcccga cgcgcgaacc gggattagtc ccgcgcgcgc acacgtggcg gccgccgacc 7980
tggtaaccgc atacgagcag acggtgaacc aggagattaa ctttcaaaaa agctttaaca 8040
accacgtgcg tacgcttgtg gcgcgcgagg aggtggctat aggactgatg catctgtggg 8100
actttgtaag cgcgctggag caaaacccaa atagcaagcc gctcatggcg cagctgttcc 8160
ttatagtgca gcacagcagg gacaacgagg cattcaggga tgcgctgcta aacatagtag 8220
agcccgaggg ccgctggctg ctcgatttga taaacatcct gcagagcata gtggtgcagg 8280
agcgcagctt gagcctggct gacaaggtgg ccgccatcaa ctattccatg cttagcctgg 8340
gcaagtttta cgcccgcaag atataccata ccccttacgt tcccatagac aaggaggtaa 8400
agatcgaggg gttctacatg cgcatggcgc tgaaggtgct taccttgagc gacgacctgg 8460
gcgtttatcg caacgagcgc atccacaagg ccgtgagcgt gagccggcgg cgcgagctca 8520
gcgaccgcga gctgatgcac agcctgcaaa gggccctggc tggcacgggc agcggcgata 8580
gagaggccga gtcctacttt gacgcgggcg ctgacctgcg ctgggcccca agccgacgcg 8640
ccctggaggc agctggggcc ggacctgggc tggcggtggc acccgcgcgc gctggcaacg 8700
tcggcggcgt ggaggaatat gacgaggacg atgagtacga gccagaggac ggcgagtact 8760
aagcggtgat gtttctgatc agatgatgca agacgcaacg gacccggcgg tgcgggcggc 8820
gctgcagagc cagccgtccg gccttaactc cacggacgac tggcgccagg tcatggaccg 8880
catcatgtcg ctgactgcgc gcaatcctga cgcgttccgg cagcagccgc aggccaaccg 8940
gctctccgca attctggaag cggtggtccc ggcgcgcgca aaccccacgc acgagaaggt 9000
gctggcgatc gtaaacgcgc tggccgaaaa cagggccatc cggcccgacg aggccggcct 9060
ggtctacgac gcgctgcttc agcgcgtggc tcgttacaac agcggcaacg tgcagaccaa 9120
cctggaccgg ctggtggggg atgtgcgcga ggccgtggcg cagcgtgagc gcgcgcagca 9180
gcagggcaac ctgggctcca tggttgcact aaacgccttc ctgagtacac agcccgccaa 9240
cgtgccgcgg ggacaggagg actacaccaa ctttgtgagc gcactgcggc taatggtgac 9300
tgagacaccg caaagtgagg tgtaccagtc tgggccagac tattttttcc agaccagtag 9360
acaaggcctg cagaccgtaa acctgagcca ggctttcaaa aacttgcagg ggctgtgggg 9420
ggtgcgggct cccacaggcg accgcgcgac cgtgtctagc ttgctgacgc ccaactcgcg 9480
cctgttgctg ctgctaatag cgcccttcac ggacagtggc agcgtgtccc gggacacata 9540
cctaggtcac ttgctgacac tgtaccgcga ggccataggt caggcgcatg tggacgagca 9600
tactttccag gagattacaa gtgtcagccg cgcgctgggg caggaggaca cgggcagcct 9660
ggaggcaacc ctaaactacc tgctgaccaa ccggcggcag aagatcccct cgttgcacag 9720
tttaaacagc gaggaggagc gcattttgcg ctacgtgcag cagagcgtga gccttaacct 9780
gatgcgcgac ggggtaacgc ccagcgtggc gctggacatg accgcgcgca acatggaacc 9840
gggcatgtat gcctcaaacc ggccgtttat caaccgccta atggactact tgcatcgcgc 9900
ggccgccgtg aaccccgagt atttcaccaa tgccatcttg aacccgcact ggctaccgcc 9960
ccctggtttc tacaccgggg gattcgaggt gcccgagggt aacgatggat tcctctggga 10020
cgacatagac gacagcgtgt tttccccgca accgcagacc ctgctagagt tgcaacagcg 10080
cgagcaggca gaggcggcgc tgcgaaagga aagcttccgc aggccaagca gcttgtccga 10140
tctaggcgct gcggccccgc ggtcagatgc tagtagccca tttccaagct tgatagggtc 10200
tcttaccagc actcgcacca cccgcccgcg cctgctgggc gaggaggagt acctaaacaa 10260
ctcgctgctg cagccgcagc gcgaaaaaaa cctgcctccg gcatttccca acaacgggat 10320
agagagccta gtggacaaga tgagtagatg gaagacgtac gcgcaggagc acagggacgt 10380
gccaggcccg cgcccgccca cccgtcgtca aaggcacgac cgtcagcggg gtctggtgtg 10440
ggaggacgat gactcggcag acgacagcag cgtcctggat ttgggaggga gtggcaaccc 10500
gtttgcgcac cttcgcccca ggctggggag aatgttttaa aaaaaaaaaa gcatgatgca 10560
aaataaaaaa ctcaccaagg ccatggcacc gagcgttggt tttcttgtat tccccttagt 10620
atgcggcgcg cggcgatgta tgaggaaggt cctcctccct cctacgagag tgtggtgagc 10680
gcggcgccag tggcggcggc gctgggttct cccttcgatg ctcccctgga cccgccgttt 10740
gtgcctccgc ggtacctgcg gcctaccggg gggagaaaca gcatccgtta ctctgagttg 10800
gcacccctat tcgacaccac ccgtgtgtac ctggtggaca acaagtcaac ggatgtggca 10860
tccctgaact accagaacga ccacagcaac tttctgacca cggtcattca aaacaatgac 10920
tacagcccgg gggaggcaag cacacagacc atcaatcttg acgaccggtc gcactggggc 10980
ggcgacctga aaaccatcct gcataccaac atgccaaatg tgaacgagtt catgtttacc 11040
aataagttta aggcgcgggt gatggtgtcg cgcttgccta ctaaggacaa tcaggtggag 11100
ctgaaatacg agtgggtgga gttcacgctg cccgagggca actactccga gaccatgacc 11160
atagacctta tgaacaacgc gatcgtggag cactacttga aagtgggcag acagaacggg 11220
gttctggaaa gcgacatcgg ggtaaagttt gacacccgca acttcagact ggggtttgac 11280
cccgtcactg gtcttgtcat gcctggggta tatacaaacg aagccttcca tccagacatc 11340
attttgctgc caggatgcgg ggtggacttc acccacagcc gcctgagcaa cttgttgggc 11400
atccgcaagc ggcaaccctt ccaggagggc tttaggatca cctacgatga tctggagggt 11460
ggtaacattc ccgcactgtt ggatgtggac gcctaccagg cgagcttgaa agatgacacc 11520
gaacagggcg ggggtggcgc aggcggcagc aacagcagtg gcagcggcgc ggaagagaac 11580
tccaacgcgg cagccgcggc aatgcagccg gtggaggaca tgaacgatca tgccattcgc 11640
ggcgacacct ttgccacacg ggctgaggag aagcgcgctg aggccgaagc agcggccgaa 11700
gctgccgccc ccgctgcgca acccgaggtc gagaagcctc agaagaaacc ggtgatcaaa 11760
cccctgacag aggacagcaa gaaacgcagt tacaacctaa taagcaatga cagcaccttc 11820
acccagtacc gcagctggta ccttgcatac aactacggcg accctcagac cggaatccgc 11880
tcatggaccc tgctttgcac tcctgacgta acctgcggct cggagcaggt ctactggtcg 11940
ttgccagaca tgatgcaaga ccccgtgacc ttccgctcca cgcgccagat cagcaacttt 12000
ccggtggtgg gcgccgagct gttgcccgtg cactccaaga gcttctacaa cgaccaggcc 12060
gtctactccc aactcatccg ccagtttacc tctctgaccc acgtgttcaa tcgctttccc 12120
gagaaccaga ttttggcgcg cccgccagcc cccaccatca ccaccgtcag tgaaaacgtt 12180
cctgctctca cagatcacgg gacgctaccg ctgcgcaaca gcatcggagg agtccagcga 12240
gtgaccatta ctgacgccag acgccgcacc tgcccctacg tttacaaggc cctgggcata 12300
gtctcgccgc gcgtcctatc gagccgcact ttttgagcaa gcatgtccat ccttatatcg 12360
cccagcaata acacaggctg gggcctgcgc ttcccaagca agatgtttgg cggggccaag 12420
aagcgctccg accaacaccc agtgcgcgtg cgcgggcact accgcgcgcc ctggggcgcg 12480
cacaaacgcg gccgcactgg gcgcaccacc gtcgatgacg ccatcgacgc ggtggtggag 12540
gaggcgcgca actacacgcc cacgccgcca ccagtgtcca cagtggacgc ggccattcag 12600
accgtggtgc gcggagcccg gcgctatgct aaaatgaaga gacggcggag gcgcgtagca 12660
cgtcgccacc gccgccgacc cggcactgcc gcccaacgcg cggcggcggc cctgcttaac 12720
cgcgcacgtc gcaccggccg acgggcggcc atgcgggccg ctcgaaggct ggccgcgggt 12780
attgtcactg tgccccccag gtccaggcga cgagcggccg ccgcagcagc cgcggccatt 12840
agtgctatga ctcagggtcg caggggcaac gtgtattggg tgcgcgactc ggttagcggc 12900
ctgcgcgtgc ccgtgcgcac ccgccccccg cgcaactaga ttgcaagaaa aaactactta 12960
gactcgtact gttgtatgta tccagcggcg gcggcgcgca acgaagctat gtccaagcgc 13020
aaaatcaaag aagagatgct ccaggtcatc gcgccggaga tctatggccc cccgaagaag 13080
gaagagcagg attacaagcc ccgaaagcta aagcgggtca aaaagaaaaa gaaagatgat 13140
gatgatgaac ttgacgacga ggtggaactg ctgcacgcta ccgcgcccag gcgacgggta 13200
cagtggaaag gtcgacgcgt aaaacgtgtt ttgcgacccg gcaccaccgt agtctttacg 13260
cccggtgagc gctccacccg cacctacaag cgcgtgtatg atgaggtgta cggcgacgag 13320
gacctgcttg agcaggccaa cgagcgcctc ggggagtttg cctacggaaa gcggcataag 13380
gacatgctgg cgttgccgct ggacgagggc aacccaacac ctagcctaaa gcccgtaaca 13440
ctgcagcagg tgctgcccgc gcttgcaccg tccgaagaaa agcgcggcct aaagcgcgag 13500
tctggtgact tggcacccac cgtgcagctg atggtaccca agcgccagcg actggaagat 13560
gtcttggaaa aaatgaccgt ggaacctggg ctggagcccg aggtccgcgt gcggccaatc 13620
aagcaggtgg cgccgggact gggcgtgcag accgtggacg ttcagatacc cactaccagt 13680
agcaccagta ttgccaccgc cacagagggc atggagacac aaacgtcccc ggttgcctca 13740
gcggtggcgg atgccgcggt gcaggcggtc gctgcggccg cgtccaagac ctctacggag 13800
gtgcaaacgg acccgtggat gtttcgcgtt tcagcccccc ggcgcccgcg cggttcgagg 13860
aagtacggcg ccgccagcgc gctactgccc gaatatgccc tacatccttc cattgcgcct 13920
acccccggct atcgtggcta cacctaccgc cccagaagac gagcaactac ccgacgccga 13980
accaccactg gaacccgccg ccgccgtcgc cgtcgccagc ccgtgctggc cccgatttcc 14040
gtgcgcaggg tggctcgcga aggaggcagg accctggtgc tgccaacagc gcgctaccac 14100
cccagcatcg tttaaaagcc ggtctttgtg gttcttgcag atatggccct cacctgccgc 14160
ctccgtttcc cggtgccggg attccgagga agaatgcacc gtaggagggg catggccggc 14220
cacggcctga cgggcggcat gcgtcgtgcg caccaccggc ggcggcgcgc gtcgcaccgt 14280
cgcatgcgcg gcggtatcct gcccctcctt attccactga tcgccgcggc gattggcgcc 14340
gtgcccggaa ttgcatccgt ggccttgcag gcgcagagac actgattaaa aacaagttgc 14400
atgtggaaaa atcaaaataa aaagtctgga ctctcacgct cgcttggtcc tgtaactatt 14460
ttgtagaatg gaagacatca actttgcgtc tctggccccg cgacacggct cgcgcccgtt 14520
catgggaaac tggcaagata tcggcaccag caatatgagc ggtggcgcct tcagctgggg 14580
ctcgctgtgg agcggcatta aaaatttcgg ttccaccgtt aagaactatg gcagcaaggc 14640
ctggaacagc agcacaggcc agatgctgag ggataagttg aaagagcaaa atttccaaca 14700
aaaggtggta gatggcctgg cctctggcat tagcggggtg gtggacctgg ccaaccaggc 14760
agtgcaaaat aagattaaca gtaagcttga tccccgccct cccgtagagg agcctccacc 14820
ggccgtggag acagtgtctc cagaggggcg tggcgaaaag cgtccgcgcc ccgacaggga 14880
agaaactctg gtgacgcaaa tagacgagcc tccctcgtac gaggaggcac taaagcaagg 14940
cctgcccacc acccgtccca tcgcgcccat ggctaccgga gtgctgggcc agcacacacc 15000
cgtaacgctg gacctgcctc cccccgccga cacccagcag aaacctgtgc tgccaggccc 15060
gaccgccgtt gttgtaaccc gtcctagccg cgcgtccctg cgccgcgccg ccagcggtcc 15120
gcgatcgttg cggcccgtag ccagtggcaa ctggcaaagc acactgaaca gcatcgtggg 15180
tctgggggtg caatccctga agcgccgacg atgcttctga atagctaacg tgtcgtatgt 15240
gtgtcatgta tgcgtccatg tcgccgccag aggagctgct gagccgccgc gcgcccgctt 15300
tccaagatgg ctaccccttc gatgatgccg cagtggtctt acatgcacat ctcgggccag 15360
gacgcctcgg agtacctgag ccccgggctg gtgcagtttg cccgcgccac cgagacgtac 15420
ttcagcctga ataacaagtt tagaaacccc acggtggcgc ctacgcacga cgtgaccaca 15480
gaccggtccc agcgtttgac gctgcggttc atccctgtgg accgtgagga tactgcgtac 15540
tcgtacaagg cgcggttcac cctagctgtg ggtgataacc gtgtgctgga catggcttcc 15600
acgtactttg acatccgcgg cgtgctggac aggggcccta cttttaagcc ctactctggc 15660
actgcctaca acgccctggc tcccaagggt gccccaaatc cttgcgaatg ggatgaagct 15720
gctactgctc ttgaaataaa cctagaagaa gaggacgatg acaacgaaga cgaagtagac 15780
gagcaagctg agcagcaaaa aactcacgta tttgggcagg cgccttattc tggtataaat 15840
attacaaagg agggtattca aataggtgtc gaaggtcaaa cacctaaata tgccgataaa 15900
acatttcaac ctgaacctca aataggagaa tctcagtggt acgaaactga aattaatcat 15960
gcagctggga gagtccttaa aaagactacc ccaatgaaac catgttacgg ttcatatgca 16020
aaacccacaa atgaaaatgg agggcaaggc attcttgtaa agcaacaaaa tggaaagcta 16080
gaaagtcaag tggaaatgca atttttctca actactgagg cgaccgcagg caatggtgat 16140
aacttgactc ctaaagtggt attgtacagt gaagatgtag atatagaaac cccagacact 16200
catatttctt acatgcccac tattaaggaa ggtaactcac gagaactaat gggccaacaa 16260
tctatgccca acaggcctaa ttacattgct tttagggaca attttattgg tctaatgtat 16320
tacaacagca cgggtaatat gggtgttctg gcgggccaag catcgcagtt gaatgctgtt 16380
gtagatttgc aagacagaaa cacagagctt tcataccagc ttttgcttga ttccattggt 16440
gatagaacca ggtacttttc tatgtggaat caggctgttg acagctatga tccagatgtt 16500
agaattattg aaaatcatgg aactgaagat gaacttccaa attactgctt tccactggga 16560
ggtgtgatta atacagagac tcttaccaag gtaaaaccta aaacaggtca ggaaaatgga 16620
tgggaaaaag atgctacaga attttcagat aaaaatgaaa taagagttgg aaataatttt 16680
gccatggaaa tcaatctaaa tgccaacctg tggagaaatt tcctgtactc caacatagcg 16740
ctgtatttgc ccgacaagct aaagtacagt ccttccaacg taaaaatttc tgataaccca 16800
aacacctacg actacatgaa caagcgagtg gtggctcccg ggttagtgga ctgctacatt 16860
aaccttggag cacgctggtc ccttgactat atggacaacg tcaacccatt taaccaccac 16920
cgcaatgctg gcctgcgcta ccgctcaatg ttgctgggca atggtcgcta tgtgcccttc 16980
cacatccagg tgcctcagaa gttctttgcc attaaaaacc tccttctcct gccgggctca 17040
tacacctacg agtggaactt caggaaggat gttaacatgg ttctgcagag ctccctagga 17100
aatgacctaa gggttgacgg agccagcatt aagtttgata gcatttgcct ttacgccacc 17160
ttcttcccca tggcccacaa caccgcctcc acgcttgagg ccatgcttag aaacgacacc 17220
aacgaccagt cctttaacga ctatctctcc gccgccaaca tgctctaccc tatacccgcc 17280
aacgctacca acgtgcccat atccatcccc tcccgcaact gggcggcttt ccgcggctgg 17340
gccttcacgc gccttaagac taaggaaacc ccatcactgg gctcgggcta cgacccttat 17400
tacacctact ctggctctat accctaccta gatggaacct tttacctcaa ccacaccttt 17460
aagaaggtgg ccattacctt tgactcttct gtcagctggc ctggcaatga ccgcctgctt 17520
acccccaacg agtttgaaat taagcgctca gttgacgggg agggttacaa cgttgcccag 17580
tgtaacatga ccaaagactg gttcctggta caaatgctag ctaactacaa cattggctac 17640
cagggcttct atatcccaga gagctacaag gaccgcatgt actccttctt tagaaacttc 17700
cagcccatga gccgtcaggt ggtggatgat actaaataca aggactacca acaggtgggc 17760
atcctacacc aacacaacaa ctctggattt gttggctacc ttgcccccac catgcgcgaa 17820
ggacaggcct accctgctaa cttcccctat ccgcttatag gcaagaccgc agttgacagc 17880
attacccaga aaaagtttct ttgcgatcgc accctttggc gcatcccatt ctccagtaac 17940
tttatgtcca tgggcgcact cacagacctg ggccaaaacc ttctctacgc caactccgcc 18000
cacgcgctag acatgacttt tgaggtggat cccatggacg agcccaccct tctttatgtt 18060
ttgtttgaag tctttgacgt ggtccgtgtg caccggccgc accgcggcgt catcgaaacc 18120
gtgtacctgc gcacgccctt ctcggccggc aacgccacaa cataaagaag caagcaacat 18180
caacaacagc tgccgccatg ggctccagtg agcaggaact gaaagccatt gtcaaagatc 18240
ttggttgtgg gccatatttt ttgggcacct atgacaagcg ctttccaggc tttgtttctc 18300
cacacaagct cgcctgcgcc atagtcaata cggccggtcg cgagactggg ggcgtacact 18360
ggatggcctt tgcctggaac ccgcactcaa aaacatgcta cctctttgag ccctttggct 18420
tttctgacca gcgactcaag caggtttacc agtttgagta cgagtcactc ctgcgccgta 18480
gcgccattgc ttcttccccc gaccgctgta taacgctgga aaagtccacc caaagcgtac 18540
aggggcccaa ctcggccgcc tgtggactat tctgctgcat gtttctccac gcctttgcca 18600
actggcccca aactcccatg gatcacaacc ccaccatgaa ccttattacc ggggtaccca 18660
actccatgct caacagtccc caggtacagc ccaccctgcg tcgcaaccag gaacagctct 18720
acagcttcct ggagcgccac tcgccctact tccgcagcca cagtgcgcag attaggagcg 18780
ccacttcttt ttgtcacttg aaaaacatgt aaaaataatg tactagagac actttcaata 18840
aaggcaaatg cttttatttg tacactctcg ggtgattatt tacccccacc cttgccgtct 18900
gcgccgttta aaaatcaaag gggttctgcc gcgcatcgct atgcgccact ggcagggaca 18960
cgttgcgata ctggtgttta gtgctccact taaactcagg cacaaccatc cgcggcagct 19020
cggtgaagtt ttcactccac aggctgcgca ccatcaccaa cgcgtttagc aggtcgggcg 19080
ccgatatctt gaagtcgcag ttggggcctc cgccctgcgc gcgcgagttg cgatacacag 19140
ggttgcagca ctggaacact atcagcgccg ggtggtgcac gctggccagc acgctcttgt 19200
cggagatcag atccgcgtcc aggtcctccg cgttgctcag ggcgaacgga gtcaactttg 19260
gtagctgcct tcccaaaaag ggcgcgtgcc caggctttga gttgcactcg caccgtagtg 19320
gcatcaaaag gtgaccgtgc ccggtctggg cgttaggata cagcgcctgc ataaaagcct 19380
tgatctgctt aaaagccacc tgagcctttg cgccttcaga gaagaacatg ccgcaagact 19440
tgccggaaaa ctgattggcc ggacaggccg cgtcgtgcac gcagcacctt gcgtcggtgt 19500
tggagatctg caccacattt cggccccacc ggttcttcac gatcttggcc ttgctagact 19560
gctccttcag cgcgcgctgc ccgttttcgc tcgtcacatc catttcaatc acgtgctcct 19620
tatttatcat aatgcttccg tgtagacact taagctcgcc ttcgatctca gcgcagcggt 19680
gcagccacaa cgcgcagccc gtgggctcgt gatgcttgta ggtcacctct gcaaacgact 19740
gcaggtacgc ctgcaggaat cgccccatca tcgtcacaaa ggtcttgttg ctggtgaagg 19800
tcagctgcaa cccgcggtgc tcctcgttca gccaggtctt gcatacggcc gccagagctt 19860
ccacttggtc aggcagtagt ttgaagttcg cctttagatc gttatccacg tggtacttgt 19920
ccatcagcgc gcgcgcagcc tccatgccct tctcccacgc agacacgatc ggcacactca 19980
gcgggttcat caccgtaatt tcactttccg cttcgctggg ctcttcctct tcctcttgcg 20040
tccgcatacc acgcgccact gggtcgtctt cattcagccg ccgcactgtg cgcttacctc 20100
ctttgccatg cttgattagc accggtgggt tgctgaaacc caccatttgt agcgccacat 20160
cttctctttc ttcctcgctg tccacgatta cctctggtga tggcgggcgc tcgggcttgg 20220
gagaagggcg cttctttttc ttcttgggcg caatggccaa atccgccgcc gaggtcgatg 20280
gccgcgggct gggtgtgcgc ggcaccagcg cgtcttgtga tgagtcttcc tcgtcctcgg 20340
actcgatacg ccgcctcatc cgcttttttg ggggcgcccg gggaggcggc ggcgacgggg 20400
acggggacga cacgtcctcc atggttgggg gacgtcgcgc cgcaccgcgt ccgcgctcgg 20460
gggtggtttc gcgctgctcc tcttcccgac tggccatttc cttctcctat aggcagaaaa 20520
agatcatgga gtcagtcgag aagaaggaca gcctaaccgc cccctctgag ttcgccacca 20580
ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca cccccgcttg 20640
aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac gacgaggacc 20700
gctcagtacc aacagaggat aaaaagcaag accaggacaa cgcagaggca aacgaggaac 20760
aagtcgggcg gggggacgaa aggcatggcg actacctaga tgtgggagac gacgtgctgt 20820
tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag cgcagcgatg 20880
tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctattc tcaccgcgcg 20940
taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc aacttctacc 21000
ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa aactgcaaga 21060
tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc ttgcggcagg 21120
gcgctgtcat acctgatatc gcctcgctca acgaagtgcc aaaaatcttt gagggtcttg 21180
gacgcgacga gaagcgcgcg gcaaacgctc tgcaacagga aaacagcgaa aatgaaagtc 21240
actctggagt gttggtggaa ctcgagggtg acaacgcgcg cctagccgta ctaaaacgca 21300
gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag gtcatgagca 21360
cagtcatgag tgagctgatc gtgcgccgtg cgcagcccct ggagagggat gcaaatttgc 21420
aagaacaaac agaggagggc ctacccgcag ttggcgacga gcagctagcg cgctggcttc 21480
aaacgcgcga gcctgccgac ttggaggagc gacgcaaact aatgatggcc gcagtgctcg 21540
ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg cagcgcaagc 21600
tagaggaaac attgcactac acctttcgac agggctacgt acgccaggcc tgcaagatct 21660
ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa aaccgccttg 21720
ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac gtccgcgact 21780
gcgtttactt atttctatgc tacacctggc agacggccat gggcgtttgg cagcagtgct 21840
tggaggagtg caacctcaag gagctgcaga aactgctaaa gcaaaacttg aaggacctat 21900
ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacatcatt ttccccgaac 21960
gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc atgttgcaga 22020
actttaggaa ctttatccta gagcgctcag gaatcttgcc cgccacctgc tgtgcacttc 22080
ctagcgactt tgtgcccatt aagtaccgcg aatgccctcc gccgctttgg ggccactgct 22140
accttctgca gctagccaac taccttgcct accactctga cataatggaa gacgtgagcg 22200
gtgacggtct actggagtgt cactgtcgct gcaacctatg caccccgcac cgctccctgg 22260
tttgcaattc gcagctgctt aacgaaagtc aaattatcgg tacctttgag ctgcagggtc 22320
cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg ctgtggacgt 22380
cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt aggttctacg 22440
aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc cagggccaca 22500
ttcttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta cgaaagggac 22560
ggggggttta cttggacccc cagtccggcg aggagctcaa cccaatcccc ccgccgccgc 22620
agccctatca gcagcagccg cgggcccttg cttcccagga tggcacccaa aaagaagctg 22680
cagctgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc agaggaggtt 22740
ttggacgagg aggaggagga catgatggaa gactgggaga gcctagacga ggaagcttcc 22800
gaggtcgaag aggtgtcaga cgaaacaccg tcaccctcgg tcgcattccc ctcgccggcg 22860
ccccagaaat cggcaaccgg ttccagcatg gctacaacct ccgctcctca ggcgccgccg 22920
gcactgcccg ttcgccgacc caaccgtaga tgggacacca ctggaaccag ggccggtaag 22980
tccaagcagc cgccgccgtt agcccaagag caacaacagc gccaaggcta ccgctcatgg 23040
cgcgggcaca agaacgccat agttgcttgc ttgcaagact gtgggggcaa catctccttc 23100
gcccgccgct ttcttctcta ccatcacggc gtggccttcc cccgtaacat cctgcattac 23160
taccgtcatc tctacagccc atactgcacc ggcggcagcg gcagcggcag caacagcagc 23220
ggccacacag aagcaaaggc gaccggatag caagactctg acaaagccca agaaatccac 23280
agcggcggca gcagcaggag gaggagcgct gcgtctggcg cccaacgaac ccgtatcgac 23340
ccgcgagctt agaaacagga tttttcccac tctgtatgct atatttcaac agagcagggg 23400
ccaagaacaa gagctgaaaa taaaaaacag gtctctgcga tccctcaccc gcagctgcct 23460
gtatcacaaa agcgaagatc agcttcggcg cacgctggaa gacgcggagg ctctcttcag 23520
taaatactgc gcgctgactc ttaaggacta gtttcgcgcc ctttctcaaa tttaagcgcg 23580
aaaactacgt catctccagc ggccacaccc ggcgccagca cctgtcgtca gcgccattat 23640
gagcaaggaa attcccacgc cctacatgtg gagttaccag ccacaaatgg gacttgcggc 23700
tggagctgcc caagactact caacccgaat aaactacatg agcgcgggac cccacatgat 23760
atcccgggtc aacggaatcc gcgcccaccg aaaccgaatt ctcttggaac aggcggctat 23820
taccaccaca cctcgtaata accttaatcc ccgtagttgg cccgctgccc tggtgtacca 23880
ggaaagtccc gctcccacca ctgtggtact tcccagagac gcccaggccg aagttcagat 23940
gactaactca ggggcgcagc ttgcgggcgg ctttcgtcac agggtgcggt cgcccgggca 24000
gggtataact cacctgacaa tcagagggcg aggtattcag ctcaacgacg agtcggtgag 24060
ctcctcgctt ggtctccgtc cggacgggac atttcagatc ggcggcgccg gccgtccttc 24120
attcacgcct cgtcaggcaa tcctaactct gcagacctcg tcctctgagc cgcgctctgg 24180
aggcattgga actctgcaat ttattgagga gtttgtgcca tcggtctact ttaacccctt 24240
ctcgggacct cccggccact atccggatca atttattcct aactttgacg cggtaaagga 24300
ctcggcggac ggctacgact gaatgttaag tggagaggca gagcaactgc gcctgaaaca 24360
cctggtccac tgtcgccgcc acaagtgctt tgcccgcgac tccggtgagt tttgctactt 24420
tgaattgccc gaggatcata tcgagggccc ggcgcacggc gtccggctta ccgcccaggg 24480
agagcttgcc cgtagcctga ttcgggagtt tacccagcgc cccctgctag ttgagcggga 24540
caggggaccc tgtgttctca ctgtgatttg caactgtcct aaccttggat tacatcaaga 24600
tcctctagtt ataactagag tacccgggga tcttattccc tttaactaat aaaaaaaaat 24660
aataaagcat cacttactta aaatcagtta gcaaatttct gtccagttta ttcagcagca 24720
cctccttgcc ctcctcccag ctctggtatt gcagcttcct cctggctgca aactttctcc 24780
acaatctaaa tggaatgtca gtttcctcct gttcctgtcc atccgcaccc actatcttca 24840
tgttgttgca gatgaagcgc gcaagaccgt ctgaagatac cttcaacccc gtgtatccat 24900
atgacacgga aaccggtcct ccaactgtgc cttttcttac tcctcccttt gtatccccca 24960
atgggtttca agagagtccc cctggggtac tctctttgcg cctatccgaa cctctagtta 25020
cctccaatgg catgcttgcg ctcaaaatgg gcaacggcct ctctctggac gaggccggca 25080
accttacctc ccaaaatgta accactgtga gcccacctct caaaaaaacc aagtcaaaca 25140
taaacctgga aatatctgca cccctcacag ttacctcaga agccctaact gtggctgccg 25200
ccgcacctct aatggtcgcg ggcaacacac tcaccatgca atcacaggcc ccgctaaccg 25260
tgcacgactc caaacttagc attgccaccc aaggacccct cacagtgtca gaaggaaagc 25320
tagccctgca aacatcaggc cccctcacca ccaccgatag cagtaccctt actatcactg 25380
cctcaccccc tctaactact gccactggta gcttgggcat tgacttgaaa gagcccattt 25440
atacacaaaa tggaaaacta ggactaaagt acggggctcc tttgcatgta acagacgacc 25500
taaacacttt gaccgtagca actggtccag gtgtgactat taataatact tccttgcaaa 25560
ctaaagttac tggagccttg ggttttgatt cacaaggcaa tatgcaactt aatgtagcag 25620
gaggactaag gattgattct caaaacagac gccttatact tgatgttagt tatccgtttg 25680
atgctcaaaa ccaactaaat ctaagactag gacagggccc tctttttata aactcagccc 25740
acaacttgga tattaactac aacaaaggcc tttacttgtt tacagcttca aacaattcca 25800
aaaagcttga ggttaaccta agcactgcca aggggttgat gtttgacgct acagccatag 25860
ccattaatgc aggagatggg cttgaatttg gttcacctaa tgcaccaaac acaaatcccc 25920
tcaaaacaaa aattggccat ggcctagaat ttgattcaaa caaggctatg gttcctaaac 25980
taggaactgg ccttagtttt gacagcacag gtgccattac agtaggaaac aaaaataatg 26040
ataagctaac tttgtggacc acaccagctc catctcctaa ctgtagacta aatgcagaga 26100
aagatgctaa actcactttg gtcttaacaa aatgtggcag tcaaatactt gctacagttt 26160
cagttttggc tgttaaaggc agtttggctc caatatctgg aacagttcaa agtgctcatc 26220
ttattataag atttgacgaa aatggagtgc tactaaacaa ttccttcctg gacccagaat 26280
attggaactt tagaaatgga gatcttactg aaggcacagc ctatacaaac gctgttggat 26340
ttatgcctaa cctatcagct tatccaaaat ctcacggtaa aactgccaaa agtaacattg 26400
tcagtcaagt ttacttaaac ggagacaaaa ctaaacctgt aacactaacc attacactaa 26460
acggtacaca ggaaacagga gacacaactc caagtgcata ctctatgtca ttttcatggg 26520
actggtctgg ccacaactac attaatgaaa tatttgccac atcctcttac actttttcat 26580
acattgccca agaataaaga atcgtttgtg ttatgtttca acgtgtttat ttttcaattg 26640
cagaaaattt caagtcattt ttcattcagt agtatagccc caccaccaca tagcttatac 26700
agatcaccgt accttaatca aactcacaga accctagtat tcaacctgcc acctccctcc 26760
caacacacag agtacacagt cctttctccc cggctggcct taaaaagcat catatcatgg 26820
gtaacagaca tattcttagg tgttatattc cacacggttt cctgtcgagc caaacgctca 26880
tcagtgatat taataaactc cccgggcagc tcacttaagt tcatgtcgct gtccagctgc 26940
tgagccacag gctgctgtcc aacttgcggt tgcttaacgg gcggcgaagg agaagtccac 27000
gcctacatgg gggtagagtc ataatcgtgc atcaggatag ggcggtggtg ctgcagcagc 27060
gcgcgaataa actgctgccg ccgccgctcc gtcctgcagg aatacaacat ggcagtggtc 27120
tcctcagcga tgattcgcac cgcccgcagc ataaggcgcc ttgtcctccg ggcacagcag 27180
cgcaccctga tctcacttaa atcagcacag taactgcagc acagcaccac aatattgttc 27240
aaaatcccac agtgcaaggc gctgtatcca aagctcatgg cggggaccac agaacccacg 27300
tggccatcat accacaagcg caggtagatt aagtggcgac ccctcataaa cacgctggac 27360
ataaacatta cctcttttgg catgttgtaa ttcaccacct cccggtacca tataaacctc 27420
tgattaaaca tggcgccatc caccaccatc ctaaaccagc tggccaaaac ctgcccgccg 27480
gctatacact gcagggaacc gggactggaa caatgacagt ggagagccca ggactcgtaa 27540
ccatggatca tcatgctcgt catgatatca atgttggcac aacacaggca cacgtgcata 27600
cacttcctca ggattacaag ctcctcccgc gttagaacca tatcccaggg aacaacccat 27660
tcctgaatca gcgtaaatcc cacactgcag ggaagacctc gcacgtaact cacgttgtgc 27720
attgtcaaag tgttacattc gggcagcagc ggatgatcct ccagtatggt agcgcgggtt 27780
tctgtctcaa aaggaggtag acgatcccta ctgtacggag tgcgccgaga caaccgagat 27840
cgtgttggtc gtagtgtcat gccaaatgga acgccggacg tagtcatatt tcctgaagca 27900
aaaccaggtg cgggcgtgac aaacagatct gcgtctccgg tctcgccgct tagatcgctc 27960
tgtgtagtag ttgtagtata tccactctct caaagcatcc aggcgccccc tggcttcggg 28020
ttctatgtaa actccttcat gcgccgctgc cctgataaca tccaccaccg cagaataagc 28080
cacacccagc caacctacac attcgttctg cgagtcacac acgggaggag cgggaagagc 28140
tggaagaacc atgttttttt ttttattcca aaagattatc caaaacctca aaatgaagat 28200
ctattaagtg aacgcgctcc cctccggtgg cgtggtcaaa ctctacagcc aaagaacaga 28260
taatggcatt tgtaagatgt tgcacaatgg cttccaaaag gcaaacggcc ctcacgtcca 28320
agtggacgta aaggctaaac ccttcagggt gaatctcctc tataaacatt ccagcacctt 28380
caaccatgcc caaataattc tcatctcgcc accttctcaa tatatctcta agcaaatccc 28440
gaatattaag tccggccatt gtaaaaatct gctccagagc gccctccacc ttcagcctca 28500
agcagcgaat catgattgca aaaattcagg ttcctcacag acctgtataa gattcaaaag 28560
cggaacatta acaaaaatac cgcgatcccg taggtccctt cgcagggcca gctgaacata 28620
atcgtgcagg tctgcacgga ccagcgcggc cacttccccg ccaggaacct tgacaaaaga 28680
acccacactg attatgacac gcatactcgg agctatgcta accagcgtag ccccgatgta 28740
agctttcaat agcttgttgc atgggcggcg atataaaatg caaggtgctg ctcaaaaaat 28800
caggcaaagc ctcgcgcaaa aaagaaagca catcgtagtc atgctcatgc agataaaggc 28860
aggtaagctc cggaaccacc acagaaaaag acaccatttt tctctcaaac atgtctgcgg 28920
gtttctgcat aaacacaaaa taaaataaca aaaaaacatt taaacattag aagcctgtct 28980
tacaacagga aaaacaaccc ttataagcat aagacggact acggccatgc cggcgtgacc 29040
gtaaaaaaac tggtcaccgt gattaaaaag caccaccgac agctcctcgg tcatgtccgg 29100
agtcataatg taagactcgg taaacacatc aggttgattc atcggtcagt gctaaaaagc 29160
gaccgaaata gcccggggga atacataccc gcaggcgtag agacaacatt acagccccca 29220
taggaggtat aacaaaatta ataggagaga aaaacacata aacacctgaa aaaccctcct 29280
gcctaggcaa aatagcaccc tcccgctcca gaacaacata cagcgcttca cagcggcagc 29340
ctaacagtca gccttaccag taaaaaagaa aacctattaa aaaaacacca ctcgacacgg 29400
caccagctca atcagtcaca gtgtaaaaaa gggccaagtg cagagcgagt atatatagga 29460
ctaaaaaatg acgtaacggt taaagtccac aaaaaacacc cagaaaaccg cacgcgaacc 29520
tacgcccaga aacgaaagcc aaaaaaccca caacttcctc aaatcgtcac ttccgttttc 29580
ccacgttacg taacttccca ttttaagaaa actacaattc ccaacacata caagttactc 29640
cgccctaaaa cctacgtcac ccgccccgtt cccacgcccc gcgccacgtc acaaactcca 29700
ccccctcatt atcatattgg cttcaatcca aaataaggta tattattgat gatgttaatt 29760
aacatgcatg gatccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 29820
gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 29880
ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 29940
acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 30000
cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 30060
caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 30120
gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 30180
tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 30240
aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 30300
ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 30360
cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 30420
tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 30480
tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 30540
ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 30600
aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 30660
aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 30720
aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 30780
gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 30840
gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 30900
caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 30960
ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 31020
attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 31080
ccattgctgc agccatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga 31140
aagccagtcc gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga 31200
caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat 31260
agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct 31320
ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct 31380
gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg 31440
aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 31500
actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 31560
ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg 31620
aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 31680
ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 31740
tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 31800
tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 31860
gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 31920
aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg 31980
atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 32040
tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 32100
tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 32160
tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 32220
tcttctgaat tttgttaaaa tttttgttaa atcagctcat tttttaacca ataggccgaa 32280
atcggcaaca tcccttataa atcaaaagaa tagaccgcga tagggttgag tgttgttcca 32340
gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc 32400
gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttgcggtcg 32460
aggtgccgta aagctctaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg 32520
ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg 32580
gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgcgcg cttaatgcgc 32640
cgctacaggg cgcgtccatt cgccattcag gatcgaatta attcttaatt aacatcatca 32700
ataatatacc ttattttgga ttgaagccaa tatgataatg agggggtgga gtttgtgacg 32760
tggcgcgggg cgtgggaacg gggcgggtga cgtagtagtg tggcggaagt gtgatgttgc 32820
aagtgtggcg gaacacatgt aagcgacgga tgtggcaaaa gtgacgtttt tggtgtgcgc 32880
cggtgtacac aggaagtgac aattttcgcg cggttttagg cggatgttgt agtaaatttg 32940
ggcgtaaccg agtaagattt ggccattttc gcgggaaaac tgaataagag gaagtgaaat 33000
ctgaataatt ttgtgttact catagcgcgt aatactgcga tctatacatt gaatcaatat 33060
tggcaattag ccatattagt cattggttat atagcataaa tcaatattgg ctattggcca 33120
ttgcatacgt tgtatctata tcataatatg tacatttata ttggctcatg tccaatatga 33180
ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta 33240
gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 33300
tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 33360
ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg 33420
gcagtacatc aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa 33480
tggcccgcct ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac 33540
atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg 33600
cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 33660
agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg 33720
ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta 33780
gtgaaccgtc agatcctcac tctcttccgc atcgctgtct gcgagggcca gctgttgggc 33840
tcgcggttga ggacaaactc ttcgcggtct ttccagtact cttggatcgg aaacccgtcg 33900
gcctccgaac ggtactccgc caccgaggga cctgagccag tccgcatcga ccggatcgga 33960
aaacctctcg agaaaggcgt ctaaccagtc acagtcgcaa ggtaggctga gcaccgtggc 34020
gggcggcagc gggtggcggt cggggttgtt tctggcggag gtgctgctga tgatgtaatt 34080
aaagtaggcg gtcttgagcc ggcggatggt cgaggtgagg tgtggcaggc ttgagatcca 34140
gctgttgggg tgagtactcc ctctcaaaag cgggcatgac ttctgcgcta agattgtcag 34200
tttccaaaaa cgaggaggat ttgatattca cctggcccga tctggccata cacttgagtg 34260
acaatgacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caagtttgga 34320
agatccccat caattactac ttgcttcctg taggaatcct ctattgttgg atcatattcg 34380
tccacaaaat gattctgaat tagctgtatc gtcaaggcac tcttgcaagc ttcatcatgg 34440
ccgacagcaa aagttatcag gcatgcacct ggtagctagt ctttaaacca atagattgca 34500
tcggtttaaa aggcaagacc gtcaaattgc gggaaagggg tcaacagccg ttcagtacca 34560
agtctcaggg gaaactttga gatggccttg caaagggtat ggtaataagc tgacggacat 34620
ggtcctaacc acgcagccaa gtcctaagtc aacagatctt ctgttgatat ggatgcagtt 34680
cacagactaa atgtcggtcg gggaagatgt attcttctca taagatatag tcggacctct 34740
ccttaatggg agctagcgga tgaagtgatg caacactgga gccgctggga actaatttgt 34800
atgcgaaagt atattgatta gttttggagt actcgcgaca tcatcacgtc gttttacaac 34860
gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 34920
gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 34980
caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg 35040
gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca 35100
aactggcaga tgcacggtta cgatgcgccc atctacacca acgtgaccta tcccattacg 35160
gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat 35220
gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttaactcg 35280
gcgtttcatc tgtggtgcaa cgggcgctgg gtcggttacg gccaggacag tcgtttgccg 35340
tctgaatttg acctgagcgc atttttacgc gccggagaaa accgcctcgc ggtgatggtg 35400
ctgcgttgga gtgacggcag ttatctggaa gatcaggata tgtggcggat gagcggcatt 35460
ttccgtgacg tctcgttgct gcataaaccg actacacaaa tcagcgattt ccatgttgcc 35520
actcgcttta atgatgattt cagccgcgct gtactggagg ctgaagttca gatgtgcggc 35580
gagttgcgtg actacctacg ggtaacagtt tctttatggc agggtgaaac gcaggtcgcc 35640
agcggcaccg cgcctttcgg cggtgaaatt atcgatgagc gtggtggtta tgccgatcgc 35700
gtcacactac gtctgaacgt cgaaaacccg aaactgtgga gcgccgaaat cccgaatctc 35760
tatcgtgcgg tggttgaact gcacaccgcc gacggcacgc tgattgaagc agaagcctgc 35820
gatgtcggtt tccgcgaggt gcggattgaa aatggtctgc tgctgctgaa cggcaagccg 35880
ttgctgattc gaggcgttaa ccgtcacgag catcatcctc tgcatggtca ggtcatggat 35940
gagcagacga tggtgcagga tatcctgctg atgaagcaga acaactttaa cgccgtgcgc 36000
tgttcgcatt atccgaacca tccgctgtgg tacacgctgt gcgaccgcta cggcctgtat 36060
gtggtggatg aagccaatat tgaaacccac ggcatggtgc caatgaatcg tctgaccgat 36120
gatccgcgct ggctaccggc gatgagcgaa cgcgtaacgc gaatggtgca gcgcgatcgt 36180
aatcacccga gtgtgatcat ctggtcgctg gggaatgaat caggccacgg cgctaatcac 36240
gacgcgctgt atcgctggat caaatctgtc gatccttccc gcccggtgca gtatgaaggc 36300
ggcggagccg acaccacggc caccgatatt atttgcccga tgtacgcgcg cgtggatgaa 36360
gaccagccct tcccggctgt gccgaaatgg tccatcaaaa aatggctttc gctacctgga 36420
gagacgcgcc cgctgatcct ttgcgaatac gcccacgcga tgggtaacag tcttggcggt 36480
ttcgctaaat actggcaggc gtttcgtcag tatccccgtt tacagggcgg cttcgtctgg 36540
gactgggtgg atcagtcgct gattaaatat gatgaaaacg gcaacccgtg gtcggcttac 36600
ggcggtgatt ttggcgatac gccgaacgat cgccagttct gtatgaacgg tctggtcttt 36660
gccgaccgca cgccgcatcc agcgctgacg gaagcaaaac accagcagca gtttttccag 36720
ttccgtttat ccgggcaaac catcgaagtg accagcgaat acctgttccg tcatagcgat 36780
aacgagctcc tgcactggat ggtggcgctg gatggtaagc cgctggcaag cggtgaagtg 36840
cctctggatg tcgctccaca aggtaaacag ttgattgaac tgcctgaact accgcagccg 36900
gagagcgccg ggcaactctg gctcacagta cgcgtagtgc aaccgaacgc gaccgcatgg 36960
tcagaagccg ggcacatcag cgcctggcag cagtggcgtc tggcggaaaa cctcagtgtg 37020
acgctccccg ccgcgtccca cgccatcccg catctgacca ccagcgaaat ggatttttgc 37080
atcgagctgg gtaataagcg ttggcaattt aaccgccagt caggctttct ttcacagatg 37140
tggattggcg ataaaaaaca actgctgacg ccgctgcgcg atcagttcac ccgtgcaccg 37200
ctggataacg acattggcgt aagtgaagcg acccgcattg accctaacgc ctgggtcgaa 37260
cgctggaagg cggcgggcca ttaccaggcc gaagcagcgt tgttgcagtg cacggcagat 37320
acacttgctg atgcggtgct gattacgacc gctcacgcgt ggcagcatca ggggaaaacc 37380
ttatttatca gccggaaaac ctaccggatt gatggtagtg gtcaaatggc gattaccgtt 37440
gatgttgaag tggcgagcga tacaccgcat ccggcgcgga ttggcctgaa ctgccagctg 37500
gcgcaggtag cagagcgggt aaactggctc ggattagggc cgcaagaaaa ctatcccgac 37560
cgccttactg ccgcctgttt tgaccgctgg gatctgccat tgtcagacat gtataccccg 37620
tacgtcttcc cgagcgaaaa cggtctgcgc tgcgggacgc gcgaattgaa ttatggccca 37680
caccagtggc gcggcgactt ccagttcaac atcagccgct acagtcaaca gcaactgatg 37740
gaaaccagcc atcgccatct gctgcacgcg gaagaaggca catggctgaa tatcgacggt 37800
ttccatatgg ggattggtgg cgacgactcc tggagcccgt cagtatcggc ggaattccag 37860
ctgagcgccg gtcgctacca ttaccagttg gtctggtgtc aaaaataatt cgaaggcgaa 37920
agatcccaac gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat 37980
tacacatggc atggatgaac tgtacaactg aggatccccc gacctcgacc tctggctaat 38040
aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa 38100
ggacatatgg gagggcaaat catttggtcg agatccctcg gagatcggat ctgggcgtgg 38160
38160
Claims (2)
- 서열번호 27로 표시되는 AS100KRib2인 Kras G12V RNA를 표적으로 하여 특이적으로 인지할 수 있는 트랜스-스플라이싱 그룹 I 리보자임.
- 제1항에 따른 리보자임을 유효성분으로 포함하는 항암용 약학 조성물.
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KR1020120039745A KR101153844B1 (ko) | 2012-04-17 | 2012-04-17 | Kras G12V RNA를 특이적으로 인지할 수 있는 트랜스-스플라이싱 그룹 I 리보자임 |
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