KR20220037964A - 재조합 발생이 최소화된 유전자치료 벡터, 상기 벡터를 포함하는 재조합 레트로바이러스 및 상기 재조합 레트로바이러스를 포함하는 암의 예방 또는 치료용 약학 조성물 - Google Patents

재조합 발생이 최소화된 유전자치료 벡터, 상기 벡터를 포함하는 재조합 레트로바이러스 및 상기 재조합 레트로바이러스를 포함하는 암의 예방 또는 치료용 약학 조성물 Download PDF

Info

Publication number
KR20220037964A
KR20220037964A KR1020210118834A KR20210118834A KR20220037964A KR 20220037964 A KR20220037964 A KR 20220037964A KR 1020210118834 A KR1020210118834 A KR 1020210118834A KR 20210118834 A KR20210118834 A KR 20210118834A KR 20220037964 A KR20220037964 A KR 20220037964A
Authority
KR
South Korea
Prior art keywords
gene
vector
cancer
recombination
seq
Prior art date
Application number
KR1020210118834A
Other languages
English (en)
Inventor
김연수
강문경
김수진
Original Assignee
(주)알티큐어
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by (주)알티큐어 filed Critical (주)알티큐어
Priority to JP2023518119A priority Critical patent/JP7412832B2/ja
Priority to CN202180063680.XA priority patent/CN116323924A/zh
Priority to GB2304267.4A priority patent/GB2614168B/en
Priority to PCT/KR2021/012776 priority patent/WO2022060155A1/ko
Publication of KR20220037964A publication Critical patent/KR20220037964A/ko
Priority to US18/185,656 priority patent/US11970708B2/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C12N9/1211—Thymidine kinase (2.7.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04001—Cytosine deaminase (3.5.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

본 발명은 재조합 발생이 최소화된 유전자치료 벡터에 관한 것으로, 바이러스 복제 시 치료 유전자를 지속적으로 발현하는 레트로바이러스 벡터 바이러스 생산 및 감염에 큰 문제점이 되는 재조합 발생을 최소화하고자, MCMV 프로모터를 반복서열 기반으로 잘라주어 절단된 MCMV 프로모터를 제작하고, 상기 절단된 MCMV 프로모터를 도입하여 벡터를 제작하였으며, 절단된 MCMV 프로모터가 도입된 벡터는 여러 번 배양하여도 재조합이 발생하지 않고, 치료 단백질 발현이 지속되며, 상기 벡터를 포함하는 바이러스로 형질감염된 세포에서 전구 약물을 투여한 경우 세포 사멸이 효과적으로 일어남을 확인하였으므로, 본 발명의 재조합 발생이 최소화된 벡터는 암의 치료에 유용하게 사용될 수 있다.

Description

재조합 발생이 최소화된 유전자치료 벡터, 상기 벡터를 포함하는 재조합 레트로바이러스 및 상기 재조합 레트로바이러스를 포함하는 암의 예방 또는 치료용 약학 조성물{Gene therapy vector with minimizing recombination, recombinant retrovirus comprising the vector and pharmaceutical composition for preventing or treating cancer comprising the recombinant retrovirus}
본 발명은 효율적인 암의 치료를 위해 치료 유전자로서 티미딘 키나아제(thymidine kinase; HSV-TK), 효모 시토신 디아미나제(yeast cytosine deaminase; yCD), 인간 CD19 유전자 또는 과립구-대식세포 콜로니-자극 인자(granulocyte-macrophage colony-stimulating factor, GM-CSF)를 포함하고 상기 치료 유전자의 발현율은 높게 유지하면서 재조합 발생은 최소화시킨 minimal MCMV 프로모터를 포함하는 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터에 관한 것이다.
유전자 치료란, 환자의 세포 또는 조직에서 질환을 유발하는 비정상 유전자를 대체하거나 유전자나 질병을 치료하는데 도움이 되는 유전자를 삽입하여 질환을 치료하는 기술을 통칭한다. 유전자 치료제의 개발 초기에는 외부의 DNA를 표적 세포의 염색체 내에 삽입하여 특정 유전자를 발현하도록 만드는 것이 유전자 치료의 주된 개념이었다. 그러나 최근에는 안티센스 올리고디옥시뉴클레오티드, siRNA 등을 이용하여 특정 질환과 관련된 유전자의 발현을 억제하는 안티센스 요법도 범주에 포함된다.
이러한 유전자 치료는 지금까지의 치료법과는 전혀 다른 개념의 접근 방법으로 질환의 근본 원인을 분자 수준에서 규명하여 치료할 수 있다. 또한, 염기서열 특이적인 작용이기 때문에 주요 질환과 관련된 유전자를 제거함으로써 다른 치료법에서 문제시되는 불필요한 부작용을 최소화할 수 있다. 또한, 이와 같이 유전자를 표적으로 하는 방법은 발현의 수준을 조절하고자 하는 유전자의 염기서열만 알면 치료제의 제작에서 별다른 최적화가 필요하지 않아, 항체나 화합물 치료제에 비해 제조과정이 매우 간단하다. 또한, 타 치료제가 표적화 하기 어려운 표적도 질병의 원인이 되는 유전자만 알면 얼마든지 표적화 할 수 있어, 차세대 치료제로서 잠재 능력이 충분하다. 이와 관련하여 기존의 의료기술로 치료가 어려운 난치병, 암, 에이즈, 유전질환, 신경계질환 등에 유전자 치료를 적용하여 치료 가능성을 높인 여러 연구 결과들이 있으며 실제 임상 실험도 진행되고 있다(YOUNG et al, 2006).
유전자 치료제는 유전자 전달체와 치료 유전자로 구성된다. 유전자를 생체 내로 전달하기 위한 도구인 유전자 전달체는 크게 바이러스성 전달체와 비바이러스성 전달체로 나눌 수 있다. 바이러스성 전달체는 바이러스 유전자의 대부분, 혹은 일부 필수 유전자를 없애서 스스로 복제할 수 없게 만들고 여기에 치료 유전자를 대신 삽입하여 제조된다(Lotze MT et al., Cancer Gene Therapy, 9:692-699, 2002). 바이러스성 전달체는 고효율로 유전자를 전달할 수 있으나, 바이러스의 종류에 따라 대량생산의 어려움, 면역반응 유발, 독성, 또는 복제가능 바이러스의 출현 등과 같은 문제점을 갖는다. 현재 유전자 치료제 개발에 활용되고 있는 주요 바이러스성 전달체로는 레트로바이러스, 렌티 바이러스, 아데노 바이러스, 아데노 부속 바이러스(AAV), 허피스 심플렉스 바이러스 및 폭스바이러스 등이 있다. 한편, 비바이러스성 전달체는 면역반응을 유도하지 않으며 독성이 낮고 대량생산이 용이하지만 유전자 전달효율이 낮고 그 발현이 일시적이다.
임상에서 많이 쓰이는 바이러스성 전달체 중 하나인 레트로바이러스 벡터는 1990년 미국 국립보건원에서 실시된 최초의 유전자 치료의 임상시험에 사용된 벡터로서 치료 유전자를 안정하게 삽입하기에 가장 유용한 벡터로 여겨지고 있다.
자가복제가 제한된 복제불능의 레트로바이러스 벡터는 비교적 큰 유전자를 삽입할 수 있으며, 역가도 106~107 pfu/㎖ 정도로서 대상 세포에의 감염에도 큰 문제가 없다. 또한, 포장 세포주(packaging cell line)가 개발되어 있어 제조방법이 용이하다. 게다가, 레트로바이러스 벡터는 레트로바이러스의 플라스미드 내로 치료 유전자를 삽입하고 이를 포장 세포에 감염시켜 재조합 바이러스를 생성시키고, 이를 대상 세포에 감염시키는 방식으로 규모확대(scale-up)가 가능하다. 그러나 염색체로의 삽입 과정에서 유전자 삽입에 따른 돌연변이가 일어날 수 있다.
한편, 암세포와 같은 증식성 세포에서 자가복제가 가능한 복제가능 레트로바이러스 벡터는 지놈(genome) 안정성에서 많은 논란이 있으며, 유전자 치료를 위한 자가복제 바이러스 벡터로서 개발시 외래도입이 가능한 유전자 크기가 약 1.3 kb로 제한되어 다양한 치료유전자의 도입이 어렵다(J. of virology, Vol.75, 6989-6998, 2001).
항암 유전자 치료제에 사용되는 치료 유전자로서 허피스 심플렉스 바이러스 티미딘 키나아제 또는 시토신 디아미나제와 같이 전구 약물 투여에 의해 암세포의 자살을 유도하는 유전자, 인터루킨-12 또는 GM-CSF 등과 같이 면역반응을 촉진할 수 있는 사이토카인 유전자, CEA 또는 Her-2 등과 같이 종양특이적인 항원유전자가 많이 사용되고 있다(Gottesman MM, Cancer Gene Therapy, 10:501-508, 2003). 자살 유전자는 암세포로 전달된 후 암을 사멸시키고, 사이토카인 유전자 또는 종양특이적 항원 유전자는 암에 대한 면역반응을 활성화 시켜 암세포를 공격한다.
최근, 악성종양에 대해 선택적으로 항종양효과를 나타내는 효소/전구 약물 합성기법의 연구가 활발하게 진행되고 있다. 실제로, 자살 유전자를 암 조직에서 발현시키고, 이의 전구체를 생체에 전신적으로 투여하면 정상세포에서는 독성이 나타나지 않고 치료 유전자가 발현된 종양세포에서만 전구체가 독성물질로 전환되어 종양세포를 파괴한다.
가장 널리 사용되고 있는 자살 유전자 중 하나는 허피스 심플렉스 바이러스 티미딘 키나아제(HSV-TK)이다. 이는 세포에 무해한 간사이클로비르(ganciclovir, GCV)라는 전구약물을 효소반응을 통해 세포독성을 가지는 물질로 바꿈으로써 자살유전자를 가지는 세포뿐만 아니라, 간극연접(gap junction)을 통해 인접한 세포의 사멸도 유도하는 방관자 효과(bystander effect)를 갖는다. 이는 임상 3상 까지 수행되어 효능과 안정성이 입증되었다(human gene therapy, 4:725-731, 1993; molecular therapy, 1:195-203, 2000).
또 다른 자살 유전자는 효모 시토신 디아미나제(yCD)로, 5-플루오로사이토신(5-FC)을 강력한 항암제인 5-플루오로우라실(5-FU)로 탈아민화시킨다. 5-FU는 5-플루오로우리딘 삼인산(5-FUTP)과 5-플루오로데옥시우리딘 일인산(5-FdUMP)으로 대사된다. 리보핵산으로 융합된 5-FUTP는 리보솜 리보핵산과 전달자 리보핵산의 합성 과정을 방해하고, 5-FdUMP는 티미딘 합성효소를 비가역적으로 저해함으로써, DNA 합성을 억제한다. 또한, yCD가 전달되지 않은 주변 세포도 사멸하는 방관자 효과(bystand effect)가 있다. 따라서 TK 또는 yCD가 발현되는 종양세포는 GCV와 5-FC와 같은 전구약물을 독성 대사물로 전환 시켜 선택적으로 암을 사멸시킨다.
최근 암을 치료하기 위한 면역 치료제에 대한 연구가 많이 수행되고 있으며, CAR-T와 같이 암 세포에서 특이적으로 발현하는 항원을 표적하는 수용체(receptor)를 바이러스 벡터에 적재하여 전달하는 면역 치료(Immunotherapy)방법이 개발되어 임상에서도 적용되고 있다. 이러한 면역치료 방법은 체내 면역세포들의 특성들을 이용하여 부작용을 최대한 줄이며, 환자의 몸이 암세포와 맞서 싸우도록 면역 반응을 강화시킬 수 있다. 암 세포에서 특이적으로 발현하는 항원 유전자의 예로는 CD19(Cluster of Differentiation 19), CEA(Carcinoembryonic Antigen) 또는 HER2(human epidermal growth factor receptor 2) 등이 있다. 암 항원 유전자 CD19은 주로 혈액암에서 특이적으로 발현하며, 총 556개의 아미노산로 구성되어 있는 95kDa 크기의 막관통(transmenbrane) 당단백질로, 세포질(cytosolic) C-말단, 세포외(extracellular) N-말단, 막관통(transmembrane) 도메인으로 구성되어 있다. 이중 세포외(extracellular) N-말단은 신호전달 펩타이드로 CAR와 결합하는 역할을 하고, 세포질(cytosolic) C-말단의 Y391, Y482/Y513 타이로신 잔기는 각각 Vav PLC(phospholipase C)와 PI3K(phosphoinositide 3-kinase)/Lyn과 같은 세포내 신호전달 기작에 관여하여 광범위한 영향을 미친다.
GM-CSF(granulocyte-macrophage colony-stimulating factor, 과립구 대식세포 콜로니 자극 인자)는 과립구의 증식과 생성뿐만 아니라 백혈구 성장인자 기능을 하는 사이토카인이며, 대식세포의 수를 급격히 늘려 감염에 대항할 수 있게 하므로 면역반응을 증가시키게 된다.
두 종류 이상의 치료 유전자를 유전자 치료에 동시에 적용하는 기술은 치료 효율 측면에서 뛰어나며, 특히 특정 유전자 치료에 내성을 나타내는 경우에 유용하다. 관련하여, 근래 TK 및 CD 투여에 의한 치료에 내성을 나타내는 암이 보고된바, TK 및 CD를 동시에 암 조직에서 발현시킬 수 있는 유전자 치료 벡터 시스템은 큰 이점을 지닌다. 그러나 HSV-TK 및 CD를 모두 복제가능 레트로바이러스 벡터(Replicating- Retrovirus Vector; RRV)에 도입하면 지놈 크기가 약 10 kb 이상이 되어 단일 레트로바이러스 벡터에 삽입하는 것은 사실상 불가능하다. 또한, 유전자 치료를 위한 복제가능 레트로바이러스 벡터는 원래 레트로바이러스가 가진 지놈 RNA 외에 외래 유전자가 도입되기 때문에 지놈 RNA의 크기가 증가하고 비상동 염기서열이 추가되며 유전자 재조합이 일어나 치료 유전자가 소실될 가능성이 높아 벡터의 구축이 어렵다.
이러한 문제점을 해결하기 위하여 본 발명자들은 복제가능 레트로바이러스에 포함되어 있는 gag-pol-env 게놈 크기를 줄여 바이러스의 안정성을 유지하고, 다른 치료 유전자의 도입이 가능하도록 하나의 게놈으로 구성된 gag-pol-env 벡터를 gag-pol과 env 유전자가 각각 별개의 벡터에 포함되도록 이중 복제가능 레트로바이러스 벡터를 제작하였다.
쥐 사이토메갈로바이러스(murine cytomegalovirus, MCMV) IE 유전자의 프로모터는 특정 세포에서 HCMV IE 유전자의 프로모터에 비해 수 배 내지 수십 배 높은 발현을 유도하며(Lafemina R et al, J Gen Virol., 69, 355-374 (1988)), 다양한 세포에서 균일하게 안정적인 발현을 유도함이 알려진 바 있다(Aiba-Masago S et al., Am J Pathol. 154, 735-743 (1999)). 특히, MCMV 주요 즉각 조기 프로모터(major immediate-early promoter, MIEP) 부위에서 상류 영역이 제거된 것이 영장류와 마우스 세포에서 매우 강력한 발현을 유도함이 보고되었다(Kim and Risser, J. Virol. 67, 239-248 (1993); 및 Kim, Biochem. Biophys. Res. Comm., 203, 1152-1159 (1994)). 또한, 대한민국 등록특허 10-0423022에는 인간, 마우스 진핵세포에서 MCMV 프로모터가 유전자의 발현을 강력하고 안정하게 유도하므로 동물용 발현벡터로 사용될 수 있음을 개시하고 있다.
그러나 MCMV 프로모터 내의 반복 염기서열로 인하여 바이러스 복제 시 MCMV 염기서열 일부가 소실되거나, MCMV 프로모터를 기점으로 다른 위치의 바이러스 벡터 염기서열이 함께 소실되어 재조합된 바이러스가 생성되며, 이는 치료 유전자를 지속적으로 발현하는 레트로바이러스 벡터 바이러스 생산 및 감염에 큰 문제점이 된다.
이에, 본 발명자들은 재조합이 일어나지 않는 유전자 치료용 바이러스 벡터를 개발하던 중, HSV-TK, hopt-yCD, hCD19 또는 GM-CSF 유전자를 치료 유전자로서 포함하며, MCMV 프로모터 내의 반복적인 염기서열을 기점으로 프로모터를 잘라주어 모두 4종의 변이체(variants)를 제작하였고, 상기 절단된 MCMV 프로모터가 도입된 벡터는 바이러스 감염 중 재조합이 일어나지 않아 치료 유전자의 소실이 없는 복제 가능한 레트로바이러스 벡터를 개발하였다. 상기 벡터는 바이러스의 재조합이 발생하지 않고 치료 유전자 발현 및 약물 감수성이 우수함을 확인하여 본 발명을 완성하였다.
본 발명의 목적은, 치료 유전자로서 티미딘 키나아제, 시토신 디아미나제, 인간 CD19 유전자 또는 과립구 대식세포 콜로니 자극인자, 및 MCMV 프로모터를 포함하는 재조합 발생이 최소화된 암의 치료를 위한 재조합 복제가능 레트로바이러스 벡터를 제공하는 것이다.
본 발명의 다른 목적은, 상기 레트로바이러스 벡터를 포함하는 재조합 레트로바이러스 및 상기 재조합 레트로바이러스가 감염된 숙주세포를 제공하는 것이다.
본 발명의 또 다른 목적은, 상기 재조합 레트로바이러스를 포함하는 암 치료용 약학 조성물 및 유전자 전달 조성물을 제공하는 것이다.
본 발명의 또 다른 목적은, 상기 복제가능 재조합 레트로바이러스 벡터의 제조방법을 제공하는 것이다.
상기 목적을 달성하기 위해, 본 발명은
MuLV의 Gag-Pol 유전자, sEF1α 프로모터 또는 MCMV 프로모터, 및 제1 치료 유전자를 포함하는 제1 재조합 발현벡터; 및 바이러스의 Env 유전자, MCMV 프로모터 및 제2 치료 유전자를 포함하는 제2 재조합 발현벡터를 포함하는 재조합 발생이 최소화된 복제가능 재조합 레트로바이러스 벡터를 제공한다.
또한, 본 발명은 상기 벡터를 포함하는 재조합 레트로바이러스를 제공한다.
또한, 본 발명은 상기 재조합 레트로바이러스로 형질감염된 숙주 세포를 제공한다.
또한, 본 발명은 상기 재조합 레트로바이러스를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다.
또한, 본 발명은 상기 재조합 레트로바이러스를 포함하는 암 치료용 유전자 전달 조성물을 제공한다.
또한, 본 발명은 1) MuLV의 Gag-Pol 유전자, sEF1α 프로모터 또는 MCMV 프로모터, 및 제1 치료 유전자를 포함하는 제1 재조합 발현벡터를 제조하는 단계; 및
2) 바이러스의 Env 유전자, MCMV 프로모터 및 제2 치료 유전자를 포함하는 제2 재조합 발현벡터를 제조하는 단계를 포함하는 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법을 제공한다.
본 발명은 재조합 발생이 최소화된 유전자치료 벡터에 관한 것으로, 바이러스 복제 시 치료 유전자를 지속적으로 발현하는 레트로바이러스 벡터 바이러스 생산 및 감염에 큰 문제점이 되는 재조합 발생을 최소화하고자, MCMV 프로모터를 반복서열 기반으로 잘라주어 절단된 MCMV 프로모터를 제작하고, 상기 절단된 MCMV 프로모터를 도입하여 벡터를 제작하였으며, 절단된 MCMV 프로모터가 도입된 벡터는 여러 번 배양하여도 재조합이 발생하지 않고, 치료 단백질 발현이 지속되며, 상기 벡터를 포함하는 바이러스로 형질감염된 세포에서 전구 약물을 투여한 경우 세포 사멸이 효과적으로 일어남을 확인하였으므로, 본 발명의 재조합 발생이 최소화된 벡터는 암의 치료에 유용하게 사용될 수 있다.
도 1은 복제가능 레트로바이러스 벡터의 감염능 및 재조합 유형을 확인하기 위해 디자인한 GaLV env-pMCMV-yCD/gag-pol-pMCMV-HSV1-TK 벡터의 모식도이다.
도 2는 GaLV env-pMCMV-yCD/gag-pol-pMCMV-HSV1-TK 벡터의 감염능 및 재조합 유형을 확인하기 위한 실험 과정을 나타낸 모식도이다.
도 3은 GaLV env-pMCMV-yCD/gag-pol-pMCMV-HSV1-TK 벡터의 재조합 유형을 확인하기 위해 수행한 중합효소연쇄반응에 사용한 프라이머의 위치를 나타낸 모식도이다.
도 4는 spRRVe-yCD:env 벡터의 재조합 발생 여부를 확인하기 위해 중합효소연쇄반응을 수행한 것으로, 바이러스 증식 및 감염은 진행되었으나 바이러스 감염 후 증폭 과정에서 재조합이 발생하였음을 확인한 도이다.
도 5는 GaLV env, MCMV 프로모터, yCD, 3’염기서열 내에서 재조합이 발생한 유형을 확인한 도이다.
도 6은 MCMV 프로모터 염기서열 내의 4군데 반복적인 염기서열(1. AACAGGAAA, 2. GGGACTTTCCAATGGGTTTTGCCCAGTACA, 3. TGGGTTTTTCC, 4. GTACTTTCCCA)을 나타낸 도이다.
도 7은 절단한 MCMV 프로모터를 GaLV Env에 도입한 spRRVe-TK, spRRVe-F1-TK, spRRVe-F2-TK, spRRVe-F3-TK, spRRVe-F4-TK 벡터의 구조를 나타낸 도이다.
도 8은 절단한 MCMV 프로모터를 MuLV Env에 도입한 sRRVe-TK, sRRVe-F1-TK, sRRVe-F2-TK, sRRVe-F3-TK, sRRVe-F4-TK 벡터의 구조를 나타낸 도이다.
도 9는 sRRVgp-sEF1a-hopt-yCD 벡터 및 spRRVe-TK, spRRVe-F1-TK, spRRVe-F2-TK, spRRVe-F3-TK, spRRVe-F4-TK 벡터에서 재조합 여부를 확인하기 위한 프라이머의 위치 및 재조합 여부를 나타낸 도이다.
도 10은 spRRVe-TK, spRRVe-F1-TK, spRRVe-F2-TK, spRRVe-F3-TK, spRRVe-F4-TK 벡터에서 티미딘 키나아제(TK) 단백질이 지속적으로 발현함을 확인한 도이다.
도 11은 spRRVe-F4-TK/sRRVgp-sEF1a-hopt-yCD 벡터에서 spRRVe-F4-TK 벡터의 재조합 발생이 일어나지 않고, 효모 시토신 디아미나제 단백질이 지속적으로 발현됨을 확인한 도이다.
도 12는 sRRVgp-sEF1a-hopt-yCD 벡터 및 spRRVe-TK, spRRVe-F1-TK, spRRVe-F2-TK, spRRVe-F3-TK, spRRVe-F4-TK 벡터에서 전구 약물인 GCV와 5-FC를 처리하였을 때, 상기 벡터에서 발현되는 티미딘 키나아제 및 효모 시토신 디아미나제로 인해 세포 사멸이 유도됨을 확인한 도이다.
도 13은 sRRVgp-sEF1a-hopt-yCD 벡터 및 sRRVe-TK, sRRVe-F1-TK, sRRVe-F2-TK, sRRVe-F3-TK, sRRVe-F4-TK 벡터에서 재조합 여부를 확인하기 위한 프라이머의 위치 및 재조합 여부를 나타낸 도이다.
도 14는 sRRVgp-F4-hopt-yCD 벡터 및 sRRVe-F4-TK 벡터에서 재조합 여부를 확인하기 위한 프라이머의 위치 및 재조합 여부를 나타낸 도이다.
도 15는 sRRVe-TK, sRRVe-F1-TK, sRRVe-F2-TK, sRRVe-F3-TK, sRRVe-F4-TK 벡터에서 티미딘 키나아제(TK) 단백질이 지속적으로 발현함을 확인한 도이다.
도 16은 sRRVgp-sEF1a-hopt-yCD 벡터 및 sRRVe-TK, sRRVe-F1-TK, sRRVe-F2-TK, sRRVe-F3-TK, sRRVe-F4-TK 벡터에서 전구 약물인 GCV를 처리하였을 때, 상기 벡터에서 발현되는 티미딘 키나아제로 인해 세포 사멸이 유도됨을 확인한 도이다.
도 17은 sRRVgp-sEF1a-hopt-yCD 벡터 및 sRRVe-F4-TK 벡터를 포함하는 바이러스를 형질감염시킨 세포에서 세포 사멸에 따른 세포 생존능을 정량적으로 나타낸 도이다.
도 18은 sRRVe-F4-TK/sRRVgp-F4-hCD19 벡터와 재조합 발생 여부를 확인하기 위한 프라이머의 증폭 위치를 나타낸 도이다.
도 19는 sRRVgp-F4-hCD19 벡터가 passage 3부터 재조합이 발생하였음을 PCR로 확인한 도이다.
도 20은 sRRVe-F4-hCD19/sRRVgp-F4-hopt-yCD 벡터와 재조합 발생 여부를 확인하기 위한 프라이머의 증폭 위치를 나타낸 도이다.
도 21은 sRRVe-F4-hCD19 벡터가 재조합이 발생하지 않았음을 PCR로 확인한 도이다.
도 22는 sRRVe-F4-TK 벡터와 sRRVgp-F4-hCD19t 벡터의 모식도이다.
도 23은 TK가 도입된 sRRVe-F4-TK 벡터와 hCD19t이 도입된 sRRVgp-F4-hCD19t 벡터가 재조합이 발생하지 않았음을 PCR로 확인한 도이다.
도 24는 sRRVgp-F4-hopt-yCD 벡터와 sRRVe-F4-hCD19t 벡터의 모식도이다.
도 25는 hopt-yCD가 도입된 sRRVgp-F4-hopt-yCD 벡터와 hCD19t가 도입된 sRRVe-F4-hCD19t 벡터가 재조합이 발생하지 않았음을 PCR로 확인한 도이다.
도 26은 sRRVgp-F4-mGM-CSF 벡터와 sRRVe-F4-hCD19t 벡터의 모식도이다.
도 27은 mGM-CSF이 도입된 sRRVgp-F4-mGM-CSF 벡터와 hCD19t가 도입된 sRRVe-F4-hCD19t 벡터가 재조합이 발생하지 않았음을 PCR로 확인한 도이다
도 28은 sRRVgp-F4-hGM-CSF 벡터와 sRRVe-F4-hCD19 벡터의 모식도이다.
도 29는 sRRVgp-F4-hGM-CSF 벡터가 구조의 불안전성으로 복제가 온전히 일어나지 않음을 PCR로 확인한 도이다.
도 30은 sRRVgp-F4-hopt-GM-CSF 벡터와 sRRVe-F4-hCD19 벡터의 모식도이다.
도 31은 sRRVgp-F4-hopt-GM-CSF 벡터와 sRRVe-F4-hCD19t 벡터의 모식도이다.
도 32는 hopt-GM-CSF가 도입된 sRRVgp-F4-hopt-GM-CSF 벡터와 hCD19이 도입된 sRRVe-F4-hCD19가 바이러스 복제 동안 재조합이 발생하지 않았음을 PCR로 확인한 도이다.
도 33은 hopt-GM-CSF가 도입된 sRRVgp-F4-hopt-GM-CSF 벡터와 hCD19t이 도입된 sRRVe-F4-hCD19t가 바이러스 복제 동안 재조합이 발생하지 않았음을 PCR로 확인한 도이다.
이하, 본 발명을 상세히 설명한다.
본 발명은 MuLV(Murine Leukemia virus)의 Gag-Pol 유전자, sEF1α 프로모터 또는 MCMV 프로모터, 및 제1 치료 유전자를 포함하는 제1 재조합 발현벡터; 및 바이러스의 Env 유전자, MCMV 프로모터 및 제2 치료 유전자를 포함하는 제2 재조합 발현벡터;를 포함하는 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터를 제공한다.
상기 MCMV 프로모터는 서열번호 3의 염기서열을 갖는 646bp의 폴리뉴클레오티드이다.
상기 MCMV 프로모터는 쥐 사이토메갈로바이러스(murine cytomegalovirus, MCMV) 프로모터로서 특정 세포에서 인간 사이토메갈로바이러스(HCMV) IE 유전자의 프로모터에 비해 수 배 내지 수십 배 높은 발현을 유도하며(Lafemina R et al, J Gen Virol., 69, 355-374 (1988)), 다양한 세포에서 균일하게 안정적인 발현을 유도함이 알려진바 있다(Aiba-Masago S et al., Am J Pathol. 154, 735-743 (1999)). 특히, MCMV 주 즉시 초기 프로모터(major immediate-early promoter, MIEP) 부위에서 상류 영역이 제거된 것이 영장류와 마우스 세포에서 매우 강력한 발현을 유도함이 보고되었다(Kim and Risser, J. Virol. 67, 239-248 (1993); 및 Kim, Biochem. Biophys. Res. Comm., 203, 1152-1159 (1994)).
그러나 상기 MCMV 프로모터는 프로모터 내의 4군데 반복적인 염기서열로 인해 MCMV 프로모터를 기점으로 다른 부위의 바이러스 벡터 염기서열이 소실되거나 프로모터 내에서 염기서열 일부가 소실되어 재조합이 발생하여 치료 유전자가 소실되어 치료 유전자를 지속적으로 발현하는 레트로바이러스 벡터 바이러스 생산 및 감염에 큰 문제점이 된다.
따라서, 재조합 발생을 최소화하기 위해 상기 MCMV 프로모터는 절단된 MCMV 프로모터인 것을 특징으로 한다.
상기 절단된 MCMV 프로모터는 서열번호 4, 서열번호 5, 서열번호 6 및 서열번호 7의 염기서열을 갖는 폴리뉴클레오티드로 구성된 군으로부터 선택되는 어느 하나일 수 있고, 바람직하게는 서열번호 5, 서열번호 6 및 서열번호 7의 염기서열을 갖는 폴리뉴클레오티드로 구성된 군으로부터 선택되는 어느 하나일 수 있으며, 더 바람직하게는 서열번호 6 또는 서열번호 7의 염기서열을 갖는 폴리뉴클레오티드일 수 있으며, 더욱 바람직하게는 서열번호 7의 염기서열을 갖는 폴리뉴클레오티드일 수 있다.
본 발명의 구체적인 실시예에서, MCMV 프로모터 전장 서열, GaLV env, 효모 시토신 디아미나제 단백질을 코딩하는 유전자를 포함하는 벡터(spRRVe-yCD) 및 MCMV 프로모터 전장 서열, gag-pol 유전자, 티미딘 키나아제(TK) 단백질을 코딩하는 유전자를 포함하는 벡터(sRRVgp-TK)에서 MCMV 프로모터 내의 반복되는 염기서열로 인해 유전자 재조합이 발생하였음을 확인하였다(도 1 내지 도 6).
상기 sEF1α(short elongation factor 1α) 프로모터는 서열번호 18의 염기서열을 갖는 폴리뉴클레오티드이다.
상기 바이러스의 Env 유전자는 긴팔원숭이 백혈병 바이러스(Gibbon ape Leukemia virus; GaLV), 암포트로픽 쥐 백혈병 바이러스(amphotropic MuLV), 친이종 쥐 백혈병 바이러스(xenotropic MuLV), 고양이 내재성 바이러스(RD114), 수포성 구내염바이러스(vesicular stomatitis virus; VSV) 및 홍역바이러스(measles virus; MV)의 Env 유전자로 이루어진 군으로부터 유래되는 어느 하나이다. 상기 폴리뉴클레오티드는 상술한 바와 같은 특징을 갖는 변이체를 포함할 수 있다.
상기 GaLV의 Env 유전자는 서열번호 19의 염기서열을 갖는 폴리뉴클레오티드이다.
상기 MuLV의 Env 유전자는 서열번호 20의 염기서열을 갖는 폴리뉴클레오티드이다.
상기 제1 치료 유전자 또는 제2 치료 유전자는 전구 약물의 투여에 의해 암 세포의 자살을 유도하는 자살 유전자, 인터루킨-12 또는 GM-CSF과 같이 면역 반응을 촉진할 수 있는 사이토카인 유전자 및 CD19, CEA 또는 HER2 같이 종양 특이적인 암 항원 유전자로 구성된 군으로부터 선택되는 어느 하나일 수 있다.
상기 자살 유전자는 티미딘 키나아제(thymidine kinase, TK) 유전자 또는 효모 시토신 디아미나제(yeast cytosine deaminase, yCD) 유전자일 수 있다.
상기 제1 치료유전자 및 제2 치료 유전자는 티미딘 키나아제(thymidine kinase, TK) 유전자, 효모 시토신 디아미나제(yCD) 유전자 및 인간 CD19 유전자로 이루어진 군으로부터 선택되는 어느 하나 이상이다.
상기 티미딘 키나아제 유전자는 서열번호 21의 염기서열을 갖는 폴리뉴클레오티드이다. 상기 폴리뉴클레오티드는 티미딘 키나아제 단백질의 아미노산 서열을 코딩하는 폴리뉴클레오티드 서열뿐만 아니라, 상기 폴리뉴클레오티드와 실질적으로 동일한 염기서열을 갖는 폴리뉴클레오티드 및 이의 단편을 포함할 수 있다. 실질적으로 동일한 염기서열을 갖는 폴리뉴클레오티드는 본 발명의 폴리뉴클레오티드와 80％ 이상, 구체적으로는 90％ 이상, 더욱 구체적으로는 95％ 이상의 상동성을 가질 수 있다. 상술한 바와 같이, 본 발명의 폴리뉴클레오티드는 이와 동등한 활성을 갖는 단백질을 코딩하는 한, 하나 이상의 염기서열이 치환, 결실 또는 삽입된 변이체를 포함할 수 있다.
상기 효모 시토신 디아미나제(yCD) 유전자는 서열번호 22의 염기서열을 갖는 폴리뉴클레오티드이다.
상기 시토신 디아미나제 유전자는 인간 코돈으로 최적화된 것일 수 있다.
본 명세서에서 사용된 용어, "인간 코돈으로 최적화된 것"은 숙주 세포 내에서 DNA가 단백질로 전사 및 번역될 때 아미노산을 지정하는 코돈 사이에 숙주에 따라 선호도가 높은 코돈이 존재하는데, 사람의 코돈으로 치환함으로써 그 핵산이 암호화하는 아미노산 또는 단백질의 발현 효율을 증가시키는 것을 의미한다.
상기 티미딘 키나아제 또는 효모 시토신 디아미나제 유전자는 전구체 약물을 활성화 시킨다. 상기 전구체 약물이 간사이클로비르(GCV) 및 5-플루오로사이토신(5-FC)으로 구성된 군으로부터 선택되는 어느 하나 이상이다. 본 발명의 일 실시예에서, 티미딘 키나아제 유전자는 간사이클로비르를, 효모 시토신 디아미나제 유전자는 5-플루오로사이토신을 활성화 시킬 수 있다.
상기 과립구 대식세포 콜로니 자극인자(GM-CSF)는 인간 코돈으로 최적화된 것일 수 있다.
본 명세서에서 사용된 용어, "인간 코돈으로 최적화된 것"은 숙주 세포 내에서 DNA가 단백질로 전사 및 번역될 때 아미노산을 지정하는 코돈 사이에 숙주에 따라 선호도가 높은 코돈이 존재하는데, 사람의 코돈으로 치환함으로써 그 핵산이 암호화하는 아미노산 또는 단백질의 발현 효율을 증가시키는 것을 의미한다.
상기 인간 CD19(Cluster of Differentiation 19) 유전자는 세포질 도메인의 아미노산이 제거된 절단된 인간 CD19 유전자일 수 있고, 제거된 세포질 도메인의 아미노산은 233개일 수 있다.
상기 인간 CD19 유전자는 서열번호 43 또는 서열번호 53의 염기서열을 갖는 폴리뉴클레오티드이다.
상기 gag 유전자는 레트로바이러스 코어를 구성하는 4종의 단백질을 암호화하는 폴리뉴클레오티드일 수 있다. 한편, 상기 pol 유전자는 레트로바이러스의 역전사 효소를 암호화하는 폴리뉴클레오티드이고, 상기 env 유전자는 레트로바이러스의 외피 당단백질을 암호화하는 폴리뉴클레오티드이다.
상기 MuLV-Gag 유전자는 쥐 백혈병 바이러스(Murine Leukemia virus)의 Gag 유전자로서 서열번호 23의 염기서열로 구성되는 폴리뉴클레오티드일 수 있다. 상기 MuLV-Pol 유전자는 쥐 백혈병 바이러스의 Pol 유전자로서 서열번호 24의 염기서열로 구성되는 폴리뉴클레오티드일 수 있다. 상기 MuLV의 Gag-Pol 유전자는 서열번호 23 및 서열번호 24의 염기서열이 융합된 염기서열로 구성되는 폴리뉴클레오티드일 수 있다.
본 명세서에서 사용된 용어, "복제가능"은 특정 유전자를 포함하는 바이러스 게놈(genome)이 형질 도입되거나 동물 세포나 특정 유전자가 포함된 바이러스 벡터가 감염된 세포에서 상기 바이러스 벡터가 스스로 복제할 수 있다는 것을 의미한다.
본 명세서에서 사용된 용어, "복제가능 레트로바이러스 벡터"는 비용균성 바이러스를 생산하는 벡터로서, 핵막이 깨지는 틈을 타 핵 안으로 들어가기 때문에 분열하는 세포, 즉 암세포에 특이적으로 감염될 수 있어 삽입된 유전자가 다른 정상세포에서 발현되는 것을 막을 수 있다. 따라서, 상기 벡터는 암세포로 안전하게 유전자를 전달할 수 있고, 바이러스의 복제가 가능하기 때문에 유전자의 전달 효율도 증대시킬 수 있다.
본 발명의 구체적인 실시예에서, 재조합 발생을 최소화하기 위해 MCMV 프로모터 내의 반복적인 염기서열을 제거한 각각 470bp, 337bp, 237bp, 160bp의 크기를 가지는 절단된 MCMV 프로모터 4가지를 제작하였다(표 2). 또한, 상기 절단된 MCMV 프로모터가 도입된 GaLV env 기반 벡터(spRRVe-TK, spRRVe-F1-TK, spRRVe-F2-TK, spRRVe-F3-TK, spRRVe-F4-TK) 및 MuLV 기반 벡터(sRRVe-TK, sRRVe-F1-TK, sRRVe-F2-TK, sRRVe-F3-TK, sRRVe-F4-TK)을 제작하고, 상기 티미딘 키나아제 단백질을 발현하는 벡터와 함께 도입되는 벡터로서 효모 시토신 디아미나제 단백질을 발현하는 gag-pol-sEF1α-hopt-yCD로 구성된 sRRVe-sEF1α-hopt-yCD 벡터를 제작하고(도 7 및 도 8), 재조합 여부를 확인한 결과, sRRVe-sEF1α-hopt-yCD 벡터에서는 재조합이 발생하지 않았고(도 9 참조), MCMV 프로모터 내의 반복 서열을 대부분 제거한 spRRVe-F4-TK 벡터에서는 재조합이 거의 발생하지 않음을 확인하였다(도 10, 도 11 및 도 13). 또한, sRRVe-sEF1α-hopt-yCD 벡터의 sEF1α를 MCMV의 F4 절단 프로모터로 치환한 벡터와 sRRVgp-F4-hopt-yCD의 조합에서도 재조합이 일어나지 않음을 확인하였다(도 14 참조). 또한, 인간 CD19 유전자를 치료 유전자로 도입한 sRRVgp-F4-hopt-yCD/sRRVe-F4-hCD19 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 20 및 도 21 참조). 또한, 인간 CD19 유전자의 세포질 도메인의 아미노산이 절단된 hCD19t를 치료 유전자로 도입한 sRRVe-F4-hCD19t 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 23 및 도 25 참조). 또한, 마우스 GM-CSF를 치료 유전자로 도입한 sRRVgp-F4-mGM-CSF 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 27 참조). 또한, 코돈 최적화된 인간 GM-CSF를 치료 유전자로 도입한 sRRVgp-F4-hopt-GM-CSF 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 32 및 도 33 참조).
따라서, 본 발명의 벡터는 재조합 발생이 최소화되어 치료 유전자가 소실되지 않고 안정적으로 발현시키는데에 유용하게 사용될 수 있다.
또한, 본 발명은 상기 벡터를 포함하는 재조합 레트로바이러스를 제공한다. 한편, 상기 재조합 레트로바이러스는 MuLV의 Gag-Pol 유전자, sEF1α 프로모터 또는 MCMV 프로모터, 및 제1 치료 유전자를 포함하는 제1 재조합 발현벡터; 및 바이러스의 Env 유전자, MCMV 프로모터 및 제2 치료 유전자를 포함하는 제2 재조합 발현벡터는 각각 또는 함께 재조합 레트로바이러스에 포함될 수 있다.
본 발명의 구체적인 실시예에서, 재조합 발생을 최소화하기 위해 MCMV 프로모터 내의 반복적인 염기서열을 제거한 각각 470bp, 337bp, 237bp, 160bp의 크기를 가지는 절단된 MCMV 프로모터 4가지를 제작하였다(표 2). 또한, 상기 절단된 MCMV 프로모터가 도입된 GaLV env 기반 벡터(spRRVe-TK, spRRVe-F1-TK, spRRVe-F2-TK, spRRVe-F3-TK, spRRVe-F4-TK) 및 MuLV 기반 벡터(sRRVe-TK, sRRVe-F1-TK, sRRVe-F2-TK, sRRVe-F3-TK, sRRVe-F4-TK)을 제작하고, 상기 티미딘 키나아제 단백질을 발현하는 벡터와 함께 도입되는 벡터로서 효모 시토신 디아미나제 단백질을 발현하는 gag-pol-sEF1α-hopt-yCD로 구성된 sRRVe-sEF1α-hopt-yCD 벡터를 제작하고(도 7 및 도 8), 재조합 여부를 확인한 결과, sRRVe-sEF1α-hopt-yCD 벡터에서는 재조합이 발생하지 않았고(도 9 참조), MCMV 프로모터 내의 반복 서열을 대부분 제거한 spRRVe-F4-TK 벡터에서는 재조합이 거의 발생하지 않음을 확인하였다(도 10, 도 11 및 도 13). 또한, sRRVe-sEF1α-hopt-yCD 벡터의 sEF1α를 MCMV의 F4 절단 프로모터로 치환한 벡터와 sRRVgp-F4-hopt-yCD의 조합에서도 재조합이 일어나지 않음을 확인하였다(도 14 참조). 또한, 인간 CD19 유전자를 치료 유전자로 도입한 sRRVgp-F4-hopt-yCD/sRRVe-F4-hCD19 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 20 및 도 21 참조). 또한, 인간 CD19 유전자의 세포질 도메인의 아미노산이 절단된 hCD19t를 치료 유전자로 도입한 sRRVe-F4-hCD19t 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 23 및 도 25 참조). 또한, 마우스 GM-CSF를 치료 유전자로 도입한 sRRVgp-F4-mGM-CSF 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 27 참조). 또한, 코돈 최적화된 인간 GM-CSF를 치료 유전자로 도입한 sRRVgp-F4-hopt-GM-CSF 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 32 및 도 33 참조).
따라서, 본 발명의 재조합 레트로 바이러스는 재조합 발생이 최소화되어 치료 유전자가 소실되지 않고 안정적으로 발현시키는데에 유용하게 사용될 수 있다.
또한, 본 발명은 상기 재조합 레트로바이러스로 형질감염된 숙주 세포를 제공한다.
상기 숙주 세포는 NS/O 골수종 세포(NS/O myeloma cell), 인간 293T 세포, 중국 햄스터 난소 세포(CHO cell), HeLa 세포, CapT 세포(인간 양수 유래 세포) COS 세포, 케나인 D17 세포, 마우스 NIH/3T3, 레트로바이러스 패키징 세포주, 인간 중간엽 줄기세포 또는 펠라인 PG4 세포일 수 있다. 본 발명의 일 실시예에서, 상기 숙주 세포는 인간 293T 세포일 수 있다.
형질감염은 재조합 레트로바이러스 벡터 플라스미드가 형질도입된 세포에서 생산된 재조합 바이러스를 상기 세포들에 감염시키는 방법으로 수행된다.
상기 형질도입은 당업계에 공지된 방법에 따라 수행될 수 있다. 일례로, 리포펙타민 방법, 미세 주입법, 칼슘 포스페이트 침전법, 전기천공법, 리포좀-매개 형질감염법, DEAE-덱스트란 처리법 및 유전자 밤바드먼트로 이루어진 군으로부터 선택되는 어느 하나 이상의 방법으로 수행될 수 있다. 본 발명의 일 실시예에서, 상기 형질도입은 리포펙타민 방법으로 수행될 수 있다.
상기 형질감염된 세포는 동물세포의 배양에 통상적으로 이용되는 배지를 사용하여 배양될 수 있다. 일례로, 상기 배지는 Eagles's MEM, a-MEM, Iscove'sMEM, 199 배지, CMRL 1066, RPMI 1640, F12, F10, DMEM, DMEM과 F12의 혼합배지, Way-mouth's MB752/1, McCoy's 5A 및 MCDB 시리즈 배지로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다. 본 발명의 일 실시예에서, 상기 배지는 DMEM일 수 있다.
또한, 본 발명은 상기 재조합 레트로바이러스를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다.
한편, 상기 재조합 레트로바이러스는 MuLV(Murine Leukemia virus)의 Gag-Pol 유전자, sEF1α 프로모터 및 효모 시토신 디아미나제(yeast cytosine deaminase, yCD) 유전자를 포함하는 제1 재조합 발현벡터; 및 바이러스의 Env 유전자, MCMV 프로모터 및 티미딘 키나아제(thymidine kinase) 유전자를 포함하는 제2 재조합 발현벡터는 각각 또는 함께 재조합 레트로바이러스에 포함될 수 있다.
상기 레트로바이러스는 분열 중인 모든 세포를 표적할 수 있으며, 구체적으로 상기 세포는 암세포일 수 있다. 상기 암세포는 점액상 세포 암종, 둥근 세포암종, 국소적 진행 종양, 전이성 암, 어윙 (Ewing) 육종, 암전이, 림프성 전이, 편평상피 세포 암종, 식도 편평 상피 세포 암종, 경구 암종, 다발성 골수종, 급성 림프구성 백혈병, 급성 비림프구성 백혈병, 만성 림프구 백혈병, 만성 골수구 백혈병, 모양 세포성 백혈병, 유출 림프종 (체강계 림프종), 흉선 림프종 폐암, 소 세포 폐암종, 피부 T 세포 림프종, 호지킨 림프종, 비-호지킨성 림프종, 부신 피질 암, ACTH-생성 종양, 비소세포 폐암, 유방암, 소 세포 암종, 도관 암종, 위암, 결장암, 결장직장암, 결장직장 종양 형성과 관련된 용종, 췌장암, 간암, 방광암, 1차 표면 방광 종양, 방광의 침습성 전이 세포 방광암종, 근 침윤성 방광암, 전립선암, 대장암, 신장암, 간암, 식도암, 난소 암종, 자궁경부암, 자궁내막암, 융모암, 난소암, 원발성 복막상피 신생물, 자궁 경관암종, 질암, 외음부암, 자궁암, 난포 중 고형 종양, 고환암, 음경암, 신장 세포 암종, 뇌암, 두경부암, 구경부암, 신경아세포종, 뇌간 신경교종, 신경교종, 중추신경계 중 전이성 종양 세포 침윤, 골종, 골육종, 악성 흑색종, 인간 피부 케라티노사이트의 종양 진행, 편평상피 세포 암종, 갑상선 암, 망막아종, 신경아세포종, 중피종, 빌름스 종양, 담낭암, 영양아세포 종양, 혈관주위세포종, 또는 카포시 육종과 같은 암으로부터 유래된 세포를 포함할 수 있다.
본 발명의 구체적인 실시예에서, 본 발명자들은 GaLV env 기반 벡터(spRRVe-TK, spRRVe-F1-TK, spRRVe-F2-TK, spRRVe-F3-TK, spRRVe-F4-TK) 및 MuLV 기반 벡터(sRRVe-TK, sRRVe-F1-TK, sRRVe-F2-TK, sRRVe-F3-TK, sRRVe-F4-TK)에서 치료 유전자로 사용되는 티미딘 키나아제 및 효모 시토신 디아미나제가 안정적으로 발현되는 것을 확인하였으며, 상기 치료 유전자가 전구 약물인 GCV 및 5-FC에 작용하여 세포를 사멸할 수 있음을 확인하였다.
따라서, 본 발명에 따른 벡터를 포함하는 재조합 레트로바이러스는 암의 예방 또는 치료 용도로 유용하게 사용될 수 있다.
본 발명의 약학 조성물은 비경구제제로 제형화 될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제 등의 주사제가 포함될 수 있다.
비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.
비경구 투여는 피부 외용 또는 복강 내 주사, 직장 내 주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부내 주사 주입방식 중 선택될 수 있다.
본 발명에 따른 조성물은 약제학적으로 유효한 양으로 투여된다. 이는 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물 등에 따라 달라질 수 있다. 본 발명의 조성물은 단독 또는 다른 치료제와 병용하여 투여될 수 있다. 병용 투여시, 투여는 순차적 또는 동시일 수 있다.
상기 조성물의 유효 용량은 체중 1㎏당 재조합 바이러스의 경우에는 1011 내지 1013 바이러스 입자 (108 내지 1010 IU)/㎏, 세포의 경우에는 103 내지 106 세포/㎏이고, 단회 투여로 진행한다.
본 발명에 따른 약학 조성물은 조성물 전체 중량에 대하여 유효성분인 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터를 10 내지 95 중량%로 포함할 수 있다. 또한, 본 발명의 약학 조성물은 상기 유효성분 이외에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 추가로 포함할 수 있다.
또한, 본 발명은 상기 재조합 레트로바이러스를 포함하는 암 치료용 유전자 전달 조성물을 제공한다.
한편, 상기 재조합 레트로바이러스는 MuLV(Murine Leukemia virus)의 Gag-Pol 유전자, sEF1α 프로모터 또는 MCMV 프로모터, 및 제1 치료 유전자를 포함하는 제1 재조합 발현벡터; 및 바이러스의 Env 유전자, MCMV 프로모터 및 제2 치료 유전자를 포함하는 제2 재조합 발현벡터는 각각 또는 함께 재조합 레트로바이러스에 포함될 수 있다.
상기 암은 상술한 바와 같은 암을 포함할 수 있다.
본 명세서에서 사용된 용어, "유전자 전달 조성물"은 유전자를 표적 세포 내로 전달할 수 있는 조성물을 의미한다.
본 발명의 구체적인 실시예에서, 본 발명자들은 GaLV env 기반 벡터(spRRVe-TK, spRRVe-F1-TK, spRRVe-F2-TK, spRRVe-F3-TK, spRRVe-F4-TK) 및 MuLV 기반 벡터(sRRVe-TK, sRRVe-F1-TK, sRRVe-F2-TK, sRRVe-F3-TK, sRRVe-F4-TK)에서 치료 유전자로 사용되는 티미딘 키나아제 및 효모 시토신 디아미나제가 안정적으로 발현되는 것을 확인하였으며, 상기 치료 유전자가 전구 약물인 GCV 및 5-FC에 작용하여 세포를 사멸할 수 있음을 확인하였다.
따라서, 본 발명에 따른 벡터를 포함하는 재조합 레트로바이러스는 암 치료용 유전자의 전달 용도로 유용하게 사용될 수 있다.
또한, 본 발명은
1) MuLV(Murine Leukemia virus)의 Gag-Pol 유전자, sEF1α 프로모터 또는 MCMV 프로모터, 및 제1 치료 유전자를 포함하는 제1 재조합 발현벡터를 제조하는 단계; 및
2) 바이러스의 Env 유전자, MCMV 프로모터 및 제2 치료 유전자를 포함하는 제2 재조합 발현벡터를 제조하는 단계를 포함하는 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법을 제공한다.
상기 벡터는 상술한 바와 같은 특징을 갖는다. 일례로, 상기 MCMV 프로모터는 서열번호 3, 서열번호 4, 서열번호 5, 서열번호 6 및 서열번호 7의 염기서열을 갖는 폴리뉴클레오티드로 구성된 군으로부터 선택되고, 상기 sEF1α 프로모터가 서열번호 18의 염기서열을 갖는 폴리뉴클레오티드이며, 상기 바이러스의 Env 유전자는 서열번호 19 또는 서열번호 20의 염기서열을 갖는 폴리뉴클레오티드이다.
또한, 상기 제1 치료 유전자 및 제2 치료 유전자는 티미딘 키나아제(thymidine kinase, TK) 유전자, 효모 시토신 디아미나제(yCD) 유전자 및 인간 CD19 유전자로 이루어진 군으로부터 선택되는 어느 하나 이상이다.
또한, 상기 티미딘 키나아제 유전자는 서열번호 21의 염기서열을 갖는 폴리뉴클레오티드, 상기 효모 시토신 디아미나제(yCD) 유전자가 서열번호 22의 염기서열을 갖는 폴리뉴클레오티드, 상기 인간 CD19 유전자가 서열번호 43의 염기서열을 갖는 폴리뉴클레오티드이다.
상기 티미딘 키나아제 유전자는 전구체 약물인 간사이클로비르를, 상기 시토신 디아미나제 유전자는 전구체 약물인 5-플루오로사이토신을 활성화 시킬 수 있다.
본 발명의 구체적인 실시예에서, 본 발명의 구체적인 실시예에서, 재조합 발생을 최소화하기 위해 MCMV 프로모터 내의 반복적인 염기서열을 제거한 각각 470bp, 337bp, 237bp, 160bp의 크기를 가지는 절단된 MCMV 프로모터 4가지를 제작하였다(표 2). 또한, 상기 절단된 MCMV 프로모터가 도입된 GaLV env 기반 벡터(spRRVe-TK, spRRVe-F1-TK, spRRVe-F2-TK, spRRVe-F3-TK, spRRVe-F4-TK) 및 MuLV 기반 벡터(sRRVe-TK, sRRVe-F1-TK, sRRVe-F2-TK, sRRVe-F3-TK, sRRVe-F4-TK)을 제작하고, 상기 티미딘 키나아제 단백질을 발현하는 벡터와 함께 도입되는 벡터로서 효모 시토신 디아미나제 단백질을 발현하는 gag-pol-sEF1α-hopt-yCD로 구성된 sRRVe-sEF1α-hopt-yCD 벡터를 제작하고(도 7 및 도 8), 재조합 여부를 확인한 결과, sRRVe-sEF1α-hopt-yCD 벡터에서는 재조합이 발생하지 않았고(도 9 참조), MCMV 프로모터 내의 반복 서열을 대부분 제거한 spRRVe-F4-TK 벡터에서는 재조합이 거의 발생하지 않음을 확인하였다(도 10, 도 11 및 도 13). 또한, sRRVe-sEF1α-hopt-yCD 벡터의 sEF1α를 MCMV의 F4 절단 프로모터로 치환한 벡터와 sRRVgp-F4-hopt-yCD의 조합에서도 재조합이 일어나지 않음을 확인하였다도 14 참조). 또한, 인간 CD19 유전자를 치료 유전자로 도입한 sRRVgp-F4-hopt-yCD/sRRVe-F4-hCD19 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 20 및 도 21 참조). 또한, 인간 CD19 유전자의 세포질 도메인의 아미노산이 절단된 hCD19t를 치료 유전자로 도입한 sRRVe-F4-hCD19t 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 23 및 도 25 참조). 또한, 마우스 GM-CSF를 치료 유전자로 도입한 sRRVgp-F4-mGM-CSF 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 27 참조). 또한, 코돈 최적화된 인간 GM-CSF를 치료 유전자로 도입한 sRRVgp-F4-hopt-GM-CSF 벡터의 경우에도 재조합이 발생하지 않음을 확인하였다(도 32 및 도 33 참조).
따라서, 본 발명의 제조방법에 의해 제조된 벡터는 재조합 발생이 최소화되어 치료 유전자가 소실되지 않고 안정적으로 발현시키는데에 유용하게 사용될 수 있다.
이하, 본 발명을 하기 실시예에 의해 상세히 설명한다.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.
< 실시예 1> 복제가능 레트로바이러스 벡터의 구축
<1-1> 바이러스 벡터의 제작
gag, pol 및 env의 유전자가 하나의 지놈으로 합성되는 기존의 RRV 벡터는 레트로바이러스를 구성하는 유전자 외에 1.3 kb를 초과하는 외래유전자를 삽입할 경우, 지놈의 크기가 증가하여 벡터 구조가 불안정해지고, 온전한 형태의 바이러스 벡터를 증식할 수 없으므로, gag-pol 유전자와 env 유전자가 각각 독립된 벡터에서 발현하도록 두 개의 벡터를 제작하였으며, env는 포유동물 감염에 친화적인 MuLV(murine leukemia virus) 의 env 유전자 또는 영장류 감염에 친화적인 GaLV(Gibbon ape Leukemia virus)의 env 유전자로 치환하였다. 치료 유전자로서 HSV-TK(herpes simplex virus thymidine kinase) 유전자를 gag-pol 벡터에, yCD(yeast cytosine deaminase) 유전자를 GaLV-env 벡터에 각각 클로닝 하고, 유전자 발현 조절을 위한 프로모터로는 쥐 사이토메갈로바이러스(murine cytomegalovirus, MCMV) 프로모터를 사용하여 벡터를 구축하였다.
구체적으로, 바이러스 벡터는 하기와 같은 방법으로 제작하였다.
1. spRRVe-yCD env vector(서열번호 25): (도 1a)
기 구축한 spRRVeMCMV-MCS (GaLVEnv-MCMV-MCS-3'-LTR) 벡터의 MCS(multi-cloning site)내에 존재하는 PmeI을 잘라 CIAP 처리하여 잘려진 벡터의 양 말단을 blunt 형태로 준비한다. yCD가 삽입되어 있는 pcDNA-yCD 벡터를 XhoI-HindIII로 잘라 yCD 유전자를 회수한 후 T4 DNA polymerase를 처리하여 양 말단을 blunt 형태로 만든다. 이후 spRRVeMCMV(PmeI, CIAP)벡터와 yCD(XhoI-HindIII, T4 DNA polymerase)를 T4 DNA ligase를 이용하여 라이게이션하여 spRRVe-yCD를 제작하였다.
2. sRRVgp-TK:gag-pol vector(서열번호 26): (도 1b)
프로모터와 transgene이 없는 sRRVgp (레트로바이러스 gag-pol를 수반하는 벡터) 벡터를 구축하였다. 이후 gag-pol과 3'-LTR 사이의 EcoRI 자리에 MCMV-TK를 도입하고자 하였다. 한 번의 클로닝 과정으로 sRRVgp-TK를 완성할 수 없었으므로 먼저 MCMV를 클로닝하였고, 이후 MCMV 아래에 TK를 도입하여 최종 산물인 sRRVgp-TK를 완성하였다. 방법은 다음과 같다.
sRRVgp의 gag-pol과 3'-LTR 사이의 EcoRI 자리에 MCMV를 클로닝하기 위해 MCMV 프로모터를 PCR로 증폭하였다.
MCMV-F-EcoRI: 5'-cgGAATTCAACAGGAAAGTCCCATTGGA-3’(서열번호 47)
MCMV-R-PmeI-EcoRI: 5'-cgGAATTCGTTTAAACCTGCGTTCTACGGTGGTCAGA-3' (서열번호 48) 증폭된 MCMV 프로모터 산물을 EcoRI으로 자르고 sRRVgp 벡터를 EcoRI, CIAP를 처리하여 회수한 후 T4 DNA ligase로 라이게이션하여 sRRVgpMCMV를 완성하였다. 이후 sRRVgpMCMV 벡터의 PmeI 자리에 TK 유전자를 클로닝하고자 TK 유전자를 PmeI이 포함되도록 PCR로 증폭하였다.
TK-F-PmeI: 5'-cgGTTTAAACATGGCTTCGTACCCCTGCCATC-3'(서열번호 49)
TK-R-PmeI: 5'-CGGTTTAAACTCAGTTAGCCTCCCCCATCTCC-3'(서열번호 50) sRRVgpMCMV를 PmeI, CIAP 처리하여 회수하고, TK 유전자를 PmeI으로 잘라 회수한 후 T4 DNA ligase로 라이게이션하여 최종 산물인 sRRVgp-TK를 제작하였다.
<1-2> 바이러스의 생산
MCMV 프로모터 조절 아래 치료 유전자가 발현되는 복제가능 레트로바이러스 벡터의 감염능 및 재조합 유형을 확인하기 위해 상기 실시예 1-1에서 제조한 벡터로 바이러스를 도 2의 모식도에 나타난 과정대로 생산하였다.
구체적으로, 상기 실시예 1-1에서 제조한 벡터를 293T 세포에 일시적 형질도입하여 바이러스를 생산하였으며, 2E7 gc(genome copies)의 바이러스를 뇌종양 세포주 U87MG에 감염하였고, 초기 감염 3일 후 배양중인 상층액을 취하여 새로운 U87MG에 재감염시켰으며, 감염 중인 U87MG 세포주는 회수하여 지노믹 DNA를 분리하였다.
<1-3> spRRVe - yCD:env 벡터의 재조합 여부 확인
유전자치료용 복제가능 레트로바이러스 벡터는 도입된 외래 유전자에 의해 지놈 RNA의 크기가 증가하고 비상동 염기서열이 추가되어 재조합이 일어날 가능성이 높다. 따라서 효율적이고 안정적인 유전자 치료용 복제가능 레트로바이러스 벡터를 구축하기 위해서는 개발단계에서 재조합 여부 및 그 정도를 확인하는 것이 매우 중요하다. 상기 실시예 1-2에서 생산한 바이러스에서 추출한 지노믹 DNA를 이용하여 중합효소연쇄반응(PCR)을 수행하여 바이러스 증식 및 감염이 진행되었는지 여부 및 바이러스 감염 후 증폭 과정에서 재조합 발생 여부를 확인하였다.
구체적으로, env 벡터를 특이적으로 증폭할 수 있는 하기 표 1의 프라이머를 이용하여 중합효소연쇄반응을 진행하였다. 도 3에 나타난 바와 같이, spRRVe-yCD:env 벡터는 GaLV env와 3' 말단을 표적부위로 증폭하는 프라이머를 이용하여 지노믹 DNA PCR를 수행하여 예상되는 크기로 유전자가 증폭되는지를 확인하였다. PCR 반응 튜브에 100ng의 지놈 DNA, 1X 반응 버퍼, 0.25 mM dNTP, 0.2 pmol 정방향 프라이머, 0.2 pmol 역방향 프라이머, 0.2 unit Taq 중합효소를 넣고 최종 부피가 20㎕이 되도록 멸균된 증류수를 첨가하였다. 이후, 하기 표 1의 조건으로 PCR을 수행한 후, 1% 아가로오스 젤에 증폭된 DNA를 로딩하여 PCR 증폭 산물을 확인함으로써 바이러스의 감염능 및 재조합 발생 여부를 확인하였다.
서열(5'→3') 서열번호
GaLV 1624F GACTCAGTCAGCAAGTTAGAG 1
MFGSacIR CAATCGGAGGACTGGCGCCCCGAGTGA 2
그 결과, 도 4에 나타난 바와 같이, spRRVe-yCD:env 벡터의 중합효소연쇄반응을 수행한 결과, 초기감염 3일과 passage 1 내지 3까지 PCR 산물이 검출되었으므로 바이러스 증식 및 감염은 진행된 것으로 확인되었다. 그러나 passage 2부터 기대하는 2,337 bp 증폭 산물보다 작은 다수의 PCR 산물이 확인되므로 바이러스 감염 후 증폭 과정에서 재조합이 발생하였음을 확인할 수 있었다. 구체적으로, passage 1까지는 단일 PCR 밴드만 관찰할 수 있었으나, passage 2 및 passage 3에서는 여러 개의 PCR 밴드가 증폭된 것을 확인할 수 있었다.
<1-4> spRRVe - yCD:env 벡터의 재조합 유형 분석
레트로바이러스는 역전사 과정을 통하여 합성이 되므로, 이러한 과정 중 지놈 서열의 불안정성이 다양한 유형의 재조합 유형을 만들어 낼 수 있다. 따라서, 상기 실시예 1-3에서 spRRVe-yCD:env 벡터의 지노믹 DNA를 이용한 PCR 반응 후, p2 및 3단계 재조합 밴드의 PCR 산물(도 4의 붉은색 박스로 표시한 부분의 밴드)을 회수하여 유전자 분석을 위해 pGEM-T 벡터에 클로닝하고, 클론을 정제하여 유전자 염기서열을 분석하여 재조합 유형을 분석하였다.
그 결과, 도 5에 나타낸 바와 같이, 회수된 PCR 산물은 GaLV env, MCMV 프로모터, yCD, 3'-LTR 염기서열 내에서 재조합이 발생한 클론을 확인할 수 있었다. 특히, 도 6에 나타난 MCMV 프로모터 내의 4군데 반복적인 염기서열(1. AACAGGAAA, 2. GGGACTTTCCAATGGGTTTTGCCCAGTACA, 3. TGGGTTTTTCC, 4. GTACTTTCCCA) 로 인해 MCMV 프로모터를 기점으로 다른 부위의 바이러스 벡터 염기서열이 소실되거나 프로모터 내에서 염기서열 일부가 소실되어 재조합이 발생하였음을 확인할 수 있었다. 따라서, 재조합이 발생하지 않도록 MCMV의 반복 서열을 최소화하고 치료 유전자의 발현이 용이하도록 MCMV 프로모터 서열을 잘라내어 각각 크기가 다른 4 종류의 프로모터를 디자인하고자 하였다.
< 실시예 2> 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 구축
<2-1> 재조합 발생이 최소화된 벡터 구축을 위한 절단된 형태의 MCMV 프로모터의 제작
MCMV 프로모터 내의 반복적인 염기서열로 인해 발생하는 복제가능 레트로바이러스 벡터의 재조합을 극복하기 위해, MCMV 프로모터 내의 반복 서열을 제거하여절단된 형태의 MCMV 프로모터를 제작하고자 하였다.
구체적으로, 초기에 사용하였던 약 646 bp의 MCMV 프로모터를 반복서열 기반으로 잘라주어 모두 4종류의 절단된 MCMV 프로모터를 제작하였다(표 2). 이후 본 연구실에서 기 구축한 sRRVe-TK와 spRRVe-TK의 646 bp MCMV 프로모터 자리에 MCMV F1(470 bp), F2(337 bp), F3(237 bp), F4(160 bp)를 도입하였다. 하기 표 3에 기재된 제한효소 자리가 포함된 증폭 프라이머를 이용한 PCR을 통하여 절단된 MCMV 프로모터를 확보하였다.
MCMV 프로모터 (5'→3')
MCMV
(서열번호 3, 646 bp)		 AACAGGAAAGTTCCATTGGAGCCAAGTACATTGAGTCAATAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATGGGAGGTAAGCCAATGGGTTTTTCCCATTACTGGCACGTATACTGAGTCATTAGGGACTTTCCAATGGGTTTTGCCCAGTACATAAGGTCAATAGGGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACGTACATAAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCTCGAGCCAATACACGTCAATGGGAAGTGAAAGGGCAGCCAAAACGTAACACCGCCCCGGTTTTCCCTGGAAATTCCATATTGGCACGCATTCTATTGGCTGAGCTGCGTTCACGTGGGTATAAGAGGCGCGACCAGCGTCGGTACCGTCGCAGTCTTCGGTCTGACCACCGTAGAACGCAGA
MCMV F1
(서열번호 4, 470 bp)		 GGGTGAATCAACAGGAAAGTCCCATTGGAGCCAAGTACACTGAGTCAATAGGGACTTTCCATTGGGTTTTGCCCAGTACAAAAGGTCAATAGGGGGTGAGTCAATGGGTTTTTCCCATTATTGGCACGTACATAAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCTCGAGCCAATACACGTCAATGGGAAGTGAAAGGGCAGCCAAAACGTAACACCGCCCCGGTTTTCCCTGGAAATTCCATATTGGCACGCATTCTATTGGCTGAGCTGCGTTCACGTGGGTATAAGAGGCGCGACCAGCGTCGGTACCGTCGCAGTCTTCGGTCTGACCACCGTAGAACGCAGA
MCMV F2
(서열번호 5, 337 bp)		 AAGGTCAATAGGGGTGAGTCATTGGGTTTTTCCAGCCAATTTAATTAAAACGCCATGTACTTTCCCACCATTGACGTCAATGGGCTATTGAAACTAATGCAACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCTCGAGCCAATACACGTCAATGGGAAGTGAAAGGGCAGCCAAAACGTAACACCGCCCCGGTTTTCCCTGGAAATTCCATATTGGCACGCATTCTATTGGCTGAGCTGCGTTCACGTGGGTATAAGAGGCGCGACCAGCGTCGGTACCGTCGCAGTCTTCGGTCTGACCACCGTAGAACGCAGA
MCMV F3
(서열번호 6, 237 bp)		 AACGTGACCTTTAAACGGTACTTTCCCATAGCTGATTAATGGGAAAGTACCGTTCTCGAGCCAATACACGTCAATGGGAAGTGAAAGGGCAGCCAAAACGTAACACCGCCCCGGTTTTCCCTGGAAATTCCATATTGGCACGCATTCTATTGGCTGAGCTGCGTTCACGTGGGTATAAGAGGCGCGACCAGCGTCGGTACCGTCGCAGTCTTCGGTCTGACCACCGTAGAACGCAGA
MCMV F4
(서열번호 7, 161 bp) 		GGAAGTGAAAGGGCAGCCAAAACGTAACACCGCCCCGGTTTTCCCTGGAAATTCCATATTGGCACGCATTCTATTGGCTGAGCTGCGTTCACGTGGGTATAAGAGGCGCGACCAGCGTCGGTACCGTCGCAGTCTTCGGTCTGACCACCGTAGAACGCAGA
프라이머 서열(5'→3') 서열번호
sRRVe(MuLV)-based TK MCMV F1-PmeI cggtttaaacGGGTGAATCAACAGGAAAGTCCC 8
MCMV F2-PmeI cggtttaaacAAGGTCAATAGGGGTGAGTCAT 9
MCMV F3-PmeI cggtttaaacAACGTGACCTTTAAACGGTACT 10
MCMV F4-PmeI cggtttaaacGGAAGTGAAAGGGCAGCCAAA 11
MCMV-R-NotI cggcggccgcTCTGCGTTCTACGGTGGTCAGACC 12
spRRVe(GaLV)-based-TK MCMV F1-PmeI cggtttaaacGGGTGAATCAACAGGAAAGTCCC 8
MCMV F2-PmeI cggtttaaacAAGGTCAATAGGGGTGAGTCAT 9
MCMV F3-PmeI cggtttaaacAACGTGACCTTTAAACGGTACT 10
MCMV F4-PmeI cggtttaaacGGAAGTGAAAGGGCAGCCAAA 11
MCMV-R-BamHI cgggatccTCTGCGTTCTACGGTGGTCAGACC 13
<2-2> 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터( spRRVe ( GaLV )-TK/sRRVgp-sEF1a-hopt-yCD)의 제작
PmeI-BamHI 제한효소로 잘라 spRRVe(GaLV)-TK의 프로모터 자리에 클로닝하여 spRRVe-TK(서열번호 27), spRRVe-F1-TK(서열번호 28), spRRVe-F2-TK(서열번호 29), spRRVe-F3-TK(서열번호 30), spRRVe-F4-TK 벡터(서열번호 31)를 완성하였다(도 7). 이후 구축된 split-dual RRV 벡터의 유효성 및 재조합 발생 유무를 확인하기 위해 sEF1a 프로모터로 조절되는 hopt-yCD를 발현하는 gag-pol 벡터 조합(sRRVgp-sEF1a-hopt-yCD, 서열번호 32)으로 바이러스를 합성한 후, U87MG 세포에 감염시킨 후 재조합 발생 여부를 확인하였다.
<2-3> 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터(sRRVe(MuLV)-TK/sRRVgp-sEF1a-hopt-yCD)의 제작
PmeI-NotI 제한효소로 잘라 sRRVe(MuLV)-TK의 프로모터 자리에 클로닝하여 sRRVe-TK(서열번호 33), sRRVe-F1-TK(서열번호 34), sRRVe-F2-TK(서열번호 35), sRRVe-F3-TK(서열번호 36), sRRVe-F4-TK(서열번호 37) 벡터를 완성하였다(도 8). 이후 구축된 split-dual RRV 벡터의 유효성 및 재조합 발생 유무를 확인하기 위해 sEF1a 프로모터로 조절되는 hopt-yCD를 발현하는 gag-pol 벡터 조합(sRRVgp-sEF1a-hopt-yCD)으로 바이러스를 합성한 후, U87MG 세포에 감염시킨 후 재조합 발생 여부를 확인하였다.
< 실시예 3> 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터(spRRVe(GaLV)-TK/sRRVgp-sEF1a-hopt-yCD)의 재조합 유형 확인
상기 실시예 2-2에서 제작한 벡터의 재조합 유형을 상기 실시예 1-3과 동일한 방법으로 확인하였다.
구체적으로, 구축된 GaLV env 기반 spRRVe-TK, spRRVe-F1-TK, spRRVe-F2-TK, spRRVe-F3-TK, spRRVe-F4-TK 벡터를 sRRVgp-sEF1a-hopt-yCD 벡터와 병용하여 293T 세포에서 바이러스를 합성한 후, 상기 실시예 1-3과 동일한 방법으로 재조합 유형 분석 시험을 진행하였다. env 벡터 특이적 증폭은 하기 표 4에 나타난 GaLV1932F와 MFGSacIR 프라이머를 사용하였고, gag-pol 벡터 특이적 증폭은 pol7130F와 MFGSacIR 프라이머를 사용하였다.
그 결과, 도 9에 나타난 바와 같이, 지노믹 디엔에이 PCR 분석 결과 sRRVgp-sEF1a-hopt-yCD 벡터는 5개 set에서 모두 재조합 발생이 일어나지 않았다. 반면 spRRVe-TK 벡터는 p1부터 재조합이 발생하기 시작하였고, spRRVe-F1-TK, spRRVe-F2-TK는 p4부터, spRRVe-F3-TK는 p5부터 재조합이 일어나기 시작했고, spRRVe-F4-TK는 p10까지 재조합이 발생하지 않았다. 다만, p6 내지 p10의 PCR 산물에서 증폭 밴드 외에 아래에 작은 밴드는 gag-pol 벡터의 완전한 hopt-yCD가 env 벡터의 TK 자리에 상호호환(reciprocal)적으로 재조합되어 삽입되었음을 유전자 염기서열 분석법으로 확인하였다.
프라이머 서열(5'→3') 서열번호
GaLV 1624F GACTCAGTCAGCAAGTTAGAG 1
GaLV 1932F GTTGCTCATCCTCGGGCCATG 14
Am1801F ATCATTGACCCTGGCCCTTC 15
pol7130F CGGCCCGGCACTCATTGGGAG 16
MuLV4194F AGCAAGCTATTGGCCACTG 17
MFGSacIR CAATCGGAGGACTGGCGCCCCGAGTGA 2
< 실시예 4> 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터(spRRVe(GaLV)-TK/sRRVgp-sEF1a-hopt-yCD)를 포함하는 바이러스로 형질감염된 세포에서 티미딘 키나아제(TK) 및 효모 시토신 디아미나제 ( yCD ) 단백질의 발현 수준 확인
이후 재조합 시험 단계에서 확보한 U87MG 세포를 대상으로 티미딘 키나아제(HSV1-TK)와 효모 시토신 디아미나제(yCD) 단백질 발현 수준을 웨스턴 블럿을 통하여 확인하였다.
구체적으로, 도 2에 나타난 바와 같이 인간 뇌종양 세포주 U87MG에 레트로바이러스 벡터 조합으로 0.1 MOI를 감염한 후 3일 후에 바이러스가 포함된 세포배양 상층액을 취하여 새로운 뇌종양 세포주 U87MG에 순차 감염하며, 초기 감염 3일 후, p1(passage 1)~ p10의 세포를 수확하여 100㎕의 T-PER Tissue extraction reagent (PIERCE, 78510) + 1x Proteinase Inhibitor로 재현탁하였다. 이후 3번의 freezing-thawing으로 세포를 깨고, 13,000 rpm에서 10분간 원심분리하여 세포질 상층액을 회수하였다. BCA를 이용하여 단백질 양을 정량 한 후, 20㎍의 세포 용해물을 취하여 SDS sample loading dye가 1%가 되도록 혼합하였다. 100℃ heat block에서 5분간 반응한 후, 얼음으로 옮겨 2분간 반응하였다. 이후 10% (TK), 13.5% (yCD) SDS-PAGE 겔에 로딩하여 단백질을 전기 영동하였다. 전기영동이 끝난 후 90V에서 2시간 동안 NC (nitrocellulose) 막으로 단백질을 transfer 한 후, blocking solution(5% skim milk in 1X TBS-T)에 넣어 상온에서 1시간 반응하였다. 이후 primary antibody (anti-rabbit-TK : Santa Cruze sc-28038, 1:500 dilution, anti-sheep yCD : Thermo Fisher Scientific PA1-85365, 1:500, anti-mouse b-actin : Sigma A2228, 1:1000)를 1% skim milk/1X TBS-T를 넣어 막에 부은 후, 4℃ 냉장고에서 밤새도록 반응시켰다. 이후 상온에서 1x TBS-T로 10분간 4번 수세한 후 Horseradish peroxidase(HRP)가 접합된 이차 항체를 넣어 상온에서 1시간 반응하였다. 이후 상온에서 1x TBS-T로 10분간 4번 수세한 후 ECL (enhanced chemiluminescence, Bio-rad Cat No. 170-5062) 용액을 넣어 반응한 후, 화학발광 이미징 시스템 장비에서(ChemiDoc, Biorad CA)에서 분석하였다.
그 결과, 도 10 및 도 11에 나타난 바와 같이, 지노믹 디엔에이 PCR 결과와 일치하게 프로모터 크기가 줄어들수록 티미딘 키나아제(TK) 단백질 발현이 안정적으로 지속되었으며, gag-pol 벡터(sRRVgp-sEF1a-hopt-yCD) 내의 효모 시토신 디아미나제(yCD) 단백질도 지속적으로 발현함을 확인할 수 있었다.
< 실시예 5> 티미딘 키나아제 및 효모 시토신 디아미나제의 전구 약물 투여시 세포 사멸 확인
상기 실시예 2-2에서 생산된 spRRVe(GaLV)-TK/sRRVgp-sEF1a-hopt-yCD 바이러스의 간사이클로비르(GCV) 및 5-플루오로사이토신(5-FC)에 대한 약물감수성을 확인하였다.
구체적으로, spRRVe-TK/sRRVgp-sEF1a-hopt-yCD 바이러스를 PLUS reagent 시약(invitrogen) 및 리포펙타민(invitrogen)을 이용하여 293T 세포주에 공형질도입(co-transfection)하였다. 2일 후 바이러스의 상층액을 회수하고, 전날 6웰에 1.5×105개로 계대한 U-87MG 세포에 바이러스와 8㎍/㎖ 농도의 폴리브랜을 첨가하여 8시간 동안 감염시켰다. 감염 5일 후(postinfection 5d), 세포의 상층액을 취하여 전날 6웰에 1.5×105개로 계대한 U-87MG 세포에 재감염 하였으며(p1), 이후 같은 방법으로 p4까지 순차감염 하였다. 각 감염 단계의 세포에 트립신-EDTA를 처리하여 단일세포로 만들어, 12웰 플레이트에 1.5×105개로 계대하였고, 계대한 다음날부터 30㎍/㎖ 농도의 GCV와 1 mM 농도의 5-FC를 각각 5일 또는 8일 동안 처리하여 세포 사멸을 확인하였다.
그 결과, 도 12에 나타난 바와 같이, 바이러스 감염 세포에 전구 약물인(pro-drug) 30㎍/㎖ 간사이클로비르(GCV)와 1 mM 5-플루오르사이토신(5-FC)를 처리하였을 때 세포가 사멸함을 확인할 수 있었다.
< 실시예 6> 절단된 형태의 MCMV 프로모터가 포함된 MuLV env 기반 벡터의 재조합 유형 확인
spRRVe-sEF1a-TK/sRRVgp-sEF1a-hopt-yCD 조합의 재조합 발생 여부 분석 시험에서 gag-pol 벡터는 재조합이 잘 일어나지 않음을 확인한 바 있으나, gag-pol 벡터의 완전한 hopt-yCD가 env 벡터의 TK 자리에 상호호환(reciprocal)적으로 재조합되어 삽입되었음을 확인한 바 있다. 이는 gag-pol 벡터와 env 벡터에서 사용하는 프로모터가 동일한 경우, 프로모터 간의 상동 재조합으로(homologous recombination) 발생하는 현상으로, 치료유전자 발현에는 영향을 미치지 않는다. 반면 sRRVgp-sEF1a-hopt-yCD / spRRVe-F4-TK 조합의 경우 프로모터 간의 비상동 재조합 발생이 일어나 유효한 치료유전자 발현에 영향을 미칠 경우의 수가 발생하므로 이러한 재조합 발생을 최소화하고자 sRRVgp-sEF1a-hopt-yCD의 sEF1a 자리에 F4 프로모터를 도입하여 sRRVgp-F4-hopt-yCD를 제작하고, sRRVgp-F4-hopt-yCD/sRRV3 sRRVe-F4-TK 벡터의 재조합 유형을 상기 실시예 1-3과 동일한 방법으로 확인하였다.
구체적으로, sRRVgp-F4-hopt-yCD 벡터는 sRRVgp-sEF1a-TK 벡터를 EcoRI로 처리하여 sEF1a-TK를 생성시키고, MCMV F4를 클로닝하기 위해 다른 치료 유전자를 클로닝할 수 있도록 제한효소 인식서열을 넣은 하기 표 5의 MCMV(F4)-EcoRI-F 및 MCMV-NotI-MluI-EcoRI-R 프라이머를 사용하여 spRRVe-TK를 주형으로 하여 PCR을 수행하였다. 이후, sRRVgp-sEF1a-TK와 MCMV F4 PCR 산물을 EcoRI으로 자른 후 클로닝하여 sRRVgp-MCMV F4 벡터를 제작하였다. 이후 hopt-yCD를 하기 표 5의 hopt-yCD-NotI-F 및 hopt-yCD-NotI-R 프라이머를 제작하여 sRRVgp-sEF1a-hopt-yCD 벡터를 주형으로 하여 PCR을 수행하였다.
프라이머 서열(5'→3' 서열번호
MCMV(F4)-EcoRI-F cggaattcGGAAGTGAAAGGGCAGCCAAA 38
MCMV-NotI-MluI-EcoRI-R cggaattcacgcgtgcgcggccgcTCTGCGTTCTACGGTGGTCAGACC 39
hopt-yCD-NotI-F cggcggccgcATGGTTACTGGGGGAATGGC 40
hopt-yCD-MluI-R acgcgtTTATTCCCCGATGTCTTCGAA 41
이후, sRRVgp-MCMV F4 벡터와 hopt-yCD PCR 산물을 NotI- MluI을 처리하여 회수한 후 클로닝하여 sRRVgp-F4-hopt-yCD 벡터를 완성하였다.
구축된 MuLV env 기반 sRRVe-F4-TK 벡터를 sRRVgp-F4-hopt-yCD 벡터와 병용하여 293T에서 바이러스를 합성한 후, 상기 실시예 1-3과 동일한 방법으로 재조합 유형 분석 시험을 진행하였다. env 벡터 특이적 증폭은 상기 표 4에 나타난 Am1801F와 MFGSacIR 프라이머를 사용하였고, gag-pol 벡터 특이적 증폭은 pol7130F와 MFGSacIR 프라이머를 사용하였다.
그 결과, 도 13에 나타난 바와 같이, 지노믹 디엔에이 PCR 분석 결과 gag-pol 벡터는 5개 세트에서 모두 재조합 발생이 일어나지 않았다. 반면 sRRVe-TK 벡터는 p2부터 작은 크기가 소실된 재조합이 발생하기 시작하였고, sRRVe-F1-TK, sRRVe-F2-TK는 p7부터, sRRVe-F3-TK는 p8부터 재조합이 일어나기 시작했음을 확인 할 수 있었다. 반면 sRRVe-F4-TK/sRRVgp-F4-hopt-yCD 벡터 조합에서 sRRVe-F4-TK는 p10까지 재조합이 발생하지 않았으며, p15까지 지속적 감염을 진행한 후 재조합 발생 여부를 확인한 결과에서도 재조합이 일어나지 않음을 확인할 수 있었다(도 14).
즉, MuLV env 벡터 기반 RRV 벡터도 MCMV 프로모터 크기가 작아질수록 재조합 발생 빈도가 줄어들었고, MCMV 프로모터의 반복 서열을 대부분 제거한 sRRVe-F4-TK 벡터에서는 재조합 발생이 일어나지 않음을 확인할 수 있어 절단된 MCMV 프로모터를 포함하는 복제 가능한 레트로바이러스 벡터는 재조합이 일어나지 않아 치료 유전자가 소실되지 않고 표적 세포로 전달될 수 있음을 확인하였다.
< 실시예 7> 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터(sRRVe(MuLV)-TK/sRRVgp-sEF1a-hopt-yCD)를 포함하는 바이러스로 형질감염된 세포에서 티미딘 키나아제(TK) 단백질의 발현 수준 확인
이후 sRRVe-F4-TK 이후 재조합 시험 단계에서 확보한 U87MG 세포를 대상으로 TK 단백질 발현 수준을 상기 실시예 4에 기재된 것과 동일한 방법 및 조건으로 웨스턴 블럿을 수행하여 확인하였다. yCD 단백질은 gag-pol 벡터 내에서 재조합이 일어나지 않아 안정적으로 발현함을 확인하였기 때문에, TK 단백질만 발현 수준을 확인하였다.
그 결과, 도 15에 나타난 바와 같이, 지노믹 디엔에이 PCR 결과와 유의하게 프로모터 크기가 줄어들수록 티미딘 키나아제(TK) 단백질 발현이 안정적으로 지속됨을 확인할 수 있었다.
< 실시예 8> 티미딘 키나아제(TK) 및 효모 시토신 디아미나제(yCD)의 전구 약물 투여시 세포 사멸 확인
상기 실시예 6에서 생산된 sRRVe(MuLV)-TK/sRRVgp-F4-hopt-yCD 벡터를 포함하는 바이러스의 간사이클로비르(GCV) 및 5-플루오로사이토신(5-FC)에 대한 약물감수성을 확인하였다.
구체적으로, spRRVe-F4-hopt-yCD/sRRVgp-TK, spRRVe-F4-hopt-yCD/sRRVgp-F1-TK, spRRVe-F4-hopt-yCD/sRRVgp-F2-TK, spRRVe-F4-hopt-yCD/sRRVgp-F3-TK 및 spRRVe-F4-hopt-yCD/sRRVgp-F4-TK 벡터를 포함하는 바이러스를 PLUS reagent 시약(invitrogen) 및 리포펙타민(invitrogen)을 이용하여 293T 세포주에 공형질도입(co-transfection)하였다. 2일 후 바이러스의 상층액을 회수하고, 전날 6웰에 1.5×105개로 계대한 U-87MG 세포에 바이러스와 8㎍/㎖ 농도의 폴리브랜을 첨가하여 8시간 동안 감염시켰다. 감염 5일 후(postinfection 5d), 세포의 상층액을 취하여 전날 6웰에 1.5×105개로 계대한 U-87MG 세포에 재감염 하였으며(p1), 이후 같은 방법으로 p4까지 순차감염 하였다. 각 감염 단계의 세포에 트립신-EDTA를 처리하여 단일세포로 만들어, 12웰 플레이트에 1.5×105개로 계대하였고, 계대한 다음날부터 30㎍/㎖ 농도의 GCV와 1 mM 농도의 5-FC를 각각 5일 또는 8일 동안 처리하여 세포 사멸을 확인하였다.
그 결과, 도 16에 나타난 바와 같이, 바이러스 감염 세포에 전구 약물인(pro-drug) 30㎍/㎖ GCV와 1mM 5-FC를 처리하였을 때 세포가 사멸함을 광학 현미경 하에서 확인할 수 있었고, 2E7 genome copies의 sRRVe-F4-TK/sRRVgp-F4-hopt-yCD 바이러스를 U87MG에 7일간 감염한 후, 30㎍/㎖ GCV와 1mM 5-FC를 처리하여 세포 사멸을 정량적으로 평가한 결과 GCV를 처리한 경우 감염 4일 후까지 세포 생존능이 약 80% 감소하였으며, 감염 10일 후에는 약 10%의 세포만 생존하여 약 90% 이상의 세포를 사멸시킨 것을 확인할 수 있었고, 5-FC를 처리한 경우 감염 2일 후에 세포 생존능이 약 40% 감소하였으며, 감염 8일 후에는 전체 세포의 약 40%만 생존하여 약 60% 이상의 세포를 사멸시킨 것을 확인할 수 있었다(도 17).
< 실시예 9> 암 항원 유전자인 인간 CD19가 도입된 재조합 발생이 최소화된 자가 복제 레트로바이러스 벡터 구축
<9-1> sRRVgp -F4- hCD19 / sRRVe -F4-TK 벡터의 제작 및 재조합 발생 여부 확인
최근 암을 치료하기 위한 면역 치료제에 대한 연구가 많이 수행되고 있으며, 실제로 CAR-T와 같이 암세포에서 특이적으로 발현하는 항원을 표적하는 수용체(receptor)를 바이러스 벡터에 적재하여 전달하는 면역 치료(Immunotherapy)방법이 개발되었고 임상에서도 적용중이다. 이러한 면역치료 방법은 체내 면역세포들의 특성들을 이용하여 부작용을 최대한 줄이며, 환자의 몸이 암세포와 맞서 싸우도록 면역 반응을 강화시킬 수 있다. 그 중 능동적 면역치료법은 암세포가 갖고 있는 암 특이적 항원(tumor-specific antigen)을 암환자에게 투여하여 면역체계를 활성화 시킴으로써 생체 내 면역기능을 활발하게 만들어 암세포를 공격하는 것이다. 따라서 인간 CD19 유전자를 자가복제 레트로바이러스 벡터에 도입하여 재조합 발생이 최소화된 암의 유전자 치료 벡터로 활용하고자 하였다.
구체적으로, hCD19 변이체2(variant 2)를 sRRVgp-F4-hopt-yCD의 hopt-yCD 자리에 클로닝하기 위해 제한효소 자리가 포함된 NotI-hCD19-MluI 유전자를 합성하였다. 이후 hCD19과 sRRVgp-F4-hopt-yCD을 각각 NotI과 MluI으로 잘라 회수한 후 클로닝하여 sRRVgp-F4-hCD19 벡터(서열번호 44)를 제작하였다. 도 18에 나타난 바와 같이, sRRVe-F4-TK(서열번호 37)와 sRRVgp-F4-hCD19 조합으로 바이러스를 합성하여 바이러스 벡터의 역가를 정량 하였으며, 0.1 MOI의 바이러스를 취하여 뇌종양 세포주 U87-MG 세포에 감염한 후 13번의 계대를 거쳤고, 각 계대 단계마다 감염 세포주를 대상으로 지노믹 디엔에이를 정제하였다. 이후 하기 표 6의 프라이머를 이용하여 지노믹 디엔에이를 대상으로 PCR을 수행하여 재조합 변이 유형을 분석하였다.
그 결과, 도 19에 나타난 바와 같이, hCD19이 도입된 벡터가 p3부터 재조합 PCR 밴드가 증폭되었음을 확인할 수 있었다.
프라이머 서열(5'→3') 서열번호
AmEnv961F ACAAGACCCAAGAATGTTGGCT 42
Am 1801F ATCATTGACCCTGGCCCTTC 15
pol 7130F CGGCCCGGCACTCATTGGGAG 16
MFGSacIR CAATCGGAGGACTGGCGCCCCGAGTGA 2
<9-2> sRRVgp -F4-hopt-yCD/ sRRVe -F4- hCD19 벡터의 제작 및 재조합 발생 여부 확인
상기 실시예 9-1에서 나타난 바와 같이, sRRVgp-F4-hCD19/sRRVe-F4-TK 벡터 조합은 바이러스 복제 동안 hCD19이 삽입된 sRRVgp-F4-hCD19 벡터에서 재조합이 발생함을 확인하였으므로, 앞서 바이러스 복제 과정에서 재조합이 발생하지 않았음을 확인한 sRRVgp-F4-hopt-yCD gag-pol 벡터를 선택하였고, sRRVe-F4-TK 벡터의 TK 유전자를 hCD19로 치환한 sRRVe-F4-hCD19 벡터를 제작하였다.
구체적으로, hCD19 변이체2(variant 2)를 이용하여 sRRVe-F4-TK의 TK 자리에 hCD19을 클로닝하기 위해 제한효소 자리를 포함한 NotI-hCD19-MluI 유전자를 합성하였다. 이후 hCD19과 sRRVgp-F4-TK을 NotI-MluI으로 잘라 회수한 후 클로닝하여 sRRVe-F4-hCD19 벡터를 제작하였다. 도 20에 나타난 바와 같이, sRRVe-F4-hCD19(서열번호 45)와 sRRVgp-F4-hopt-yCD(서열번호 46) 조합으로 바이러스를 합성한 후, 바이러스 벡터의 역가를 정량하여 0.1 MOI의 바이러스를 취하고 상기 표 6의 프라이머를 이용하여 재조합 변이 유형 분석 실험을 진행하였다.
도 21에 표기한 바와 같이 env 벡터의 복제 및 지놈 안정성을 확인 할 수 있는 프라이머를 이용하여 PCR를 수행하였으며, 그 결과 AmEnv961F/MFGSacIR 증폭산물의 크기는 3,460bp이었고, AmEnv1801F/MFGSacIR는 증폭산물의 크기는 3,030bp 이었다. 또한 gag-pol 벡터의 복제 및 지놈 안정성을 확인 할 수 있는 pol7130R/MFGSacIR 프라이머를 사용한 증폭산물의 크기는 2,092bp 이었으므로 hCD19이 도입된 벡터 sRRVe-F4-hCD19과 sRRVgp-F4-hopt-yCD 벡터 조합에서는 바이러스 복제 시 재조합이 발생하지 않았음을 확인 할 수 있었다.
<실시예 10> 암 항원 유전자인 인간 CD19t(truncated human CD19, hCD19t)가 도입된 재조합 발생이 최소화된 자가 복제 레트로바이러스 벡터 구축
<10-1> sRRVgp-F4-hCD19t 벡터의 제작 및 재조합 발생 여부 확인
CD19 N-말단/CAR-T 기작 외에 CD19의 C-말단에 의해 발생할 수 있는 세포 내 신호전달을 억제하기 위하여, 세포질 도메인의 233개 아미노산이 제거되고 323개 아미노산으로 구성된 truncated CD19(CD19t)을 자가복제 레트로바이러스 벡터에 도입하여 재조합 발생이 최소화된 암 유전자 치료 벡터로 활용하고자 하였다. CD19t는 세포외(extracellular) N-말단, 막관통(transmembrane) 도메인, 19개의 아미노산만 남은 세포질(cytosolic) C-말단으로 구성된다.
sRRVgp-F4-hCD19t 벡터를 하기와 같이 구축하였다. hCD19t를 확보하기 위해 hCD19 변이체 2(variant 2)를 주형으로 하기 표 7의 프라이머를 이용하여 hCD19t를 증폭하였다.
프라이머 서열(5'→3' 서열번호
hCD19t-NotI-F cggcggccgcATGCCACCTCCTCGCCTCCTCTTC 51
hCD19t-MluI-R caacgcgtTCATCTTTTCCTCCTCAGGACCAGG 52
클로닝을 용이하게 하기 위해 5' 쪽으로는 NotI, 3' 쪽으로는 MluI 제한효소 자리를 넣어 프라이머를 제작하였다. 이후 sRRVgp-F4-hopt-yCD 벡터의 hopt-yCD 자리에 hCD19t를 클로닝하고자 벡터를 NotI-MluI으로 자르고, 증폭된 hCD19t를 NotI-MluI으로 잘라 회수한 후 벡터에 도입하여 sRRVgp-F4-hCD19t 벡터(서열번호 54)를 제작하였다(도 22). 그 후, sRRVe-F4-TK(서열번호 37) 벡터와 sRRVgp-F4-hCD19t 벡터의 조합으로 바이러스를 합성한 후, 바이러스 벡터의 역가를 정량하여 0.1 MOI의 바이러스를 취하고 상기 표 6의 프라이머를 이용하여 재조합 변이 유형 분석 실험을 진행하였다.
그 결과, 도 23에 나타난 바와 같이, AmEnv961F/MFGSacIR 프라이머에 의한 sRRVe-F4-TK 벡터의 증폭산물 2,946bp와 pol7130R/MFGSacIR 프라이머에 의한 sRRVgp-F4-hCD19t 증폭산물 2,591bp가 바이러스 복제 및 재감염 과정인 passage 12까지 지속적으로 증폭됨을 확인하였다. 이러한 결과로 passage 12까지 바이러스 복제 및 재감염이 지속적으로 진행되었음을 확인할 수 있었다. 상기 결과는 TK가 도입된 sRRVe-F4-TK 벡터뿐 아니라 hCD19t이 도입된 sRRVe-F4-hCD19t 벡터 모두 바이러스 복제 동안 지놈 안전성(genome stability)이 우수하다는 것을 제시한다.
<10-2> sRRVe-F4-hCD19t 벡터의 제작 및 재조합 발생 여부 확인
sRRVe-F4-hCD19t 벡터를 하기와 같이 구축하였다. hCD19t를 확보하기 위해 hCD19 변이체2 (variant 2)를 주형으로 하여 하기 표 8의 프라이머를 이용하여 hCD19t를 증폭하였다.
프라이머 서열(5'→3' 서열번호
hCD19t-NotI-F cggcggccgcATGCCACCTCCTCGCCTCCTCTTC 51
hCD19t-SalI-R tggtcgacTCATCTTTTCCTCCTCAGGACCAGG 55
클로닝을 용이하게 하기 위해 5' 쪽으로는 NotI, 3' 쪽으로는 SalI 제한효소 자리를 넣었다. 이후 sRRVe-F4-hCD19 벡터의 hCD19 자리에 hCD19t를 클로닝하고자 벡터를 NotI-SalI으로 자르고, 증폭된 hCD19t를 NotI-SalI으로 잘라 회수한 후 벡터에 도입하여 sRRVe-F4-hCD19t 벡터(서열번호 56)를 제작하였다(도 24). 그 후, sRRVgp-F4-hopt-yCD(서열번호 46)와 sRRVe-F4-hCD19t 벡터 조합으로 바이러스를 합성한 후, 바이러스 벡터의 역가를 정량하여 0.1 MOI의 바이러스를 취하고 상기 표 6의 프라이머를 이용하여 재조합 변이 유형 분석 실험을 진행하였다.
그 결과, 도 25에 나타난 바와 같이, passage 12까지 AmEnv961F/MFGSacIR 프라이머에 의한 sRRVe-F4-hCD19t 벡터의 증폭산물 2,760bp와 pol7130R/MFGSacIR 프라이머에 의한 sRRVgp-F4-hopt-yCD 증폭산물 2,089bp이 확인 되었으므로 바이러스 복제 및 재감염이 지속적으로 진행되었음을 확인할 수 있었다. 상기 결과는 hopt-yCD이 도입된 sRRVgp-F4-hopt-yCD 벡터뿐 아니라 hCD19t가 도입된 sRRVe-F4-hCD19t 벡터 모두 바이러스 복제 동안 지놈 안전성(genome stability)이 우수하다는 것을 제시한다.
<실시예 11> 과립구-대식세포 콜로니 자극인자(Granulocyte-macrophage colony-stimulating factor, GM-CSF)가 도입된 재조합 발생이 최소화된 자가 복제 레트로바이러스 벡터 구축
<11-1> sRRVgp-F4-mGM-CSF 벡터의 제작 및 재조합 발생 여부 확인
치료 유전자 및 truncated hCD19를 적재하는 자가복제 레트로바이러스 벡터를 암 환자를 대상으로 적용 시 환자의 면역 증대를 촉진하기 위하여 자가복제 레트로 바이러스 벡터에 마우스 GM-CSF와 인간 GM-CSF를 하기와 같이 도입하였다.
먼저, sRRVgp-F4-mGM-CSF 벡터를 구축하고자 하였다. 마우스 GM-CSF(mGM-CSF)를 확보하기 위해 기 구축된 spRRVe-mGM-CSF(서열번호 57)를 주형으로 하기 표 9의 프라이머를 이용하여 mGM-CSF를 증폭하였다.
프라이머 서열(5'→3' 서열번호
mGM-CSF-F-NotI cggcggccgcAGGATGTGGCTGCAGAATTT 58
mGM-CSF-R-MluI tggtcgacTCATTTTTGGACTGGTTTTT 59
이 때 클로닝을 용이하게 하기 위해 5' 쪽으로는 NotI, 3' 쪽으로는 MluI 제한효소 자리를 넣었다. 이후 sRRVe-F4-hopt-yCD 벡터의 hopt-yCD 자리에 mGM-CSF를 클로닝하고자 벡터를 NotI-MluI으로 자르고, 증폭된 mGM-CSF 를 NotI-MluI으로 잘라 회수한 후 클로닝하여 sRRVgp-F4-mGM-CSF 벡터(서열번호 60)를 제작하였다(도 26). 그 후, sRRVgp-F4-mGM-CSF와 sRRVe-F4-hCD19t(서열번호 56) 벡터 조합으로 바이러스를 합성한 후, 바이러스 벡터의 역가를 정량하여 0.1 MOI의 바이러스를 취하고 상기 표 6의 프라이머를 이용하여 재조합 변이 유형 분석 실험을 진행하였다.
그 결과, 도 27에 나타난 바와 같이, AmEnv961F/MFGSacIR 프라이머에 의한 sRRVe-F4-hCD19t 벡터의 증폭산물 2,760bp와 pol7130R/MFGSacIR 프라이머에 의한 sRRVgp-F4-mGM-CSF 증폭산물 2,041bp가 바이러스 복제 및 재감염 과정인 passage12까지 지속적으로 증폭됨을 확인하였다. 상기 결과는 hCD19t가 도입된 sRRVe-F4-hCD19t 벡터뿐 아니라 mGM-CSF이 도입된 sRRVgp-F4-mGM-CSF 벡터 모두 바이러스 복제 동안 지놈 안전성(genome stability)이 우수하다는 것을 제시한다.
<11-2> sRRVgp-F4-hGM-CSF 벡터의 제작 및 재조합 발생 여부 확인
sRRVgp-F4-hGM-CSF 벡터를 구축하고자 hGM-CSF를 확보하기 위해 기 구축된 spRRVe-hGM-CSF(서열번호 61)를 주형으로 하여 하기 표 10의 프라이머를 이용하여 hGM-CSF를 증폭하였다.
프라이머 서열(5'→3' 서열번호
hGM-CSF-F-NotI cggcggccgcAGGATGTGGCTGCAGAGC 62
hGM-CSF-R-MluI tggtcgacTCACTCCTGGACTGGCTCCC 63
클로닝을 용이하게 하기 위해 5' 쪽으로는 NotI, 3' 쪽으로는 MluI 제한효소 자리를 넣었다. 이후 sRRVe-F4-hopt-yCD 벡터의 hopt-yCD 자리에 hGM-CSF를 클로닝하고자 벡터를 NotI-MluI으로 자르고, 증폭된 hGM-CSF를 NotI-MluI으로 잘라 회수한 후 클로닝하여 sRRVgp-F4-hGM-CSF 벡터(서열번호 64)를 제작하였다(도 28). 그 후, sRRVgp-F4-hGM-CSF와 sRRVe-F4-hCD19t(서열번호 56) 벡터 조합으로 바이러스를 합성한 후, 바이러스 벡터의 역가를 정량하여 0.1 MOI의 바이러스를 취하고 상기 표 6의 프라이머를 이용하여 재조합 변이 유형 분석 실험을 진행하였다.
그 결과, 도 29에 나타난 바와 같이, AmEnv961F/MFGSacIR 프라이머에 의한 sRRVe-F4-hCD19 벡터의 증폭산물 3,460bp은 passage 6까지 지속적으로 증폭되었으나, pol7130R/MFGSacIR 프라이머에 의한 sRRVgp-F4-hGM-CSF는 증폭 산물이 초기 감염부터 ~passage 6까지 2가지 형태의 증폭 산물로 관찰되므로 초기 감염부터 지놈 구조의 불안전성으로 복제가 온전히 일어나지 않음을 알 수 있었다.
<11-3> hGM-CSF의 인간 코돈 최적화 염기서열 확보
상기 실시예 11-2에서 재조합 변이로 인해 복제가 온전히 일어나지 않은 문제점을 해결하기 위하여 hGM-CSF 염기서열을 인간 코돈 최적화(human codon optimization) 염기서열로 변환하고자 하였다.
인간 코돈 염기서열은 국내 ㈜코스모젠텍에 의뢰하여 염기서열 확보 및 합성을 진행하였다. 이 때 자가복제 레트로 바이러스 벡터에 클로닝을 용이하게 하기 위해 5'쪽에 NotI과 3'쪽에 MluI 제한효소 자리를 넣어 합성하였다(서열번호 65).
<11-4> sRRVgp-F4-hopt-GM-CSF 벡터의 제작 및 재조합 발생 여부 확인
sRRVgp-F4-hopt-GM-CSF 벡터를 구축하고자, NotI과 MluI으로 자른 sRRVe-F4-hopt-yCD 벡터의 hopt-yCD 자리에 상기 실시예 11-3에서 합성한 hopt-GM-CSF를 클로닝하여 sRRVgp-F4-hopt-GM-CSF 벡터(서열번호 66)를 제작하였다(도 30). 그 후, sRRVgp-F4-hopt-GM-CSF와 sRRVe-F4-hCD19(서열번호 45) 벡터(도 30), sRRVgp-F4-hopt-GM-CSF와 sRRVe-F4-hCD19t(서열번호 56) 조합(도 31)으로 바이러스를 합성한 후, 바이러스 벡터의 역가를 정량하여 0.1 MOI의 바이러스를 취하고 상기 표 6의 프라이머를 이용하여 재조합 변이 유형 분석 실험을 진행하였다.
그 결과, 도 32에 나타난 바와 같이, AmEnv961F/MFGSacIR 프라이머에 의한 sRRVe-F4-hCD19 벡터의 증폭산물 3,460bp과 pol7130R/MFGSacIR 프라이머에 의한 sRRVgp-F4-hopt-GM-CSF 증폭산물 2,047bp가 바이러스 복제 및 재감염 과정인 passage 12까지 지속적으로 증폭됨을 확인하였다. 상기 결과는 hCD19가 도입된 sRRVe-F4-hCD19 벡터뿐 아니라 hopt-GM-CSF이 도입된 sRRVgp-F4-hopt-GM-CSF 벡터 모두 바이러스 복제 동안 지놈 안전성(genome stability)이 우수함을 제시한다.
또한, 도 33에 나타난 바와 같이, AmEnv961F/MFGSacIR 프라이머에 의한 sRRVe-F4-hCD19t 벡터의 증폭산물 2,760bp과 pol7130R/MFGSacIR 프라이머에 의한 sRRVgp-F4-hopt-GM-CSF 증폭산물 2,047bp가 바이러스 복제 및 재감염 과정인 passage 12까지 지속적으로 증폭됨을 확인하였다. 상기 결과는 hCD19t가 도입된 sRRVe-F4-hCD19t 벡터뿐 아니라 hopt-GM-CSF이 도입된 sRRVgp-F4-hopt-GM-CSF 벡터 모두 바이러스 복제 동안 지놈 안전성(genome stability)이 우수함을 제시한다.
<110> RTCURE Co., Ltd <120> Gene therapy vector with minimizing recombination, recombinant retrovirus comprising the vector and pharmaceutical composition for preventing or treating cancer comprising the recombinant retrovirus <130> 2021P-07-028 <150> KR 10-2020-0120797 <151> 2020-09-18 <160> 66 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GaLV 1624F <400> 1 gactcagtca gcaagttaga g 21 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> MFGSacIR <400> 2 caatcggagg actggcgccc cgagtga 27 <210> 3 <211> 646 <212> DNA <213> Artificial Sequence <220> <223> MCMV promoter <400> 3 aacaggaaag ttccattgga gccaagtaca ttgagtcaat agggactttc caatgggttt 60 tgcccagtac ataaggtcaa tgggaggtaa gccaatgggt ttttcccatt actggcacgt 120 atactgagtc attagggact ttccaatggg ttttgcccag tacataaggt caataggggt 180 gaatcaacag gaaagtccca ttggagccaa gtacactgag tcaataggga ctttccattg 240 ggttttgccc agtacaaaag gtcaataggg ggtgagtcaa tgggtttttc ccattattgg 300 cacgtacata aggtcaatag gggtgagtca ttgggttttt ccagccaatt taattaaaac 360 gccatgtact ttcccaccat tgacgtcaat gggctattga aactaatgca acgtgacctt 420 taaacggtac tttcccatag ctgattaatg ggaaagtacc gttctcgagc caatacacgt 480 caatgggaag tgaaagggca gccaaaacgt aacaccgccc cggttttccc tggaaattcc 540 atattggcac gcattctatt ggctgagctg cgttcacgtg ggtataagag gcgcgaccag 600 cgtcggtacc gtcgcagtct tcggtctgac caccgtagaa cgcaga 646 <210> 4 <211> 470 <212> DNA <213> Artificial Sequence <220> <223> MCMV F1 promoter <400> 4 gggtgaatca acaggaaagt cccattggag ccaagtacac tgagtcaata gggactttcc 60 attgggtttt gcccagtaca aaaggtcaat agggggtgag tcaatgggtt tttcccatta 120 ttggcacgta cataaggtca ataggggtga gtcattgggt ttttccagcc aatttaatta 180 aaacgccatg tactttccca ccattgacgt caatgggcta ttgaaactaa tgcaacgtga 240 cctttaaacg gtactttccc atagctgatt aatgggaaag taccgttctc gagccaatac 300 acgtcaatgg gaagtgaaag ggcagccaaa acgtaacacc gccccggttt tccctggaaa 360 ttccatattg gcacgcattc tattggctga gctgcgttca cgtgggtata agaggcgcga 420 ccagcgtcgg taccgtcgca gtcttcggtc tgaccaccgt agaacgcaga 470 <210> 5 <211> 337 <212> DNA <213> Artificial Sequence <220> <223> MCMV F2 promoter <400> 5 aaggtcaata ggggtgagtc attgggtttt tccagccaat ttaattaaaa cgccatgtac 60 tttcccacca ttgacgtcaa tgggctattg aaactaatgc aacgtgacct ttaaacggta 120 ctttcccata gctgattaat gggaaagtac cgttctcgag ccaatacacg tcaatgggaa 180 gtgaaagggc agccaaaacg taacaccgcc ccggttttcc ctggaaattc catattggca 240 cgcattctat tggctgagct gcgttcacgt gggtataaga ggcgcgacca gcgtcggtac 300 cgtcgcagtc ttcggtctga ccaccgtaga acgcaga 337 <210> 6 <211> 237 <212> DNA <213> Artificial Sequence <220> <223> MCMV F3 promoter <400> 6 aacgtgacct ttaaacggta ctttcccata gctgattaat gggaaagtac cgttctcgag 60 ccaatacacg tcaatgggaa gtgaaagggc agccaaaacg taacaccgcc ccggttttcc 120 ctggaaattc catattggca cgcattctat tggctgagct gcgttcacgt gggtataaga 180 ggcgcgacca gcgtcggtac cgtcgcagtc ttcggtctga ccaccgtaga acgcaga 237 <210> 7 <211> 161 <212> DNA <213> Artificial Sequence <220> <223> MCMV F4 promoter <400> 7 ggaagtgaaa gggcagccaa aacgtaacac cgccccggtt ttccctggaa attccatatt 60 ggcacgcatt ctattggctg agctgcgttc acgtgggtat aagaggcgcg accagcgtcg 120 gtaccgtcgc agtcttcggt ctgaccaccg tagaacgcag a 161 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> MCMV F1-PmeI <400> 8 cggtttaaac gggtgaatca acaggaaagt ccc 33 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> MCMV F2-PmeI <400> 9 cggtttaaac aaggtcaata ggggtgagtc at 32 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> MCMV F3-PmeI <400> 10 cggtttaaac aacgtgacct ttaaacggta ct 32 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> MCMV F4-PmeI <400> 11 cggtttaaac ggaagtgaaa gggcagccaa a 31 <210> 12 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> MCMV-R-NotI <400> 12 cggcggccgc tctgcgttct acggtggtca gacc 34 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> MCMV R-BamHI <400> 13 cgggatcctc tgcgttctac ggtggtcaga cc 32 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GaLV 1932F <400> 14 gttgctcatc ctcgggccat g 21 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Am1801F <400> 15 atcattgacc ctggcccttc 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pol7130F <400> 16 cggcccggca ctcattggga g 21 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> MuLV4194F <400> 17 agcaagctat tggccactg 19 <210> 18 <211> 212 <212> DNA <213> Artificial Sequence <220> <223> sEF1alpha promoter <400> 18 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat 60 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 120 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 180 tttttcgcaa cgggtttgcc gccagaacac ag 212 <210> 19 <211> 2058 <212> DNA <213> Artificial Sequence <220> <223> GaLV env <400> 19 atggtattgc tgcctgggtc catgcttctc acctcaaacc tgcaccacct tcggcaccag 60 atgagtcctg ggagctggaa aagactgatc atcctcttaa gctgcgtatt cggcggcggc 120 gggacgagtc tgcaaaataa gaacccccac cagcccatga ccctcacttg gcaggtactg 180 tcccaaactg gagacgttgt ctgggataca aaggcagtcc agcccccttg gacttggtgg 240 cccacactta aacctgatgt atgtgccttg gcggctagtc ttgagtcctg ggatatcccg 300 ggaaccgatg tctcgtcctc taaacgagtc agacctccgg actcagacta tactgccgct 360 tataagcaaa tcacctgggg agccataggg tgcagctacc ctcgggctag gactagaatg 420 gcaagctcta ccttctacgt atgtccccgg gatggccgga ccctttcaga agctagaagg 480 tgcggggggc tagaatccct atactgtaaa gaatgggatt gtgagaccac ggggaccggt 540 tattggctat ctaaatcctc aaaagacctc ataactgtaa aatgggacca aaatagcgaa 600 tggactcaaa aatttcaaca gtgtcaccag accggctggt gtaaccccct taaaatagat 660 ttcacagaca aaggaaaatt atccaaggac tggataacgg gaaaaacctg gggattaaga 720 ttctatgtgt ctggacatcc aggcgtacag ttcaccattc gcttaaaaat caccaacatg 780 ccagctgtgg cagtaggtcc tgacctcgtc cttgtggaac aaggacctcc tagaacgtcc 840 ctcgctctcc cacctcctct tcccccaagg gaagcgccac cgccatctct ccccgactct 900 aactccacag ccctggcgac tagtgcacaa actcccacgg tgagaaaaac aattgttacc 960 ctaaacactc cgcctcccac cacaggcgac agactttttg atcttgtgca gggggccttc 1020 ctaaccttaa atgctaccaa cccaggggcc actgagtctt gctggctttg tttggccatg 1080 ggcccccctt attatgaagc aatagcctca tcaggagagg tcgcctactc caccgacctt 1140 gaccggtgcc gctgggggac ccaaggaaag ctcaccctca ctgaggtctc aggacacggg 1200 ttgtgcatag gaaaggtgcc ctttacccat cagcatctct gcaatcagac cctatccatc 1260 aattcctccg gagaccatca gtatctgctc ccctccaacc atagctggtg ggcttgcagc 1320 actggcctca ccccttgcct ctccacctca gtttttaatc agactagaga tttctgtatc 1380 caggtccagc tgattcctcg catctattac tatcctgaag aagttttgtt acaggcctat 1440 gacaattctc accccaggac taaaagagag gctgtctcac ttaccctagc tgttttactg 1500 gggttgggaa tcacggcggg aataggtact ggttcaactg ccttaattaa aggacctata 1560 gacctccagc aaggcctgac aagcctccag atcgccatag atgctgacct ccgggccctc 1620 caagactcag tcagcaagtt agaggactca ctgacttccc tgtccgaggt agtgctccaa 1680 aataggagag gccttgactt gctgtttcta aaagaaggtg gcctctgtgc ggccctaaag 1740 gaagagtgct gtttttacat agaccactca ggtgcagtac gggactccat gaaaaaactc 1800 aaagaaaaac tggataaaag acagttagag cgccagaaaa gccaaaactg gtatgaagga 1860 tggttcaata actccccttg gttcactacc ctgctatcaa ccatcgctgg gcccctatta 1920 ctcctccttc tgttgctcat cctcgggcca tgcatcatca ataagttagt tcaattcatc 1980 aatgatagga taagtgcagt taaaattctg gtccttagac aaaaatatca ggccctagag 2040 aacgaaggta acctttaa 2058 <210> 20 <211> 1965 <212> DNA <213> Artificial Sequence <220> <223> MuLV env <400> 20 atggcgcgtt caacgctctc aaaaccccct caagataaga ttaacccgtg gaagccctta 60 atagtcatgg gagtcctgtt aggagtaggg atggcagaga gcccccatca ggtctttaat 120 gtaacctgga gagtcaccaa cctgatgact gggcgtaccg ccaatgccac ctccctcctg 180 ggaactgtac aagatgcctt cccaaaatta tattttgatc tatgtgatct ggtcggagag 240 gagtgggacc cttcagacca ggaaccgtat gtcgggtatg gctgcaagta ccccgcaggg 300 agacagcgga cccggacttt tgacttttac gtgtgccctg ggcataccgt aaagtcgggg 360 tgtgggggac caggagaggg ctactgtggt aaatgggggt gtgaaaccac cggacaggct 420 tactggaagc ccacatcatc gtgggaccta atctccctta agcgcggtaa caccccctgg 480 gacacgggat gctctaaagt tgcctgtggc ccctgctacg acctctccaa agtatccaat 540 tccttccaag gggctactcg agggggcaga tgcaaccctc tagtcctaga attcactgat 600 gcaggaaaaa aggctaactg ggacgggccc aaatcgtggg gactgagact gtaccggaca 660 ggaacagatc ctattaccat gttctccctg acccggcagg tccttaatgt gggaccccga 720 gtccccatag ggcccaaccc agtattaccc gaccaaagac tcccttcctc accaatagag 780 attgtaccgg ctccacagcc acctagcccc ctcaatacca gttacccccc ttccactacc 840 agtacaccct caacctcccc tacaagtcca agtgtcccac agccaccccc aggaactgga 900 gatagactac tagctctagt caaaggagcc tatcaggcgc ttaacctcac caatcccgac 960 aagacccaag aatgttggct gtgcttagtg tcgggacctc cttattacga aggagtagcg 1020 gtcgtgggca cttataccaa tcattccacc gctccggcca actgtacggc cacttcccaa 1080 cataagctta ccctatctga agtgacagga cagggcctat gcatgggggc agtacctaaa 1140 actcaccagg ccttatgtaa caccacccaa agcgccggct caggatccta ctaccttgca 1200 gcacccgccg gaacaatgtg ggcttgcagc actggattga ctccctgctt gtccaccacg 1260 gtgctcaatc taaccacaga ttattgtgta ttagttgaac tctggcccag agtaatttac 1320 cactcccccg attatatgta tggtcagctt gaacagcgta ccaaatataa aagagagcca 1380 gtatcattga ccctggccct tctactagga ggattaacca tgggagggat tgcagctgga 1440 atagggacgg ggaccactgc cttaattaaa acccagcagt ttgagcagct tcatgccgct 1500 atccagacag acctcaacga agtcgaaaag tcaattacca acctagaaaa gtcactgacc 1560 tcgttgtctg aagtagtcct acagaaccgc agaggcctag atttgctatt cctaaaggag 1620 ggaggtctct gcgcagccct aaaagaagaa tgttgttttt atgcagacca cacggggcta 1680 gtgagagaca gcatggccaa attaagagaa aggcttaatc agagacaaaa actatttgag 1740 acaggccaag gatggttcga agggctgttt aatagatccc cctggtttac caccttaatc 1800 tccaccatca tgggacctct aatagtactc ttactgatct tactctttgg accttgcatt 1860 ctcaatcgat tagtccaatt tgttaaagac aggatatcag tggtccaggc tctagttttg 1920 actcaacaat atcaccagct gaagcctata gagtacgagc catag 1965 <210> 21 <211> 1131 <212> DNA <213> Artificial Sequence <220> <223> Thymidine kinase encoding sequence <400> 21 atggcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 60 ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 120 cgcctggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc tcacgggatg 180 gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 240 gtacccgagc cgatgactta ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc 300 tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360 atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 420 cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct caccctcatc 480 ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata ccttatgggc 540 agcatgacct cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 600 acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660 cagcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 720 ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggcggga ggattgggga 780 cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 840 cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggccccc 900 aacggcgacc tgtacaacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 960 cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 1020 ctgcaactta cctccgggat ggtccagacc cacgtcacca cccccggctc cataccgacg 1080 atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaactg a 1131 <210> 22 <211> 477 <212> DNA <213> Artificial Sequence <220> <223> Human-codon optimized yeast cytosine deaminase encoding sequence <400> 22 atggttactg ggggaatggc atctaagtgg gatcagaaag gtatggacat cgcttatgaa 60 gaggctgctc tcggctacaa agagggtgga gtgcctatcg gagggtgcct gatcaacaac 120 aaggacggca gtgtgctggg gaggggccac aatatgaggt tccaaaaagg ctccgccact 180 ctccacgggg aaattagtac cctcgagaat tgcggacgat tggaagggaa ggtgtacaag 240 gatacaacac tgtacaccac cctgtcaccc tgtgatatgt gcacaggcgc cattatcatg 300 tacggaatcc ctagatgtgt cgtgggggag aatgtaaact tcaaaagtaa gggggagaaa 360 tatctccaga cccgggggca cgaagtcgtc gttgtggacg atgaacggtg taagaagatc 420 atgaagcagt ttatcgatga gaggccccag gactggttcg aagacatcgg ggaataa 477 <210> 23 <211> 1617 <212> DNA <213> Artificial Sequence <220> <223> MuLV gag <400> 23 atgggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaa agatgtcgag 60 cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttgggttac cttctgctct 120 gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc 180 atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc agaccaggtc 240 ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt caagcccttt 300 gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc ccttgaacct 360 cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc tctaggcgcc 420 aaacctaaac ctcaagttct ttctgacagt ggggggccgc tcatcgacct acttacagaa 480 gaccccccgc cttataggga cccaagacca cccccttccg acagggacgg aaatggtgga 540 gaagcgaccc ctgcgggaga ggcaccggac ccctccccaa tggcatctcg cctacgtggg 600 agacgggagc cccctgtggc cgactccact acctcgcagg cattccccct ccgcgcagga 660 ggaaacggac agcttcaata ctggccgttc tcctcttctg acctttacaa ctggaaaaat 720 aataaccctt ctttttctga agatccaggt aaactgacag ctctgatcga gtctgttctc 780 atcacccatc agcccacctg ggacgactgt cagcagctgt tggggactct gctgaccgga 840 gaagaaaaac aacgggtgct cttagaggct agaaaggcgg tgcggggcga tgatgggcgc 900 cccactcaac tgcccaatga agtcgatgcc gcttttcccc tcgagcgccc agactgggat 960 tacaccaccc aggcaggtag gaaccaccta gtccactatc gccagttgct cctagcgggt 1020 ctccaaaacg cgggcagaag ccccaccaat ttggccaagg taaaaggaat aacacaaggg 1080 cccaatgagt ctccctcggc cttcctagag agacttaagg aagcctatcg caggtacact 1140 ccttatgacc ctgaggaccc agggcaagaa actaatgtgt ctatgtcttt catttggcag 1200 tctgccccag acattgggag aaagttagag aggttagaag atttaaaaaa caagacgctt 1260 ggagatttgg ttagagaggc agaaaagatc tttaataaac gagaaacccc ggaagaaaga 1320 gaggaacgta tcaggagaga aacagaggaa aaagaagaac gccgtaggac agaggatgag 1380 cagaaagaga aagaaagaga tcgtaggaga catagagaga tgagcaagct attggccact 1440 gtcgttagtg gacagaaaca ggatagacag ggaggagaac gaaggaggtc ccaactcgat 1500 cgcgaccagt gtgcctactg caaagaaaag gggcactggg ctaaagattg tcccaagaaa 1560 ccacgaggac ctcggggacc aagaccccag acctccctcc tgaccctaga tgactag 1617 <210> 24 <211> 3635 <212> DNA <213> Artificial Sequence <220> <223> MuLV pol <400> 24 ggaggtcagg gtcaggagcc cccccctgaa cccaggataa ccctcaaagt cggggggcaa 60 cccgtcacct tcctggtaga tactggggcc caacactccg tgctgaccca aaatcctgga 120 cccctaagtg ataagtctgc ctgggtccaa ggggctactg gaggaaagcg gtatcgctgg 180 accacggatc gcaaagtaca tctagctacc ggtaaggtca cccactcttt cctccatgta 240 ccagactgtc cctatcctct gttaggaaga gatttgctga ctaaactaaa agcccaaatc 300 cactttgagg gatcaggagc tcaggttatg ggaccaatgg ggcagcccct gcaagtgttg 360 accctaaata tagaagatga gcatcggcta catgagacct caaaagagcc agatgtttct 420 ctagggtcca catggctgtc tgattttcct caggcctggg cggaaaccgg gggcatggga 480 ctggcagttc gccaagctcc tctgatcata cctctgaaag caacctctac ccccgtgtcc 540 ataaaacaat accccatgtc acaagaagcc agactgggga tcaagcccca catacagaga 600 ctgttggacc agggaatact ggtaccctgc cagtccccct ggaacacgcc cctgctaccc 660 gttaagaaac cagggactaa tgattatagg cctgtccagg atctgagaga agtcaacaag 720 cgggtggaag acatccaccc caccgtgccc aacccttaca acctcttgag cgggctccca 780 ccgtcccacc agtggtacac tgtgcttgat ttaaaggatg cctttttctg cctgagactc 840 caccccacca gtcagcctct cttcgccttt gagtggagag atccagagat gggaatctca 900 ggacaattga cctggaccag actcccacag ggtttcaaaa acagtcccac cctgtttgat 960 gaggcactgc acagagacct agcagacttc cggatccagc acccagactt gatcctgcta 1020 cagtacgtgg atgacttact gctggccgcc acttctgagc tagactgcca acaaggtact 1080 cgggccctgt tacaaaccct agggaacctc gggtatcggg cctcggccaa gaaagcccaa 1140 atttgccaga aacaggtcaa gtatctgggg tatcttctaa aagagggtca gagatggctg 1200 actgaggcca gaaaagagac tgtgatgggg cagcctactc cgaagacccc tcgacaacta 1260 agggagttcc tagggacggc aggcttctgt cgcctctgga tccctgggtt tgcagaaatg 1320 gcagccccct tgtaccctct caccaaaacg gggactctgt ttaattgggg cccagaccaa 1380 caaaaggcct atcaagaaat caagcaagct cttctaactg ccccagccct ggggttgcca 1440 gatttgacta agccctttga actctttgtc gacgagaagc agggctacgc caaaggtgtc 1500 ctaacgcaaa aactgggacc ttggcgtcgg ccggtggcct acctgtccaa aaagctagac 1560 ccagtagcag ctgggtggcc cccttgccta cggatggtag cagccattgc cgtactgaca 1620 aaggatgcag gcaagctaac catgggacag ccactagtca ttctggcccc ccatgcagta 1680 gaggcactag tcaaacaacc ccccgaccgc tggctttcca acgcccggat gactcactat 1740 caggccttgc ttttggacac ggaccgggtc cagttcggac cggtggtagc cctgaacccg 1800 gctacgctgc tcccactgcc tgaggaaggg ctgcaacaca actgccttga tatcctggcc 1860 gaagcccacg gaacccgacc cgacctaacg gaccagccgc tcccagacgc cgaccacacc 1920 tggtacacgg atggaagcag tctcttacaa gagggacagc gtaaggcggg agctgcggtg 1980 accaccgaga ccgaggtaat ctgggctaaa gccctgccag ccgggacatc cgctcagcgg 2040 gctgaactga tagcactcac ccaggcccta aagatggcag aaggtaagaa gctaaatgtt 2100 tatactgata gccgttatgc ttttgctact gcccatatcc atggagaaat atacagaagg 2160 cgtgggttgc tcacatcaga aggcaaagag atcaaaaata aagacgagat cttggcccta 2220 ctaaaagccc tctttctgcc caaaagactt agcataatcc attgtccagg acatcaaaag 2280 ggacacagcg ccgaggctag aggcaaccgg atggctgacc aagcggcccg aaaggcagcc 2340 atcacagaga ctccagacac ctctaccctc ctcatagaaa attcatcacc ctacacctca 2400 gaacattttc attacacagt gactgatata aaggacctaa ccaagttggg ggccatttat 2460 gataaaacaa agaagtattg ggtctaccaa ggaaaacctg tgatgcctga ccagtttact 2520 tttgaattat tagactttct tcatcagctg actcacctca gcttctcaaa aatgaaggct 2580 ctcctagaga gaagccacag tccctactac atgctgaacc gggatcgaac actcaaaaat 2640 atcactgaga cctgcaaagc ttgtgcacaa gtcaacgcca gcaagtctgc cgttaaacag 2700 ggaactaggg tccgcgggca tcggcccggc actcattggg agatcgattt caccgagata 2760 aagcccggat tgtatggcta taaatatctt ctagttttta tagatacctt ttctggctgg 2820 atagaagcct tcccaaccaa gaaagaaacc gccaaggtcg taaccaagaa gctactagag 2880 gagatcttcc ccaggttcgg catgcctcag gtattgggaa ctgacaatgg gcctgccttc 2940 gtctccaagg tgagtcagac agtggccgat ctgttgggga ttgattggaa attacattgt 3000 gcatacagac cccaaagctc aggccaggta gaaagaatga atagaaccat caaggagact 3060 ttaactaaat taacgcttgc aactggctct agagactggg tgctcctact ccccttagcc 3120 ctgtaccgag cccgcaacac gccgggcccc catggcctca ccccatatga gatcttatat 3180 ggggcacccc cgccccttgt aaacttccct gaccctgaca tgacaagagt tactaacagc 3240 ccctctctcc aagctcactt acaggctctc tacttagtcc agcacgaagt ctggagacct 3300 ctggcggcag cctaccaaga acaactggac cgaccggtgg tacctcaccc ttaccgagtc 3360 ggcgacacag tgtgggtccg ccgacaccag actaagaacc tagaacctcg ctggaaagga 3420 ccttacacag tcctgctgac cacccccacc gccctcaaag tagacggcat cgcagcttgg 3480 atacacgccg cccacgtgaa ggctgccgac cccgggggtg gaccatcctc tagactgaca 3540 tggcgcgttc aacgctctca aaacccctta aaaataaggt taacccgcga ggccccctaa 3600 tccccttaat tcttctgatg ctcagagggg tcagt 3635 <210> 25 <211> 9694 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-yCD env vector <400> 25 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacaattc 1800 gatcatacct ggtgttgctg actaccccga ccgcggtaaa agtcgatggt attgctgcct 1860 gggtccatgc ttctcacctc aaacctgcac caccttcggc accagatgag tcctgggagc 1920 tggaaaagac tgatcatcct cttaagctgc gtattcggcg gcggcgggac gagtctgcaa 1980 aataagaacc cccaccagcc catgaccctc acttggcagg tactgtccca aactggagac 2040 gttgtctggg atacaaaggc agtccagccc ccttggactt ggtggcccac acttaaacct 2100 gatgtatgtg ccttggcggc tagtcttgag tcctgggata tcccgggaac cgatgtctcg 2160 tcctctaaac gagtcagacc tccggactca gactatactg ccgcttataa gcaaatcacc 2220 tggggagcca tagggtgcag ctaccctcgg gctaggacta gaatggcaag ctctaccttc 2280 tacgtatgtc cccgggatgg ccggaccctt tcagaagcta gaaggtgcgg ggggctagaa 2340 tccctatact gtaaagaatg ggattgtgag accacgggga ccggttattg gctatctaaa 2400 tcctcaaaag acctcataac tgtaaaatgg gaccaaaata gcgaatggac tcaaaaattt 2460 caacagtgtc accagaccgg ctggtgtaac ccccttaaaa tagatttcac agacaaagga 2520 aaattatcca aggactggat aacgggaaaa acctggggat taagattcta tgtgtctgga 2580 catccaggcg tacagttcac cattcgctta aaaatcacca acatgccagc tgtggcagta 2640 ggtcctgacc tcgtccttgt ggaacaagga cctcctagaa cgtccctcgc tctcccacct 2700 cctcttcccc caagggaagc gccaccgcca tctctccccg actctaactc cacagccctg 2760 gcgactagtg cacaaactcc cacggtgaga aaaacaattg ttaccctaaa cactccgcct 2820 cccaccacag gcgacagact ttttgatctt gtgcaggggg ccttcctaac cttaaatgct 2880 accaacccag gggccactga gtcttgctgg ctttgtttgg ccatgggccc cccttattat 2940 gaagcaatag cctcatcagg agaggtcgcc tactccaccg accttgaccg gtgccgctgg 3000 gggacccaag gaaagctcac cctcactgag gtctcaggac acgggttgtg cataggaaag 3060 gtgcccttta cccatcagca tctctgcaat cagaccctat ccatcaattc ctccggagac 3120 catcagtatc tgctcccctc caaccatagc tggtgggctt gcagcactgg cctcacccct 3180 tgcctctcca cctcagtttt taatcagact agagatttct gtatccaggt ccagctgatt 3240 cctcgcatct attactatcc tgaagaagtt ttgttacagg cctatgacaa ttctcacccc 3300 aggactaaaa gagaggctgt ctcacttacc ctagctgttt tactggggtt gggaatcacg 3360 gcgggaatag gtactggttc aactgcctta attaaaggac ctatagacct ccagcaaggc 3420 ctgacaagcc tccagatcgc catagatgct gacctccggg ccctccaaga ctcagtcagc 3480 aagttagagg actcactgac ttccctgtcc gaggtagtgc tccaaaatag gagaggcctt 3540 gacttgctgt ttctaaaaga aggtggcctc tgtgcggccc taaaggaaga gtgctgtttt 3600 tacatagacc actcaggtgc agtacgggac tccatgaaaa aactcaaaga aaaactggat 3660 aaaagacagt tagagcgcca gaaaagccaa aactggtatg aaggatggtt caataactcc 3720 ccttggttca ctaccctgct atcaaccatc gctgggcccc tattactcct ccttctgttg 3780 ctcatcctcg ggccatgcat catcaataag ttagttcaat tcatcaatga taggataagt 3840 gcagttaaaa ttctggtcct tagacaaaaa tatcaggccc tagagaacga aggtaacctt 3900 taattttgct ctaagattag agctattcac aagagaaatg gggaatcact agtgaattaa 3960 accgataagc gtttttttca ataacaggaa agtcccattg gagccaagta cattgagtca 4020 atagggactt tccaatgggt tttgcccagt acataaggtc aatgggaggt aagccaatgg 4080 gtttttccca ttactggcac gtatactgag tcattaggga ctttccaatg ggttttgccc 4140 agtacataag gtcaataggg gtgaatcaac aggaaagtcc cattggagcc aagtacactg 4200 agtcaatagg gactttccat tgggttttgc ccagtacaaa aggtcaatag ggggtgagtc 4260 aatgggtttt tcccattatt ggcacgtaca taaggtcaat aggggtgagt cattgggttt 4320 ttccagccaa tttaattaaa acgccatgta ctttcccacc attgacgtca atgggctatt 4380 gaaactaatg caacgtgacc tttaaacggt actttcccat agctgattaa tgggaaagta 4440 ccgttctcga gccaatacac gtcaatggga agtgaaaggg cagccaaaac gtaacaccgc 4500 cccggttttc cctggaaatt ccatattggc acgcattcta ttggctgagc tgcgttctac 4560 gtgggtataa gaggcgcgac cagcgtcggt accgtcgcag tcttcggtct gaccaccgta 4620 gaacgcagaa ctctagaact agtcgggatc cgcgttaaca tttgcggccg ctttagttta 4680 gcttatggtg acagggggaa tggcaagcaa gtgggatcag aagggtatgg acattgccta 4740 tgaggaggcg gccttaggtt acaaagaggg tggtgttcct attggcggat gtcttatcaa 4800 taacaaagac ggaagtgttc tcggtcgtgg tcacaacatg agatttcaaa agggatccgc 4860 cacactacat ggtgagatct ccactttgga aaactgtggg agattagagg gcaaagtgta 4920 caaagatacc actttgtata cgacgctgtc tccatgcgac atgtgtacag gtgccatcat 4980 catgtatggt attccacgct gtgttgtcgg tgagaacgtt aatttcaaaa gtaagggcga 5040 gaaatattta caaactagag gtcacgaggt tgttgttgtt gacgatgaga ggtgtaaaaa 5100 gatcatgaaa caatttatcg atgaaagacc tcaggattgg tttgaagata ttggtgagta 5160 gctcgaaaac gcgtcgaccc atcgatggta ccagatccgg attagtccaa tttgttaaag 5220 acaggatatc agtggtccag gctctagttt tgactcaaca atatcaccag ctgaagccta 5280 tagagtacga gccatagata aaataaaaga ttttatttag tctccagaaa aaggggggaa 5340 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 5400 ggaaaaatac ataactgaga atagagaagt tcagatcaag gtcaggaaca gatggaacag 5460 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa 5520 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc 5580 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag 5640 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg 5700 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat 5760 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg 5820 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct 5880 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca cacatgcagc 5940 atgtatcaaa attaatttgg ttttttttct taagtattta cattaaatgg ccatagtact 6000 taaagttaca ttggcttcct tgaaataaac atggagtatt cagaatgtgt cataaatatt 6060 tctaatttta agatagtatc tccattggct ttctactttt tcttttattt ttttttgtcc 6120 tctgtcttcc atttgttgtt gttgttgttt gtttgtttgt ttgttggttg gttggttaat 6180 ttttttttaa agatcctaca ctatagttca agctagacta ttagctactc tgtaacccag 6240 ggtgaccttg aagtcatggg tagcctgctg ttttagcctt cccacatcta agattacagg 6300 tatgagctat catttttggt atatgattga ttgattgatt gatgtgtgtg tgtgtgattg 6360 tgtttgtgtg tgtgactgtg aaaatgtgtg tatgggtgtg tgtgaatgtg tgtatgtatg 6420 tgtgtgtgtg agtgtgtgtg tgtgtgtgtg catgtgtgtg tgtgtgactg tgtctatgtg 6480 tatgactgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgttgtgaaa 6540 aaatattcta tggtagtgag agccaacgct ccggctcagg tgtcaggttg gtttttgaga 6600 cagagtcttt cacttagctt ggaattcact ggccgtcgtt ttacaacgtc gtgactggga 6660 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 6720 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 6780 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg 6840 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc 6900 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 6960 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 7020 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt 7080 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 7140 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 7200 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 7260 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 7320 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 7380 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 7440 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 7500 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 7560 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 7620 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 7680 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 7740 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 7800 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 7860 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 7920 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 7980 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 8040 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 8100 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 8160 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 8220 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 8280 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 8340 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 8400 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 8460 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 8520 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 8580 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 8640 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 8700 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 8760 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 8820 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 8880 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 8940 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 9000 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 9060 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg 9120 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct 9180 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta 9240 tgaccatgat tacgccaagc tttgctccta ggagtttcct aatacttccc aaactcaaat 9300 atataaagca tttgacttgt tctatgccct agggggcggg gggaagctaa gccagctttt 9360 tttaacattt aaaatgttaa ttccatttta aatgcacaga tgtttttatt tcataagggt 9420 ttcaatgtgc atgaatgctg caatattcct gttaccaaag ctagtataaa taaaaataga 9480 taaacgtgga aattacttag agtttctgtc attaacgttt ccttcctcag ttgacaacat 9540 aaatgcgctg ctgagcaagc cagtttgcat ctgtcaggat caatttccca ttatgccagt 9600 catattaatt actagtcaat tagttgattt ttatttttga catatacatg tgaatgaaag 9660 accccacctg taggtttggc aagctagctt aagt 9694 <210> 26 <211> 11162 <212> DNA <213> Artificial Sequence <220> <223> sRRVgp-TK:gag-pol vector <400> 26 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 60 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 480 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 600 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 660 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 720 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 780 atcaatctaa agtatatatg agtaacctga tcaggactct tccttttcat gaacaataaa 840 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 900 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 960 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 1020 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1080 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 1140 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 1200 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 1260 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 1320 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 1380 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 1440 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 1500 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 1560 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 1620 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 1680 tgagtttttc taagaatttg tgaatgaaag accccacctg taggtttggc aagctagctt 1740 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat 1800 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1860 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1920 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1980 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2040 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2100 gcgcttatgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2160 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2220 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2280 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2340 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt 2400 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg 2460 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc 2520 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg 2580 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc 2640 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg 2700 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt 2760 tgtctgagaa tatgggccag actgttacca ctcccttaag tttgacctta ggtcactgga 2820 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 2880 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 2940 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 3000 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 3060 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 3120 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 3180 ctctaggcgc caaacctaaa cctcaagttc tttctgacag tggggggccg ctcatcgacc 3240 tacttacaga agaccccccg ccttataggg acccaagacc acccccttcc gacagggacg 3300 gaaatggtgg agaagcgacc cctgcgggag aggcaccgga cccctcccca atggcatctc 3360 gcctacgtgg gagacgggag ccccctgtgg ccgactccac tacctcgcag gcattccccc 3420 tccgcgcagg aggaaacgga cagcttcaat actggccgtt ctcctcttct gacctttaca 3480 actggaaaaa taataaccct tctttttctg aagatccagg taaactgaca gctctgatcg 3540 agtctgttct catcacccat cagcccacct gggacgactg tcagcagctg ttggggactc 3600 tgctgaccgg agaagaaaaa caacgggtgc tcttagaggc tagaaaggcg gtgcggggcg 3660 atgatgggcg ccccactcaa ctgcccaatg aagtcgatgc cgcttttccc ctcgagcgcc 3720 cagactggga ttacaccacc caggcaggta ggaaccacct agtccactat cgccagttgc 3780 tcctagcggg tctccaaaac gcgggcagaa gccccaccaa tttggccaag gtaaaaggaa 3840 taacacaagg gcccaatgag tctccctcgg ccttcctaga gagacttaag gaagcctatc 3900 gcaggtacac tccttatgac cctgaggacc cagggcaaga aactaatgtg tctatgtctt 3960 tcatttggca gtctgcccca gacattggga gaaagttaga gaggttagaa gatttaaaaa 4020 acaagacgct tggagatttg gttagagagg cagaaaagat ctttaataaa cgagaaaccc 4080 cggaagaaag agaggaacgt atcaggagag aaacagagga aaaagaagaa cgccgtagga 4140 cagaggatga gcagaaagag aaagaaagag atcgtaggag acatagagag atgagcaagc 4200 tattggccac tgtcgttagt ggacagaaac aggatagaca gggaggagaa cgaaggaggt 4260 cccaactcga tcgcgaccag tgtgcctact gcaaagaaaa ggggcactgg gctaaagatt 4320 gtcccaagaa accacgagga cctcggggac caagacccca gacctccctc ctgaccctag 4380 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 4440 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 4500 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 4560 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 4620 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 4680 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 4740 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 4800 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 4860 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 4920 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 4980 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 5040 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 5100 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 5160 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 5220 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 5280 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 5340 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 5400 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 5460 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 5520 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 5580 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 5640 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 5700 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 5760 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 5820 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 5880 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 5940 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 6000 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 6060 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 6120 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 6180 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 6240 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 6300 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 6360 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 6420 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 6480 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 6540 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 6600 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 6660 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 6720 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 6780 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 6840 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 6900 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 6960 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 7020 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 7080 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 7140 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 7200 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 7260 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 7320 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 7380 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 7440 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 7500 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 7560 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 7620 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 7680 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 7740 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 7800 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 7860 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 7920 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 7980 ccccctaatc cccttaattc ttctgatgct cagaggggtc agtaaacgaa ttcaacagga 8040 aagttccatt ggagccaagt acattgagtc aatagggact ttccaatggg ttttgcccag 8100 tacataaggt caatgggagg taagccaatg ggtttttccc attactggca cgtatactga 8160 gtcattaggg actttccaat gggttttgcc cagtacataa ggtcaatagg ggtgaatcaa 8220 caggaaagtc ccattggagc caagtacact gagtcaatag ggactttcca ttgggttttg 8280 cccagtacaa aaggtcaata gggggtgagt caatgggttt ttcccattat tggcacgtac 8340 ataaggtcaa taggggtgag tcattgggtt tttccagcca atttaattaa aacgccatgt 8400 actttcccac cattgacgtc aatgggctat tgaaactaat gcaacgtgac ctttaaacgg 8460 tactttccca tagctgatta atgggaaagt accgttctcg agccaataca cgtcaatggg 8520 aagtgaaagg gcagccaaaa cgtaacaccg ccccggtttt ccctggaaat tccatattgg 8580 cacgcattct attggctgag ctgcgttcta cgtgggtata agaggcgcga ccagcgtcgg 8640 taccgtcgca gtcttcggtc tgaccaccgt agaacgcaga gtttgatccg cgttaacatt 8700 tgcggccgct ttagtttcat ggcttcgtac ccctgccatc aacacgcgtc tgcgttcgac 8760 caggctgcgc gttctcgcgg ccatagcaac cgacgtacgg cgttgcgccc tcgccggcag 8820 caagaagcca cggaagtccg cctggagcag aaaatgccca cgctactgcg ggtttatata 8880 gacggtcctc acgggatggg gaaaaccacc accacgcaac tgctggtggc cctgggttcg 8940 cgcgacgata tcgtctacgt acccgagccg atgacttact ggcaggtgct gggggcttcc 9000 gagacaatcg cgaacatcta caccacacaa caccgcctcg accagggtga gatatcggcc 9060 ggggacgcgg cggtggtaat gacaagcgcc cagataacaa tgggcatgcc ttatgccgtg 9120 accgacgccg ttctggctcc tcatatcggg ggggaggctg ggagctcaca tgccccgccc 9180 ccggccctca ccctcatctt cgaccgccat cccatcgccg ccctcctgtg ctacccggcc 9240 gcgcgatacc ttatgggcag catgaccccc caggccgtgc tggcgttcgt ggccctcatc 9300 ccgccgacct tgcccggcac aaacatcgtg ttgggggccc ttccggagga cagacacatc 9360 gaccgcctgg ccaaacgcca gcgccccggc gagcggcttg acctggctat gctggccgcg 9420 attcgccgcg tttacgggct gcttgccaat acggtgcggt atctgcaggg cggcgggtcg 9480 tggcgggagg attggggaca gctttcgggg acggccgtgc cgccccaggg tgccgagccc 9540 cagagcaacg cgggcccacg accccatatc ggggacacgt tatttaccct gtttcgggcc 9600 cccgagttgc tggcccccaa cggcgacctg tacaacgtgt ttgcctgggc cttggacgtc 9660 ttggccaaac gcctccgtcc catgcacgtc tttatcctgg attacgacca atcgcccgcc 9720 ggctgccggg acgccctgct gcaacttacc tccgggatgg tccagaccca cgtcaccacc 9780 cccggctcca taccgacgat ctgcgacctg gcgcgcacgt ttgcccggga gatgggggag 9840 gctaactgaa acacggaagg ggtaccgaat tcctcgaaaa cgcgtcgaaa acgaattcat 9900 aaaataaaag attttatttt gtctccagaa aaagggggga atgaaagacc ccacctgtag 9960 gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata cataactgag 10020 aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg ggccaaacag 10080 gatagctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg gaacagctga 10140 atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac 10200 agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc agatgtttcc 10260 agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc 10320 ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct 10380 cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt atccaataaa 10440 ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag 10500 tgattgacta cccgtcagcg ggggtctttc attgttactt aaagttacat tggcttcctt 10560 gaaataaaca tggagtattc agaatgtgtc ataaatattt ctaattttaa gatagtatct 10620 ccattggctt tctacttttt cttttatttt tttttgtcct ctgtcttcca tttgttgttg 10680 ttgttgtttg tttgtttgtt tgttggttgg ttggttaatt tttttttaaa gatcctacac 10740 tatagttcaa gctagactat tagctactct gtaacccagg gtgaccttga agtcatgggt 10800 agcctgctgt tttagccttc ccacatctaa gattacaggt atgagctatc atttttggta 10860 tattgattga ttgattgatt gatgtgtgtg tgtgtgattg tgtttgtgtg tgtgactgtg 10920 aaaatgtgtg tatgggtgtg tgtgaatgtg tgtatgtatg tgtgtgtgtg agtgtgtgtg 10980 tgtgtgtgtg catgtgtgtg tgtgtgactg tgtctatgtg tatgactgtg tgtgtgtgtg 11040 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgttgtgaaa aaatattcta tggtagtgag 11100 agccaacgct ccggctcagg tgtcaggttg gtttttgaga cagagtcttt cacttagctt 11160 gg 11162 <210> 27 <211> 10383 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-TK vector <400> 27 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacaattc 1800 gatcatacct ggtgttgctg actaccccga ccgcggtaaa agtcgatggt attgctgcct 1860 gggtccatgc ttctcacctc aaacctgcac caccttcggc accagatgag tcctgggagc 1920 tggaaaagac tgatcatcct cttaagctgc gtattcggcg gcggcgggac gagtctgcaa 1980 aataagaacc cccaccagcc catgaccctc acttggcagg tactgtccca aactggagac 2040 gttgtctggg atacaaaggc agtccagccc ccttggactt ggtggcccac acttaaacct 2100 gatgtatgtg ccttggcggc tagtcttgag tcctgggata tcccgggaac cgatgtctcg 2160 tcctctaaac gagtcagacc tccggactca gactatactg ccgcttataa gcaaatcacc 2220 tggggagcca tagggtgcag ctaccctcgg gctaggacta gaatggcaag ctctaccttc 2280 tacgtatgtc cccgggatgg ccggaccctt tcagaagcta gaaggtgcgg ggggctagaa 2340 tccctatact gtaaagaatg ggattgtgag accacgggga ccggttattg gctatctaaa 2400 tcctcaaaag acctcataac tgtaaaatgg gaccaaaata gcgaatggac tcaaaaattt 2460 caacagtgtc accagaccgg ctggtgtaac ccccttaaaa tagatttcac agacaaagga 2520 aaattatcca aggactggat aacgggaaaa acctggggat taagattcta tgtgtctgga 2580 catccaggcg tacagttcac cattcgctta aaaatcacca acatgccagc tgtggcagta 2640 ggtcctgacc tcgtccttgt ggaacaagga cctcctagaa cgtccctcgc tctcccacct 2700 cctcttcccc caagggaagc gccaccgcca tctctccccg actctaactc cacagccctg 2760 gcgactagtg cacaaactcc cacggtgaga aaaacaattg ttaccctaaa cactccgcct 2820 cccaccacag gcgacagact ttttgatctt gtgcaggggg ccttcctaac cttaaatgct 2880 accaacccag gggccactga gtcttgctgg ctttgtttgg ccatgggccc cccttattat 2940 gaagcaatag cctcatcagg agaggtcgcc tactccaccg accttgaccg gtgccgctgg 3000 gggacccaag gaaagctcac cctcactgag gtctcaggac acgggttgtg cataggaaag 3060 gtgcccttta cccatcagca tctctgcaat cagaccctat ccatcaattc ctccggagac 3120 catcagtatc tgctcccctc caaccatagc tggtgggctt gcagcactgg cctcacccct 3180 tgcctctcca cctcagtttt taatcagact agagatttct gtatccaggt ccagctgatt 3240 cctcgcatct attactatcc tgaagaagtt ttgttacagg cctatgacaa ttctcacccc 3300 aggactaaaa gagaggctgt ctcacttacc ctagctgttt tactggggtt gggaatcacg 3360 gcgggaatag gtactggttc aactgcctta attaaaggac ctatagacct ccagcaaggc 3420 ctgacaagcc tccagatcgc catagatgct gacctccggg ccctccaaga ctcagtcagc 3480 aagttagagg actcactgac ttccctgtcc gaggtagtgc tccaaaatag gagaggcctt 3540 gacttgctgt ttctaaaaga aggtggcctc tgtgcggccc taaaggaaga gtgctgtttt 3600 tacatagacc actcaggtgc agtacgggac tccatgaaaa aactcaaaga aaaactggat 3660 aaaagacagt tagagcgcca gaaaagccaa aactggtatg aaggatggtt caataactcc 3720 ccttggttca ctaccctgct atcaaccatc gctgggcccc tattactcct ccttctgttg 3780 ctcatcctcg ggccatgcat catcaataag ttagttcaat tcatcaatga taggataagt 3840 gcagttaaaa ttctggtcct tagacaaaaa tatcaggccc tagagaacga aggtaacctt 3900 taattttgct ctaagattag agctattcac aagagaaatg gggaatcact agtgaattaa 3960 accgataagc gtttttttca ataacaggaa agtcccattg gagccaagta cattgagtca 4020 atagggactt tccaatgggt tttgcccagt acataaggtc aatgggaggt aagccaatgg 4080 gtttttccca ttactggcac gtatactgag tcattaggga ctttccaatg ggttttgccc 4140 agtacataag gtcaataggg gtgaatcaac aggaaagtcc cattggagcc aagtacactg 4200 agtcaatagg gactttccat tgggttttgc ccagtacaaa aggtcaatag ggggtgagtc 4260 aatgggtttt tcccattatt ggcacgtaca taaggtcaat aggggtgagt cattgggttt 4320 ttccagccaa tttaattaaa acgccatgta ctttcccacc attgacgtca atgggctatt 4380 gaaactaatg caacgtgacc tttaaacggt actttcccat agctgattaa tgggaaagta 4440 ccgttctcga gccaatacac gtcaatgggg aagtgaaagg gcagccaaac gtaacacgcc 4500 ccggtttccc ctggaaattc atattggcac gcattctatt ggctgagctg cgttctacgt 4560 gggtataaga ggcgcgacca gcgtcggtac cgtcgcagtc ttcggtctga ccaccgtaga 4620 acgcagaact ctagaactag tggatccgcg ttaacatttg cggccgcttt agtttcatgg 4680 cttcgtaccc ctgccatcaa cacgcgtctg cgttcgacca ggctgcgcgt tctcgcggcc 4740 atagcaaccg acgtacggcg ttgcgccctc gccggcagca agaagccacg gaagtccgcc 4800 tggagcagaa aatgcccacg ctactgcggg tttatataga cggtcctcac gggatgggga 4860 aaaccaccac cacgcaactg ctggtggccc tgggttcgcg cgacgatatc gtctacgtac 4920 ccgagccgat gacttactgg caggtgctgg gggcttccga gacaatcgcg aacatctaca 4980 ccacacaaca ccgcctcgac cagggtgaga tatcggccgg ggacgcggcg gtggtaatga 5040 caagcgccca gataacaatg ggcatgcctt atgccgtgac cgacgccgtt ctggctcctc 5100 atatcggggg ggaggctggg agctcacatg ccccgccccc ggccctcacc ctcatcttcg 5160 accgccatcc catcgccgcc ctcctgtgct acccggccgc gcgatacctt atgggcagca 5220 tgacccccca ggccgtgctg gcgttcgtgg ccctcatccc gccgaccttg cccggcacaa 5280 acatcgtgtt gggggccctt ccggaggaca gacacatcga ccgcctggcc aaacgccagc 5340 gccccggcga gcggcttgac ctggctatgc tggccgcgat tcgccgcgtt tacgggctgc 5400 ttgccattta cgggctgctt gccaatacgg tgcggtatct gcagggcggc gggtcgtggc 5460 gggaggattg gggacagctt tcggggacgg ccgtgccgcc ccagggtgcc gagccccaga 5520 gcaacgcggg cccacgaccc catatcgggg acacgttatt taccctgttt cgggcccccg 5580 agttgctggc ccccaacggc gacctgtaca acgtgtttgc ctgggccttg gacgtcttgg 5640 ccaaacgcct ccgtcccatg cacgtcttta tcctggatta cgaccaatcg cccgccggct 5700 gccgggacgc cctgctgcaa cttacctccg ggatggtcca gacccacgtc accacccccg 5760 gctccatacc gacgatctgc gacctggcgc gcacgtttgc ccgggagatg ggggaggcta 5820 actgaaacac ggaaggggta ccgaattcct cgaaaacgcg tcgacccatc gatggtacca 5880 gatccggatt agtccaattt gttaaagaca ggatatcagt ggtccaggct ctagttttga 5940 ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa taaaagattt 6000 tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag 6060 cttaagtaac gccattttgc aaggcatgga aaaatacata actgagaata ganaagttca 6120 gatcaaggtc aggaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa 6180 gcagttcctg ccccggctca gggccaagaa cagatggaac agctgaatat gggccaaaca 6240 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga 6300 tgcggtccag ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg 6360 acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt 6420 cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact cggggcgcca 6480 gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg 6540 catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg 6600 tcagcggggg tctttcacac atgcagcatg tatcaaaatt aatttggttt tttttcttaa 6660 gtatttacat taaatggcca tagtacttaa agttacattg gcttccttga aataaacatg 6720 gagtattcag aatgtgtcat aaatatttct aattttaaga tagtatctcc attggctttc 6780 tactttttct tttatttttt tttgtcctct gtcttccatt tgttgttgtt gttgtttgtt 6840 tgtttgtttg ttggttggtt ggttaatttt tttttaaaga tcctacacta tagttcaagc 6900 tagactatta gctactctgt aacccagggt gaccttgaag tcatgggtag cctgctgttt 6960 tagccttccc acatctaaga ttacaggtat gagctatcat ttttggtata tgattgattg 7020 attgattgat gtgtgtgtgt gtgattgtgt ttgtgtgtgt gactgtgaaa atgtgtgtat 7080 gggtgtgtgt gaatgtgtgt atgtatgtgt gtgtgtgagt gtgtgtgtgt gtgtgtgcat 7140 gtgtgtgtgt gtgactgtgt ctatgtgtat gactgtgtgt gtgtgtgtgt gtgtgtgtgt 7200 gtgtgtgtgt gtgtgtgtgt tgtgaaaaaa tattctatgg tagtgagagc caacgctccg 7260 gctcaggtgt caggttggtt tttgagacag agtctttcac ttagcttgga attccractg 7320 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 7380 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 7440 tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg 7500 catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc 7560 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 7620 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 7680 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 7740 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 7800 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 7860 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 7920 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 7980 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 8040 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 8100 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 8160 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 8220 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 8280 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 8340 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 8400 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 8460 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 8520 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 8580 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 8640 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 8700 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 8760 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 8820 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 8880 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 8940 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 9000 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 9060 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac 9120 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 9180 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 9240 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 9300 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 9360 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 9420 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 9480 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 9540 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 9600 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 9660 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca 9720 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 9780 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat 9840 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 9900 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct ttgctcctag 9960 gagtttccta atacttccca aactcaaata tataaagcat ttgacttgtt ctatgcccta 10020 gggggcgggg ggaagctaag ccagcttttt ttaacattta aaatgttaat tccattttaa 10080 atgcacagat gtttttattt cataagggtt tcaatgtgca tgaatgctgc aatattcctg 10140 ttaccaaagc tagtataaat aaaaatagat aaacgtggaa attacttaga gtttctgtca 10200 ttaacgtttc cttcctcagt tgacaacata aatgcgctgc tgagcaagcc agtttgcatc 10260 tgtcaggatc aatttcccat tatgccagtc atattaatta ctagtcaatt agttgatttt 10320 tatttttgac atatacatgt gaatgaaaga ccccacctgt aggtttggca agctagctta 10380 agt 10383 <210> 28 <211> 9993 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-F1-TK vector <400> 28 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacaattc 1800 gatcatacct ggtgttgctg actaccccga ccgcggtaaa agtcgatggt attgctgcct 1860 gggtccatgc ttctcacctc aaacctgcac caccttcggc accagatgag tcctgggagc 1920 tggaaaagac tgatcatcct cttaagctgc gtattcggcg gcggcgggac gagtctgcaa 1980 aataagaacc cccaccagcc catgaccctc acttggcagg tactgtccca aactggagac 2040 gttgtctggg atacaaaggc agtccagccc ccttggactt ggtggcccac acttaaacct 2100 gatgtatgtg ccttggcggc tagtcttgag tcctgggata tcccgggaac cgatgtctcg 2160 tcctctaaac gagtcagacc tccggactca gactatactg ccgcttataa gcaaatcacc 2220 tggggagcca tagggtgcag ctaccctcgg gctaggacta gaatggcaag ctctaccttc 2280 tacgtatgtc cccgggatgg ccggaccctt tcagaagcta gaaggtgcgg ggggctagaa 2340 tccctatact gtaaagaatg ggattgtgag accacgggga ccggttattg gctatctaaa 2400 tcctcaaaag acctcataac tgtaaaatgg gaccaaaata gcgaatggac tcaaaaattt 2460 caacagtgtc accagaccgg ctggtgtaac ccccttaaaa tagatttcac agacaaagga 2520 aaattatcca aggactggat aacgggaaaa acctggggat taagattcta tgtgtctgga 2580 catccaggcg tacagttcac cattcgctta aaaatcacca acatgccagc tgtggcagta 2640 ggtcctgacc tcgtccttgt ggaacaagga cctcctagaa cgtccctcgc tctcccacct 2700 cctcttcccc caagggaagc gccaccgcca tctctccccg actctaactc cacagccctg 2760 gcgactagtg cacaaactcc cacggtgaga aaaacaattg ttaccctaaa cactccgcct 2820 cccaccacag gcgacagact ttttgatctt gtgcaggggg ccttcctaac cttaaatgct 2880 accaacccag gggccactga gtcttgctgg ctttgtttgg ccatgggccc cccttattat 2940 gaagcaatag cctcatcagg agaggtcgcc tactccaccg accttgaccg gtgccgctgg 3000 gggacccaag gaaagctcac cctcactgag gtctcaggac acgggttgtg cataggaaag 3060 gtgcccttta cccatcagca tctctgcaat cagaccctat ccatcaattc ctccggagac 3120 catcagtatc tgctcccctc caaccatagc tggtgggctt gcagcactgg cctcacccct 3180 tgcctctcca cctcagtttt taatcagact agagatttct gtatccaggt ccagctgatt 3240 cctcgcatct attactatcc tgaagaagtt ttgttacagg cctatgacaa ttctcacccc 3300 aggactaaaa gagaggctgt ctcacttacc ctagctgttt tactggggtt gggaatcacg 3360 gcgggaatag gtactggttc aactgcctta attaaaggac ctatagacct ccagcaaggc 3420 ctgacaagcc tccagatcgc catagatgct gacctccggg ccctccaaga ctcagtcagc 3480 aagttagagg actcactgac ttccctgtcc gaggtagtgc tccaaaatag gagaggcctt 3540 gacttgctgt ttctaaaaga aggtggcctc tgtgcggccc taaaggaaga gtgctgtttt 3600 tacatagacc actcaggtgc agtacgggac tccatgaaaa aactcaaaga aaaactggat 3660 aaaagacagt tagagcgcca gaaaagccaa aactggtatg aaggatggtt caataactcc 3720 ccttggttca ctaccctgct atcaaccatc gctgggcccc tattactcct ccttctgttg 3780 ctcatcctcg ggccatgcat catcaataag ttagttcaat tcatcaatga taggataagt 3840 gcagttaaaa ttctggtcct tagacaaaaa tatcaggccc tagagaacga aggtaacctt 3900 taacacgtga aggctgccga ccccgggggt ggaccatcct ctagactgtg ctcgacgttt 3960 aaacgggtga atcaacagga aagtcccatt ggagccaagt acactgagtc aatagggact 4020 ttccattggg ttttgcccag tacaaaaggt caataggggg tgagtcaatg ggtttttccc 4080 attattggca cgtacataag gtcaataggg gtgagtcatt gggtttttcc agccaattta 4140 attaaaacgc catgtacttt cccaccattg acgtcaatgg gctattgaaa ctaatgcaac 4200 gtgaccttta aacggtactt tcccatagct gattaatggg aaagtaccgt tctcgagcca 4260 atacacgtca atgggaagtg aaagggcagc caaaacgtaa caccgccccg gttttccctg 4320 gaaattccat attggcacgc attctattgg ctgagctgcg ttcacgtggg tataagaggc 4380 gcgaccagcg tcggtaccgt cgcagtcttc ggtctgacca ccgtagaacg cagaacgcgt 4440 atggcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 4500 ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 4560 cgcctggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc tcacgggatg 4620 gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 4680 gtacccgagc cgatgactta ctggcaggtg ctgggggctt ccgagacaat cgcgaacatc 4740 tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 4800 atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 4860 cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct caccctcatc 4920 ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcgata ccttatgggc 4980 agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 5040 acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 5100 cagcgccccg gcgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 5160 ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggcggga ggattgggga 5220 cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 5280 cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggccccc 5340 aacggcgacc tgtacaacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 5400 cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 5460 ctgcaactta cctccgggat ggtccagacc cacgtcacca cccccggctc cataccgacg 5520 atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaactg agtcgacatc 5580 gatggtacca gatccgataa aataaaaggt tttatttagt ctcctgaaaa aggggggaat 5640 gaaagacccc acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg 5700 gaaaaataca taactgagaa tagagaagtt cagatcaagg tcaggaacag atggaacagc 5760 tgaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag 5820 aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc 5880 cggctcaggg ccaagaacag atggtcccca gatgcggtcc agccctcagc agtttctaga 5940 gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga 6000 actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata 6060 aaagagccca caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt 6120 acccgtgtat ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt 6180 gggagggtct cctctgagtg attgactacc cgtcagcggg ggtctttcac acatgcagca 6240 tgtatcaaaa ttaatttggt tttttttctt aagtatttac attaaatggc catagtactt 6300 aaagttacat tggcttcctt gaaataaaca tggagtattc agaatgtgtc ataaatattt 6360 ctaattttaa gatagtatct ccattggctt tctacttttt cttttatttt tttttgtcct 6420 ctgtcttcca tttgttgttg ttgttgtttg tttgtttgtt tgttggttgg ttggttaatt 6480 tttttttaaa gatcctacac tatagttcaa gctagactat tagctactct gtaacccagg 6540 gtgaccttga agtcatgggt agcctgctgt tttagccttc ccacatctaa gattacaggt 6600 atgagctatc atttttggta tatgattgat tgattgattg atgtgtgtgt gtgtgattgt 6660 gtttgtgtgt gtgactgtga aaatgtgtgt atgggtgtgt gtgaatgtgt gtatgtatgt 6720 gtgtgtgtga gtgtgtgtgt gtgtgtgtgc atgtgtgtgt gtgtgactgt gtctatgtgt 6780 atgactgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gttgtgaaaa 6840 aatattctat ggtagtgaga gccaacgctc cggctcaggt gtcaggttgg tttttgagac 6900 agagtctttc acttagcttg gaattcactg gccgtcgttt tacaacgtcg tgactgggaa 6960 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 7020 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 7080 tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg 7140 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca 7200 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct 7260 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg 7320 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt 7380 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 7440 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 7500 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 7560 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 7620 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 7680 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 7740 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 7800 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 7860 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 7920 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 7980 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 8040 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 8100 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 8160 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 8220 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 8280 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 8340 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac 8400 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag 8460 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 8520 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 8580 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 8640 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt 8700 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 8760 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 8820 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 8880 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 8940 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 9000 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 9060 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 9120 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 9180 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 9240 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 9300 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 9360 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc 9420 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt 9480 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat 9540 gaccatgatt acgccaagct ttgctcctag gagtttccta atacttccca aactcaaata 9600 tataaagcat ttgacttgtt ctatgcccta gggggcgggg ggaagctaag ccagcttttt 9660 ttaacattta aaatgttaat tccattttaa atgcacagat gtttttattt cataagggtt 9720 tcaatgtgca tgaatgctgc aatattcctg ttaccaaagc tagtataaat aaaaatagat 9780 aaacgtggaa attacttaga gtttctgtca ttaacgtttc cttcctcagt tgacaacata 9840 aatgcgctgc tgagcaagcc agtttgcatc tgtcaggatc aatttcccat tatgccagtc 9900 atattaatta ctagtcaatt agttgatttt tatttttgac atatacatgt gaatgaaaga 9960 ccccacctgt aggtttggca agctagctta agt 9993 <210> 29 <211> 9860 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-F2-TK vector <400> 29 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacaattc 1800 gatcatacct ggtgttgctg actaccccga ccgcggtaaa agtcgatggt attgctgcct 1860 gggtccatgc ttctcacctc aaacctgcac caccttcggc accagatgag tcctgggagc 1920 tggaaaagac tgatcatcct cttaagctgc gtattcggcg gcggcgggac gagtctgcaa 1980 aataagaacc cccaccagcc catgaccctc acttggcagg tactgtccca aactggagac 2040 gttgtctggg atacaaaggc agtccagccc ccttggactt ggtggcccac acttaaacct 2100 gatgtatgtg ccttggcggc tagtcttgag tcctgggata tcccgggaac cgatgtctcg 2160 tcctctaaac gagtcagacc tccggactca gactatactg ccgcttataa gcaaatcacc 2220 tggggagcca tagggtgcag ctaccctcgg gctaggacta gaatggcaag ctctaccttc 2280 tacgtatgtc cccgggatgg ccggaccctt tcagaagcta gaaggtgcgg ggggctagaa 2340 tccctatact gtaaagaatg ggattgtgag accacgggga ccggttattg gctatctaaa 2400 tcctcaaaag acctcataac tgtaaaatgg gaccaaaata gcgaatggac tcaaaaattt 2460 caacagtgtc accagaccgg ctggtgtaac ccccttaaaa tagatttcac agacaaagga 2520 aaattatcca aggactggat aacgggaaaa acctggggat taagattcta tgtgtctgga 2580 catccaggcg tacagttcac cattcgctta aaaatcacca acatgccagc tgtggcagta 2640 ggtcctgacc tcgtccttgt ggaacaagga cctcctagaa cgtccctcgc tctcccacct 2700 cctcttcccc caagggaagc gccaccgcca tctctccccg actctaactc cacagccctg 2760 gcgactagtg cacaaactcc cacggtgaga aaaacaattg ttaccctaaa cactccgcct 2820 cccaccacag gcgacagact ttttgatctt gtgcaggggg ccttcctaac cttaaatgct 2880 accaacccag gggccactga gtcttgctgg ctttgtttgg ccatgggccc cccttattat 2940 gaagcaatag cctcatcagg agaggtcgcc tactccaccg accttgaccg gtgccgctgg 3000 gggacccaag gaaagctcac cctcactgag gtctcaggac acgggttgtg cataggaaag 3060 gtgcccttta cccatcagca tctctgcaat cagaccctat ccatcaattc ctccggagac 3120 catcagtatc tgctcccctc caaccatagc tggtgggctt gcagcactgg cctcacccct 3180 tgcctctcca cctcagtttt taatcagact agagatttct gtatccaggt ccagctgatt 3240 cctcgcatct attactatcc tgaagaagtt ttgttacagg cctatgacaa ttctcacccc 3300 aggactaaaa gagaggctgt ctcacttacc ctagctgttt tactggggtt gggaatcacg 3360 gcgggaatag gtactggttc aactgcctta attaaaggac ctatagacct ccagcaaggc 3420 ctgacaagcc tccagatcgc catagatgct gacctccggg ccctccaaga ctcagtcagc 3480 aagttagagg actcactgac ttccctgtcc gaggtagtgc tccaaaatag gagaggcctt 3540 gacttgctgt ttctaaaaga aggtggcctc tgtgcggccc taaaggaaga gtgctgtttt 3600 tacatagacc actcaggtgc agtacgggac tccatgaaaa aactcaaaga aaaactggat 3660 aaaagacagt tagagcgcca gaaaagccaa aactggtatg aaggatggtt caataactcc 3720 ccttggttca ctaccctgct atcaaccatc gctgggcccc tattactcct ccttctgttg 3780 ctcatcctcg ggccatgcat catcaataag ttagttcaat tcatcaatga taggataagt 3840 gcagttaaaa ttctggtcct tagacaaaaa tatcaggccc tagagaacga aggtaacctt 3900 taacacgtga aggctgccga ccccgggggt ggaccatcct ctagactgtg ctcgacgttt 3960 aaacaaggtc aataggggtg agtcattggg tttttccagc caatttaatt aaaacgccat 4020 gtactttccc accattgacg tcaatgggct attgaaacta atgcaacgtg acctttaaac 4080 ggtactttcc catagctgat taatgggaaa gtaccgttct cgagccaata cacgtcaatg 4140 ggaagtgaaa gggcagccaa aacgtaacac cgccccggtt ttccctggaa attccatatt 4200 ggcacgcatt ctattggctg agctgcgttc acgtgggtat aagaggcgcg accagcgtcg 4260 gtaccgtcgc agtcttcggt ctgaccaccg tagaacgcag aacgcgtatg gcttcgtacc 4320 cctgccatca acacgcgtct gcgttcgacc aggctgcgcg ttctcgcggc catagcaacc 4380 gacgtacggc gttgcgccct cgccggcagc aagaagccac ggaagtccgc ctggagcaga 4440 aaatgcccac gctactgcgg gtttatatag acggtcctca cgggatgggg aaaaccacca 4500 ccacgcaact gctggtggcc ctgggttcgc gcgacgatat cgtctacgta cccgagccga 4560 tgacttactg gcaggtgctg ggggcttccg agacaatcgc gaacatctac accacacaac 4620 accgcctcga ccagggtgag atatcggccg gggacgcggc ggtggtaatg acaagcgccc 4680 agataacaat gggcatgcct tatgccgtga ccgacgccgt tctggctcct catatcgggg 4740 gggaggctgg gagctcacat gccccgcccc cggccctcac cctcatcttc gaccgccatc 4800 ccatcgccgc cctcctgtgc tacccggccg cgcgatacct tatgggcagc atgacccccc 4860 aggccgtgct ggcgttcgtg gccctcatcc cgccgacctt gcccggcaca aacatcgtgt 4920 tgggggccct tccggaggac agacacatcg accgcctggc caaacgccag cgccccggcg 4980 agcggcttga cctggctatg ctggccgcga ttcgccgcgt ttacgggctg cttgccaata 5040 cggtgcggta tctgcagggc ggcgggtcgt ggcgggagga ttggggacag ctttcgggga 5100 cggccgtgcc gccccagggt gccgagcccc agagcaacgc gggcccacga ccccatatcg 5160 gggacacgtt atttaccctg tttcgggccc ccgagttgct ggcccccaac ggcgacctgt 5220 acaacgtgtt tgcctgggcc ttggacgtct tggccaaacg cctccgtccc atgcacgtct 5280 ttatcctgga ttacgaccaa tcgcccgccg gctgccggga cgccctgctg caacttacct 5340 ccgggatggt ccagacccac gtcaccaccc ccggctccat accgacgatc tgcgacctgg 5400 cgcgcacgtt tgcccgggag atgggggagg ctaactgagt cgacatcgat ggtaccagat 5460 ccgataaaat aaaaggtttt atttagtctc ctgaaaaagg ggggaatgaa agaccccacc 5520 tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa 5580 ctgagaatag agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca 5640 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca 5700 gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca 5760 agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg 5820 tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag 5880 ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa 5940 cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca 6000 ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct 6060 ctgagtgatt gactacccgt cagcgggggt ctttcacaca tgcagcatgt atcaaaatta 6120 atttggtttt ttttcttaag tatttacatt aaatggccat agtacttaaa gttacattgg 6180 cttccttgaa ataaacatgg agtattcaga atgtgtcata aatatttcta attttaagat 6240 agtatctcca ttggctttct actttttctt ttattttttt ttgtcctctg tcttccattt 6300 gttgttgttg ttgtttgttt gtttgtttgt tggttggttg gttaattttt ttttaaagat 6360 cctacactat agttcaagct agactattag ctactctgta acccagggtg accttgaagt 6420 catgggtagc ctgctgtttt agccttccca catctaagat tacaggtatg agctatcatt 6480 tttggtatat gattgattga ttgattgatg tgtgtgtgtg tgattgtgtt tgtgtgtgtg 6540 actgtgaaaa tgtgtgtatg ggtgtgtgtg aatgtgtgta tgtatgtgtg tgtgtgagtg 6600 tgtgtgtgtg tgtgtgcatg tgtgtgtgtg tgactgtgtc tatgtgtatg actgtgtgtg 6660 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtt gtgaaaaaat attctatggt 6720 agtgagagcc aacgctccgg ctcaggtgtc aggttggttt ttgagacaga gtctttcact 6780 tagcttggaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 6840 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 6900 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc 6960 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta 7020 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 7080 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 7140 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc 7200 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag 7260 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 7320 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 7380 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 7440 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 7500 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 7560 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 7620 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 7680 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 7740 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 7800 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 7860 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 7920 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 7980 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 8040 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 8100 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 8160 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 8220 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 8280 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 8340 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 8400 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 8460 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 8520 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 8580 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 8640 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 8700 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 8760 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 8820 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 8880 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 8940 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 9000 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 9060 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 9120 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 9180 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 9240 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 9300 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 9360 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 9420 ccaagctttg ctcctaggag tttcctaata cttcccaaac tcaaatatat aaagcatttg 9480 acttgttcta tgccctaggg ggcgggggga agctaagcca gcttttttta acatttaaaa 9540 tgttaattcc attttaaatg cacagatgtt tttatttcat aagggtttca atgtgcatga 9600 atgctgcaat attcctgtta ccaaagctag tataaataaa aatagataaa cgtggaaatt 9660 acttagagtt tctgtcatta acgtttcctt cctcagttga caacataaat gcgctgctga 9720 gcaagccagt ttgcatctgt caggatcaat ttcccattat gccagtcata ttaattacta 9780 gtcaattagt tgatttttat ttttgacata tacatgtgaa tgaaagaccc cacctgtagg 9840 tttggcaagc tagcttaagt 9860 <210> 30 <211> 9760 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-F3-TK vector <400> 30 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacaattc 1800 gatcatacct ggtgttgctg actaccccga ccgcggtaaa agtcgatggt attgctgcct 1860 gggtccatgc ttctcacctc aaacctgcac caccttcggc accagatgag tcctgggagc 1920 tggaaaagac tgatcatcct cttaagctgc gtattcggcg gcggcgggac gagtctgcaa 1980 aataagaacc cccaccagcc catgaccctc acttggcagg tactgtccca aactggagac 2040 gttgtctggg atacaaaggc agtccagccc ccttggactt ggtggcccac acttaaacct 2100 gatgtatgtg ccttggcggc tagtcttgag tcctgggata tcccgggaac cgatgtctcg 2160 tcctctaaac gagtcagacc tccggactca gactatactg ccgcttataa gcaaatcacc 2220 tggggagcca tagggtgcag ctaccctcgg gctaggacta gaatggcaag ctctaccttc 2280 tacgtatgtc cccgggatgg ccggaccctt tcagaagcta gaaggtgcgg ggggctagaa 2340 tccctatact gtaaagaatg ggattgtgag accacgggga ccggttattg gctatctaaa 2400 tcctcaaaag acctcataac tgtaaaatgg gaccaaaata gcgaatggac tcaaaaattt 2460 caacagtgtc accagaccgg ctggtgtaac ccccttaaaa tagatttcac agacaaagga 2520 aaattatcca aggactggat aacgggaaaa acctggggat taagattcta tgtgtctgga 2580 catccaggcg tacagttcac cattcgctta aaaatcacca acatgccagc tgtggcagta 2640 ggtcctgacc tcgtccttgt ggaacaagga cctcctagaa cgtccctcgc tctcccacct 2700 cctcttcccc caagggaagc gccaccgcca tctctccccg actctaactc cacagccctg 2760 gcgactagtg cacaaactcc cacggtgaga aaaacaattg ttaccctaaa cactccgcct 2820 cccaccacag gcgacagact ttttgatctt gtgcaggggg ccttcctaac cttaaatgct 2880 accaacccag gggccactga gtcttgctgg ctttgtttgg ccatgggccc cccttattat 2940 gaagcaatag cctcatcagg agaggtcgcc tactccaccg accttgaccg gtgccgctgg 3000 gggacccaag gaaagctcac cctcactgag gtctcaggac acgggttgtg cataggaaag 3060 gtgcccttta cccatcagca tctctgcaat cagaccctat ccatcaattc ctccggagac 3120 catcagtatc tgctcccctc caaccatagc tggtgggctt gcagcactgg cctcacccct 3180 tgcctctcca cctcagtttt taatcagact agagatttct gtatccaggt ccagctgatt 3240 cctcgcatct attactatcc tgaagaagtt ttgttacagg cctatgacaa ttctcacccc 3300 aggactaaaa gagaggctgt ctcacttacc ctagctgttt tactggggtt gggaatcacg 3360 gcgggaatag gtactggttc aactgcctta attaaaggac ctatagacct ccagcaaggc 3420 ctgacaagcc tccagatcgc catagatgct gacctccggg ccctccaaga ctcagtcagc 3480 aagttagagg actcactgac ttccctgtcc gaggtagtgc tccaaaatag gagaggcctt 3540 gacttgctgt ttctaaaaga aggtggcctc tgtgcggccc taaaggaaga gtgctgtttt 3600 tacatagacc actcaggtgc agtacgggac tccatgaaaa aactcaaaga aaaactggat 3660 aaaagacagt tagagcgcca gaaaagccaa aactggtatg aaggatggtt caataactcc 3720 ccttggttca ctaccctgct atcaaccatc gctgggcccc tattactcct ccttctgttg 3780 ctcatcctcg ggccatgcat catcaataag ttagttcaat tcatcaatga taggataagt 3840 gcagttaaaa ttctggtcct tagacaaaaa tatcaggccc tagagaacga aggtaacctt 3900 taacacgtga aggctgccga ccccgggggt ggaccatcct ctagactgtg ctcgacgttt 3960 aaacaacgtg acctttaaac ggtactttcc catagctgat taatgggaaa gtaccgttct 4020 cgagccaata cacgtcaatg ggaagtgaaa gggcagccaa aacgtaacac cgccccggtt 4080 ttccctggaa attccatatt ggcacgcatt ctattggctg agctgcgttc acgtgggtat 4140 aagaggcgcg accagcgtcg gtaccgtcgc agtcttcggt ctgaccaccg tagaacgcag 4200 aacgcgtatg gcttcgtacc cctgccatca acacgcgtct gcgttcgacc aggctgcgcg 4260 ttctcgcggc catagcaacc gacgtacggc gttgcgccct cgccggcagc aagaagccac 4320 ggaagtccgc ctggagcaga aaatgcccac gctactgcgg gtttatatag acggtcctca 4380 cgggatgggg aaaaccacca ccacgcaact gctggtggcc ctgggttcgc gcgacgatat 4440 cgtctacgta cccgagccga tgacttactg gcaggtgctg ggggcttccg agacaatcgc 4500 gaacatctac accacacaac accgcctcga ccagggtgag atatcggccg gggacgcggc 4560 ggtggtaatg acaagcgccc agataacaat gggcatgcct tatgccgtga ccgacgccgt 4620 tctggctcct catatcgggg gggaggctgg gagctcacat gccccgcccc cggccctcac 4680 cctcatcttc gaccgccatc ccatcgccgc cctcctgtgc tacccggccg cgcgatacct 4740 tatgggcagc atgacccccc aggccgtgct ggcgttcgtg gccctcatcc cgccgacctt 4800 gcccggcaca aacatcgtgt tgggggccct tccggaggac agacacatcg accgcctggc 4860 caaacgccag cgccccggcg agcggcttga cctggctatg ctggccgcga ttcgccgcgt 4920 ttacgggctg cttgccaata cggtgcggta tctgcagggc ggcgggtcgt ggcgggagga 4980 ttggggacag ctttcgggga cggccgtgcc gccccagggt gccgagcccc agagcaacgc 5040 gggcccacga ccccatatcg gggacacgtt atttaccctg tttcgggccc ccgagttgct 5100 ggcccccaac ggcgacctgt acaacgtgtt tgcctgggcc ttggacgtct tggccaaacg 5160 cctccgtccc atgcacgtct ttatcctgga ttacgaccaa tcgcccgccg gctgccggga 5220 cgccctgctg caacttacct ccgggatggt ccagacccac gtcaccaccc ccggctccat 5280 accgacgatc tgcgacctgg cgcgcacgtt tgcccgggag atgggggagg ctaactgagt 5340 cgacatcgat ggtaccagat ccgataaaat aaaaggtttt atttagtctc ctgaaaaagg 5400 ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca 5460 aggcatggaa aaatacataa ctgagaatag agaagttcag atcaaggtca ggaacagatg 5520 gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 5580 ggccaagaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt 5640 cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtccagc cctcagcagt 5700 ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct 5760 tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag 5820 ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg 5880 cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct 5940 gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcacaca 6000 tgcagcatgt atcaaaatta atttggtttt ttttcttaag tatttacatt aaatggccat 6060 agtacttaaa gttacattgg cttccttgaa ataaacatgg agtattcaga atgtgtcata 6120 aatatttcta attttaagat agtatctcca ttggctttct actttttctt ttattttttt 6180 ttgtcctctg tcttccattt gttgttgttg ttgtttgttt gtttgtttgt tggttggttg 6240 gttaattttt ttttaaagat cctacactat agttcaagct agactattag ctactctgta 6300 acccagggtg accttgaagt catgggtagc ctgctgtttt agccttccca catctaagat 6360 tacaggtatg agctatcatt tttggtatat gattgattga ttgattgatg tgtgtgtgtg 6420 tgattgtgtt tgtgtgtgtg actgtgaaaa tgtgtgtatg ggtgtgtgtg aatgtgtgta 6480 tgtatgtgtg tgtgtgagtg tgtgtgtgtg tgtgtgcatg tgtgtgtgtg tgactgtgtc 6540 tatgtgtatg actgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtt 6600 gtgaaaaaat attctatggt agtgagagcc aacgctccgg ctcaggtgtc aggttggttt 6660 ttgagacaga gtctttcact tagcttggaa ttcactggcc gtcgttttac aacgtcgtga 6720 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 6780 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 6840 tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 6900 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca 6960 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag 7020 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa 7080 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 7140 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 7200 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 7260 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 7320 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 7380 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 7440 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 7500 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg 7560 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 7620 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 7680 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 7740 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 7800 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 7860 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 7920 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 7980 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 8040 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 8100 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 8160 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 8220 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 8280 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 8340 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 8400 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 8460 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 8520 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 8580 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 8640 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 8700 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 8760 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 8820 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 8880 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 8940 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 9000 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 9060 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc 9120 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 9180 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt 9240 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 9300 cagctatgac catgattacg ccaagctttg ctcctaggag tttcctaata cttcccaaac 9360 tcaaatatat aaagcatttg acttgttcta tgccctaggg ggcgggggga agctaagcca 9420 gcttttttta acatttaaaa tgttaattcc attttaaatg cacagatgtt tttatttcat 9480 aagggtttca atgtgcatga atgctgcaat attcctgtta ccaaagctag tataaataaa 9540 aatagataaa cgtggaaatt acttagagtt tctgtcatta acgtttcctt cctcagttga 9600 caacataaat gcgctgctga gcaagccagt ttgcatctgt caggatcaat ttcccattat 9660 gccagtcata ttaattacta gtcaattagt tgatttttat ttttgacata tacatgtgaa 9720 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt 9760 <210> 31 <211> 9685 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-F4-TK vector <400> 31 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacaattc 1800 gatcatacct ggtgttgctg actaccccga ccgcggtaaa agtcgatggt attgctgcct 1860 gggtccatgc ttctcacctc aaacctgcac caccttcggc accagatgag tcctgggagc 1920 tggaaaagac tgatcatcct cttaagctgc gtattcggcg gcggcgggac gagtctgcaa 1980 aataagaacc cccaccagcc catgaccctc acttggcagg tactgtccca aactggagac 2040 gttgtctggg atacaaaggc agtccagccc ccttggactt ggtggcccac acttaaacct 2100 gatgtatgtg ccttggcggc tagtcttgag tcctgggata tcccgggaac cgatgtctcg 2160 tcctctaaac gagtcagacc tccggactca gactatactg ccgcttataa gcaaatcacc 2220 tggggagcca tagggtgcag ctaccctcgg gctaggacta gaatggcaag ctctaccttc 2280 tacgtatgtc cccgggatgg ccggaccctt tcagaagcta gaaggtgcgg ggggctagaa 2340 tccctatact gtaaagaatg ggattgtgag accacgggga ccggttattg gctatctaaa 2400 tcctcaaaag acctcataac tgtaaaatgg gaccaaaata gcgaatggac tcaaaaattt 2460 caacagtgtc accagaccgg ctggtgtaac ccccttaaaa tagatttcac agacaaagga 2520 aaattatcca aggactggat aacgggaaaa acctggggat taagattcta tgtgtctgga 2580 catccaggcg tacagttcac cattcgctta aaaatcacca acatgccagc tgtggcagta 2640 ggtcctgacc tcgtccttgt ggaacaagga cctcctagaa cgtccctcgc tctcccacct 2700 cctcttcccc caagggaagc gccaccgcca tctctccccg actctaactc cacagccctg 2760 gcgactagtg cacaaactcc cacggtgaga aaaacaattg ttaccctaaa cactccgcct 2820 cccaccacag gcgacagact ttttgatctt gtgcaggggg ccttcctaac cttaaatgct 2880 accaacccag gggccactga gtcttgctgg ctttgtttgg ccatgggccc cccttattat 2940 gaagcaatag cctcatcagg agaggtcgcc tactccaccg accttgaccg gtgccgctgg 3000 gggacccaag gaaagctcac cctcactgag gtctcaggac acgggttgtg cataggaaag 3060 gtgcccttta cccatcagca tctctgcaat cagaccctat ccatcaattc ctccggagac 3120 catcagtatc tgctcccctc caaccatagc tggtgggctt gcagcactgg cctcacccct 3180 tgcctctcca cctcagtttt taatcagact agagatttct gtatccaggt ccagctgatt 3240 cctcgcatct attactatcc tgaagaagtt ttgttacagg cctatgacaa ttctcacccc 3300 aggactaaaa gagaggctgt ctcacttacc ctagctgttt tactggggtt gggaatcacg 3360 gcgggaatag gtactggttc aactgcctta attaaaggac ctatagacct ccagcaaggc 3420 ctgacaagcc tccagatcgc catagatgct gacctccggg ccctccaaga ctcagtcagc 3480 aagttagagg actcactgac ttccctgtcc gaggtagtgc tccaaaatag gagaggcctt 3540 gacttgctgt ttctaaaaga aggtggcctc tgtgcggccc taaaggaaga gtgctgtttt 3600 tacatagacc actcaggtgc agtacgggac tccatgaaaa aactcaaaga aaaactggat 3660 aaaagacagt tagagcgcca gaaaagccaa aactggtatg aaggatggtt caataactcc 3720 ccttggttca ctaccctgct atcaaccatc gctgggcccc tattactcct ccttctgttg 3780 ctcatcctcg ggccatgcat catcaataag ttagttcaat tcatcaatga taggataagt 3840 gcagttaaaa ttctggtcct tagacaaaaa tatcaggccc tagagaacga aggtaacctt 3900 taacacgtga aggctgccga ccccgggggt ggaccatcct ctagactgtg ctcgacgttt 3960 aaacggaagt gaaagggcag ccaaaacgta acaccgcccc ggttttccct ggaaattcca 4020 tattggcacg cattctattg gctgagctgc gttcacgtgg gtataagagg cgcgaccagc 4080 gtcggtaccg tcgcagtctt cggtctgacc accgtagaac gcagaggatc ctatggcttc 4140 gtacccctgc catcaacacg cgtctgcgtt cgaccaggct gcgcgttctc gcggccatag 4200 caaccgacgt acggcgttgc gccctcgccg gcagcaagaa gccacggaag tccgcctgga 4260 gcagaaaatg cccacgctac tgcgggttta tatagacggt cctcacggga tggggaaaac 4320 caccaccacg caactgctgg tggccctggg ttcgcgcgac gatatcgtct acgtacccga 4380 gccgatgact tactggcagg tgctgggggc ttccgagaca atcgcgaaca tctacaccac 4440 acaacaccgc ctcgaccagg gtgagatatc ggccggggac gcggcggtgg taatgacaag 4500 cgcccagata acaatgggca tgccttatgc cgtgaccgac gccgttctgg ctcctcatat 4560 cgggggggag gctgggagct cacatgcccc gcccccggcc ctcaccctca tcttcgaccg 4620 ccatcccatc gccgccctcc tgtgctaccc ggccgcgcga taccttatgg gcagcatgac 4680 cccccaggcc gtgctggcgt tcgtggccct catcccgccg accttgcccg gcacaaacat 4740 cgtgttgggg gcccttccgg aggacagaca catcgaccgc ctggccaaac gccagcgccc 4800 cggcgagcgg cttgacctgg ctatgctggc cgcgattcgc cgcgtttacg ggctgcttgc 4860 caatacggtg cggtatctgc agggcggcgg gtcgtggcgg gaggattggg gacagctttc 4920 ggggacggcc gtgccgcccc agggtgccga gccccagagc aacgcgggcc cacgacccca 4980 tatcggggac acgttattta ccctgtttcg ggcccccgag ttgctggccc ccaacggcga 5040 cctgtacaac gtgtttgcct gggccttgga cgtcttggcc aaacgcctcc gtcccatgca 5100 cgtctttatc ctggattacg accaatcgcc cgccggctgc cgggacgccc tgctgcaact 5160 tacctccggg atggtccaga cccacgtcac cacccccggc tccataccga cgatctgcga 5220 cctggcgcgc acgtttgccc gggagatggg ggaggctaac tgagtcgaca tcgatggtac 5280 cagatccgat aaaataaaag gttttattta gtctcctgaa aaagggggga atgaaagacc 5340 ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata 5400 cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg 5460 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 5520 gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 5580 ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc 5640 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 5700 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 5760 cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt 5820 atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt 5880 ctcctctgag tgattgacta cccgtcagcg ggggtctttc acacatgcag catgtatcaa 5940 aattaatttg gttttttttc ttaagtattt acattaaatg gccatagtac ttaaagttac 6000 attggcttcc ttgaaataaa catggagtat tcagaatgtg tcataaatat ttctaatttt 6060 aagatagtat ctccattggc tttctacttt ttcttttatt tttttttgtc ctctgtcttc 6120 catttgttgt tgttgttgtt tgtttgtttg tttgttggtt ggttggttaa ttttttttta 6180 aagatcctac actatagttc aagctagact attagctact ctgtaaccca gggtgacctt 6240 gaagtcatgg gtagcctgct gttttagcct tcccacatct aagattacag gtatgagcta 6300 tcatttttgg tatatgattg attgattgat tgatgtgtgt gtgtgtgatt gtgtttgtgt 6360 gtgtgactgt gaaaatgtgt gtatgggtgt gtgtgaatgt gtgtatgtat gtgtgtgtgt 6420 gagtgtgtgt gtgtgtgtgt gcatgtgtgt gtgtgtgact gtgtctatgt gtatgactgt 6480 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttgtgaa aaaatattct 6540 atggtagtga gagccaacgc tccggctcag gtgtcaggtt ggtttttgag acagagtctt 6600 tcacttagct tggaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 6660 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 6720 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct 6780 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 6840 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 6900 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 6960 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 7020 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 7080 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 7140 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 7200 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 7260 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 7320 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 7380 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 7440 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 7500 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 7560 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 7620 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 7680 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 7740 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 7800 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 7860 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 7920 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 7980 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 8040 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 8100 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 8160 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 8220 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 8280 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 8340 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 8400 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 8460 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 8520 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 8580 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 8640 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 8700 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 8760 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 8820 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 8880 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 8940 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 9000 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 9060 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat 9120 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 9180 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga 9240 ttacgccaag ctttgctcct aggagtttcc taatacttcc caaactcaaa tatataaagc 9300 atttgacttg ttctatgccc tagggggcgg ggggaagcta agccagcttt ttttaacatt 9360 taaaatgtta attccatttt aaatgcacag atgtttttat ttcataaggg tttcaatgtg 9420 catgaatgct gcaatattcc tgttaccaaa gctagtataa ataaaaatag ataaacgtgg 9480 aaattactta gagtttctgt cattaacgtt tccttcctca gttgacaaca taaatgcgct 9540 gctgagcaag ccagtttgca tctgtcagga tcaatttccc attatgccag tcatattaat 9600 tactagtcaa ttagttgatt tttatttttg acatatacat gtgaatgaaa gaccccacct 9660 gtaggtttgg caagctagct taagt 9685 <210> 32 <211> 10008 <212> DNA <213> Artificial Sequence <220> <223> sRRVgp-sEF1a-hopt-yCD <400> 32 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 60 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 480 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 600 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 660 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 720 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 780 atcaatctaa agtatatatg agtaacctga tcaggactct tccttttcat gaacaataaa 840 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 900 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 960 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 1020 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1080 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 1140 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 1200 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 1260 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 1320 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 1380 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 1440 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 1500 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 1560 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 1620 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 1680 tgagtttttc taagaatttg tgaatgaaag accccacctg taggtttggc aagctagctt 1740 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat 1800 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1860 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1920 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1980 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2040 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2100 gcgcttatgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2160 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2220 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2280 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2340 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt 2400 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg 2460 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc 2520 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg 2580 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc 2640 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg 2700 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt 2760 tgtctgagaa tatgggccag actgttacca ctcccttaag tttgacctta ggtcactgga 2820 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 2880 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 2940 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 3000 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 3060 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 3120 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 3180 ctctaggcgc caaacctaaa cctcaagttc tttctgacag tggggggccg ctcatcgacc 3240 tacttacaga agaccccccg ccttataggg acccaagacc acccccttcc gacagggacg 3300 gaaatggtgg agaagcgacc cctgcgggag aggcaccgga cccctcccca atggcatctc 3360 gcctacgtgg gagacgggag ccccctgtgg ccgactccac tacctcgcag gcattccccc 3420 tccgcgcagg aggaaacgga cagcttcaat actggccgtt ctcctcttct gacctttaca 3480 actggaaaaa taataaccct tctttttctg aagatccagg taaactgaca gctctgatcg 3540 agtctgttct catcacccat cagcccacct gggacgactg tcagcagctg ttggggactc 3600 tgctgaccgg agaagaaaaa caacgggtgc tcttagaggc tagaaaggcg gtgcggggcg 3660 atgatgggcg ccccactcaa ctgcccaatg aagtcgatgc cgcttttccc ctcgagcgcc 3720 cagactggga ttacaccacc caggcaggta ggaaccacct agtccactat cgccagttgc 3780 tcctagcggg tctccaaaac gcgggcagaa gccccaccaa tttggccaag gtaaaaggaa 3840 taacacaagg gcccaatgag tctccctcgg ccttcctaga gagacttaag gaagcctatc 3900 gcaggtacac tccttatgac cctgaggacc cagggcaaga aactaatgtg tctatgtctt 3960 tcatttggca gtctgcccca gacattggga gaaagttaga gaggttagaa gatttaaaaa 4020 acaagacgct tggagatttg gttagagagg cagaaaagat ctttaataaa cgagaaaccc 4080 cggaagaaag agaggaacgt atcaggagag aaacagagga aaaagaagaa cgccgtagga 4140 cagaggatga gcagaaagag aaagaaagag atcgtaggag acatagagag atgagcaagc 4200 tattggccac tgtcgttagt ggacagaaac aggatagaca gggaggagaa cgaaggaggt 4260 cccaactcga tcgcgaccag tgtgcctact gcaaagaaaa ggggcactgg gctaaagatt 4320 gtcccaagaa accacgagga cctcggggac caagacccca gacctccctc ctgaccctag 4380 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 4440 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 4500 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 4560 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 4620 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 4680 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 4740 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 4800 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 4860 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 4920 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 4980 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 5040 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 5100 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 5160 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 5220 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 5280 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 5340 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 5400 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 5460 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 5520 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 5580 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 5640 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 5700 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 5760 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 5820 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 5880 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 5940 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 6000 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 6060 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 6120 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 6180 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 6240 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 6300 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 6360 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 6420 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 6480 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 6540 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 6600 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 6660 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 6720 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 6780 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 6840 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 6900 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 6960 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 7020 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 7080 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 7140 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 7200 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 7260 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 7320 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 7380 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 7440 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 7500 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 7560 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 7620 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 7680 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 7740 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 7800 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 7860 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 7920 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 7980 ccccctaatc cccttaattc ttctgatgct cagaggggtc agtaaacgaa ttcgggcaga 8040 gcgcacatcg cccgcagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc 8100 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt 8160 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg 8220 caacgggttt gccgccagaa cacaggcggc cgcatggtta ctgggggaat ggcatctaag 8280 tgggatcaga aaggtatgga catcgcttat gaagaggctg ctctcggcta caaagagggt 8340 ggagtgccta tcggagggtg cctgatcaac aacaaggacg gcagtgtgct ggggaggggc 8400 cacaatatga ggttccaaaa aggctccgcc actctccacg gggaaattag taccctcgag 8460 aattgcggac gattggaagg gaaggtgtac aaggatacaa cactgtacac caccctgtca 8520 ccctgtgata tgtgcacagg cgccattatc atgtacggaa tccctagatg tgtcgtgggg 8580 gagaatgtaa acttcaaaag taagggggag aaatatctcc agacccgggg gcacgaagtc 8640 gtcgttgtgg acgatgaacg gtgtaagaag atcatgaagc agtttatcga tgagaggccc 8700 caggactggt tcgaagacat cggggaataa gtttaaacga attcataaaa taaaagattt 8760 tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag 8820 cttaagtaac gccattttgc aaggcatgga aaaatacata actgagaata gagaagttca 8880 gatcaaggtc aggaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa 8940 gcagttcctg ccccggctca gggccaagaa cagatggaac agctgaatat gggccaaaca 9000 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga 9060 tgcggtccag ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg 9120 acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt 9180 cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact cggggcgcca 9240 gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg 9300 catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg 9360 tcagcggggg tctttcattg ttacttaaag ttacattggc ttccttgaaa taaacatgga 9420 gtattcagaa tgtgtcataa atatttctaa ttttaagata gtatctccat tggctttcta 9480 ctttttcttt tatttttttt tgtcctctgt cttccatttg ttgttgttgt tgtttgtttg 9540 tttgtttgtt ggttggttgg ttaatttttt tttaaagatc ctacactata gttcaagcta 9600 gactattagc tactctgtaa cccagggtga ccttgaagtc atgggtagcc tgctgtttta 9660 gccttcccac atctaagatt acaggtatga gctatcattt ttggtatatt gattgattga 9720 ttgattgatg tgtgtgtgtg tgattgtgtt tgtgtgtgtg actgtgaaaa tgtgtgtatg 9780 ggtgtgtgtg aatgtgtgta tgtatgtgtg tgtgtgagtg tgtgtgtgtg tgtgtgcatg 9840 tgtgtgtgtg tgactgtgtc tatgtgtatg actgtgtgtg tgtgtgtgtg tgtgtgtgtg 9900 tgtgtgtgtg tgtgtgtgtt gtgaaaaaat attctatggt agtgagagcc aacgctccgg 9960 ctcaggtgtc aggttggttt ttgagacaga gtctttcact tagcttgg 10008 <210> 33 <211> 10108 <212> DNA <213> Artificial Sequence <220> <223> sRRVe-TK vector <400> 33 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacgtgat 1800 ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 1860 agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 1920 aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 1980 aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 2040 gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 2100 acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 2160 tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 2220 ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 2280 cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 2340 cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 2400 aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 2460 aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 2520 ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 2580 tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 2640 tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 2700 tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 2760 gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 2820 cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 2880 taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 2940 tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 3000 acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 3060 gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 3120 ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 3180 atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 3240 agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 3300 ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 3360 gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 3420 aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 3480 gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 3540 aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 3600 caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 3660 caatcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac 3720 tcaacaatat caccagctga agcctataga gtacgagcca tagcacgtga aggctgccga 3780 ccccgggggt ggacatcctc tagactgcca tggcgcggat ctcgacgttt aaacaacagg 3840 aaagttccat tggagccaag tacattgagt caatagggac tttccaatgg gttttgccca 3900 gtacataagg tcaatgggag gtaagccaat gggtttttcc cattactggc acgtatactg 3960 agtcattagg gactttccaa tgggttttgc ccagtacata aggtcaatag gggtgaatca 4020 acaggaaagt cccattggag ccaagtacac tgagtcaata gggactttcc attgggtttt 4080 gcccagtaca aaaggtcaat agggggtgag tcaatgggtt tttcccatta ttggcacgta 4140 cataaggtca ataggggtga gtcattgggt ttttccagcc aatttaatta aaacgccatg 4200 tactttccca ccattgacgt caatgggcta ttgaaactaa tgcaacgtga cctttaaacg 4260 gtactttccc atagctgatt aatgggaaag taccgttctc gagccaatac acgtcaatgg 4320 gaagtgaaag ggcagccaaa acgtaacacc gccccggttt tccctggaaa ttccatattg 4380 gcacgcattc tattggctga gctgcgttct acgtgggtat aagaggcgcg accagcgtcg 4440 gtaccgtcgc agtcttcggt ctgaccaccg tagaacgcag aactctagaa ctagtggatc 4500 cgttaacatt tgcggccgct ttagtttatg gtgacatggc ttcgtacccc tgccatcaac 4560 acgcgtctgc gttcgaccag gctgcgcgtt ctcgcggcca tagcaaccga cgtacggcgt 4620 tgcgccctcg ccggcagcaa gaagccacgg aagtccgcct ggagcagaaa atgcccacgc 4680 tactgcgggt ttatatagac ggtcctcacg ggatggggaa aaccaccacc acgcaactgc 4740 tggtggccct gggttcgcgc gacgatatcg tctacgtacc cgagccgatg acttactggc 4800 aggtgctggg ggcttccgag acaatcgcga acatctacac cacacaacac cgcctcgacc 4860 agggtgagat atcggccggg gacgcggcgg tggtaatgac aagcgcccag ataacaatgg 4920 gcatgcctta tgccgtgacc gacgccgttc tggctcctca tatcgggggg gaggctggga 4980 gctcacatgc cccgcccccg gccctcaccc tcatcttcga ccgccatccc atcgccgccc 5040 tcctgtgcta cccggccgcg cgatacctta tgggcagcat gaccccccag gccgtgctgg 5100 cgttcgtggc cctcatcccg ccgaccttgc ccggcacaaa catcgtgttg ggggcccttc 5160 cggaggacag acacatcgac cgcctggcca aacgccagcg ccccggcgag cggcttgacc 5220 tggctatgct ggccgcgatt cgccgcgttt acgggctgct tgccaatacg gtgcggtatc 5280 tgcagggcgg cgggtcgtgg cgggaggatt ggggacagct ttcggggacg gccgtgccgc 5340 cccagggtgc cgagccccag agcaacgcgg gcccacgacc ccatatcggg gacacgttat 5400 ttaccctgtt tcgggccccc gagttgctgg cccccaacgg cgacctgtac aacgtgtttg 5460 cctgggcctt ggacgtcttg gccaaacgcc tccgtcccat gcacgtcttt atcctggatt 5520 acgaccaatc gcccgccggc tgccgggacg ccctgctgca acttacctcc gggatggtcc 5580 agacccacgt caccaccccc ggctccatac cgacgatctg cgacctggcg cgcacgtttg 5640 cccgggagat gggggaggct aactgatcga gaaacgcgtc gacccatcga acatcgatgg 5700 taccagatcc gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag 5760 accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa 5820 atacataact gagaatagag aagttcagat caaggtcagg aacagatgga acagctgaat 5880 atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag 5940 atggaacagc tgaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct 6000 cagggccaag aacagatggt ccccagatgc ggtccagccc tcagcagttt ctagagaacc 6060 atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa 6120 ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga 6180 gcccacaacc cctcactcgg ggcgccagtc ctccgattga ctgagtcgcc cgggtacccg 6240 tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag 6300 ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcacacatg cagcatgtat 6360 caaaattaat ttggtttttt ttcttaagta tttacattaa atggccatag tacttaaagt 6420 tacattggct tccttgaaat aaacatggag tattcagaat gtgtcataaa tatttctaat 6480 tttaagatag tatctccatt ggctttctac tttttctttt attttttttt gtcctctgtc 6540 ttccatttgt tgttgttgtt gtttgtttgt ttgtttgttg gttggttggt taattttttt 6600 ttaaagatcc tacactatag ttcaagctag actattagct actctgtaac ccagggtgac 6660 cttgaagtca tgggtagcct gctgttttag ccttcccaca tctaagatta caggtatgag 6720 ctatcatttt tggtatatga ttgattgatt gattgatgtg tgtgtgtgtg attgtgtttg 6780 tgtgtgtgac tgtgaaaatg tgtgtatggg tgtgtgtgaa tgtgtgtatg tatgtgtgtg 6840 tgtgagtgtg tgtgtgtgtg tgtgcatgtg tgtgtgtgtg actgtgtcta tgtgtatgac 6900 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgttgt gaaaaaatat 6960 tctatggtag tgagagccaa cgctccggct caggtgtcag gttggttttt gagacagagt 7020 ctttcactta gcttggaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 7080 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 7140 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg 7200 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 7260 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc 7320 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac 7380 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg 7440 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta 7500 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 7560 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 7620 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 7680 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 7740 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 7800 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 7860 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 7920 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 7980 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 8040 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 8100 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 8160 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 8220 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 8280 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 8340 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 8400 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 8460 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 8520 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 8580 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 8640 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 8700 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 8760 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 8820 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 8880 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 8940 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 9000 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 9060 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 9120 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 9180 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 9240 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 9300 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 9360 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 9420 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 9480 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 9540 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 9600 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 9660 tgattacgcc aagctttgct cctaggagtt tcctaatact tcccaaactc aaatatataa 9720 agcatttgac ttgttctatg ccctaggggg cggggggaag ctaagccagc tttttttaac 9780 atttaaaatg ttaattccat tttaaatgca cagatgtttt tatttcataa gggtttcaat 9840 gtgcatgaat gctgcaatat tcctgttacc aaagctagta taaataaaaa tagataaacg 9900 tggaaattac ttagagtttc tgtcattaac gtttccttcc tcagttgaca acataaatgc 9960 gctgctgagc aagccagttt gcatctgtca ggatcaattt cccattatgc cagtcatatt 10020 aattactagt caattagttg atttttattt ttgacatata catgtgaatg aaagacccca 10080 cctgtaggtt tggcaagcta gcttaagt 10108 <210> 34 <211> 9901 <212> DNA <213> Artificial Sequence <220> <223> sRRVe-F1-TK vector <400> 34 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacgtgat 1800 ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 1860 agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 1920 aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 1980 aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 2040 gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 2100 acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 2160 tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 2220 ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 2280 cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 2340 cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 2400 aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 2460 aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 2520 ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 2580 tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 2640 tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 2700 tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 2760 gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 2820 cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 2880 taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 2940 tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 3000 acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 3060 gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 3120 ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 3180 atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 3240 agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 3300 ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 3360 gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 3420 aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 3480 gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 3540 aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 3600 caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 3660 caatcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac 3720 tcaacaatat caccagctga agcctataga gtacgagcca tagcacgtga aggctgccga 3780 ccccgggggt ggacatcctc tagactgcca tggcgcggat ctcgacgttt aaacgggtga 3840 atcaacagga aagtcccatt ggagccaagt acactgagtc aatagggact ttccattggg 3900 ttttgcccag tacaaaaggt caataggggg tgagtcaatg ggtttttccc attattggca 3960 cgtacataag gtcaataggg gtgagtcatt gggtttttcc agccaattta attaaaacgc 4020 catgtacttt cccaccattg acgtcaatgg gctattgaaa ctaatgcaac gtgaccttta 4080 aacggtactt tcccatagct gattaatggg aaagtaccgt tctcgagcca atacacgtca 4140 atgggaagtg aaagggcagc caaaacgtaa caccgccccg gttttccctg gaaattccat 4200 attggcacgc attctattgg ctgagctgcg ttcacgtggg tataagaggc gcgaccagcg 4260 tcggtaccgt cgcagtcttc ggtctgacca ccgtagaacg cagagcggcc gctttagttt 4320 atggtgacat ggcttcgtac ccctgccatc aacacgcgtc tgcgttcgac caggctgcgc 4380 gttctcgcgg ccatagcaac cgacgtacgg cgttgcgccc tcgccggcag caagaagcca 4440 cggaagtccg cctggagcag aaaatgccca cgctactgcg ggtttatata gacggtcctc 4500 acgggatggg gaaaaccacc accacgcaac tgctggtggc cctgggttcg cgcgacgata 4560 tcgtctacgt acccgagccg atgacttact ggcaggtgct gggggcttcc gagacaatcg 4620 cgaacatcta caccacacaa caccgcctcg accagggtga gatatcggcc ggggacgcgg 4680 cggtggtaat gacaagcgcc cagataacaa tgggcatgcc ttatgccgtg accgacgccg 4740 ttctggctcc tcatatcggg ggggaggctg ggagctcaca tgccccgccc ccggccctca 4800 ccctcatctt cgaccgccat cccatcgccg ccctcctgtg ctacccggcc gcgcgatacc 4860 ttatgggcag catgaccccc caggccgtgc tggcgttcgt ggccctcatc ccgccgacct 4920 tgcccggcac aaacatcgtg ttgggggccc ttccggagga cagacacatc gaccgcctgg 4980 ccaaacgcca gcgccccggc gagcggcttg acctggctat gctggccgcg attcgccgcg 5040 tttacgggct gcttgccaat acggtgcggt atctgcaggg cggcgggtcg tggcgggagg 5100 attggggaca gctttcgggg acggccgtgc cgccccaggg tgccgagccc cagagcaacg 5160 cgggcccacg accccatatc ggggacacgt tatttaccct gtttcgggcc cccgagttgc 5220 tggcccccaa cggcgacctg tacaacgtgt ttgcctgggc cttggacgtc ttggccaaac 5280 gcctccgtcc catgcacgtc tttatcctgg attacgacca atcgcccgcc ggctgccggg 5340 acgccctgct gcaacttacc tccgggatgg tccagaccca cgtcaccacc cccggctcca 5400 taccgacgat ctgcgacctg gcgcgcacgt ttgcccggga gatgggggag gctaactgat 5460 cgagaaacgc gtcgacccat cgaacatcga tggtaccaga tccgataaaa taaaagattt 5520 tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag 5580 cttaagtaac gccattttgc aaggcatgga aaaatacata actgagaata gagaagttca 5640 gatcaaggtc aggaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa 5700 gcagttcctg ccccggctca gggccaagaa cagatggaac agctgaatat gggccaaaca 5760 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga 5820 tgcggtccag ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg 5880 acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt 5940 cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact cggggcgcca 6000 gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg 6060 catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg 6120 tcagcggggg tctttcacac atgcagcatg tatcaaaatt aatttggttt tttttcttaa 6180 gtatttacat taaatggcca tagtacttaa agttacattg gcttccttga aataaacatg 6240 gagtattcag aatgtgtcat aaatatttct aattttaaga tagtatctcc attggctttc 6300 tactttttct tttatttttt tttgtcctct gtcttccatt tgttgttgtt gttgtttgtt 6360 tgtttgtttg ttggttggtt ggttaatttt tttttaaaga tcctacacta tagttcaagc 6420 tagactatta gctactctgt aacccagggt gaccttgaag tcatgggtag cctgctgttt 6480 tagccttccc acatctaaga ttacaggtat gagctatcat ttttggtata tgattgattg 6540 attgattgat gtgtgtgtgt gtgattgtgt ttgtgtgtgt gactgtgaaa atgtgtgtat 6600 gggtgtgtgt gaatgtgtgt atgtatgtgt gtgtgtgagt gtgtgtgtgt gtgtgtgcat 6660 gtgtgtgtgt gtgactgtgt ctatgtgtat gactgtgtgt gtgtgtgtgt gtgtgtgtgt 6720 gtgtgtgtgt gtgtgtgtgt tgtgaaaaaa tattctatgg tagtgagagc caacgctccg 6780 gctcaggtgt caggttggtt tttgagacag agtctttcac ttagcttgga attcactggc 6840 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc 6900 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 6960 ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca 7020 tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc 7080 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 7140 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 7200 gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 7260 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 7320 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 7380 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 7440 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 7500 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 7560 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 7620 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 7680 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 7740 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 7800 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 7860 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 7920 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 7980 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 8040 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 8100 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 8160 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 8220 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 8280 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 8340 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 8400 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 8460 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 8520 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 8580 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 8640 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 8700 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 8760 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 8820 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 8880 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 8940 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 9000 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 9060 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 9120 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 9180 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 9240 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt 9300 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta 9360 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg 9420 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttt gctcctagga 9480 gtttcctaat acttcccaaa ctcaaatata taaagcattt gacttgttct atgccctagg 9540 gggcgggggg aagctaagcc agcttttttt aacatttaaa atgttaattc cattttaaat 9600 gcacagatgt ttttatttca taagggtttc aatgtgcatg aatgctgcaa tattcctgtt 9660 accaaagcta gtataaataa aaatagataa acgtggaaat tacttagagt ttctgtcatt 9720 aacgtttcct tcctcagttg acaacataaa tgcgctgctg agcaagccag tttgcatctg 9780 tcaggatcaa tttcccatta tgccagtcat attaattact agtcaattag ttgattttta 9840 tttttgacat atacatgtga atgaaagacc ccacctgtag gtttggcaag ctagcttaag 9900 t 9901 <210> 35 <211> 9768 <212> DNA <213> Artificial Sequence <220> <223> sRRVe-F2-TK vector <400> 35 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacgtgat 1800 ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 1860 agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 1920 aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 1980 aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 2040 gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 2100 acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 2160 tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 2220 ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 2280 cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 2340 cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 2400 aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 2460 aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 2520 ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 2580 tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 2640 tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 2700 tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 2760 gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 2820 cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 2880 taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 2940 tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 3000 acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 3060 gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 3120 ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 3180 atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 3240 agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 3300 ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 3360 gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 3420 aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 3480 gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 3540 aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 3600 caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 3660 caatcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac 3720 tcaacaatat caccagctga agcctataga gtacgagcca tagcacgtga aggctgccga 3780 ccccgggggt ggacatcctc tagactgcca tggcgcggat ctcgacgttt aaacaaggtc 3840 aataggggtg agtcattggg tttttccagc caatttaatt aaaacgccat gtactttccc 3900 accattgacg tcaatgggct attgaaacta atgcaacgtg acctttaaac ggtactttcc 3960 catagctgat taatgggaaa gtaccgttct cgagccaata cacgtcaatg ggaagtgaaa 4020 gggcagccaa aacgtaacac cgccccggtt ttccctggaa attccatatt ggcacgcatt 4080 ctattggctg agctgcgttc acgtgggtat aagaggcgcg accagcgtcg gtaccgtcgc 4140 agtcttcggt ctgaccaccg tagaacgcag agcggccgct ttagtttatg gtgacatggc 4200 ttcgtacccc tgccatcaac acgcgtctgc gttcgaccag gctgcgcgtt ctcgcggcca 4260 tagcaaccga cgtacggcgt tgcgccctcg ccggcagcaa gaagccacgg aagtccgcct 4320 ggagcagaaa atgcccacgc tactgcgggt ttatatagac ggtcctcacg ggatggggaa 4380 aaccaccacc acgcaactgc tggtggccct gggttcgcgc gacgatatcg tctacgtacc 4440 cgagccgatg acttactggc aggtgctggg ggcttccgag acaatcgcga acatctacac 4500 cacacaacac cgcctcgacc agggtgagat atcggccggg gacgcggcgg tggtaatgac 4560 aagcgcccag ataacaatgg gcatgcctta tgccgtgacc gacgccgttc tggctcctca 4620 tatcgggggg gaggctggga gctcacatgc cccgcccccg gccctcaccc tcatcttcga 4680 ccgccatccc atcgccgccc tcctgtgcta cccggccgcg cgatacctta tgggcagcat 4740 gaccccccag gccgtgctgg cgttcgtggc cctcatcccg ccgaccttgc ccggcacaaa 4800 catcgtgttg ggggcccttc cggaggacag acacatcgac cgcctggcca aacgccagcg 4860 ccccggcgag cggcttgacc tggctatgct ggccgcgatt cgccgcgttt acgggctgct 4920 tgccaatacg gtgcggtatc tgcagggcgg cgggtcgtgg cgggaggatt ggggacagct 4980 ttcggggacg gccgtgccgc cccagggtgc cgagccccag agcaacgcgg gcccacgacc 5040 ccatatcggg gacacgttat ttaccctgtt tcgggccccc gagttgctgg cccccaacgg 5100 cgacctgtac aacgtgtttg cctgggcctt ggacgtcttg gccaaacgcc tccgtcccat 5160 gcacgtcttt atcctggatt acgaccaatc gcccgccggc tgccgggacg ccctgctgca 5220 acttacctcc gggatggtcc agacccacgt caccaccccc ggctccatac cgacgatctg 5280 cgacctggcg cgcacgtttg cccgggagat gggggaggct aactgatcga gaaacgcgtc 5340 gacccatcga acatcgatgg taccagatcc gataaaataa aagattttat ttagtctcca 5400 gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt aagtaacgcc 5460 attttgcaag gcatggaaaa atacataact gagaatagag aagttcagat caaggtcagg 5520 aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc 5580 cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga tatctgtggt 5640 aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtccagccc 5700 tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc 5760 ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc 5820 tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc ctccgattga 5880 ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg 5940 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct 6000 ttcacacatg cagcatgtat caaaattaat ttggtttttt ttcttaagta tttacattaa 6060 atggccatag tacttaaagt tacattggct tccttgaaat aaacatggag tattcagaat 6120 gtgtcataaa tatttctaat tttaagatag tatctccatt ggctttctac tttttctttt 6180 attttttttt gtcctctgtc ttccatttgt tgttgttgtt gtttgtttgt ttgtttgttg 6240 gttggttggt taattttttt ttaaagatcc tacactatag ttcaagctag actattagct 6300 actctgtaac ccagggtgac cttgaagtca tgggtagcct gctgttttag ccttcccaca 6360 tctaagatta caggtatgag ctatcatttt tggtatatga ttgattgatt gattgatgtg 6420 tgtgtgtgtg attgtgtttg tgtgtgtgac tgtgaaaatg tgtgtatggg tgtgtgtgaa 6480 tgtgtgtatg tatgtgtgtg tgtgagtgtg tgtgtgtgtg tgtgcatgtg tgtgtgtgtg 6540 actgtgtcta tgtgtatgac tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 6600 tgtgtgttgt gaaaaaatat tctatggtag tgagagccaa cgctccggct caggtgtcag 6660 gttggttttt gagacagagt ctttcactta gcttggaatt cactggccgt cgttttacaa 6720 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 6780 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 6840 agcctgaatg gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 6900 tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag 6960 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc 7020 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 7080 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc 7140 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc 7200 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 7260 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc 7320 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg 7380 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat 7440 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 7500 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa 7560 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa 7620 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt 7680 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 7740 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 7800 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg 7860 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 7920 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 7980 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg 8040 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg 8100 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg 8160 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 8220 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 8280 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 8340 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 8400 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 8460 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta 8520 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 8580 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 8640 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 8700 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 8760 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 8820 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 8880 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 8940 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 9000 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 9060 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct 9120 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa 9180 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct 9240 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 9300 acaggaaaca gctatgacca tgattacgcc aagctttgct cctaggagtt tcctaatact 9360 tcccaaactc aaatatataa agcatttgac ttgttctatg ccctaggggg cggggggaag 9420 ctaagccagc tttttttaac atttaaaatg ttaattccat tttaaatgca cagatgtttt 9480 tatttcataa gggtttcaat gtgcatgaat gctgcaatat tcctgttacc aaagctagta 9540 taaataaaaa tagataaacg tggaaattac ttagagtttc tgtcattaac gtttccttcc 9600 tcagttgaca acataaatgc gctgctgagc aagccagttt gcatctgtca ggatcaattt 9660 cccattatgc cagtcatatt aattactagt caattagttg atttttattt ttgacatata 9720 catgtgaatg aaagacccca cctgtaggtt tggcaagcta gcttaagt 9768 <210> 36 <211> 9668 <212> DNA <213> Artificial Sequence <220> <223> sRRVe-F3-TK vector <400> 36 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacgtgat 1800 ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 1860 agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 1920 aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 1980 aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 2040 gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 2100 acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 2160 tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 2220 ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 2280 cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 2340 cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 2400 aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 2460 aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 2520 ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 2580 tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 2640 tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 2700 tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 2760 gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 2820 cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 2880 taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 2940 tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 3000 acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 3060 gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 3120 ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 3180 atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 3240 agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 3300 ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 3360 gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 3420 aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 3480 gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 3540 aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 3600 caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 3660 caatcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac 3720 tcaacaatat caccagctga agcctataga gtacgagcca tagcacgtga aggctgccga 3780 ccccgggggt ggacatcctc tagactgcca tggcgcggat ctcgacgttt aaacaacgtg 3840 acctttaaac ggtactttcc catagctgat taatgggaaa gtaccgttct cgagccaata 3900 cacgtcaatg ggaagtgaaa gggcagccaa aacgtaacac cgccccggtt ttccctggaa 3960 attccatatt ggcacgcatt ctattggctg agctgcgttc acgtgggtat aagaggcgcg 4020 accagcgtcg gtaccgtcgc agtcttcggt ctgaccaccg tagaacgcag agcggccgct 4080 ttagtttatg gtgacatggc ttcgtacccc tgccatcaac acgcgtctgc gttcgaccag 4140 gctgcgcgtt ctcgcggcca tagcaaccga cgtacggcgt tgcgccctcg ccggcagcaa 4200 gaagccacgg aagtccgcct ggagcagaaa atgcccacgc tactgcgggt ttatatagac 4260 ggtcctcacg ggatggggaa aaccaccacc acgcaactgc tggtggccct gggttcgcgc 4320 gacgatatcg tctacgtacc cgagccgatg acttactggc aggtgctggg ggcttccgag 4380 acaatcgcga acatctacac cacacaacac cgcctcgacc agggtgagat atcggccggg 4440 gacgcggcgg tggtaatgac aagcgcccag ataacaatgg gcatgcctta tgccgtgacc 4500 gacgccgttc tggctcctca tatcgggggg gaggctggga gctcacatgc cccgcccccg 4560 gccctcaccc tcatcttcga ccgccatccc atcgccgccc tcctgtgcta cccggccgcg 4620 cgatacctta tgggcagcat gaccccccag gccgtgctgg cgttcgtggc cctcatcccg 4680 ccgaccttgc ccggcacaaa catcgtgttg ggggcccttc cggaggacag acacatcgac 4740 cgcctggcca aacgccagcg ccccggcgag cggcttgacc tggctatgct ggccgcgatt 4800 cgccgcgttt acgggctgct tgccaatacg gtgcggtatc tgcagggcgg cgggtcgtgg 4860 cgggaggatt ggggacagct ttcggggacg gccgtgccgc cccagggtgc cgagccccag 4920 agcaacgcgg gcccacgacc ccatatcggg gacacgttat ttaccctgtt tcgggccccc 4980 gagttgctgg cccccaacgg cgacctgtac aacgtgtttg cctgggcctt ggacgtcttg 5040 gccaaacgcc tccgtcccat gcacgtcttt atcctggatt acgaccaatc gcccgccggc 5100 tgccgggacg ccctgctgca acttacctcc gggatggtcc agacccacgt caccaccccc 5160 ggctccatac cgacgatctg cgacctggcg cgcacgtttg cccgggagat gggggaggct 5220 aactgatcga gaaacgcgtc gacccatcga acatcgatgg taccagatcc gataaaataa 5280 aagattttat ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc 5340 aagctagctt aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagag 5400 aagttcagat caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct 5460 gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg 5520 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt 5580 ccccagatgc ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc 5640 cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc 5700 ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg 5760 ggcgccagtc ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt 5820 gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga 5880 ctacccgtca gcgggggtct ttcacacatg cagcatgtat caaaattaat ttggtttttt 5940 ttcttaagta tttacattaa atggccatag tacttaaagt tacattggct tccttgaaat 6000 aaacatggag tattcagaat gtgtcataaa tatttctaat tttaagatag tatctccatt 6060 ggctttctac tttttctttt attttttttt gtcctctgtc ttccatttgt tgttgttgtt 6120 gtttgtttgt ttgtttgttg gttggttggt taattttttt ttaaagatcc tacactatag 6180 ttcaagctag actattagct actctgtaac ccagggtgac cttgaagtca tgggtagcct 6240 gctgttttag ccttcccaca tctaagatta caggtatgag ctatcatttt tggtatatga 6300 ttgattgatt gattgatgtg tgtgtgtgtg attgtgtttg tgtgtgtgac tgtgaaaatg 6360 tgtgtatggg tgtgtgtgaa tgtgtgtatg tatgtgtgtg tgtgagtgtg tgtgtgtgtg 6420 tgtgcatgtg tgtgtgtgtg actgtgtcta tgtgtatgac tgtgtgtgtg tgtgtgtgtg 6480 tgtgtgtgtg tgtgtgtgtg tgtgtgttgt gaaaaaatat tctatggtag tgagagccaa 6540 cgctccggct caggtgtcag gttggttttt gagacagagt ctttcactta gcttggaatt 6600 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 6660 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 6720 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc 6780 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg 6840 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 6900 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 6960 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 7020 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 7080 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 7140 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 7200 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 7260 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 7320 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 7380 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 7440 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 7500 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 7560 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 7620 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 7680 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 7740 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 7800 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 7860 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 7920 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 7980 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 8040 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 8100 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 8160 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 8220 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 8280 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 8340 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc 8400 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 8460 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 8520 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 8580 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 8640 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 8700 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 8760 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 8820 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 8880 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 8940 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 9000 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 9060 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca 9120 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg 9180 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctttgct 9240 cctaggagtt tcctaatact tcccaaactc aaatatataa agcatttgac ttgttctatg 9300 ccctaggggg cggggggaag ctaagccagc tttttttaac atttaaaatg ttaattccat 9360 tttaaatgca cagatgtttt tatttcataa gggtttcaat gtgcatgaat gctgcaatat 9420 tcctgttacc aaagctagta taaataaaaa tagataaacg tggaaattac ttagagtttc 9480 tgtcattaac gtttccttcc tcagttgaca acataaatgc gctgctgagc aagccagttt 9540 gcatctgtca ggatcaattt cccattatgc cagtcatatt aattactagt caattagttg 9600 atttttattt ttgacatata catgtgaatg aaagacccca cctgtaggtt tggcaagcta 9660 gcttaagt 9668 <210> 37 <211> 9592 <212> DNA <213> Artificial Sequence <220> <223> sRRVe-F4-TK vector <400> 37 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacgtgat 1800 ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 1860 agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 1920 aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 1980 aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 2040 gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 2100 acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 2160 tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 2220 ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 2280 cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 2340 cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 2400 aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 2460 aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 2520 ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 2580 tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 2640 tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 2700 tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 2760 gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 2820 cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 2880 taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 2940 tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 3000 acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 3060 gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 3120 ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 3180 atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 3240 agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 3300 ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 3360 gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 3420 aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 3480 gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 3540 aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 3600 caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 3660 caatcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac 3720 tcaacaatat caccagctga agcctataga gtacgagcca tagcacgtga aggctgccga 3780 ccccgggggt ggacatcctc tagactgcca tggcgcggat ctcgacgttt aaacggaagt 3840 gaaagggcag ccaaaacgta acaccgcccc ggttttccct ggaaattcca tattggcacg 3900 cattctattg gctgagctgc gttcacgtgg gtataagagg cgcgaccagc gtcggtaccg 3960 tcgcagtctt cggtctgacc accgtagaac gcagagcggc cgctttagtt tatggtgaca 4020 tggcttcgta cccctgccat caacacgcgt ctgcgttcga ccaggctgcg cgttctcgcg 4080 gccatagcaa ccgacgtacg gcgttgcgcc ctcgccggca gcaagaagcc acggaagtcc 4140 gcctggagca gaaaatgccc acgctactgc gggtttatat agacggtcct cacgggatgg 4200 ggaaaaccac caccacgcaa ctgctggtgg ccctgggttc gcgcgacgat atcgtctacg 4260 tacccgagcc gatgacttac tggcaggtgc tgggggcttc cgagacaatc gcgaacatct 4320 acaccacaca acaccgcctc gaccagggtg agatatcggc cggggacgcg gcggtggtaa 4380 tgacaagcgc ccagataaca atgggcatgc cttatgccgt gaccgacgcc gttctggctc 4440 ctcatatcgg gggggaggct gggagctcac atgccccgcc cccggccctc accctcatct 4500 tcgaccgcca tcccatcgcc gccctcctgt gctacccggc cgcgcgatac cttatgggca 4560 gcatgacccc ccaggccgtg ctggcgttcg tggccctcat cccgccgacc ttgcccggca 4620 caaacatcgt gttgggggcc cttccggagg acagacacat cgaccgcctg gccaaacgcc 4680 agcgccccgg cgagcggctt gacctggcta tgctggccgc gattcgccgc gtttacgggc 4740 tgcttgccaa tacggtgcgg tatctgcagg gcggcgggtc gtggcgggag gattggggac 4800 agctttcggg gacggccgtg ccgccccagg gtgccgagcc ccagagcaac gcgggcccac 4860 gaccccatat cggggacacg ttatttaccc tgtttcgggc ccccgagttg ctggccccca 4920 acggcgacct gtacaacgtg tttgcctggg ccttggacgt cttggccaaa cgcctccgtc 4980 ccatgcacgt ctttatcctg gattacgacc aatcgcccgc cggctgccgg gacgccctgc 5040 tgcaacttac ctccgggatg gtccagaccc acgtcaccac ccccggctcc ataccgacga 5100 tctgcgacct ggcgcgcacg tttgcccggg agatggggga ggctaactga tcgagaaacg 5160 cgtcgaccca tcgaacatcg atggtaccag atccgataaa ataaaagatt ttatttagtc 5220 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa 5280 cgccattttg caaggcatgg aaaaatacat aactgagaat agagaagttc agatcaaggt 5340 caggaacaga tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct 5400 gccccggctc agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg 5460 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca 5520 gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat 5580 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt 5640 ctgctccccg agctcaataa aagagcccac aacccctcac tcggggcgcc agtcctccga 5700 ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact 5760 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg 5820 gtctttcaca catgcagcat gtatcaaaat taatttggtt ttttttctta agtatttaca 5880 ttaaatggcc atagtactta aagttacatt ggcttccttg aaataaacat ggagtattca 5940 gaatgtgtca taaatatttc taattttaag atagtatctc cattggcttt ctactttttc 6000 ttttattttt ttttgtcctc tgtcttccat ttgttgttgt tgttgtttgt ttgtttgttt 6060 gttggttggt tggttaattt ttttttaaag atcctacact atagttcaag ctagactatt 6120 agctactctg taacccaggg tgaccttgaa gtcatgggta gcctgctgtt ttagccttcc 6180 cacatctaag attacaggta tgagctatca tttttggtat atgattgatt gattgattga 6240 tgtgtgtgtg tgtgattgtg tttgtgtgtg tgactgtgaa aatgtgtgta tgggtgtgtg 6300 tgaatgtgtg tatgtatgtg tgtgtgtgag tgtgtgtgtg tgtgtgtgca tgtgtgtgtg 6360 tgtgactgtg tctatgtgta tgactgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 6420 tgtgtgtgtg ttgtgaaaaa atattctatg gtagtgagag ccaacgctcc ggctcaggtg 6480 tcaggttggt ttttgagaca gagtctttca cttagcttgg aattcactgg ccgtcgtttt 6540 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc 6600 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 6660 gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg 6720 tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag 6780 ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 6840 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 6900 gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa 6960 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg 7020 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 7080 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 7140 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 7200 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 7260 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 7320 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 7380 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 7440 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 7500 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 7560 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 7620 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 7680 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 7740 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 7800 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 7860 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 7920 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 7980 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 8040 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 8100 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 8160 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 8220 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 8280 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 8340 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 8400 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 8460 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 8520 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 8580 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 8640 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 8700 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 8760 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 8820 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 8880 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 8940 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg 9000 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 9060 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 9120 tcacacagga aacagctatg accatgatta cgccaagctt tgctcctagg agtttcctaa 9180 tacttcccaa actcaaatat ataaagcatt tgacttgttc tatgccctag ggggcggggg 9240 gaagctaagc cagctttttt taacatttaa aatgttaatt ccattttaaa tgcacagatg 9300 tttttatttc ataagggttt caatgtgcat gaatgctgca atattcctgt taccaaagct 9360 agtataaata aaaatagata aacgtggaaa ttacttagag tttctgtcat taacgtttcc 9420 ttcctcagtt gacaacataa atgcgctgct gagcaagcca gtttgcatct gtcaggatca 9480 atttcccatt atgccagtca tattaattac tagtcaatta gttgattttt atttttgaca 9540 tatacatgtg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gt 9592 <210> 38 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> MCMV(F4)-EcoRI-F <400> 38 cggaattcgg aagtgaaagg gcagccaaa 29 <210> 39 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> MCMV-NotI-MluI-EcoRI-R <400> 39 cggaattcac gcgtgcgcgg ccgctctgcg ttctacggtg gtcagacc 48 <210> 40 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> hopt-yCD-NotI-F <400> 40 cggcggccgc atggttactg ggggaatggc 30 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hopt-yCD-MluI-R <400> 41 acgcgtttat tccccgatgt cttcgaa 27 <210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> AmEnv961F <400> 42 acaagaccca agaatgttgg ct 22 <210> 43 <211> 1671 <212> DNA <213> Artificial Sequence <220> <223> hCD19 variant 2 <400> 43 atgccacctc ctcgcctcct cttcttcctc ctcttcctca cccccatgga agtcaggccc 60 gaggaacctc tagtggtgaa ggtggaagag ggagataacg ctgtgctgca gtgcctcaag 120 gggacctcag atggccccac tcagcagctg acctggtctc gggagtcccc gcttaaaccc 180 ttcttaaaac tcagcctggg gctgccaggc ctgggaatcc acatgaggcc cctggccatc 240 tggcttttca tcttcaacgt ctctcaacag atggggggct tctacctgtg ccagccgggg 300 cccccctctg agaaggcctg gcagcctggc tggacagtca atgtggaggg cagcggggag 360 ctgttccggt ggaatgtttc ggacctaggt ggcctgggct gtggcctgaa gaacaggtcc 420 tcagagggcc ccagctcccc ttccgggaag ctcatgagcc ccaagctgta tgtgtgggcc 480 aaagaccgcc ctgagatctg ggagggagag cctccgtgtc tcccaccgag ggacagcctg 540 aaccagagcc tcagccagga cctcaccatg gcccctggct ccacactctg gctgtcctgt 600 ggggtacccc ctgactctgt gtccaggggc cccctctcct ggacccatgt gcaccccaag 660 gggcctaagt cattgctgag cctagagctg aaggacgatc gcccggccag agatatgtgg 720 gtaatggaga cgggtctgtt gttgccccgg gccacagctc aagacgctgg aaagtattat 780 tgtcaccgtg gcaacctgac catgtcattc cacctggaga tcactgctcg gccagtacta 840 tggcactggc tgctgaggac tggtggctgg aaggtctcag ctgtgacttt ggcttatctg 900 atcttctgcc tgtgttccct tgtgggcatt cttcatcttc aaagagccct ggtcctgagg 960 aggaaaagaa agcgaatgac tgaccccacc aggagattct tcaaagtgac gcctccccca 1020 ggaagcgggc cccagaacca gtacgggaac gtgctgtctc tccccacacc cacctcaggc 1080 ctcggacgcg cccagcgttg ggccgcaggc ctggggggca ctgccccgtc ttatggaaac 1140 ccgagcagcg acgtccaggc ggatggagcc ttggggtccc ggagcccgcc gggagtgggc 1200 ccagaagaag aggaagggga gggctatgag gaacctgaca gtgaggagga ctccgagttc 1260 tatgagaacg actccaacct tgggcaggac cagctctccc aggatggcag cggctacgag 1320 aaccctgagg atgagcccct gggtcctgag gatgaagact ccttctccaa cgctgagtct 1380 tatgagaacg aggatgaaga gctgacccag ccggtcgcca ggacaatgga cttcctgagc 1440 cctcatgggt cagcctggga ccccagccgg gaagcaacct ccctggggtc ccagtcctat 1500 gaggatatga gaggaatcct gtatgcagcc ccccagctcc gctccattcg gggccagcct 1560 ggacccaatc atgaggaaga tgcagactct tatgagaaca tggataatcc cgatgggcca 1620 gacccagcct ggggaggagg gggccgcatg ggcacctgga gcaccaggtg a 1671 <210> 44 <211> 11157 <212> DNA <213> Artificial Sequence <220> <223> sRRVgp-F4-hCD19 vector <400> 44 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 60 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 480 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 600 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 660 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 720 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 780 atcaatctaa agtatatatg agtaacctga tcaggactct tccttttcat gaacaataaa 840 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 900 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 960 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 1020 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1080 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 1140 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 1200 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 1260 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 1320 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 1380 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 1440 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 1500 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 1560 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 1620 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 1680 tgagtttttc taagaatttg tgaatgaaag accccacctg taggtttggc aagctagctt 1740 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat 1800 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1860 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1920 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1980 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2040 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2100 gcgcttatgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2160 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2220 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2280 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2340 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt 2400 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg 2460 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc 2520 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg 2580 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc 2640 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg 2700 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt 2760 tgtctgagaa tatgggccag actgttacca ctcccttaag tttgacctta ggtcactgga 2820 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 2880 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 2940 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 3000 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 3060 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 3120 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 3180 ctctaggcgc caaacctaaa cctcaagttc tttctgacag tggggggccg ctcatcgacc 3240 tacttacaga agaccccccg ccttataggg acccaagacc acccccttcc gacagggacg 3300 gaaatggtgg agaagcgacc cctgcgggag aggcaccgga cccctcccca atggcatctc 3360 gcctacgtgg gagacgggag ccccctgtgg ccgactccac tacctcgcag gcattccccc 3420 tccgcgcagg aggaaacgga cagcttcaat actggccgtt ctcctcttct gacctttaca 3480 actggaaaaa taataaccct tctttttctg aagatccagg taaactgaca gctctgatcg 3540 agtctgttct catcacccat cagcccacct gggacgactg tcagcagctg ttggggactc 3600 tgctgaccgg agaagaaaaa caacgggtgc tcttagaggc tagaaaggcg gtgcggggcg 3660 atgatgggcg ccccactcaa ctgcccaatg aagtcgatgc cgcttttccc ctcgagcgcc 3720 cagactggga ttacaccacc caggcaggta ggaaccacct agtccactat cgccagttgc 3780 tcctagcggg tctccaaaac gcgggcagaa gccccaccaa tttggccaag gtaaaaggaa 3840 taacacaagg gcccaatgag tctccctcgg ccttcctaga gagacttaag gaagcctatc 3900 gcaggtacac tccttatgac cctgaggacc cagggcaaga aactaatgtg tctatgtctt 3960 tcatttggca gtctgcccca gacattggga gaaagttaga gaggttagaa gatttaaaaa 4020 acaagacgct tggagatttg gttagagagg cagaaaagat ctttaataaa cgagaaaccc 4080 cggaagaaag agaggaacgt atcaggagag aaacagagga aaaagaagaa cgccgtagga 4140 cagaggatga gcagaaagag aaagaaagag atcgtaggag acatagagag atgagcaagc 4200 tattggccac tgtcgttagt ggacagaaac aggatagaca gggaggagaa cgaaggaggt 4260 cccaactcga tcgcgaccag tgtgcctact gcaaagaaaa ggggcactgg gctaaagatt 4320 gtcccaagaa accacgagga cctcggggac caagacccca gacctccctc ctgaccctag 4380 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 4440 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 4500 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 4560 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 4620 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 4680 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 4740 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 4800 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 4860 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 4920 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 4980 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 5040 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 5100 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 5160 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 5220 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 5280 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 5340 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 5400 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 5460 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 5520 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 5580 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 5640 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 5700 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 5760 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 5820 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 5880 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 5940 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 6000 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 6060 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 6120 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 6180 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 6240 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 6300 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 6360 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 6420 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 6480 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 6540 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 6600 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 6660 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 6720 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 6780 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 6840 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 6900 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 6960 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 7020 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 7080 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 7140 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 7200 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 7260 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 7320 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 7380 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 7440 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 7500 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 7560 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 7620 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 7680 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 7740 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 7800 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 7860 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 7920 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 7980 ccccctaatc cccttaattc ttctgatgct cagaggggtc agtaaacgaa ttcggaagtg 8040 aaagggcagc caaaacgtaa caccgccccg gttttccctg gaaattccat attggcacgc 8100 attctattgg ctgagctgcg ttcacgtggg tataagaggc gcgaccagcg tcggtaccgt 8160 cgcagtcttc ggtctgacca ccgtagaacg cagagcggcc gcatgccacc tcctcgcctc 8220 ctcttcttcc tcctcttcct cacccccatg gaagtcaggc ccgaggaacc tctagtggtg 8280 aaggtggaag agggagataa cgctgtgctg cagtgcctca aggggacctc agatggcccc 8340 actcagcagc tgacctggtc tcgggagtcc ccgcttaaac ccttcttaaa actcagcctg 8400 gggctgccag gcctgggaat ccacatgagg cccctggcca tctggctttt catcttcaac 8460 gtctctcaac agatgggggg cttctacctg tgccagccgg ggcccccctc tgagaaggcc 8520 tggcagcctg gctggacagt caatgtggag ggcagcgggg agctgttccg gtggaatgtt 8580 tcggacctag gtggcctggg ctgtggcctg aagaacaggt cctcagaggg ccccagctcc 8640 ccttccggga agctcatgag ccccaagctg tatgtgtggg ccaaagaccg ccctgagatc 8700 tgggagggag agcctccgtg tctcccaccg agggacagcc tgaaccagag cctcagccag 8760 gacctcacca tggcccctgg ctccacactc tggctgtcct gtggggtacc ccctgactct 8820 gtgtccaggg gccccctctc ctggacccat gtgcacccca aggggcctaa gtcattgctg 8880 agcctagagc tgaaggacga tcgcccggcc agagatatgt gggtaatgga gacgggtctg 8940 ttgttgcccc gggccacagc tcaagacgct ggaaagtatt attgtcaccg tggcaacctg 9000 accatgtcat tccacctgga gatcactgct cggccagtac tatggcactg gctgctgagg 9060 actggtggct ggaaggtctc agctgtgact ttggcttatc tgatcttctg cctgtgttcc 9120 cttgtgggca ttcttcatct tcaaagagcc ctggtcctga ggaggaaaag aaagcgaatg 9180 actgacccca ccaggagatt cttcaaagtg acgcctcccc caggaagcgg gccccagaac 9240 cagtacggga acgtgctgtc tctccccaca cccacctcag gcctcggacg cgcccagcgt 9300 tgggccgcag gcctgggggg cactgccccg tcttatggaa acccgagcag cgacgtccag 9360 gcggatggag ccttggggtc ccggagcccg ccgggagtgg gcccagaaga agaggaaggg 9420 gagggctatg aggaacctga cagtgaggag gactccgagt tctatgagaa cgactccaac 9480 cttgggcagg accagctctc ccaggatggc agcggctacg agaaccctga ggatgagccc 9540 ctgggtcctg aggatgaaga ctccttctcc aacgctgagt cttatgagaa cgaggatgaa 9600 gagctgaccc agccggtcgc caggacaatg gacttcctga gccctcatgg gtcagcctgg 9660 gaccccagcc gggaagcaac ctccctgggg tcccagtcct atgaggatat gagaggaatc 9720 ctgtatgcag ccccccagct ccgctccatt cggggccagc ctggacccaa tcatgaggaa 9780 gatgcagact cttatgagaa catggataat cccgatgggc cagacccagc ctggggagga 9840 gggggccgca tgggcacctg gagcaccagg tgagtttaaa cacgcgtgaa ttcataaaat 9900 aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg 9960 gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa ctgagaatag 10020 agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca aacaggatat 10080 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca gctgaatatg 10140 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 10200 gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg tttccagggt 10260 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc 10320 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc 10380 ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca ataaaccctc 10440 ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt 10500 gactacccgt cagcgggggt ctttcattgt tacttaaagt tacattggct tccttgaaat 10560 aaacatggag tattcagaat gtgtcataaa tatttctaat tttaagatag tatctccatt 10620 ggctttctac tttttctttt attttttttt gtcctctgtc ttccatttgt tgttgttgtt 10680 gtttgtttgt ttgtttgttg gttggttggt taattttttt ttaaagatcc tacactatag 10740 ttcaagctag actattagct actctgtaac ccagggtgac cttgaagtca tgggtagcct 10800 gctgttttag ccttcccaca tctaagatta caggtatgag ctatcatttt tggtatattg 10860 attgattgat tgattgatgt gtgtgtgtgt gattgtgttt gtgtgtgtga ctgtgaaaat 10920 gtgtgtatgg gtgtgtgtga atgtgtgtat gtatgtgtgt gtgtgagtgt gtgtgtgtgt 10980 gtgtgcatgt gtgtgtgtgt gactgtgtct atgtgtatga ctgtgtgtgt gtgtgtgtgt 11040 gtgtgtgtgt gtgtgtgtgt gtgtgtgttg tgaaaaaata ttctatggta gtgagagcca 11100 acgctccggc tcaggtgtca ggttggtttt tgagacagag tctttcactt agcttgg 11157 <210> 45 <211> 10105 <212> DNA <213> Artificial Sequence <220> <223> sRRVe-F4-hCD19 vector <400> 45 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacgtgat 1800 ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 1860 agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 1920 aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 1980 aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 2040 gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 2100 acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 2160 tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 2220 ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 2280 cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 2340 cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 2400 aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 2460 aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 2520 ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 2580 tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 2640 tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 2700 tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 2760 gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 2820 cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 2880 taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 2940 tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 3000 acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 3060 gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 3120 ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 3180 atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 3240 agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 3300 ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 3360 gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 3420 aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 3480 gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 3540 aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 3600 caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 3660 caatcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac 3720 tcaacaatat caccagctga agcctataga gtacgagcca tagcacgtga aggctgccga 3780 ccccgggggt ggacatcctc tagactgcca tggcgcggat ctcgacgttt aaacggaagt 3840 gaaagggcag ccaaaacgta acaccgcccc ggttttccct ggaaattcca tattggcacg 3900 cattctattg gctgagctgc gttcacgtgg gtataagagg cgcgaccagc gtcggtaccg 3960 tcgcagtctt cggtctgacc accgtagaac gcagagcggc cgcatgccac ctcctcgcct 4020 cctcttcttc ctcctcttcc tcacccccat ggaagtcagg cccgaggaac ctctagtggt 4080 gaaggtggaa gagggagata acgctgtgct gcagtgcctc aaggggacct cagatggccc 4140 cactcagcag ctgacctggt ctcgggagtc cccgcttaaa cccttcttaa aactcagcct 4200 ggggctgcca ggcctgggaa tccacatgag gcccctggcc atctggcttt tcatcttcaa 4260 cgtctctcaa cagatggggg gcttctacct gtgccagccg gggcccccct ctgagaaggc 4320 ctggcagcct ggctggacag tcaatgtgga gggcagcggg gagctgttcc ggtggaatgt 4380 ttcggaccta ggtggcctgg gctgtggcct gaagaacagg tcctcagagg gccccagctc 4440 cccttccggg aagctcatga gccccaagct gtatgtgtgg gccaaagacc gccctgagat 4500 ctgggaggga gagcctccgt gtctcccacc gagggacagc ctgaaccaga gcctcagcca 4560 ggacctcacc atggcccctg gctccacact ctggctgtcc tgtggggtac cccctgactc 4620 tgtgtccagg ggccccctct cctggaccca tgtgcacccc aaggggccta agtcattgct 4680 gagcctagag ctgaaggacg atcgcccggc cagagatatg tgggtaatgg agacgggtct 4740 gttgttgccc cgggccacag ctcaagacgc tggaaagtat tattgtcacc gtggcaacct 4800 gaccatgtca ttccacctgg agatcactgc tcggccagta ctatggcact ggctgctgag 4860 gactggtggc tggaaggtct cagctgtgac tttggcttat ctgatcttct gcctgtgttc 4920 ccttgtgggc attcttcatc ttcaaagagc cctggtcctg aggaggaaaa gaaagcgaat 4980 gactgacccc accaggagat tcttcaaagt gacgcctccc ccaggaagcg ggccccagaa 5040 ccagtacggg aacgtgctgt ctctccccac acccacctca ggcctcggac gcgcccagcg 5100 ttgggccgca ggcctggggg gcactgcccc gtcttatgga aacccgagca gcgacgtcca 5160 ggcggatgga gccttggggt cccggagccc gccgggagtg ggcccagaag aagaggaagg 5220 ggagggctat gaggaacctg acagtgagga ggactccgag ttctatgaga acgactccaa 5280 ccttgggcag gaccagctct cccaggatgg cagcggctac gagaaccctg aggatgagcc 5340 cctgggtcct gaggatgaag actccttctc caacgctgag tcttatgaga acgaggatga 5400 agagctgacc cagccggtcg ccaggacaat ggacttcctg agccctcatg ggtcagcctg 5460 ggaccccagc cgggaagcaa cctccctggg gtcccagtcc tatgaggata tgagaggaat 5520 cctgtatgca gccccccagc tccgctccat tcggggccag cctggaccca atcatgagga 5580 agatgcagac tcttatgaga acatggataa tcccgatggg ccagacccag cctggggagg 5640 agggggccgc atgggcacct ggagcaccag gtgagtcgac ccatcgaaca tcgatggtac 5700 cagatccgat aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc 5760 ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaaata 5820 cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca gctgaatatg 5880 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg 5940 gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc cccggctcag 6000 ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta gagaaccatc 6060 agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt gaactaacca 6120 atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc 6180 cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg gtacccgtgt 6240 atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc ttgggagggt 6300 ctcctctgag tgattgacta cccgtcagcg ggggtctttc acacatgcag catgtatcaa 6360 aattaatttg gttttttttc ttaagtattt acattaaatg gccatagtac ttaaagttac 6420 attggcttcc ttgaaataaa catggagtat tcagaatgtg tcataaatat ttctaatttt 6480 aagatagtat ctccattggc tttctacttt ttcttttatt tttttttgtc ctctgtcttc 6540 catttgttgt tgttgttgtt tgtttgtttg tttgttggtt ggttggttaa ttttttttta 6600 aagatcctac actatagttc aagctagact attagctact ctgtaaccca gggtgacctt 6660 gaagtcatgg gtagcctgct gttttagcct tcccacatct aagattacag gtatgagcta 6720 tcatttttgg tatatgattg attgattgat tgatgtgtgt gtgtgtgatt gtgtttgtgt 6780 gtgtgactgt gaaaatgtgt gtatgggtgt gtgtgaatgt gtgtatgtat gtgtgtgtgt 6840 gagtgtgtgt gtgtgtgtgt gcatgtgtgt gtgtgtgact gtgtctatgt gtatgactgt 6900 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttgtgaa aaaatattct 6960 atggtagtga gagccaacgc tccggctcag gtgtcaggtt ggtttttgag acagagtctt 7020 tcacttagct tggaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 7080 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 7140 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct 7200 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 7260 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 7320 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 7380 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 7440 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 7500 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 7560 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 7620 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 7680 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 7740 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 7800 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 7860 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 7920 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 7980 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 8040 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 8100 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 8160 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 8220 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 8280 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 8340 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 8400 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 8460 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 8520 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 8580 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 8640 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 8700 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 8760 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 8820 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 8880 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 8940 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 9000 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 9060 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 9120 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 9180 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 9240 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 9300 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 9360 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 9420 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 9480 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat 9540 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 9600 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga 9660 ttacgccaag ctttgctcct aggagtttcc taatacttcc caaactcaaa tatataaagc 9720 atttgacttg ttctatgccc tagggggcgg ggggaagcta agccagcttt ttttaacatt 9780 taaaatgtta attccatttt aaatgcacag atgtttttat ttcataaggg tttcaatgtg 9840 catgaatgct gcaatattcc tgttaccaaa gctagtataa ataaaaatag ataaacgtgg 9900 aaattactta gagtttctgt cattaacgtt tccttcctca gttgacaaca taaatgcgct 9960 gctgagcaag ccagtttgca tctgtcagga tcaatttccc attatgccag tcatattaat 10020 tactagtcaa ttagttgatt tttatttttg acatatacat gtgaatgaaa gaccccacct 10080 gtaggtttgg caagctagct taagt 10105 <210> 46 <211> 9955 <212> DNA <213> Artificial Sequence <220> <223> sRRVgp-F4-hopt-yCD <400> 46 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 60 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 480 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 600 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 660 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 720 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 780 atcaatctaa agtatatatg agtaacctga tcaggactct tccttttcat gaacaataaa 840 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 900 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 960 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 1020 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1080 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 1140 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 1200 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 1260 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 1320 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 1380 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 1440 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 1500 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 1560 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 1620 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 1680 tgagtttttc taagaatttg tgaatgaaag accccacctg taggtttggc aagctagctt 1740 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat 1800 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1860 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1920 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1980 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2040 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2100 gcgcttatgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2160 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2220 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2280 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2340 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt 2400 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg 2460 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc 2520 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg 2580 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc 2640 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg 2700 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt 2760 tgtctgagaa tatgggccag actgttacca ctcccttaag tttgacctta ggtcactgga 2820 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 2880 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 2940 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 3000 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 3060 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 3120 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 3180 ctctaggcgc caaacctaaa cctcaagttc tttctgacag tggggggccg ctcatcgacc 3240 tacttacaga agaccccccg ccttataggg acccaagacc acccccttcc gacagggacg 3300 gaaatggtgg agaagcgacc cctgcgggag aggcaccgga cccctcccca atggcatctc 3360 gcctacgtgg gagacgggag ccccctgtgg ccgactccac tacctcgcag gcattccccc 3420 tccgcgcagg aggaaacgga cagcttcaat actggccgtt ctcctcttct gacctttaca 3480 actggaaaaa taataaccct tctttttctg aagatccagg taaactgaca gctctgatcg 3540 agtctgttct catcacccat cagcccacct gggacgactg tcagcagctg ttggggactc 3600 tgctgaccgg agaagaaaaa caacgggtgc tcttagaggc tagaaaggcg gtgcggggcg 3660 atgatgggcg ccccactcaa ctgcccaatg aagtcgatgc cgcttttccc ctcgagcgcc 3720 cagactggga ttacaccacc caggcaggta ggaaccacct agtccactat cgccagttgc 3780 tcctagcggg tctccaaaac gcgggcagaa gccccaccaa tttggccaag gtaaaaggaa 3840 taacacaagg gcccaatgag tctccctcgg ccttcctaga gagacttaag gaagcctatc 3900 gcaggtacac tccttatgac cctgaggacc cagggcaaga aactaatgtg tctatgtctt 3960 tcatttggca gtctgcccca gacattggga gaaagttaga gaggttagaa gatttaaaaa 4020 acaagacgct tggagatttg gttagagagg cagaaaagat ctttaataaa cgagaaaccc 4080 cggaagaaag agaggaacgt atcaggagag aaacagagga aaaagaagaa cgccgtagga 4140 cagaggatga gcagaaagag aaagaaagag atcgtaggag acatagagag atgagcaagc 4200 tattggccac tgtcgttagt ggacagaaac aggatagaca gggaggagaa cgaaggaggt 4260 cccaactcga tcgcgaccag tgtgcctact gcaaagaaaa ggggcactgg gctaaagatt 4320 gtcccaagaa accacgagga cctcggggac caagacccca gacctccctc ctgaccctag 4380 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 4440 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 4500 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 4560 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 4620 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 4680 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 4740 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 4800 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 4860 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 4920 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 4980 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 5040 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 5100 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 5160 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 5220 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 5280 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 5340 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 5400 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 5460 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 5520 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 5580 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 5640 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 5700 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 5760 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 5820 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 5880 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 5940 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 6000 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 6060 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 6120 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 6180 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 6240 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 6300 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 6360 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 6420 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 6480 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 6540 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 6600 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 6660 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 6720 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 6780 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 6840 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 6900 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 6960 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 7020 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 7080 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 7140 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 7200 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 7260 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 7320 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 7380 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 7440 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 7500 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 7560 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 7620 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 7680 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 7740 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 7800 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 7860 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 7920 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 7980 ccccctaatc cccttaattc ttctgatgct cagaggggtc agtaaacgaa ttcggaagtg 8040 aaagggcagc caaaacgtaa caccgccccg gttttccctg gaaattccat attggcacgc 8100 attctattgg ctgagctgcg ttcacgtggg tataagaggc gcgaccagcg tcggtaccgt 8160 cgcagtcttc ggtctgacca ccgtagaacg cagagcggcc gcatggttac tgggggaatg 8220 gcatctaagt gggatcagaa aggtatggac atcgcttatg aagaggctgc tctcggctac 8280 aaagagggtg gagtgcctat cggagggtgc ctgatcaaca acaaggacgg cagtgtgctg 8340 gggaggggcc acaatatgag gttccaaaaa ggctccgcca ctctccacgg ggaaattagt 8400 accctcgaga attgcggacg attggaaggg aaggtgtaca aggatacaac actgtacacc 8460 accctgtcac cctgtgatat gtgcacaggc gccattatca tgtacggaat ccctagatgt 8520 gtcgtggggg agaatgtaaa cttcaaaagt aagggggaga aatatctcca gacccggggg 8580 cacgaagtcg tcgttgtgga cgatgaacgg tgtaagaaga tcatgaagca gtttatcgat 8640 gagaggcccc aggactggtt cgaagacatc ggggaataaa cgcgtgaatt cataaaataa 8700 aagattttat ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc 8760 aagctagctt aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagag 8820 aagttcagat caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct 8880 gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg 8940 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt 9000 ccccagatgc ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc 9060 cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc 9120 ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg 9180 ggcgccagtc ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt 9240 gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga 9300 ctacccgtca gcgggggtct ttcattgtta cttaaagtta cattggcttc cttgaaataa 9360 acatggagta ttcagaatgt gtcataaata tttctaattt taagatagta tctccattgg 9420 ctttctactt tttcttttat ttttttttgt cctctgtctt ccatttgttg ttgttgttgt 9480 ttgtttgttt gtttgttggt tggttggtta attttttttt aaagatccta cactatagtt 9540 caagctagac tattagctac tctgtaaccc agggtgacct tgaagtcatg ggtagcctgc 9600 tgttttagcc ttcccacatc taagattaca ggtatgagct atcatttttg gtatattgat 9660 tgattgattg attgatgtgt gtgtgtgtga ttgtgtttgt gtgtgtgact gtgaaaatgt 9720 gtgtatgggt gtgtgtgaat gtgtgtatgt atgtgtgtgt gtgagtgtgt gtgtgtgtgt 9780 gtgcatgtgt gtgtgtgtga ctgtgtctat gtgtatgact gtgtgtgtgt gtgtgtgtgt 9840 gtgtgtgtgt gtgtgtgtgt gtgtgttgtg aaaaaatatt ctatggtagt gagagccaac 9900 gctccggctc aggtgtcagg ttggtttttg agacagagtc tttcacttag cttgg 9955 <210> 47 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> MCMV-F-EcoRI <400> 47 cggaattcaa caggaaagtc ccattgga 28 <210> 48 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> MCMV-R-PmeI-EcoRI <400> 48 cggaattcgt ttaaacctgc gttctacggt ggtcaga 37 <210> 49 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> TK-F-PmeI <400> 49 cggtttaaac atggcttcgt acccctgcca tc 32 <210> 50 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> TK-R-PmeI <400> 50 cggtttaaac tcagttagcc tcccccatct cc 32 <210> 51 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> hCD19t-NotI-F <400> 51 cggcggccgc atgccacctc ctcgcctcct cttc 34 <210> 52 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> hCD19t-MluI-R <400> 52 caacgcgttc atcttttcct cctcaggacc agg 33 <210> 53 <211> 972 <212> DNA <213> Artificial Sequence <220> <223> hCD19t polynucleotide sequence <400> 53 atgccacctc ctcgcctcct cttcttcctc ctcttcctca cccccatgga agtcaggccc 60 gaggaacctc tagtggtgaa ggtggaagag ggagataacg ctgtgctgca gtgcctcaag 120 gggacctcag atggccccac tcagcagctg acctggtctc gggagtcccc gcttaaaccc 180 ttcttaaaac tcagcctggg gctgccaggc ctgggaatcc acatgaggcc cctggccatc 240 tggcttttca tcttcaacgt ctctcaacag atggggggct tctacctgtg ccagccgggg 300 cccccctctg agaaggcctg gcagcctggc tggacagtca atgtggaggg cagcggggag 360 ctgttccggt ggaatgtttc ggacctaggt ggcctgggct gtggcctgaa gaacaggtcc 420 tcagagggcc ccagctcccc ttccgggaag ctcatgagcc ccaagctgta tgtgtgggcc 480 aaagaccgcc ctgagatctg ggagggagag cctccgtgtc tcccaccgag ggacagcctg 540 aaccagagcc tcagccagga cctcaccatg gcccctggct ccacactctg gctgtcctgt 600 ggggtacccc ctgactctgt gtccaggggc cccctctcct ggacccatgt gcaccccaag 660 gggcctaagt cattgctgag cctagagctg aaggacgatc gcccggccag agatatgtgg 720 gtaatggaga cgggtctgtt gttgccccgg gccacagctc aagacgctgg aaagtattat 780 tgtcaccgtg gcaacctgac catgtcattc cacctggaga tcactgctcg gccagtacta 840 tggcactggc tgctgaggac tggtggctgg aaggtctcag ctgtgacttt ggcttatctg 900 atcttctgcc tgtgttccct tgtgggcatt cttcatcttc aaagagccct ggtcctgagg 960 aggaaaagat ga 972 <210> 54 <211> 10450 <212> DNA <213> Artificial Sequence <220> <223> sRRVgp-F4-hCD19t vector <400> 54 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 60 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 480 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 600 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 660 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 720 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 780 atcaatctaa agtatatatg agtaacctga tcaggactct tccttttcat gaacaataaa 840 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 900 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 960 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 1020 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1080 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 1140 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 1200 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 1260 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 1320 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 1380 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 1440 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 1500 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 1560 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 1620 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 1680 tgagtttttc taagaatttg tgaatgaaag accccacctg taggtttggc aagctagctt 1740 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat 1800 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1860 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1920 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1980 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2040 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2100 gcgcttatgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2160 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2220 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2280 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2340 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt 2400 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg 2460 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc 2520 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg 2580 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc 2640 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg 2700 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt 2760 tgtctgagaa tatgggccag actgttacca ctcccttaag tttgacctta ggtcactgga 2820 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 2880 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 2940 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 3000 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 3060 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 3120 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 3180 ctctaggcgc caaacctaaa cctcaagttc tttctgacag tggggggccg ctcatcgacc 3240 tacttacaga agaccccccg ccttataggg acccaagacc acccccttcc gacagggacg 3300 gaaatggtgg agaagcgacc cctgcgggag aggcaccgga cccctcccca atggcatctc 3360 gcctacgtgg gagacgggag ccccctgtgg ccgactccac tacctcgcag gcattccccc 3420 tccgcgcagg aggaaacgga cagcttcaat actggccgtt ctcctcttct gacctttaca 3480 actggaaaaa taataaccct tctttttctg aagatccagg taaactgaca gctctgatcg 3540 agtctgttct catcacccat cagcccacct gggacgactg tcagcagctg ttggggactc 3600 tgctgaccgg agaagaaaaa caacgggtgc tcttagaggc tagaaaggcg gtgcggggcg 3660 atgatgggcg ccccactcaa ctgcccaatg aagtcgatgc cgcttttccc ctcgagcgcc 3720 cagactggga ttacaccacc caggcaggta ggaaccacct agtccactat cgccagttgc 3780 tcctagcggg tctccaaaac gcgggcagaa gccccaccaa tttggccaag gtaaaaggaa 3840 taacacaagg gcccaatgag tctccctcgg ccttcctaga gagacttaag gaagcctatc 3900 gcaggtacac tccttatgac cctgaggacc cagggcaaga aactaatgtg tctatgtctt 3960 tcatttggca gtctgcccca gacattggga gaaagttaga gaggttagaa gatttaaaaa 4020 acaagacgct tggagatttg gttagagagg cagaaaagat ctttaataaa cgagaaaccc 4080 cggaagaaag agaggaacgt atcaggagag aaacagagga aaaagaagaa cgccgtagga 4140 cagaggatga gcagaaagag aaagaaagag atcgtaggag acatagagag atgagcaagc 4200 tattggccac tgtcgttagt ggacagaaac aggatagaca gggaggagaa cgaaggaggt 4260 cccaactcga tcgcgaccag tgtgcctact gcaaagaaaa ggggcactgg gctaaagatt 4320 gtcccaagaa accacgagga cctcggggac caagacccca gacctccctc ctgaccctag 4380 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 4440 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 4500 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 4560 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 4620 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 4680 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 4740 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 4800 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 4860 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 4920 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 4980 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 5040 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 5100 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 5160 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 5220 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 5280 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 5340 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 5400 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 5460 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 5520 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 5580 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 5640 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 5700 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 5760 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 5820 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 5880 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 5940 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 6000 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 6060 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 6120 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 6180 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 6240 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 6300 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 6360 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 6420 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 6480 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 6540 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 6600 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 6660 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 6720 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 6780 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 6840 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 6900 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 6960 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 7020 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 7080 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 7140 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 7200 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 7260 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 7320 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 7380 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 7440 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 7500 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 7560 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 7620 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 7680 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 7740 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 7800 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 7860 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 7920 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 7980 ccccctaatc cccttaattc ttctgatgct cagaggggtc agtaaacgaa ttcggaagtg 8040 aaagggcagc caaaacgtaa caccgccccg gttttccctg gaaattccat attggcacgc 8100 attctattgg ctgagctgcg ttcacgtggg tataagaggc gcgaccagcg tcggtaccgt 8160 cgcagtcttc ggtctgacca ccgtagaacg cagagcggcc gcatgccacc tcctcgcctc 8220 ctcttcttcc tcctcttcct cacccccatg gaagtcaggc ccgaggaacc tctagtggtg 8280 aaggtggaag agggagataa cgctgtgctg cagtgcctca aggggacctc agatggcccc 8340 actcagcagc tgacctggtc tcgggagtcc ccgcttaaac ccttcttaaa actcagcctg 8400 gggctgccag gcctgggaat ccacatgagg cccctggcca tctggctttt catcttcaac 8460 gtctctcaac agatgggggg cttctacctg tgccagccgg ggcccccctc tgagaaggcc 8520 tggcagcctg gctggacagt caatgtggag ggcagcgggg agctgttccg gtggaatgtt 8580 tcggacctag gtggcctggg ctgtggcctg aagaacaggt cctcagaggg ccccagctcc 8640 ccttccggga agctcatgag ccccaagctg tatgtgtggg ccaaagaccg ccctgagatc 8700 tgggagggag agcctccgtg tctcccaccg agggacagcc tgaaccagag cctcagccag 8760 gacctcacca tggcccctgg ctccacactc tggctgtcct gtggggtacc ccctgactct 8820 gtgtccaggg gccccctctc ctggacccat gtgcacccca aggggcctaa gtcattgctg 8880 agcctagagc tgaaggacga tcgcccggcc agagatatgt gggtaatgga gacgggtctg 8940 ttgttgcccc gggccacagc tcaagacgct ggaaagtatt attgtcaccg tggcaacctg 9000 accatgtcat tccacctgga gatcactgct cggccagtac tatggcactg gctgctgagg 9060 actggtggct ggaaggtctc agctgtgact ttggcttatc tgatcttctg cctgtgttcc 9120 cttgtgggca ttcttcatct tcaaagagcc ctggtcctga ggaggaaaag atgaacgcgt 9180 gaattcataa aataaaagat tttatttagt ctccagaaaa aggggggaat gaaagacccc 9240 acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg gaaaaataca 9300 taactgagaa tagagaagtt cagatcaagg tcaggaacag atggaacagc tgaatatggg 9360 ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatgga 9420 acagctgaat atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg 9480 ccaagaacag atggtcccca gatgcggtcc agccctcagc agtttctaga gaaccatcag 9540 atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat 9600 cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca 9660 caacccctca ctcggggcgc cagtcctccg attgactgag tcgcccgggt acccgtgtat 9720 ccaataaacc ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct 9780 cctctgagtg attgactacc cgtcagcggg ggtctttcat tgttacttaa agttacattg 9840 gcttccttga aataaacatg gagtattcag aatgtgtcat aaatatttct aattttaaga 9900 tagtatctcc attggctttc tactttttct tttatttttt tttgtcctct gtcttccatt 9960 tgttgttgtt gttgtttgtt tgtttgtttg ttggttggtt ggttaatttt tttttaaaga 10020 tcctacacta tagttcaagc tagactatta gctactctgt aacccagggt gaccttgaag 10080 tcatgggtag cctgctgttt tagccttccc acatctaaga ttacaggtat gagctatcat 10140 ttttggtata ttgattgatt gattgattga tgtgtgtgtg tgtgattgtg tttgtgtgtg 10200 tgactgtgaa aatgtgtgta tgggtgtgtg tgaatgtgtg tatgtatgtg tgtgtgtgag 10260 tgtgtgtgtg tgtgtgtgca tgtgtgtgtg tgtgactgtg tctatgtgta tgactgtgtg 10320 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg ttgtgaaaaa atattctatg 10380 gtagtgagag ccaacgctcc ggctcaggtg tcaggttggt ttttgagaca gagtctttca 10440 cttagcttgg 10450 <210> 55 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> hCD19t-SalI-R <400> 55 tggtcgactc atcttttcct cctcaggacc agg 33 <210> 56 <211> 9406 <212> DNA <213> Artificial Sequence <220> <223> sRRVe-F4-hCD19t vector <400> 56 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacgtgat 1800 ggcgcgttca acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat 1860 agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg tctttaatgt 1920 aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 1980 aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga 2040 gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 2100 acagcggacc cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg 2160 tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg gacaggctta 2220 ctggaagccc acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga 2280 cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc 2340 cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat tcactgatgc 2400 aggaaaaaag gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg 2460 aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 2520 ccccataggg cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat 2580 tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt ccactaccag 2640 tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag gaactggaga 2700 tagactacta gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa 2760 gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag gagtagcggt 2820 cgtgggcact tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca 2880 taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac 2940 tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact accttgcagc 3000 acccgccgga acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt 3060 gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag taatttacca 3120 ctcccccgat tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt 3180 atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat 3240 agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc atgccgctat 3300 ccagacagac ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc 3360 gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc taaaggaggg 3420 aggtctctgc gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt 3480 gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac 3540 aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca ccttaatctc 3600 caccatcatg ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct 3660 caatcgatta gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac 3720 tcaacaatat caccagctga agcctataga gtacgagcca tagcacgtga aggctgccga 3780 ccccgggggt ggacatcctc tagactgcca tggcgcggat ctcgacgttt aaacggaagt 3840 gaaagggcag ccaaaacgta acaccgcccc ggttttccct ggaaattcca tattggcacg 3900 cattctattg gctgagctgc gttcacgtgg gtataagagg cgcgaccagc gtcggtaccg 3960 tcgcagtctt cggtctgacc accgtagaac gcagagcggc cgcatgccac ctcctcgcct 4020 cctcttcttc ctcctcttcc tcacccccat ggaagtcagg cccgaggaac ctctagtggt 4080 gaaggtggaa gagggagata acgctgtgct gcagtgcctc aaggggacct cagatggccc 4140 cactcagcag ctgacctggt ctcgggagtc cccgcttaaa cccttcttaa aactcagcct 4200 ggggctgcca ggcctgggaa tccacatgag gcccctggcc atctggcttt tcatcttcaa 4260 cgtctctcaa cagatggggg gcttctacct gtgccagccg gggcccccct ctgagaaggc 4320 ctggcagcct ggctggacag tcaatgtgga gggcagcggg gagctgttcc ggtggaatgt 4380 ttcggaccta ggtggcctgg gctgtggcct gaagaacagg tcctcagagg gccccagctc 4440 cccttccggg aagctcatga gccccaagct gtatgtgtgg gccaaagacc gccctgagat 4500 ctgggaggga gagcctccgt gtctcccacc gagggacagc ctgaaccaga gcctcagcca 4560 ggacctcacc atggcccctg gctccacact ctggctgtcc tgtggggtac cccctgactc 4620 tgtgtccagg ggccccctct cctggaccca tgtgcacccc aaggggccta agtcattgct 4680 gagcctagag ctgaaggacg atcgcccggc cagagatatg tgggtaatgg agacgggtct 4740 gttgttgccc cgggccacag ctcaagacgc tggaaagtat tattgtcacc gtggcaacct 4800 gaccatgtca ttccacctgg agatcactgc tcggccagta ctatggcact ggctgctgag 4860 gactggtggc tggaaggtct cagctgtgac tttggcttat ctgatcttct gcctgtgttc 4920 ccttgtgggc attcttcatc ttcaaagagc cctggtcctg aggaggaaaa gatgagtcga 4980 cccatcgaac atcgatggta ccagatccga taaaataaaa gattttattt agtctccaga 5040 aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat 5100 tttgcaaggc atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa 5160 cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg 5220 gctcagggcc aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa 5280 gcagttcctg ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc 5340 agcagtttct agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct 5400 gtgccttatt tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc 5460 cccgagctca ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact 5520 gagtcgcccg ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt 5580 ctcgctgttc cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt 5640 cacacatgca gcatgtatca aaattaattt ggtttttttt cttaagtatt tacattaaat 5700 ggccatagta cttaaagtta cattggcttc cttgaaataa acatggagta ttcagaatgt 5760 gtcataaata tttctaattt taagatagta tctccattgg ctttctactt tttcttttat 5820 ttttttttgt cctctgtctt ccatttgttg ttgttgttgt ttgtttgttt gtttgttggt 5880 tggttggtta attttttttt aaagatccta cactatagtt caagctagac tattagctac 5940 tctgtaaccc agggtgacct tgaagtcatg ggtagcctgc tgttttagcc ttcccacatc 6000 taagattaca ggtatgagct atcatttttg gtatatgatt gattgattga ttgatgtgtg 6060 tgtgtgtgat tgtgtttgtg tgtgtgactg tgaaaatgtg tgtatgggtg tgtgtgaatg 6120 tgtgtatgta tgtgtgtgtg tgagtgtgtg tgtgtgtgtg tgcatgtgtg tgtgtgtgac 6180 tgtgtctatg tgtatgactg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 6240 tgtgttgtga aaaaatattc tatggtagtg agagccaacg ctccggctca ggtgtcaggt 6300 tggtttttga gacagagtct ttcacttagc ttggaattca ctggccgtcg ttttacaacg 6360 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt 6420 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 6480 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 6540 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc 6600 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 6660 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 6720 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 6780 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 6840 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 6900 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 6960 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 7020 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 7080 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 7140 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 7200 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 7260 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 7320 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 7380 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 7440 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 7500 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 7560 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 7620 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 7680 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 7740 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 7800 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 7860 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 7920 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 7980 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 8040 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 8100 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 8160 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 8220 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 8280 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 8340 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 8400 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 8460 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 8520 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 8580 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 8640 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 8700 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 8760 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 8820 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 8880 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 8940 aggaaacagc tatgaccatg attacgccaa gctttgctcc taggagtttc ctaatacttc 9000 ccaaactcaa atatataaag catttgactt gttctatgcc ctagggggcg gggggaagct 9060 aagccagctt tttttaacat ttaaaatgtt aattccattt taaatgcaca gatgttttta 9120 tttcataagg gtttcaatgt gcatgaatgc tgcaatattc ctgttaccaa agctagtata 9180 aataaaaata gataaacgtg gaaattactt agagtttctg tcattaacgt ttccttcctc 9240 agttgacaac ataaatgcgc tgctgagcaa gccagtttgc atctgtcagg atcaatttcc 9300 cattatgcca gtcatattaa ttactagtca attagttgat ttttattttt gacatataca 9360 tgtgaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagt 9406 <210> 57 <211> 9535 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-mGM-CSF <400> 57 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacaattc 1800 gatcatacct ggtgttgctg actaccccga ccgcggtaaa agtcgatggt attgctgcct 1860 gggtccatgc ttctcacctc aaacctgcac caccttcggc accagatgag tcctgggagc 1920 tggaaaagac tgatcatcct cttaagctgc gtattcggcg gcggcgggac gagtctgcaa 1980 aataagaacc cccaccagcc catgaccctc acttggcagg tactgtccca aactggagac 2040 gttgtctggg atacaaaggc agtccagccc ccttggactt ggtggcccac acttaaacct 2100 gatgtatgtg ccttggcggc tagtcttgag tcctgggata tcccgggaac cgatgtctcg 2160 tcctctaaac gagtcagacc tccggactca gactatactg ccgcttataa gcaaatcacc 2220 tggggagcca tagggtgcag ctaccctcgg gctaggacta gaatggcaag ctctaccttc 2280 tacgtatgtc cccgggatgg ccggaccctt tcagaagcta gaaggtgcgg ggggctagaa 2340 tccctatact gtaaagaatg ggattgtgag accacgggga ccggttattg gctatctaaa 2400 tcctcaaaag acctcataac tgtaaaatgg gaccaaaata gcgaatggac tcaaaaattt 2460 caacagtgtc accagaccgg ctggtgtaac ccccttaaaa tagatttcac agacaaagga 2520 aaattatcca aggactggat aacgggaaaa acctggggat taagattcta tgtgtctgga 2580 catccaggcg tacagttcac cattcgctta aaaatcacca acatgccagc tgtggcagta 2640 ggtcctgacc tcgtccttgt ggaacaagga cctcctagaa cgtccctcgc tctcccacct 2700 cctcttcccc caagggaagc gccaccgcca tctctccccg actctaactc cacagccctg 2760 gcgactagtg cacaaactcc cacggtgaga aaaacaattg ttaccctaaa cactccgcct 2820 cccaccacag gcgacagact ttttgatctt gtgcaggggg ccttcctaac cttaaatgct 2880 accaacccag gggccactga gtcttgctgg ctttgtttgg ccatgggccc cccttattat 2940 gaagcaatag cctcatcagg agaggtcgcc tactccaccg accttgaccg gtgccgctgg 3000 gggacccaag gaaagctcac cctcactgag gtctcaggac acgggttgtg cataggaaag 3060 gtgcccttta cccatcagca tctctgcaat cagaccctat ccatcaattc ctccggagac 3120 catcagtatc tgctcccctc caaccatagc tggtgggctt gcagcactgg cctcacccct 3180 tgcctctcca cctcagtttt taatcagact agagatttct gtatccaggt ccagctgatt 3240 cctcgcatct attactatcc tgaagaagtt ttgttacagg cctatgacaa ttctcacccc 3300 aggactaaaa gagaggctgt ctcacttacc ctagctgttt tactggggtt gggaatcacg 3360 gcgggaatag gtactggttc aactgcctta attaaaggac ctatagacct ccagcaaggc 3420 ctgacaagcc tccagatcgc catagatgct gacctccggg ccctccaaga ctcagtcagc 3480 aagttagagg actcactgac ttccctgtcc gaggtagtgc tccaaaatag gagaggcctt 3540 gacttgctgt ttctaaaaga aggtggcctc tgtgcggccc taaaggaaga gtgctgtttt 3600 tacatagacc actcaggtgc agtacgggac tccatgaaaa aactcaaaga aaaactggat 3660 aaaagacagt tagagcgcca gaaaagccaa aactggtatg aaggatggtt caataactcc 3720 ccttggttca ctaccctgct atcaaccatc gctgggcccc tattactcct ccttctgttg 3780 ctcatcctcg ggccatgcat catcaataag ttagttcaat tcatcaatga taggataagt 3840 gcagttaaaa ttctggtcct tagacaaaaa tatcaggccc tagagaacga aggtaacctt 3900 taacacgtga aggctgccga ccccgggggt ggaccatcct ctagactgtg ctcgacgttt 3960 aaaccgataa gcgttttttt caataacagg aaagtcccat tggagccaag tacattgagt 4020 caatagggac tttccaatgg gttttgccca gtacataagg tcaatgggag gtaagccaat 4080 gggtttttcc cattactggc acgtatactg agtcattagg gactttccaa tgggttttgc 4140 ccagtacata aggtcaatag gggtgaatca acaggaaagt cccattggag ccaagtacac 4200 tgagtcaata gggactttcc attgggtttt gcccagtaca aaaggtcaat agggggtgag 4260 tcaatgggtt tttcccatta ttggcacgta cataaggtca ataggggtga gtcattgggt 4320 ttttccagcc aatttaatta aaacgccatg tactttccca ccattgacgt caatgggcta 4380 ttgaaactaa tgcaacgtga cctttaaacg gtactttccc atagctgatt aatgggaaag 4440 taccgttctc gagccaatac acgtcaatgg gaagtgaaag ggcagccaaa acgtaacacc 4500 gccccggttt tcccctggaa attccatatt ggcacgcatt ctattggctg agctgcgttc 4560 tacgtgggta taagaggcgc gaccagcgtc ggtaccgtcg cagtcttcgg tctgaccacc 4620 gtagaacgca gaactctaga actagtggat cccccgggct gcaggaattc aggatgtggc 4680 tgcagaattt acttttcctg ggcattgtgg tctacagcct ctcagcaccc acccgctcac 4740 ccatcactgt cacccggcct tggaagcatg tagaggccat caaagaagcc ctgaacctcc 4800 tggatgacat gcctgtcacg ttgaatgaag aggtagaagt cgtctctaac gagttctcct 4860 tcaagaagct aacatgtgtg cagacccgcc tgaagatatt cgagcagggt ctacggggca 4920 atttcaccaa actcaagggc gccttgaaca tgacagccag ctactaccag acatactgcc 4980 ccccaactcc ggaaacggac tgtgaaacac aagttaccac ctatgcggat ttcatagaca 5040 gccttaaaac ctttctgact gatatcccct ttgaatgcaa aaaaccagtc caaaaatgag 5100 aattcgatat caagcttatc gatggggtac cagatccgat aaaataaaag attttattta 5160 gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag 5220 taacgccatt ttgcaaggca tggaaaaata cataactgag aatagagaag ttcagatcaa 5280 ggtcaggaac agatggaaca gctgaatatg ggccaaacag gatatctgtg gtaagcagtt 5340 cctgccccgg ctcagggcca agaacagatg gaacagctga atatgggcca aacaggatat 5400 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt 5460 ccagccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga 5520 aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg 5580 cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggggc gccagtcctc 5640 cgattgactg agtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg 5700 acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg 5760 ggggtctttc acacatgcag catgtatcaa aattaatttg gttttttttc ttaagtattt 5820 acattaaatg gccatagtac ttaaagttac attggcttcc ttgaaataaa catggagtat 5880 tcagaatgtg tcataaatat ttctaatttt aagatagtat ctccattggc tttctacttt 5940 ttcttttatt tttttttgtc ctctgtcttc catttgttgt tgttgttgtt tgtttgtttg 6000 tttgttggtt ggttggttaa ttttttttta aagatcctac actatagttc aagctagact 6060 attagctact ctgtaaccca gggtgacctt gaagtcatgg gtagcctgct gttttagcct 6120 tcccacatct aagattacag gtatgagcta tcatttttgg tatatgattg attgattgat 6180 tgatgtgtgt gtgtgtgatt gtgtttgtgt gtgtgactgt gaaaatgtgt gtatgggtgt 6240 gtgtgaatgt gtgtatgtat gtgtgtgtgt gagtgtgtgt gtgtgtgtgt gcatgtgtgt 6300 gtgtgtgact gtgtctatgt gtatgactgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 6360 gtgtgtgtgt gtgttgtgaa aaaatattct atggtagtga gagccaacgc tccggctcag 6420 gtgtcaggtt ggtttttgag acagagtctt tcacttagct tggaattcac tggccgtcgt 6480 tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 6540 tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 6600 gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg 6660 cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 6720 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 6780 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 6840 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 6900 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 6960 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 7020 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 7080 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 7140 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 7200 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 7260 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 7320 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 7380 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 7440 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 7500 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 7560 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 7620 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 7680 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 7740 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 7800 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 7860 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 7920 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 7980 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 8040 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 8100 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 8160 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 8220 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 8280 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 8340 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 8400 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 8460 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 8520 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 8580 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 8640 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 8700 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 8760 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 8820 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa 8880 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 8940 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 9000 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 9060 atttcacaca ggaaacagct atgaccatga ttacgccaag ctttgctcct aggagtttcc 9120 taatacttcc caaactcaaa tatataaagc atttgacttg ttctatgccc tagggggcgg 9180 ggggaagcta agccagcttt ttttaacatt taaaatgtta attccatttt aaatgcacag 9240 atgtttttat ttcataaggg tttcaatgtg catgaatgct gcaatattcc tgttaccaaa 9300 gctagtataa ataaaaatag ataaacgtgg aaattactta gagtttctgt cattaacgtt 9360 tccttcctca gttgacaaca taaatgcgct gctgagcaag ccagtttgca tctgtcagga 9420 tcaatttccc attatgccag tcatattaat tactagtcaa ttagttgatt tttatttttg 9480 acatatacat gtgaatgaaa gaccccacct gtaggtttgg caagctagct taagt 9535 <210> 58 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> mGM-CSF-F-NotI <400> 58 cggcggccgc aggatgtggc tgcagaattt 30 <210> 59 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> mGM-CSF-R-MluI <400> 59 tggtcgactc atttttggac tggttttt 28 <210> 60 <211> 9907 <212> DNA <213> Artificial Sequence <220> <223> sRRVgp-F4-mGM-CSF vector <400> 60 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 60 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 480 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 600 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 660 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 720 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 780 atcaatctaa agtatatatg agtaacctga tcaggactct tccttttcat gaacaataaa 840 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 900 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 960 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 1020 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1080 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 1140 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 1200 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 1260 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 1320 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 1380 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 1440 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 1500 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 1560 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 1620 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 1680 tgagtttttc taagaatttg tgaatgaaag accccacctg taggtttggc aagctagctt 1740 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat 1800 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1860 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1920 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1980 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2040 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2100 gcgcttatgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2160 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2220 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2280 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2340 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt 2400 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg 2460 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc 2520 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg 2580 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc 2640 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg 2700 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt 2760 tgtctgagaa tatgggccag actgttacca ctcccttaag tttgacctta ggtcactgga 2820 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 2880 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 2940 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 3000 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 3060 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 3120 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 3180 ctctaggcgc caaacctaaa cctcaagttc tttctgacag tggggggccg ctcatcgacc 3240 tacttacaga agaccccccg ccttataggg acccaagacc acccccttcc gacagggacg 3300 gaaatggtgg agaagcgacc cctgcgggag aggcaccgga cccctcccca atggcatctc 3360 gcctacgtgg gagacgggag ccccctgtgg ccgactccac tacctcgcag gcattccccc 3420 tccgcgcagg aggaaacgga cagcttcaat actggccgtt ctcctcttct gacctttaca 3480 actggaaaaa taataaccct tctttttctg aagatccagg taaactgaca gctctgatcg 3540 agtctgttct catcacccat cagcccacct gggacgactg tcagcagctg ttggggactc 3600 tgctgaccgg agaagaaaaa caacgggtgc tcttagaggc tagaaaggcg gtgcggggcg 3660 atgatgggcg ccccactcaa ctgcccaatg aagtcgatgc cgcttttccc ctcgagcgcc 3720 cagactggga ttacaccacc caggcaggta ggaaccacct agtccactat cgccagttgc 3780 tcctagcggg tctccaaaac gcgggcagaa gccccaccaa tttggccaag gtaaaaggaa 3840 taacacaagg gcccaatgag tctccctcgg ccttcctaga gagacttaag gaagcctatc 3900 gcaggtacac tccttatgac cctgaggacc cagggcaaga aactaatgtg tctatgtctt 3960 tcatttggca gtctgcccca gacattggga gaaagttaga gaggttagaa gatttaaaaa 4020 acaagacgct tggagatttg gttagagagg cagaaaagat ctttaataaa cgagaaaccc 4080 cggaagaaag agaggaacgt atcaggagag aaacagagga aaaagaagaa cgccgtagga 4140 cagaggatga gcagaaagag aaagaaagag atcgtaggag acatagagag atgagcaagc 4200 tattggccac tgtcgttagt ggacagaaac aggatagaca gggaggagaa cgaaggaggt 4260 cccaactcga tcgcgaccag tgtgcctact gcaaagaaaa ggggcactgg gctaaagatt 4320 gtcccaagaa accacgagga cctcggggac caagacccca gacctccctc ctgaccctag 4380 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 4440 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 4500 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 4560 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 4620 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 4680 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 4740 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 4800 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 4860 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 4920 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 4980 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 5040 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 5100 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 5160 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 5220 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 5280 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 5340 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 5400 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 5460 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 5520 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 5580 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 5640 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 5700 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 5760 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 5820 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 5880 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 5940 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 6000 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 6060 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 6120 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 6180 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 6240 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 6300 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 6360 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 6420 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 6480 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 6540 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 6600 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 6660 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 6720 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 6780 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 6840 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 6900 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 6960 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 7020 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 7080 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 7140 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 7200 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 7260 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 7320 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 7380 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 7440 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 7500 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 7560 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 7620 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 7680 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 7740 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 7800 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 7860 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 7920 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 7980 ccccctaatc cccttaattc ttctgatgct cagaggggtc agtaaacgaa ttcggaagtg 8040 aaagggcagc caaaacgtaa caccgccccg gttttccctg gaaattccat attggcacgc 8100 attctattgg ctgagctgcg ttcacgtggg tataagaggc gcgaccagcg tcggtaccgt 8160 cgcagtcttc ggtctgacca ccgtagaacg cagagcggcc gcaggatgtg gctgcagaat 8220 ttacttttcc tgggcattgt ggtctacagc ctctcagcac ccacccgctc acccatcact 8280 gtcacccggc cttggaagca tgtagaggcc atcaaagaag ccctgaacct cctggatgac 8340 atgcctgtca cgttgaatga agaggtagaa gtcgtctcta acgagttctc cttcaagaag 8400 ctaacatgtg tgcagacccg cctgaagata ttcgagcagg gtctacgggg caatttcacc 8460 aaactcaagg gcgccttgaa catgacagcc agctactacc agacatactg ccccccaact 8520 ccggaaacgg actgtgaaac acaagttacc acctatgcgg atttcataga cagccttaaa 8580 acctttctga ctgatatccc ctttgaatgc aaaaaaccag tccaaaaatg aacgcgtgaa 8640 ttcataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc 8700 tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa aaatacataa 8760 ctgagaatag agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca 8820 aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca 8880 gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca 8940 agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg 9000 tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag 9060 ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa 9120 cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca 9180 ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct 9240 ctgagtgatt gactacccgt cagcgggggt ctttcattgt tacttaaagt tacattggct 9300 tccttgaaat aaacatggag tattcagaat gtgtcataaa tatttctaat tttaagatag 9360 tatctccatt ggctttctac tttttctttt attttttttt gtcctctgtc ttccatttgt 9420 tgttgttgtt gtttgtttgt ttgtttgttg gttggttggt taattttttt ttaaagatcc 9480 tacactatag ttcaagctag actattagct actctgtaac ccagggtgac cttgaagtca 9540 tgggtagcct gctgttttag ccttcccaca tctaagatta caggtatgag ctatcatttt 9600 tggtatattg attgattgat tgattgatgt gtgtgtgtgt gattgtgttt gtgtgtgtga 9660 ctgtgaaaat gtgtgtatgg gtgtgtgtga atgtgtgtat gtatgtgtgt gtgtgagtgt 9720 gtgtgtgtgt gtgtgcatgt gtgtgtgtgt gactgtgtct atgtgtatga ctgtgtgtgt 9780 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgttg tgaaaaaata ttctatggta 9840 gtgagagcca acgctccggc tcaggtgtca ggttggtttt tgagacagag tctttcactt 9900 agcttgg 9907 <210> 61 <211> 9593 <212> DNA <213> Artificial Sequence <220> <223> spRRVe-hGM-CSF <400> 61 aacgccattt tgcaaggcat ggaaaaatac ataactgaga atagaaaagt tcagatcaag 60 gtcaggaaca gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc 120 ctgccccggc tcagggccaa gaacagatgg aacagctgaa tatgggccaa acaggatatc 180 tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc 240 cagccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa 300 atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc 360 ttatgctccc cgagctcaat aaaagagccc acaacccctc actcggggcg ccagtcctcc 420 gattgactga gtcgcccggg tacccgtgta tccaataaac cctcttgcag ttgcatccga 480 cttgtggtct cgctgttcct tgggagggtc tcctctgagt gattgactac ccgtcagcgg 540 gggtctttca tttgggggct cgtccgggat cgggagaccc ctgcccaggg accaccgacc 600 caccaccggg aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct 660 atgactgatt ttatgcgcct gcgtcggtac tagttagcta actagctctg tatctggcgg 720 acccgtggtg gaactgacga gttcggaaca cccggccgca accctgggag acgtcccagg 780 gacttcgggg gccgtttttg tggcccgacc tgagtcctaa aatcccgatc gtttaggact 840 ctttggtgca ccccccttag aggagggata tgtggttctg gtaggagacg agaacctaaa 900 acagttcccg cctccgtctg aatttttgct ttcggtttgg gaccgaagcc gcgccgcgcg 960 tcttgtctgc tgcagcatcg ttctgtgttg tctctgtctg actgtgtttc tgtatttgtc 1020 tgaaaatatg ggcccgggct agcctgttac cactccctta agtttgacct taggtcactg 1080 gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga gacgttgggt 1140 taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag acggcacctt 1200 taaccgagac ctcactaccc aggttaagat caaggtcttt tcacctggcc cgcatggaca 1260 cccagaccag gtggggtaca tcgtgacctg ggaagccttg gcttttgacc cccctccctg 1320 ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg ccccgtctct 1380 cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag ccctcactcc 1440 ttctctaggc gcccccatat ggccatatga gatcttatat ggggcacccc cgccccttgt 1500 aaacttccct gaccctgaca tgacaagagt tactaacagc ccctctctcc aagctcactt 1560 acaggctctc tacttagtcc agcacgaagt ctggagacct ctggcggcag cctaccaaga 1620 acaactggac cgaccggtgg tacctcaccc ttaccgagtc ggcgacacag tgtgggtccg 1680 ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag tcctgctgac 1740 cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg cccacaattc 1800 gatcatacct ggtgttgctg actaccccga ccgcggtaaa agtcgatggt attgctgcct 1860 gggtccatgc ttctcacctc aaacctgcac caccttcggc accagatgag tcctgggagc 1920 tggaaaagac tgatcatcct cttaagctgc gtattcggcg gcggcgggac gagtctgcaa 1980 aataagaacc cccaccagcc catgaccctc acttggcagg tactgtccca aactggagac 2040 gttgtctggg atacaaaggc agtccagccc ccttggactt ggtggcccac acttaaacct 2100 gatgtatgtg ccttggcggc tagtcttgag tcctgggata tcccgggaac cgatgtctcg 2160 tcctctaaac gagtcagacc tccggactca gactatactg ccgcttataa gcaaatcacc 2220 tggggagcca tagggtgcag ctaccctcgg gctaggacta gaatggcaag ctctaccttc 2280 tacgtatgtc cccgggatgg ccggaccctt tcagaagcta gaaggtgcgg ggggctagaa 2340 tccctatact gtaaagaatg ggattgtgag accacgggga ccggttattg gctatctaaa 2400 tcctcaaaag acctcataac tgtaaaatgg gaccaaaata gcgaatggac tcaaaaattt 2460 caacagtgtc accagaccgg ctggtgtaac ccccttaaaa tagatttcac agacaaagga 2520 aaattatcca aggactggat aacgggaaaa acctggggat taagattcta tgtgtctgga 2580 catccaggcg tacagttcac cattcgctta aaaatcacca acatgccagc tgtggcagta 2640 ggtcctgacc tcgtccttgt ggaacaagga cctcctagaa cgtccctcgc tctcccacct 2700 cctcttcccc caagggaagc gccaccgcca tctctccccg actctaactc cacagccctg 2760 gcgactagtg cacaaactcc cacggtgaga aaaacaattg ttaccctaaa cactccgcct 2820 cccaccacag gcgacagact ttttgatctt gtgcaggggg ccttcctaac cttaaatgct 2880 accaacccag gggccactga gtcttgctgg ctttgtttgg ccatgggccc cccttattat 2940 gaagcaatag cctcatcagg agaggtcgcc tactccaccg accttgaccg gtgccgctgg 3000 gggacccaag gaaagctcac cctcactgag gtctcaggac acgggttgtg cataggaaag 3060 gtgcccttta cccatcagca tctctgcaat cagaccctat ccatcaattc ctccggagac 3120 catcagtatc tgctcccctc caaccatagc tggtgggctt gcagcactgg cctcacccct 3180 tgcctctcca cctcagtttt taatcagact agagatttct gtatccaggt ccagctgatt 3240 cctcgcatct attactatcc tgaagaagtt ttgttacagg cctatgacaa ttctcacccc 3300 aggactaaaa gagaggctgt ctcacttacc ctagctgttt tactggggtt gggaatcacg 3360 gcgggaatag gtactggttc aactgcctta attaaaggac ctatagacct ccagcaaggc 3420 ctgacaagcc tccagatcgc catagatgct gacctccggg ccctccaaga ctcagtcagc 3480 aagttagagg actcactgac ttccctgtcc gaggtagtgc tccaaaatag gagaggcctt 3540 gacttgctgt ttctaaaaga aggtggcctc tgtgcggccc taaaggaaga gtgctgtttt 3600 tacatagacc actcaggtgc agtacgggac tccatgaaaa aactcaaaga aaaactggat 3660 aaaagacagt tagagcgcca gaaaagccaa aactggtatg aaggatggtt caataactcc 3720 ccttggttca ctaccctgct atcaaccatc gctgggcccc tattactcct ccttctgttg 3780 ctcatcctcg ggccatgcat catcaataag ttagttcaat tcatcaatga taggataagt 3840 gcagttaaaa ttctggtcct tagacaaaaa tatcaggccc tagagaacga aggtaacctt 3900 taattttgct ctaagattag agctattcac aagagaaatg gggatcacta gtgaattaaa 3960 caacaggaaa gttccattgg agccaagtac attgagtcaa tagggacttt ccaatgggtt 4020 ttgcccagta cataaggtca atgggaggta agccaatggg tttttcccat tactggcacg 4080 tatactgagt cattagggac tttccaatgg gttttgccca gtacataagg tcaatagggg 4140 tgaatcaaca ggaaagtccc attggagcca agtacactga gtcaataggg actttccatt 4200 gggttttgcc cagtacaaaa ggtcaatagg gggtgagtca atgggttttt cccattattg 4260 gcacgtacat aaggtcaata ggggtgagtc attgggtttt tccagccaat ttaattaaaa 4320 cgccatgtac tttcccacca ttgacgtcaa tgggctattg aaactaatgc aacgtgacct 4380 ttaaacggta ctttcccata gctgattaat gggaaagtac cgttctcgag ccaatacacg 4440 tcaatgggaa gtgaaagggc agccaaaacg taacaccgcc ccggttttcc ctggaaattc 4500 catattggca cgcattctat tggctgagct gcgttctacg tgggtataag aggcgcgacc 4560 agcgtcggta ccgtcgcagt cttcggtctg accaccgtag aacgcagaac tctagaacta 4620 gtcgggatcc gttaacatgt ggctgcagag cctgctgctc ttgggcactg tggcctgcag 4680 catctctgca cccgcccgct cgcccagccc cagcacgcag ccctgggagc atgtgaatgc 4740 catccaggag gcccggcgtc tcctgaacct gagtagagac actgctgctg agatgaatga 4800 aacagtagaa gtcatctcag aaatgtttga cctccaggag ccgacctgcc tacagacccg 4860 cctggagctg tacaagcagg gcctgcgggg cagcctcacc aagctcaagg gccccttgac 4920 catgatggcc agccactaca agcagcactg ccctccaacc ccggaaactt cctgtgcaac 4980 ccagattatc acctttgaaa gtttcaaaga gaacctgaag gactttctgc ttgtcatccc 5040 ctttgactgc tgggagccag tccaggagtg agttaacatc gatggggtac cagatccgga 5100 ttagtccaat ttgttaaaga caggatatca gtggtccagg ctctagtttt gactcaacaa 5160 tatcaccagc tgaagcctat agagtacgag ccatagataa aataaaagat tttatttagt 5220 ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta 5280 acgccatttt gcaaggcatg gaaaaataca taactgagaa tagagaagtt cagatcaagg 5340 tcaggaacag atggaacagc tgaatatggg ccaaacagga tatctgtggt aagcagttcc 5400 tgccccggct cagggccaag aacagatgga acagctgaat atgggccaaa caggatatct 5460 gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggtcccca gatgcggtcc 5520 agccctcagc agtttctaga gaaccatcag atgtttccag ggtgccccaa ggacctgaaa 5580 tgaccctgtg ccttatttga actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct 5640 tctgctcccc gagctcaata aaagagccca caacccctca ctcggggcgc cagtcctccg 5700 attgactgag tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt tgcatccgac 5760 ttgtggtctc gctgttcctt gggagggtct cctctgagtg attgactacc cgtcagcggg 5820 ggtctttcac acatgcagca tgtatcaaaa ttaatttggt tttttttctt aagtatttac 5880 attaaatggc catagtactt aaagttacat tggcttcctt gaaataaaca tggagtattc 5940 agaatgtgtc ataaatattt ctaattttaa gatagtatct ccattggctt tctacttttt 6000 cttttatttt tttttgtcct ctgtcttcca tttgttgttg ttgttgtttg tttgtttgtt 6060 tgttggttgg ttggttaatt tttttttaaa gatcctacac tatagttcaa gctagactat 6120 tagctactct gtaacccagg gtgaccttga agtcatgggt agcctgctgt tttagccttc 6180 ccacatctaa gattacaggt atgagctatc atttttggta tatgattgat tgattgattg 6240 atgtgtgtgt gtgtgattgt gtttgtgtgt gtgactgtga aaatgtgtgt atgggtgtgt 6300 gtgaatgtgt gtatgtatgt gtgtgtgtga gtgtgtgtgt gtgtgtgtgc atgtgtgtgt 6360 gtgtgactgt gtctatgtgt atgactgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 6420 gtgtgtgtgt gttgtgaaaa aatattctat ggtagtgaga gccaacgctc cggctcaggt 6480 gtcaggttgg tttttgagac agagtctttc acttagcttg gaattcactg gccgtcgttt 6540 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 6600 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 6660 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg 6720 gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 6780 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 6840 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 6900 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta 6960 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 7020 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 7080 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 7140 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 7200 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 7260 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 7320 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 7380 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 7440 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 7500 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 7560 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 7620 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 7680 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 7740 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 7800 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 7860 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 7920 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 7980 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 8040 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 8100 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 8160 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 8220 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 8280 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 8340 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 8400 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 8460 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 8520 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 8580 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 8640 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 8700 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 8760 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 8820 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 8880 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 8940 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 9000 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 9060 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 9120 ttcacacagg aaacagctat gaccatgatt acgccaagct ttgctcctag gagtttccta 9180 atacttccca aactcaaata tataaagcat ttgacttgtt ctatgcccta gggggcgggg 9240 ggaagctaag ccagcttttt ttaacattta aaatgttaat tccattttaa atgcacagat 9300 gtttttattt cataagggtt tcaatgtgca tgaatgctgc aatattcctg ttaccaaagc 9360 tagtataaat aaaaatagat aaacgtggaa attacttaga gtttctgtca ttaacgtttc 9420 cttcctcagt tgacaacata aatgcgctgc tgagcaagcc agtttgcatc tgtcaggatc 9480 aatttcccat tatgccagtc atattaatta ctagtcaatt agttgatttt tatttttgac 9540 atatacatgt gaatgaaaga ccccacctgt aggtttggca agctagctta agt 9593 <210> 62 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hGM-CSF-F-NotI <400> 62 cggcggccgc aggatgtggc tgcagagc 28 <210> 63 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hGM-CSF-R-MluI <400> 63 tggtcgactc actcctggac tggctccc 28 <210> 64 <211> 9916 <212> DNA <213> Artificial Sequence <220> <223> sRRVgp-F4-hGM-CSF vector <400> 64 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 60 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 480 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 600 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 660 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 720 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 780 atcaatctaa agtatatatg agtaacctga tcaggactct tccttttcat gaacaataaa 840 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 900 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 960 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 1020 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1080 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 1140 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 1200 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 1260 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 1320 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 1380 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 1440 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 1500 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 1560 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 1620 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 1680 tgagtttttc taagaatttg tgaatgaaag accccacctg taggtttggc aagctagctt 1740 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat 1800 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1860 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1920 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1980 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2040 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2100 gcgcttatgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2160 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2220 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2280 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2340 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt 2400 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg 2460 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc 2520 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg 2580 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc 2640 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg 2700 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt 2760 tgtctgagaa tatgggccag actgttacca ctcccttaag tttgacctta ggtcactgga 2820 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 2880 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 2940 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 3000 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 3060 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 3120 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 3180 ctctaggcgc caaacctaaa cctcaagttc tttctgacag tggggggccg ctcatcgacc 3240 tacttacaga agaccccccg ccttataggg acccaagacc acccccttcc gacagggacg 3300 gaaatggtgg agaagcgacc cctgcgggag aggcaccgga cccctcccca atggcatctc 3360 gcctacgtgg gagacgggag ccccctgtgg ccgactccac tacctcgcag gcattccccc 3420 tccgcgcagg aggaaacgga cagcttcaat actggccgtt ctcctcttct gacctttaca 3480 actggaaaaa taataaccct tctttttctg aagatccagg taaactgaca gctctgatcg 3540 agtctgttct catcacccat cagcccacct gggacgactg tcagcagctg ttggggactc 3600 tgctgaccgg agaagaaaaa caacgggtgc tcttagaggc tagaaaggcg gtgcggggcg 3660 atgatgggcg ccccactcaa ctgcccaatg aagtcgatgc cgcttttccc ctcgagcgcc 3720 cagactggga ttacaccacc caggcaggta ggaaccacct agtccactat cgccagttgc 3780 tcctagcggg tctccaaaac gcgggcagaa gccccaccaa tttggccaag gtaaaaggaa 3840 taacacaagg gcccaatgag tctccctcgg ccttcctaga gagacttaag gaagcctatc 3900 gcaggtacac tccttatgac cctgaggacc cagggcaaga aactaatgtg tctatgtctt 3960 tcatttggca gtctgcccca gacattggga gaaagttaga gaggttagaa gatttaaaaa 4020 acaagacgct tggagatttg gttagagagg cagaaaagat ctttaataaa cgagaaaccc 4080 cggaagaaag agaggaacgt atcaggagag aaacagagga aaaagaagaa cgccgtagga 4140 cagaggatga gcagaaagag aaagaaagag atcgtaggag acatagagag atgagcaagc 4200 tattggccac tgtcgttagt ggacagaaac aggatagaca gggaggagaa cgaaggaggt 4260 cccaactcga tcgcgaccag tgtgcctact gcaaagaaaa ggggcactgg gctaaagatt 4320 gtcccaagaa accacgagga cctcggggac caagacccca gacctccctc ctgaccctag 4380 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 4440 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 4500 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 4560 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 4620 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 4680 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 4740 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 4800 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 4860 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 4920 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 4980 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 5040 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 5100 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 5160 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 5220 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 5280 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 5340 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 5400 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 5460 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 5520 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 5580 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 5640 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 5700 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 5760 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 5820 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 5880 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 5940 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 6000 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 6060 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 6120 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 6180 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 6240 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 6300 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 6360 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 6420 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 6480 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 6540 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 6600 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 6660 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 6720 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 6780 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 6840 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 6900 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 6960 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 7020 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 7080 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 7140 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 7200 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 7260 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 7320 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 7380 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 7440 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 7500 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 7560 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 7620 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 7680 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 7740 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 7800 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 7860 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 7920 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 7980 ccccctaatc cccttaattc ttctgatgct cagaggggtc agtaaacgaa ttcggaagtg 8040 aaagggcagc caaaacgtaa caccgccccg gttttccctg gaaattccat attggcacgc 8100 attctattgg ctgagctgcg ttcacgtggg tataagaggc gcgaccagcg tcggtaccgt 8160 cgcagtcttc ggtctgacca ccgtagaacg cagagcggcc gcaggatgtg gctgcagagc 8220 ctgctgctct tgggcactgt ggcctgcagc atctctgcac ccgcccgctc gcccagcccc 8280 agcacgcagc cctgggagca tgtgaatgcc atccaggagg cccggcgtct cctgaacctg 8340 agtagagaca ctgctgctga gatgaatgaa acagtagaag tcatctcaga aatgtttgac 8400 ctccaggagc cgacctgcct acagacccgc ctggagctgt acaagcaggg cctgcggggc 8460 agcctcacca agctcaaggg ccccttgacc atgatggcca gccactacaa gcagcactgc 8520 cctccaaccc cggaaacttc ctgtgcaacc cagattatca cctttgaaag tttcaaagag 8580 aacctgaagg actttctgct tgtcatcccc tttgactgct gggagccagt ccaggagtga 8640 acgcgtgaat tcataaaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa 8700 gaccccacct gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa 8760 aatacataac tgagaataga gaagttcaga tcaaggtcag gaacagatgg aacagctgaa 8820 tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca 8880 gatggaacag ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc 8940 tcagggccaa gaacagatgg tccccagatg cggtccagcc ctcagcagtt tctagagaac 9000 catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta 9060 accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag 9120 agcccacaac ccctcactcg gggcgccagt cctccgattg actgagtcgc ccgggtaccc 9180 gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga 9240 gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcattgtt acttaaagtt 9300 acattggctt ccttgaaata aacatggagt attcagaatg tgtcataaat atttctaatt 9360 ttaagatagt atctccattg gctttctact ttttctttta tttttttttg tcctctgtct 9420 tccatttgtt gttgttgttg tttgtttgtt tgtttgttgg ttggttggtt aatttttttt 9480 taaagatcct acactatagt tcaagctaga ctattagcta ctctgtaacc cagggtgacc 9540 ttgaagtcat gggtagcctg ctgttttagc cttcccacat ctaagattac aggtatgagc 9600 tatcattttt ggtatattga ttgattgatt gattgatgtg tgtgtgtgtg attgtgtttg 9660 tgtgtgtgac tgtgaaaatg tgtgtatggg tgtgtgtgaa tgtgtgtatg tatgtgtgtg 9720 tgtgagtgtg tgtgtgtgtg tgtgcatgtg tgtgtgtgtg actgtgtcta tgtgtatgac 9780 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgttgt gaaaaaatat 9840 tctatggtag tgagagccaa cgctccggct caggtgtcag gttggttttt gagacagagt 9900 ctttcactta gcttgg 9916 <210> 65 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> human codon optimized hGM-CSF polynucleotide sequence <400> 65 atgtggcttc aaagtttgtt gctccttgga accgtcgctt gctctattag cgcacccgca 60 cgcagcccaa gtccttccac gcagccttgg gaacatgtta atgcgatcca agaagcacgg 120 cgcctcttga atctttcccg agacacggcg gcagaaatga atgagacagt tgaagtcatt 180 tcagagatgt ttgatctcca agaacctact tgtttgcaga ctcgactcga actttacaag 240 cagggtctcc ggggaagttt gaccaagctc aagggtcccc ttacgatgat ggcgtcccac 300 tacaagcaac actgcccccc gacaccagaa acgtcatgtg ctacccaaat cattaccttt 360 gaatcattca aggagaacct caaggatttt ctgttggtca taccattcga ctgttgggag 420 cccgtgcagg aatga 435 <210> 66 <211> 9913 <212> DNA <213> Artificial Sequence <220> <223> sRRVgp-F4-hopt-GM-CSF vector <400> 66 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 60 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 120 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 180 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 240 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 300 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 360 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 420 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 480 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 540 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 600 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 660 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 720 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 780 atcaatctaa agtatatatg agtaacctga tcaggactct tccttttcat gaacaataaa 840 actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac 900 gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 960 ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 1020 tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 1080 gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 1140 ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca 1200 ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 1260 gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg 1320 tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga 1380 cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt 1440 ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 1500 ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 1560 tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 1620 tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga 1680 tgagtttttc taagaatttg tgaatgaaag accccacctg taggtttggc aagctagctt 1740 aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagaa aagttcagat 1800 caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca 1860 gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga 1920 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1980 ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 2040 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 2100 gcgcttatgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc 2160 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat 2220 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca 2280 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc 2340 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt 2400 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg 2460 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc 2520 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg 2580 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc 2640 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg 2700 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt 2760 tgtctgagaa tatgggccag actgttacca ctcccttaag tttgacctta ggtcactgga 2820 aagatgtcga gcggatcgct cacaaccagt cggtagatgt caagaagaga cgttgggtta 2880 ccttctgctc tgcagaatgg ccaaccttta acgtcggatg gccgcgagac ggcaccttta 2940 accgagacct catcacccag gttaagatca aggtcttttc acctggcccg catggacacc 3000 cagaccaggt cccctacatc gtgacctggg aagccttggc ttttgacccc cctccctggg 3060 tcaagccctt tgtacaccct aagcctccgc ctcctcttcc tccatccgcc ccgtctctcc 3120 cccttgaacc tcctcgttcg accccgcctc gatcctccct ttatccagcc ctcactcctt 3180 ctctaggcgc caaacctaaa cctcaagttc tttctgacag tggggggccg ctcatcgacc 3240 tacttacaga agaccccccg ccttataggg acccaagacc acccccttcc gacagggacg 3300 gaaatggtgg agaagcgacc cctgcgggag aggcaccgga cccctcccca atggcatctc 3360 gcctacgtgg gagacgggag ccccctgtgg ccgactccac tacctcgcag gcattccccc 3420 tccgcgcagg aggaaacgga cagcttcaat actggccgtt ctcctcttct gacctttaca 3480 actggaaaaa taataaccct tctttttctg aagatccagg taaactgaca gctctgatcg 3540 agtctgttct catcacccat cagcccacct gggacgactg tcagcagctg ttggggactc 3600 tgctgaccgg agaagaaaaa caacgggtgc tcttagaggc tagaaaggcg gtgcggggcg 3660 atgatgggcg ccccactcaa ctgcccaatg aagtcgatgc cgcttttccc ctcgagcgcc 3720 cagactggga ttacaccacc caggcaggta ggaaccacct agtccactat cgccagttgc 3780 tcctagcggg tctccaaaac gcgggcagaa gccccaccaa tttggccaag gtaaaaggaa 3840 taacacaagg gcccaatgag tctccctcgg ccttcctaga gagacttaag gaagcctatc 3900 gcaggtacac tccttatgac cctgaggacc cagggcaaga aactaatgtg tctatgtctt 3960 tcatttggca gtctgcccca gacattggga gaaagttaga gaggttagaa gatttaaaaa 4020 acaagacgct tggagatttg gttagagagg cagaaaagat ctttaataaa cgagaaaccc 4080 cggaagaaag agaggaacgt atcaggagag aaacagagga aaaagaagaa cgccgtagga 4140 cagaggatga gcagaaagag aaagaaagag atcgtaggag acatagagag atgagcaagc 4200 tattggccac tgtcgttagt ggacagaaac aggatagaca gggaggagaa cgaaggaggt 4260 cccaactcga tcgcgaccag tgtgcctact gcaaagaaaa ggggcactgg gctaaagatt 4320 gtcccaagaa accacgagga cctcggggac caagacccca gacctccctc ctgaccctag 4380 atgactaggg aggtcagggt caggagcccc cccctgaacc caggataacc ctcaaagtcg 4440 gggggcaacc cgtcaccttc ctggtagata ctggggccca acactccgtg ctgacccaaa 4500 atcctggacc cctaagtgat aagtctgcct gggtccaagg ggctactgga ggaaagcggt 4560 atcgctggac cacggatcgc aaagtacatc tagctaccgg taaggtcacc cactctttcc 4620 tccatgtacc agactgtccc tatcctctgt taggaagaga tttgctgact aaactaaaag 4680 cccaaatcca ctttgaggga tcaggagctc aggttatggg accaatgggg cagcccctgc 4740 aagtgttgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag 4800 atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg 4860 gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc 4920 ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca 4980 tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc 5040 tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag 5100 tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg 5160 ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc 5220 tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg 5280 gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc 5340 tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga 5400 tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 5460 aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 5520 aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 5580 gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 5640 gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 5700 cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 5760 cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 5820 ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 5880 aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 5940 agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 6000 tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 6060 atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 6120 ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 6180 tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 6240 tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 6300 accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 6360 ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 6420 ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 6480 taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 6540 acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 6600 tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 6660 atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 6720 aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccct 6780 acacctcaga acattttcat tacacagtga ctgatataaa ggacctaacc aagttggggg 6840 ccatttatga taaaacaaag aagtattggg tctaccaagg aaaacctgtg atgcctgacc 6900 agtttacttt tgaattatta gactttcttc atcagctgac tcacctcagc ttctcaaaaa 6960 tgaaggctct cctagagaga agccacagtc cctactacat gctgaaccgg gatcgaacac 7020 tcaaaaatat cactgagacc tgcaaagctt gtgcacaagt caacgccagc aagtctgccg 7080 ttaaacaggg aactagggtc cgcgggcatc ggcccggcac tcattgggag atcgatttca 7140 ccgagataaa gcccggattg tatggctata aatatcttct agtttttata gatacctttt 7200 ctggctggat agaagccttc ccaaccaaga aagaaaccgc caaggtcgta accaagaagc 7260 tactagagga gatcttcccc aggttcggca tgcctcaggt attgggaact gacaatgggc 7320 ctgccttcgt ctccaaggtg agtcagacag tggccgatct gttggggatt gattggaaat 7380 tacattgtgc atacagaccc caaagctcag gccaggtaga aagaatgaat agaaccatca 7440 aggagacttt aactaaatta acgcttgcaa ctggctctag agactgggtg ctcctactcc 7500 ccttagccct gtaccgagcc cgcaacacgc cgggccccca tggcctcacc ccatatgaga 7560 tcttatatgg ggcacccccg ccccttgtaa acttccctga ccctgacatg acaagagtta 7620 ctaacagccc ctctctccaa gctcacttac aggctctcta cttagtccag cacgaagtct 7680 ggagacctct ggcggcagcc taccaagaac aactggaccg accggtggta cctcaccctt 7740 accgagtcgg cgacacagtg tgggtccgcc gacaccagac taagaaccta gaacctcgct 7800 ggaaaggacc ttacacagtc ctgctgacca cccccaccgc cctcaaagta gacggcatcg 7860 cagcttggat acacgccgcc cacgtgaagg ctgccgaccc cgggggtgga ccatcctcta 7920 gactgacatg gcgcgttcaa cgctctcaaa accccttaaa aataaggtta acccgcgagg 7980 ccccctaatc cccttaattc ttctgatgct cagaggggtc agtaaacgaa ttcggaagtg 8040 aaagggcagc caaaacgtaa caccgccccg gttttccctg gaaattccat attggcacgc 8100 attctattgg ctgagctgcg ttcacgtggg tataagaggc gcgaccagcg tcggtaccgt 8160 cgcagtcttc ggtctgacca ccgtagaacg cagagcggcc gcatgtggct tcaaagtttg 8220 ttgctccttg gaaccgtcgc ttgctctatt agcgcacccg cacgcagccc aagtccttcc 8280 acgcagcctt gggaacatgt taatgcgatc caagaagcac ggcgcctctt gaatctttcc 8340 cgagacacgg cggcagaaat gaatgagaca gttgaagtca tttcagagat gtttgatctc 8400 caagaaccta cttgtttgca gactcgactc gaactttaca agcagggtct ccggggaagt 8460 ttgaccaagc tcaagggtcc ccttacgatg atggcgtccc actacaagca acactgcccc 8520 ccgacaccag aaacgtcatg tgctacccaa atcattacct ttgaatcatt caaggagaac 8580 ctcaaggatt ttctgttggt cataccattc gactgttggg agcccgtgca ggaatgaacg 8640 cgtgaattca taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac 8700 cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat 8760 acataactga gaatagagaa gttcagatca aggtcaggaa cagatggaac agctgaatat 8820 gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat 8880 ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca 8940 gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat 9000 cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc 9060 aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc 9120 ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg 9180 tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg 9240 tctcctctga gtgattgact acccgtcagc gggggtcttt cattgttact taaagttaca 9300 ttggcttcct tgaaataaac atggagtatt cagaatgtgt cataaatatt tctaatttta 9360 agatagtatc tccattggct ttctactttt tcttttattt ttttttgtcc tctgtcttcc 9420 atttgttgtt gttgttgttt gtttgtttgt ttgttggttg gttggttaat ttttttttaa 9480 agatcctaca ctatagttca agctagacta ttagctactc tgtaacccag ggtgaccttg 9540 aagtcatggg tagcctgctg ttttagcctt cccacatcta agattacagg tatgagctat 9600 catttttggt atattgattg attgattgat tgatgtgtgt gtgtgtgatt gtgtttgtgt 9660 gtgtgactgt gaaaatgtgt gtatgggtgt gtgtgaatgt gtgtatgtat gtgtgtgtgt 9720 gagtgtgtgt gtgtgtgtgt gcatgtgtgt gtgtgtgact gtgtctatgt gtatgactgt 9780 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgttgtgaa aaaatattct 9840 atggtagtga gagccaacgc tccggctcag gtgtcaggtt ggtttttgag acagagtctt 9900 tcacttagct tgg 9913

Claims (38)

  1. Gag-Pol 유전자, sEF1α(short elongation factor 1α) 프로모터 또는 MCMV (murine cytomegalovirus) 프로모터, 및 제1 치료 유전자를 포함하는 제1 재조합 발현벡터; 및
    바이러스의 Env 유전자, MCMV 프로모터 및 제2 치료 유전자를 포함하는 제2 재조합 발현벡터; 를 포함하는 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  2. 제1항에 있어서, 상기 Gag-Pol 유전자는 긴팔원숭이 백혈병 바이러스(Gibbon ape Leukemia virus; GaLV) 또는 암포트로픽 쥐 백혈병 바이러스(amphotropic MuLV)의 Gag-Pol 유전자인 것인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  3. 제1항에 있어서, 상기 MCMV 프로모터는 서열번호 3의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  4. 제1항에 있어서, 상기 MCMV 프로모터는 절단된 MCMV 프로모터인 것을 특징으로 하는, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  5. 제4항에 있어서, 상기 절단된 MCMV 프로모터는 서열번호 4, 서열번호 5, 서열번호 6 및 서열번호 7의 염기서열을 갖는 폴리뉴클레오티드로 구성된 군으로부터 선택되는 어느 하나인 것인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  6. 제1항에 있어서, 상기 sEF1α 프로모터가 서열번호 18의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  7. 제1항에 있어서, 상기 바이러스의 Env 유전자는 긴팔원숭이 백혈병 바이러스(Gibbon ape Leukemia virus; GaLV), 암포트로픽 쥐 백혈병 바이러스(amphotropic MuLV), 친이종 쥐 백혈병 바이러스(xenotropic MuLV), 고양이 내재성 바이러스(RD114), 수포성 구내염바이러스(vesicular stomatitis virus; VSV) 및 홍역바이러스(measles virus; MV)의 Env 유전자로 이루어진 군으로부터 유래되는 어느 하나인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  8. 제7항에 있어서, GaLV의 Env 유전자가 서열번호 19의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  9. 제7항에 있어서, MuLV의 Env 유전자가 서열번호 20의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  10. 제1항에 있어서, 상기 제1 치료 유전자 및 제2 치료 유전자는 자살 유전자, 사이토카인 유전자 및 암 항원 유전자로 이루어진 군으로부터 선택되는 어느 하나인 것인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  11. 제10항에 있어서, 상기 자살 유전자는 티미딘 키나아제(thymidine kinase, TK) 유전자 또는 효모 시토신 디아미나제(yCD) 유전자인 것인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  12. 제11항에 있어서, 상기 티미딘 키나아제 유전자가 서열번호 21의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  13. 제11항에 있어서, 상기 효모 시토신 디아미나제(yCD) 유전자가 서열번호 22의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  14. 제10항에 있어서 상기 사이토카인 유전자는 과립구 대식세포 콜로니 자극 인자(Granulocyte-macrophage colony-stimulating factor, GM-CSF)인 것인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  15. 제14항에 있어서, 상기 과립구 대식세포 콜로니 자극 인자는 인간 코돈 최적화된, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  16. 제15항에 있어서, 상기 과립구 대식세포 콜로니 자극 인자는 서열번호 65의 염기서열을 포함하는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  17. 제10항에 있어서 상기 암 항원 유전자는 인간 CD19(Cluster of Differentiation 19), CEA(Carcinoembryonic Antigen) 또는 HER2(human epidermal growth factor receptor 2)인 것인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  18. 제17항에 있어서, 상기 인간 CD19 유전자가 서열번호 43의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제 가능 레트로바이러스 벡터.
  19. 제17항에 있어서, 상기 인간 CD19 유전자는 세포질 도메인의 아미노산이 제거된 절단된 인간 CD19(truncated hCD19) 유전자인 것인, 재조합 발생이 최소화된 복제 가능 레트로바이러스 벡터.
  20. 제17항에 있어서, 상기 절단된 인간 CD19(truncated hCD19) 유전자는 서열번호 53의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제 가능 레트로바이러스 벡터.
  21. 제12항 또는 제13항에 있어서, 상기 티미딘 키나아제 또는 효모 시토신 디아미나제 유전자가 전구체 약물을 활성화 시키는, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  22. 제21항에 있어서, 상기 전구체 약물이 간사이클로비르(GCV) 및 5-플루오로사이토신(5-FC)으로 구성된 군으로부터 선택되는 어느 하나 이상인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터.
  23. 제1항 내지 제20항 중 어느 한 항의 벡터를 포함하는 재조합 레트로바이러스.
  24. 제23항의 재조합 레트로바이러스로 형질감염된 분리된 숙주 세포.
  25. 제24항에 있어서, 상기 숙주 세포는 NS/O 골수종 세포(NS/O myeloma cell), 인간 293T 세포, 중국 햄스터 난소 세포(CHO cell), HeLa 세포, CapT 세포(인간 양수 유래 세포) COS 세포, 케나인 D17 세포, 마우스 NIH/3T3, 레트로바이러스 패키징 세포주, 인간 중간엽 줄기세포 및 펠라인 PG4 세포로 구성된 군으로부터 선택되는 어느 하나인 것인, 재조합 레트로바이러스로 형질감염된 숙주 세포.
  26. 제23항의 재조합 레트로바이러스를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물.
  27. 제24항의 재조합 레트로바이러스로 형질감염된 숙주 세포를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물.
  28. 제26항 또는 제27항에 있어서, 상기 암이 점액상 세포 암종, 둥근 세포 암종, 국소적 진행 종양, 전이성 암, 어윙(Ewing) 육종, 암전이, 림프성 전이, 편평상피 세포 암종, 식도 편평상피 세포 암종, 경구 암종, 다발성골수종, 급성 림프구성 백혈병, 급성 비림프구성 백혈병, 만성 림프구 백혈병, 만성 골수구 백혈병, 모양 세포성 백혈병, 유출 림프종 (체강계 림프종), 흉선 림프종 폐암, 소세포 폐암종, 피부 T 세포 림프종, 호지킨 림프종, 비-호지킨성 림프종, 부신 피질 암, ACTH-생성 종양, 비소세포 폐암, 유방암, 소세포 암종, 도관 암종, 위암, 결장암, 결장직장암, 결장직장 종양 형성과 관련된 용종, 췌장암, 간암, 방광암, 1차 표면 방광 종양, 방광의 침습성 전이 세포 방광암종, 근 침윤성 방광암, 전립선암, 대장암, 신장암, 간암, 식도암, 난소 암종, 자궁경부암, 자궁내막암, 융모암, 난소암, 원발성 복막상피 신생물, 자궁 경관 암종, 질암, 외음부암, 자궁암, 난포 중 고형 종양, 고환암, 음경암, 신장 세포 암종, 뇌암, 두경부암, 구경부암, 신경아세포종, 뇌간 신경교종, 신경교종, 중추신경계 중 전이성 종양 세포 침윤, 골종, 골육종, 악성 흑색종, 인간 피부 케라티노사이트의 종양 진행, 편평상피 세포 암종, 갑상선 암, 망막아종, 신경아세포종, 중피종, 빌름스 종양, 담낭암, 영양아세포 종양, 혈관주위세포종, 또는 카포시 육종인, 암의 예방 또는 치료용 약학 조성물.
  29. 제23항의 재조합 레트로바이러스를 포함하는 암 치료용 유전자 전달 조성물.
  30. 1) MuLV(Murine Leukemia virus)의 Gag-Pol 유전자, sEF1α 프로모터 또는 MCMV 프로모터, 및 제1 치료 유전자를 포함하는 제1 재조합 발현벡터를 제조하는 단계; 및
    2) 바이러스의 Env 유전자, MCMV 프로모터 및 제2 치료 유전자를 포함하는 제2 재조합 발현벡터를 제조하는 단계;를 포함하는 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법.
  31. 제30항에 있어서, 상기 MCMV 프로모터는 서열번호 3, 서열번호 4, 서열번호 5, 서열번호 6 및 서열번호 7의 염기서열을 갖는 폴리뉴클레오티드로 구성된 군으로부터 선택되는 어느 하나인 것인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법.
  32. 제30항에 있어서, 상기 sEF1α 프로모터가 서열번호 18의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법.
  33. 제30항에 있어서, 상기 바이러스의 Env 유전자는 서열번호 19 또는 서열번호 20의 염기서열을 갖는, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법.
  34. 제30항에 있어서, 상기 제1 치료 유전자는 티미딘 키나아제(thymidine kinase, TK) 유전자, 효모 시토신 디아미나제(yCD) 유전자 및 인간 CD19 유전자로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법.
  35. 제30항에 있어서, 제2 치료 유전자는 티미딘 키나아제(thymidine kinase, TK) 유전자, 효모 시토신 디아미나제(yCD) 유전자 및 인간 CD19 유전자로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법.
  36. 제34항 또는 제35항에 있어서, 상기 티미딘 키나아제 유전자가 서열번호 21의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법.
  37. 제34항 또는 제35항에 있어서, 상기 효모 시토신 디아미나제(yCD) 유전자가 서열번호 22의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법.
  38. 제34항 또는 제35항에 있어서, 상기 인간 CD19 유전자가 서열번호 43의 염기서열을 갖는 폴리뉴클레오티드인, 재조합 발생이 최소화된 복제가능 레트로바이러스 벡터의 제조방법.





KR1020210118834A 2020-09-18 2021-09-07 재조합 발생이 최소화된 유전자치료 벡터, 상기 벡터를 포함하는 재조합 레트로바이러스 및 상기 재조합 레트로바이러스를 포함하는 암의 예방 또는 치료용 약학 조성물 KR20220037964A (ko)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2023518119A JP7412832B2 (ja) 2020-09-18 2021-09-17 組換え発生が最小限に抑えられた遺伝子治療用ベクター、ベクターを含む組換えレトロウイルス、及び組換えレトロウイルスを含む、癌を予防又は治療するための医薬組成物
CN202180063680.XA CN116323924A (zh) 2020-09-18 2021-09-17 具有最小化重组发生的基因疗法载体、包含载体的重组逆转录病毒和用于预防或治疗癌症的包含重组逆转录病毒的药物组合物
GB2304267.4A GB2614168B (en) 2020-09-18 2021-09-17 Gene therapy vector with minimized recombination occurrence, recombinant retrovirus comprising vector, and pharmaceutical composition for preventing or
PCT/KR2021/012776 WO2022060155A1 (ko) 2020-09-18 2021-09-17 재조합 발생이 최소화된 유전자치료 벡터, 상기 벡터를 포함하는 재조합 레트로바이러스 및 상기 재조합 레트로바이러스를 포함하는 암의 예방 또는 치료용 약학 조성물
US18/185,656 US11970708B2 (en) 2020-09-18 2023-03-17 Gene therapy vector with minimizing recombination, recombinant retrovirus comprising the vector, and pharmaceutical composition for preventing or treating cancer comprising the recombinant retrovirus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20200120797 2020-09-18
KR1020200120797 2020-09-18

Publications (1)

Publication Number Publication Date
KR20220037964A true KR20220037964A (ko) 2022-03-25

Family

ID=80935574

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020210118834A KR20220037964A (ko) 2020-09-18 2021-09-07 재조합 발생이 최소화된 유전자치료 벡터, 상기 벡터를 포함하는 재조합 레트로바이러스 및 상기 재조합 레트로바이러스를 포함하는 암의 예방 또는 치료용 약학 조성물

Country Status (1)

Country Link
KR (1) KR20220037964A (ko)

Similar Documents

Publication Publication Date Title
CN111344395A (zh) 产生经修饰的自然杀伤细胞的方法及使用方法
KR102451510B1 (ko) Pd-1 호밍 엔도뉴클레아제 변이체, 조성물 및 사용 방법
AU774643B2 (en) Compositions and methods for use in recombinational cloning of nucleic acids
KR20200064129A (ko) 트랜스제닉 선택 방법 및 조성물
KR102604096B1 (ko) 윌슨병을 치료하기 위한 유전자 치료
CN105960413B (zh) 人工dna-结合蛋白及其用途
KR20200032174A (ko) 강화된 키메라 항원 수용체 및 이의 용도
DK2768848T3 (en) METHODS AND PROCEDURES FOR EXPRESSION AND SECRETARY OF PEPTIDES AND PROTEINS
KR20170108946A (ko) Fc 수용체-유사 5를 표적화하는 키메라 항원 수용체 및 그의 용도
KR20180097631A (ko) 핵산을 와우 및 전정 세포에 전달하기 위한 물질 및 방법
JP2023036921A (ja) 蝸牛および前庭細胞に核酸を送達するための物質および方法
KR20170002445A (ko) 이중 항체 구축물을 발현하는 aav를 포함하는 조성물 및 이들의 용도
CN107580503B (zh) 用于治疗细菌感染的杀菌剂与亲溶酶体碱化剂的组合
KR20210005179A (ko) 헤모글로빈-관련 돌연변이를 편집하기 위한 상동성-지정 복구 주형 설계 및 전달
CN112912112A (zh) 肝特异性核酸调节元件以及其方法及用途
CN112041334A (zh) 人foxp3在经基因编辑的t细胞中的表达
CN112262214A (zh) 病毒载体及包装细胞系
CN110785179A (zh) Wiskott-Aldrich综合征和X连锁血小板减少症中的治疗性基因组编辑
CN116083398B (zh) 分离的Cas13蛋白及其应用
KR20220041214A (ko) Il-1 수퍼패밀리의 사이토카인의 시공적 제한 활성으로 무장된 면역반응성 세포
CN115768890A (zh) 通过分子和物理启动对t细胞免疫疗法的热控制
CN107988259B (zh) SmartBac杆状病毒表达系统及其应用
KR20210151785A (ko) 비바이러스성 dna 벡터 및 fviii 치료제 발현을 위한 이의 용도
KR20240032025A (ko) 내이의 세포 유형-특이적 유전자 발현을 위한 조성물 및 방법
KR20220142502A (ko) 근육 특이적 핵산 조절 요소 및 이의 방법 및 용도

Legal Events

Date Code Title Description
E902 Notification of reason for refusal