PT1197567E - Caracterização da função de genes utilizando inibição de arn de cadeia dupla - Google Patents

Caracterização da função de genes utilizando inibição de arn de cadeia dupla Download PDF

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PT1197567E
PT1197567E PT01129274T PT01129274T PT1197567E PT 1197567 E PT1197567 E PT 1197567E PT 01129274 T PT01129274 T PT 01129274T PT 01129274 T PT01129274 T PT 01129274T PT 1197567 E PT1197567 E PT 1197567E
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dna
promoter
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elegans
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Geert Plaetinck
Christ Platteeuw
Katherine Mortier
Thierry Bogaert
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Devgen Nv
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Description

ΕΡ 1 197 567/ΡΤ
DESCRIÇÃO
"Caracterização da função de genes utilizando inibição de ARN de cadeia dupla" A presente invenção refere-se à caracterização ou identificação da função de genes utilizando inibição de ARN de cadeia dupla (ARNcdi) e métodos de identificação do ADN responsável pela indução de um fenótipo especifico numa célula e um método para atribuir a função a sequências de genes conhecidos.
Recentemente foi descrito na Nature vol.391, pp.806-811, Fevereiro 98, que a introdução de um ARN de cadeia dupla numa célula resulta numa interferência potente e especifica com expressão de genes endógenos na célula e que esta interferência é substancialmente mais eficaz do que proporcionar cada uma das cadeias de ARN individualmente conforme proposto na tecnologia anti-sentido. Foi também verificado que esta redução especifica da actividade do gene ocorreu no verme nematóide Caenorhabditis elegans (C.elegans) quando o ARN foi introduzido no genoma ou na cavidade corporal do verme.
Os presentes inventores utilizaram esta técnica e aplicaram-na para definir métodos novos e inventivos para atribuir funções a genes ou fragmentos de ADN, que foram sequenciados em vários projectos, tais como, por exemplo, o projecto do genoma humano e a que ainda é preciso ser atribuída uma função particular, e para uso na identificação do ADN responsável por conferir um fenótipo particular.
Em WO 90/14090 descreve-se um método de produção de ARN de cadeia dupla in vitro por transcrição de cadeias molde clonadas em vectores pGEM. São preparadas duas cadeias simples separadas de ARN, em reacções de transcrição in vitro separadas, que subsequentemente são hibridadas para produzir ARN de cadeia dupla.
De acordo com a invenção, é proporcionado um método de introdução de ARNcd ou ADN capaz de produzir ARNcd num organismo não humano, método esse que compreende a alimentação do referido organismo com um microorganismo adequado 2 ΕΡ 1 197 567/ΡΤ compreendendo um vector de expressão compreendendo um promotor ou promotores orientados relativamente a uma sequência de ADN de modo a que sejam capazes de iniciar a transcrição da citada sequência de ADN em ARN de cadeia dupla após liqação de um factor de transcrição apropriado aos referidos promotor ou promotores ou a alimentação do citado organismo directamente com um tal vector de expressão.
Um método de identificação do ADN responsável por conferir um fenótipo numa célula pode compreender a) a construção de uma biblioteca de ADNc ou genómica do ADN da citada célula numa orientação relativamente a um promotor(es) capaz de promover a transcrição do citado ADNc ou ADN em ARN de cadeia dupla (cd) após ligação de um factor de transcrição apropriado ao(s) citado(s) promotor(es), b) introdução da citada biblioteca numa ou mais das citadas células contendo o citado factor de transcrição, e c) identificação e isolamento de um fenótipo desejado da citada célula contendo a citada biblioteca e identificação do ADN ou fragmento de ADNc da citada biblioteca responsável por conferir o citado fenótipo. A biblioteca pode ser organizada em agrupamentos hierárquicos conforme descrito com mais detalhes nos exemplos proporcionados, antes da etapa b) de modo a incluir, por exemplo, famílias de genes.
Um método para atribuir a função a uma sequência de ADN conhecida, pode compreender a) a identificação de um homólogo(s) do citado ADN numa célula, b) o isolamento do homólogo (s) do ADN relevante ou de um seu fragmento a partir da citada célula, c) a clonagem do citado homólogo ou fragmento num vector apropriado numa orientação relativamente a um promotor(es) capaz de promover a transcrição do ARNcd após ligação de um factor de transcrição apropriado ao citado promotor(es), d) a introdução do citado vector na citada célula da etapa a) contendo o citado factor de transcrição, e e) a identificação do fenótipo da citada célula em comparação com o tipo selvagem.
Na invenção, a sequência de nucleótidos ou de ADN pode ser proporcionada numa orientação com sentido ou anti-sentido relativamente a um promotor simples que possui as propriedades definidas acima, ou alternativamente pode ser proporcionado entre dois promotores idênticos. Em ambos os métodos acima 3 ΕΡ 1 197 567/ΡΤ descritos, ο ARNcd é proporcionado pela transcrição iniciada pelo promotor após ligação do seu factor de transcrição apropriado. A célula de acordo com o citado método pode ser derivada de, ou pode estar contida num organismo. Quando a célula está contida num organismo, o organismo pode ser adaptado para expressar o factor de transcrição apropriado. 0 organismo pode ser qualquer entre uma planta, um animal, um fungo ou uma levedura, mas preferencialmente pode ser o verme nematóide C. elegans, que pode ser qualquer entre um tipo selvagem, um mutante nuc-1 ou pha-ts de C. elegans ou uma combinação das citadas mutações. Numa concretização alternativa, a biblioteca de ADNc ou ADN ou o homólogo do ADN ou o seu fragmento, podem de forma vantajosa ser submetidos a transfecção ou transformados num microrganismo, tal como uma célula bacteriana ou de levedura, que pode ser introduzidos no organismo, que é preferencialmente o verme nematóide C. elegans. Nesta concretização da invenção o microrganismo pode ser adaptado para expressar o factor de transcrição apropriado. Preferencialmente, o microrganismo é E.coli.
Em cada aspecto dos métodos descritos, a biblioteca de ADN, o homólogo de ADN ou o fragmento de ADN, podem ser construídos num vector de ADN adequado que contém uma sequência de nucleótidos que codifica o citado factor de transcrição. Alternativamente, o citado factor de transcrição é codificado por um vector adicional. Numa outra alternativa adicional, a célula ou o organismo podem expressar ou ser adaptados para expressar o citado factor de transcrição. Preferencialmente, qualquer um dos vectores utilizados no método de acordo com a invenção contém um marcador seleccionável que pode ser, por exemplo, uma sequência de nucleótidos codificando sup-35 ou um seu fragmento. A sequência de nucleótidos pode estar orientada em relação a um promotor de modo a que a ligação de um factor de transcrição ao promotor inicie a transcrição do ADN num ARN de cadeia dupla. A figura 10 ilustra os vectores e a orientação da sequência de ADN que permitem a produção de ARN de dupla cadeia em C. elegans. Deste modo, numa concretização, o ADN está localizado entre dois promotores num vector capaz de expressar ARNcd após ligação de um factor de transcrição apropriado aos citados promotores. Alternativamente, o vector 4 ΕΡ 1 197 567/ΡΤ compreende duas cópias da sequência de ADN organizadas numa orientação com sentido e anti-sentido relativamente ao promotor e cujo marcador é seleccionável quando contido num C. elegans mutante pha-1. Preferencialmente, os promotores são qualquer um dos promotores de T7, T3 ou SP6 e o factor de transcrição contém a polimerase apropriada.
Preferencialmente, o marcador seleccionável compreende uma sequência de nucleótidos capaz de inibir ou impedir a expressão de um gene na citada célula e em que o gene é responsável por conferir em fenótipo conhecido. Esta sequência de nucleótidos pode ser parte de, ou idêntica ao citado gene que confere o citado fenótipo, e esta sequência de nucleótidos está ela própria orientada em relação a um promotor(es) apropriado(s) capaz(es) de iniciar a transcrição de ARN de cadeia dupla após ligação de um factor de transcrição apropriado ao citado promotor(es). Alternativamente, a sequência de nucleótidos pode ser parte de, ou idêntica à citada sequência génica que confere o citado fenótipo, e esta sequência de nucleótidos é tal que permite a integração do citado vector adequado ou adicional por recombinação homóloga no genoma da citada célula e após a citada integração, a citada sequência de nucleótidos é capaz de inibir a expressão de citada sequência do gene que confere o citado fenótipo.
Nesta concretização a citada sequência de nucleótidos compreende codões de paragem suficientes para impedir a tradução da citada sequência de nucleótidos após a sua integração no citado genoma.
De forma vantajosa, no citado método podem ser adicionados compostos à citada célula ou organismo com o objectivo de pesquisa dos fenótipos desejados, tais como por exemplo, resistência ou sensibilidade ao composto, quando comparado com o tipo selvagem. Os promotores são preferencialmente indutiveis. 0 factor de transcrição pode ser, em algumas concretizações, derivado de fagos, tal como por exemplo, uma polimerase de T7 conduzida por um promotor fágico. Contudo, quando é utilizado C. elegans, pode ser utilizado um promotor especifico do verme ou do tecido, tal como por exemplo, let858, SERCA, UL6, myo-2 ou myo-3. Preferencialmente, a estirpe de E.coli é uma ARNase III e ainda mais preferencialmente uma estirpe negativa para ARNase. 5 ΕΡ 1 197 567/ΡΤ
Um método de geração de um organismo não humano transgénico contendo um factor de transcrição exógeno e um transgene contendo um promotor operativamente ligado ao fragmento de ADN que é expresso após ligação do citado factor de transcrição, pode compreender: a) proporcionar um primeiro organismo transgénico contendo uma primeira construção incorporando ADN que codifica um factor de transcrição exógeno e um segundo organismo transgénico contendo uma segunda construção incluindo pelo menos um promotor operativamente ligado a uma sequência de ADN desejada que é expressa após ligação do factor de transcrição do citado primeiro organismo transgénico; b) cruzar os citados primeiro e segundo organismos transgénicos e seleccionar a progénie que expressa a citada sequência de ADN desejada. Numa concretização os citados primeiro e segundo organismos transgénicos são gerados transformando as citadas primeira e segunda construções nos respectivos microrganismos para subsequente alimentação ao respectivo organismo. Preferencialmente, a citada segunda construção contém a citada sequência de ADN desejada numa orientação relativamente ao citado promotor, que lhe permite iniciar a transcrição do citado ADN em ARNcd após ligação do citado factor de transcrição. Nesta concretização a citada segunda construção contém dois promotores que flanqueiam a citada sequência de ADN desejada, promotores estes que podem iniciar a transcrição da citada sequência de ADN em ARNcd após ligação do citado factor de transcrição aos citados promotores. Alternativamente, a citada sequência de ADN é proporcionada numa orientação com sentido e anti-sentido relativamente ao citado promotor para produzir ARNcd após ligação do factor de transcrição aos promotores. Em cada uma destas concretizações a primeira e/ou a segunda construções podem preferencialmente ser proporcionadas com um gene repórter operativamente ligado a um promotor que é capaz de iniciar a transcrição do citado repórter após ligação do citado factor de transcrição. Preferencialmente, o gene repórter codifica qualquer entre luciferase, proteína verde fluorescente, β-galactosidade ou β-lactamase.
Um método de validação de clones identificados em experiências com vectores de dois híbridos em levedura, experiências essas que são bem conhecidas dos peritos na especialidade e experiências essas que foram primeiro propostas por Chien e col. (1991) para detectar interacções 6 ΕΡ 1 197 567/ΡΤ proteína-proteína, pode compreender proporcionar uma construção incluindo o ADN que codifica uma proteína identificada numa experiência com vectores de dois híbridos, construção esta que é tal que o citado ADN é proporcionado numa orientação relativamente a um ou mais promotores capazes de promover a transcrição do citado ADN em ARN de cadeia dupla após ligação de um factor de transcrição apropriado aos citados promotores, transformar uma célula, tal como uma célula bacteriana ou alternativamente transformar um organismo contendo o citado factor de transcrição, com as citadas construções, e identificar uma mudança fenotípica na citada célula ou organismo, que pode ser C. elegans ou semelhante, em comparação com o tipo selvagem. Preferencialmente, o factor de transcrição é indutível na célula ou no organismo. Mais uma vez a sequência de ADN pode estar localizada entre dois promotores ou em ambas orientações, com sentido e anti-sentido, relativamente a um único promotor, conforme descrito acima. Preferencialmente, o promotor é um promotor de polimerase fágica e o citado factor de transcrição é uma ARN-polimerase, e preferencialmente polimerases de T7. Estão também incluídos no âmbito da presente invenção os vectores utilizados para transformar as citadas células ou organismos e as próprias células ou organismos.
Um método para aliviar a infestação de plantas por pragas pode compreender: a) a identificação de uma sequência de ADN da citada praga que é crítica para a sua sobrevivência, crescimento, proliferação ou reprodução, b) a clonagem da citada sequência da etapa a) , ou um fragmento, num vector apropriado relativamente a um ou mais promotores capazes de transcrever a citada sequência em ARN ou ARNcd após ligação de um factor de transcrição apropriado aos citados promotores, e c) a introdução do citado vector na planta.
Deste modo, de forma vantajosa, o método proporciona um mecanismo particularmente selectivo para aliviar a infestação por pragas, e em alguns casos infestação parasitária, de plantas, tal que, quando a praga se alimenta da planta, irá digerir o ARNcd expresso na planta, deste modo inibindo a expressão do ADN na praga que é crítico para o seu crescimento, sobrevivência, proliferação ou reprodução. Numa concretização preferida, a praga pode ser qualquer entre Tylenchulus ssp., Radopholus ssp., Rhadinaphelenchus ssp., 7 ΕΡ 1 197 567/ΡΤ
Heterodera ssp., Rotylenchulus ssp., Pratylenchus ssp., Belonolaimus ssp., Canjanus ssp., Meloidogyne ssp., Globodera ssp., Nacobbus ssp, Ditylenchus ssp., Aphelenchoides ssp., Hircshmenniella ssp., Anguina ssp., Hoplolaimus ssp., Heliotylenchus ssp., Criconemella ssp., Xiphinema ssp., Longidorus ssp., Trichodorus ssp., Paratrichodorus ssp., Aphelenchs ssp. A sequência de ADN ou seu fragmento, de acordo com este aspecto da invenção, pode ser clonado entre dois promotores específicos de tecidos, tais como dois promotores específicos da raiz. 0 vector utilizado em cada um dos métodos aqui descritos para a construção da citada biblioteca, contém dois promotores idênticos orientados de tal forma que são capazes de iniciar a transcrição da sequência de ADN localizada entre os citados promotores, em ARNcd, após ligação de um factor de transcrição apropriado aos citados promotores. A sequência de ADN pode, por exemplo, incluir um local de clonagem múltipla. Preferencialmente, o vector de expressão contém uma sequência de nucleótidos que codifica um marcador seleccionável. Numa concretização a sequência de nucleótidos que codifica o citado marcador seleccionável está localizada entre dois promotores idênticos orientados de tal forma que são capazes de iniciar a transcrição do ADN localizado entre os citados promotores, em ARN de cadeia dupla, após ligação de um factor de transcrição apropriado aos citados promotores. Preferencialmente, o marcador seleccionável contém uma sequência de nucleótidos que codifica sup-35, para introdução em C. elegans apresentando uma mutação pha-1.
Preferencialmente, o factor de transcrição contém uma polimerase fágica que se liga ao seu promotor correspondente ou um promotor específico de C. elegans e ainda mais preferencialmente polimerase de T7. Preferencialmente, o vector inclui um local de clonagem múltipla entre os citados promotores idênticos.
Um vector de expressão para expressar um factor de transcrição apropriado pode compreender uma sequência de nucleótidos que codifica o citado factor de transcrição ligado operativamente a sequências de controlo de expressão adequadas. Preferencialmente, as sequências de controlo de expressão incluem promotores que são promotores específicos de 8 ΕΡ 1 197 567/ΡΤ tecidos ou genéricos, constitutivos, indutíveis, ou suas combinações. Preferencialmente, o factor de transcrição contém uma polimerase fágica, e preferencialmente uma ARN-polimerase de T7, T3 ou SP6.
Um sistema de selecção para identificar a transformação de uma célula ou organismo com um vector como aqui descrito, sistema este que contém um vector como aqui descrito, onde o citado marcador seleccionável contém uma sequência de nucleótidos capaz de inibir ou impedir a expressão de um gene na citada célula ou organismo, gene esse que é responsável por conferir um fenótipo conhecido. Preferencialmente, a citada sequência de nucleótidos corresponde a uma parte de, ou é idêntica ao citado gene que confere o citado fenótipo conhecido, e essa sequência de nucleótidos está ela própria localizada entre dois promotores idênticos capazes de iniciar a transcrição de ARN de dupla cadeia após ligação de um factor de transcrição apropriado. Alternativamente, a sequência de nucleótidos compreende uma sequência de nucleótidos que é parte de, ou idêntica à citada sequência génica que confere um fenótipo conhecido à citada célula ou organismo, e que é tal que, após a integração do citado vector por recombinação homóloga no cromossoma da citada célula ou organismo da citada sequência, inibe a expressão da citada sequência génica que confere o citado fenótipo conhecido. Preferencialmente, de acordo com esta concretização a sequência de nucleótidos contém codões de paragem suficientes para impedir a tradução da sequência de nucleótidos após integração no citado cromossoma.
Preferencialmente, a sequência génica conhecida contém um gene sup-35 ou um seu fragmento que é seleccionável por identificação da progénie que cresce a uma temperatura acima de 25 °C após introdução num verme C. elegans mutante pha-1 et123ts.
Um método para atribuir a função a uma sequência de ADN de um organismo multicelular pode compreender: a) proporcionar i) uma construção que contém o citado fragmento de ADN clonado entre dois promotores capazes de promover a transcrição no citado organismo multicelular, num organismo multicelular capaz de iniciar a transcrição do citado promotor; b) identificar o fenótipo do citado organismo multicelular comparativamente com o tipo selvagem. 9 ΕΡ 1 197 567/ΡΤ A presente invenção pode ser mais claramente compreendida pelos seguintes exemplos que são puramente exemplificativos com referência às figuras anexas, nas quais: a Figura 1: é uma sequência de nucleótidos de plasmideo PGN1 de acordo com a presente invenção; a Figura 2: é uma sequência de nucleótidos do plasmideo PGN100 de acordo com a presente invenção; a Figura 3: é uma representação esquemática dos vectores utilizados e do regime de transformação utilizado nos métodos aqui descritos; a Figura 4: é uma ilustração de um vector de expressão utilizado de acordo com a invenção; a Figura 5: é uma ilustração esquemática dos vectores de expressão de ARN-po 1 imer a s e de T7 utilizados para transformar C. elegans; a Figura 6: é uma ilustração do plasmideo PGN1 . a Figura 7: é uma representação diagramática de um vector melhorado para a inibição do ARNcd que codifica ARNcd de sup-35; a Figura 8: é uma ilustração de um vector para a integração no genoma de C. elegans; a Figura 9: é uma ilustração da posição de uma sequência(s) de ADN relativamente a um promotor apropriado para iniciar a expressão de ARNcd a partir da(s) sequência(s) de ADN; a Figura 10: é uma representação do plasmideo pGN108; a Figura 11: é uma representação do plasmideo pGN105; a Figura 12: é uma representação do plasmideo pGN400; a Figura 13: é uma representação do plasmideo pGN401; a Figura 14: é uma representação do plasmideo pGNUO; 10 ΕΡ 1 197 567/ΡΤ a Figura 15: é uma representação do plasmídeo pAS2 com promotores de T7/T3/SP6 directos e inversos; a Figura 16: é uma representação do plasmídeo pGAD424 com promotores de T7/T3/SP6 directos e inversos; a Figura 17: é uma representação do plasmídeo pAS2-cyh2- HA+both-T7-final; a Figura 18: é uma representação do plasmídeo pGAD424-without-FULL-ICE-BOTH-T7; a Figura 19: (a) é uma representação do plasmídeo pGN205 e (b) é uma representação do plasmídeo pGN207;
Exemplo A: Construção de uma biblioteca de ADNc ordenada e agrupada hierarquicamente e suas aplicações.
Uma biblioteca reunida e ordenada de forma aleatória: O vector é um vector de E. coli contendo dois promotores de T7, com um local de clonagem múltipla (MCS) no meio. Os dois promotores estão orientados um para o outro, e para o MCS. Na presença da ARN-polimerase de T7, expresso em E. coli, C. elegans ou qualquer outro organismo, o ARN será produzido, começando a partir dos dois promotores de T7. Como estes estão orientados em sentidos opostos, ambas as cadeias de ARN serão produzidas a partir do ADN inserido (clonado) no MCS entre os dois promotores o que resulta na geração do ARN de cadeia dupla (ARNcd) após ligação da ARN-polimerase de T7.
Uma biblioteca ADNc de C. elegans é construída no MCS utilizando técnicas de biologia molecular padrão. A biblioteca é transformada em E. coli, e a E. coli resultante é crescida em cultura e é armazenada em placas de 96 poços. Neste estádio, o ADN do plasmídeo pode ser isolado e armazenado em placas de 96 poços correspondentes às das colónias de E. coli. Aproximadamente 100 000 colónias são classificadas. Deste modo, a biblioteca irá abrigar aproximadamente 5 vezes a variação total de ADNc expresso de C. elegans, o que proporciona a oportunidade para sequências com pouca expressão estarem presentes na biblioteca. Isto resultará em aproximadamente 1041 placas com 96 poços. As placas são agrupadas hierarquicamente, conforme necessário. Para o 11 ΕΡ 1 197 567/ΡΤ presente agrupamento dos clones, são dispostas numa fila de 10 a 100. Se o agrupamento hierárguico for por 8 ou 12 (números mais convenientes já gue as placas de 96 poços possuem uma grelha de 8 a 12), isto resultará em aproximadamente 87 placas de múltiplos poços e aproximadamente 8352 poços. Se o agrupamento hierárguico é por 96 poços, gue é uma placa completa, isto resulta em aproximadamente 11 placas e aproximadamente 1041 poços. Em qualquer estádio do agrupamento hierárquico, o ADN do plasmideo pode ser isolado, o que seria menos elaborado quando menos placas são utilizadas, mas resultará numa perda de complexidade embora isto não seja o caso no agrupamento por 12. 0 agrupamento do ADN pode também ser realizado com o ADN original.
As experiências abaixo descrevem como deve ser realizado o agrupamento hierárquico, tanto para o ADN como para a biblioteca de E. coli.
Uma biblioteca ordenada para tecnologia de ARNi, contendo todos os genes do genoma de C. elegans, com suas aplicações
Como o projecto de sequenciação do genoma se está aproximando do fim, esta informação pode ser utilizada na aplicação da tecnologia de inibição de ARN de T7. Cada gene do genoma de C. elegans pode ser clonado utilizando a tecnologia PCR. De preferência, os exões serão clonados com um comprimento minimo de 500 pb. Se os exões são muito pequenos, serão isolados fragmentos menores por PCR, ou mesmo partes de intrões e exões vizinhos serão isoladas com a tecnologia PCR de modo que é clonada no mínimo uma parte suficiente da região traduzida do gene. Para isso, necessitam de ser realizadas no mínimo 17000 reacções de PCR. Esta colecção de produtos de PCR será clonada num vector de T7 conforme descrito (dois promotores de T7 orientados um para o outro com um local de clonagem múltipla entre eles). Cada produto de PCR é clonado independentemente, ou pode ser utilizado para gerar uma biblioteca aleatória, análoga à biblioteca de ADNc descrita. Se cada produto de PCR for clonado individualmente, ADN da bactéria resultante e do plasmideo podem ser agrupados de várias formas. Inicialmente, esta colecção de produtos de PCR clonados individualmente no vector de T7 de ARNi pode ser agrupada aleatoriamente, conforme descrito na biblioteca aleatória. Este agrupamento pode também ser feito de uma forma 12 ΕΡ 1 197 567/ΡΤ mais racional. Por exemplo, os genes do genoma de C. elegans podem ser analisados utilizando ferramentas bio-informáticas (em silico-biologia). Vários genes do genoma irão pertencer a uma família de genes, ou irão possuir homólogos no genoma. Estes membros da família de genes irão ser agrupados, ou os membros, sendo homólogos irão ser agrupados. Desta forma, o número total de cerca de 17000 clones é reduzido para uma quantidade mais utilizável. Esta biblioteca pode ser utilizada para pesquisar fenótipos nos métodos de acordo com a invenção. 0 fenótipo resultante proporciona uma descrição funcional para o gene ou família do gene ou homólogos do gene do genoma de C. elegans. Como a biblioteca consiste numa parte de todos os genes no genoma, este método permite a descrição do genoma completo em termos fenótipo-funcionais. Para isto, o ARN de cadeia dupla (ARNcd) precisa de ser introduzido no verme. Esta introdução de clones individuais, ou de clones agrupados, sendo agrupados de forma aleatória ou sendo agrupados de forma racional, pode ser alcançada de várias formas conforme descrito.
Exemplo de um vector para a expressão de ARNi de cadeia dupla
Qualquer vector contendo um promotor de T7 pode ser utilizado, e que contém um local de clonagem múltipla (há vários disponíveis comercialmente). São projectados iniciadores contendo o promotor de T7 e um iniciador com a cadeia complementar inversa, ambas com as extremidades apropriadas. Estes iniciadores podem ser hibridados, e se bem projectados, clonados no vector de eleição. A sequência mínima de um promotor de T7 é TAATACGACTCACTATAGGGCGA. Embora qualquer vector possa ser utilizado para a construção de um vector de expressão de T7 segue-se um exemplo de como alcançar o com o vector pGEM-3zf(-). - o vector pGEM-3zf(+) (PROMEGA) foi digerido com HindIII e Sall. os iniciadores oGNl e oGN2 foram misturados numa concentração final de 1 pg/30 μΐ, fervidos e arrefecidos lentamente até atingir a temperatura ambiente. - 0 iniciador foi ligado ao vector utilizando procedimentos de ligação padrão. O vector resultante é pGNl (mostrado na Figura 1) e contém dois promotores de T7 orientados um para o 13 ΕΡ 1 197 567/ΡΤ outro, e possui um local de clonagem múltipla entre eles. As sequências de oGNl e oGN2 são:
- oGNl: AGC TGT AAT ACG ACT CAC TAT AGG GCG AGA AGC TT
- oGN2: TCG AAA GCT TCT CGC ATA ATA GTG AGT CGT ATT AC
Exemplo de construção de uma biblioteca 0 ARN pode ser isolado de todos os organismos que sejam sensíveis ao ARNi. Em geral, o ARN isolado é depois copiado num ADNc de dupla cadeia, e subsequentemente preparado em vectores apropriados para clonagem. Existem vários procedimentos e podem ser adquiridos kits de biologia molecular de várias empresas, incluindo Promega, Clontech, Boehringer Mannheim, BRL, etc. que possilita permitem:
- o isolamento de ARN - eventualmente pode ser isolado o ARN poliA (várias técnicas e Kits disponíveis) - síntese da primeira cadeia com transcriptase inversa de AMV, iniciadores hexaméricos aleatórios e/ou iniciador oligo (dT)
- síntese da segunda cadeia com ARNase H, ADN-Polimerase I - extremidades lisas com ADN-polimerase de T4 - adição de um adaptador com ADN-ligase de T4 - eventualmente tratamento com polinucleótido-quinase de T4 - clonagem do ADNc no vector A mistura de ligação resultante pode ser considerada como a biblioteca de ADNc. A ligação contém todo o ADNc do procedimento ligado ao vector de interesse. Para ordenar a biblioteca, a ligação necessita de ser transformada em estirpes de E. coli.
Aplicação desta biblioteca de £. coli ou de ADN estirpe que produz ARN de T7: - uma estirpe padrão é BL21 (DE3): F-ompT lon]hsds (r-m-; e Estirpe B de E. coli) À(DE3). Eventualmente variantes de 14 ΕΡ 1 197 567/ΡΤ BL21 (DE3) podem ser utilizadas, embora seja utilizada BL21 (DE3) pLysS. - qualquer outra estirpe de E. coli que produza a ARN-polimerase de T7, que possa estar disponível necessita de ser construída. Isto pode ser gerado facilmente utilizando um fago, que está disponível comercialmente; neste caso, é utilizado o vector XCE6 (proporcionado pela Promega). Quase todas as estirpes de E. coli podem ser transf ectadas com esse fago e irão produzir ARN-polimerase de T7. - uma E. coli mutante para ARNase III. Várias estirpes estão em princípio disponíveis, escolhemos numa primeira experiência utilizar a estirpe AB301-105: rna-19, suc-11, bio-3, gdhA2, his95, rnc=-105, relAl, spoTl, metBl (Kinder e colab. 1973 Mol. Gen. Genet. 126:53), mas outras estirpes podem ser mais adequadas. Esta estirpe é infectada com XCE6 e assim será formada uma variante que produz T7.
Vermes C. elegans do tipo selvagem podem ser crescidos sobre os agrupamento de bactérias. A bactéria expressa a ARN-polimerase de T7. Isto resulta em grandes quantidades de ARNcd no intestino de C. elegans, que se irá difundir no organismo e resulta na inibição de expressão. Esta biblioteca pode agora ser utilizada para a pesquisa de vários fenótipos. Esta técnica possui a vantagem de ser mais rápida para detectar genes relevantes em certas vias, do que a tecnologia conhecida para C. elegans. Além disso, se for encontrado um fenótipo interessante, o gene responsável pode ser clonado facilmente.
Utilizando o agrupamento hierárquico, pode-se facilmente encontrar numa segunda pesquisa o clone relevante do agrupamento. O ADN inserido deste clone pode então ser sequenciado. Esta experiência resulta em dados bioquímicos e genéticos numa etapa.
Estirpes de C. elegans do tipo selvagem podem ser combinadas com compostos para pesquisar o fenótipo, a resistência a fármacos e a sensibilidade a fármacos. A estirpe de C. elegans pode ser uma estirpe mutante, pesquisando um fenótipo melhorado, um fenótipo reduzido ou um novo fenótipo. A estirpe de C. elegans pode ser uma estirpe mutante, e a 15 ΕΡ 1 197 567/ΡΤ pesquisa da biblioteca pode ser combinada com compostos. Logo, pode-se pesquisar resistência a fármacos, sensibilidade a fármacos, fenótipo melhorado, fenótipo reduzido ou um novo fenótipo. A estirpe da E. coli pode ser qualquer estirpe que expresse ARN-polimerase de T7, como BL21 (DE3), por exemplo, mas a formação do ARN de cadeia dupla pode ser melhorada utilizando uma estirpe de E. coli especial que seja negativa para ARNase III. A ARNase III reconhece ansas especificas no ARNcd. Eventualmente, pode ser utilizada uma estirpe de E. coli que tenha eliminadas ARNases que não a ARNase III ou pode ser utilizada uma E. coli que tenha eliminado uma ou mais ARNAses. A expressão da ARN-polimerase de T7 na maioria das estirpes e construções de E. coli conhecidas que estão disponíveis para gerar estirpes de E. coli que produzem ARN-polimerase de T7, geralmente compreende um promotor indutível. Desta forma, a produção de ARN-polimerase de T7 é regulada, e consequentemente a produção do ARNcd. De forma vantajosa, este aspecto pode ser utilizado para alimentar "por pulsos" os vermes C. elegans em estádios específicos do crescimento. Os vermes são crescidos nas estirpes de E. coli não induzidas. Quando o verme atinge o estádio de interesse, a produção de ARN de T7 na bactéria é induzida. Isto permite o estudo da função de qualquer gene em qualquer ponto do ciclo de vida do animal.
Pesquisa da biblioteca quanto a homólogos de supostos genes humanos de interesse e atribuição da função a estes genes
Centenas de genes foram isolados em vários projectos, sejam projectos genómicos, arranjos diferenciais expressos, estudos de hibridação, etc. A biblioteca de ADNc descrita pode proporcionar uma forma para validar e/ou atribuir uma função a estes genes de uma forma rápida e eficiente. Antes de tudo, o homólogo ou homólogos do verme ou os genes precisam ser identificados por ferramentas bio-infomáticas (em silico-biologia). Iniciadores PCR são desenvolvidos e o fragmento de ADNc é isolado utilizando tecnologia de PCR. A PCR pode ser realizada com agrupamentos hierárquicos. 0 agrupamento positivo ou poços individuais portadores da bactéria que possui o ADNc apropriado, são alimentados ao C. elegans e o fenótipo é classificado. 16 ΕΡ 1 197 567/ΡΤ A PCR pode ser realizada com ADNc isolado de C. elegans. 0 ADN resultante pode ser clonado no vector de T7 e transformado na E. coli que produz o ARNcd com que os vermes C. elegans são então alimentados. Dependendo de que maneira seja mais rápida e mais fiável, precisa ser feita uma escolha.
Se o qene pertencer a uma família de qenes, o verme pode precisar ser alimentado com uma mistura de bactérias, cada uma portadora de uma parte do membro da família de qenes. Estirpes de E. coli, condições de crescimento e combinações com compostos podem ser realizados conforme descrito acima.
Se é utilizada uma biblioteca racional em que todos os genes de C. elegans são clonados de uma forma organizada e estruturada, o homólogo C. elegans e eventualmente os outros homólogos, ortólogos e membros da família de genes podem ser facilmente identificados na biblioteca utilizando a sílico-biologia. Não está envolvida PCR nesta etapa, e podem ser isolados as bactérias e/ou o ADN com os quais o verme vai ser crescido.
Exemplos: A ideia da série de experiências foi testar o vector de ARNi a as várias estirpes de E. coli que foram construídas. 1) construção de um plasmídeo de teste
Qualquer ADNc que proporciona um fenótipo claro no verme quando, anulado, ou usado numa experiência de ARNi, pode ser utilizado. Sabe-se que unc-22 é um bom candidato, mas são possíveis outros genes. Optámos por um sistema sensível que pode ser utilizado num estádio posterior. 0 sistema foi testado com sup-35 num fundo pha-1. 0 exão 5 do sup-35 foi isolado por PCR e clonado no vector pGNl de promotor de T7. 0 vector resultante foi designado pGN2. Os vermes mutantes pha-1 (e2123) não podem produzir progénie a temperaturas maiores do que 25°C. Isto deve-se a um problema de desenvolvimento na embriogénese. Quando o sup-35 é anulado, ou inibido nesta estirpe, a progénie pode crescer a esta temperatura. A combinação de vermes mutantes pha-1 e ARNi de sup-35 é um bom sistema para validar as várias opções. 17 ΕΡ 1 197 567/ΡΤ 2) Teste do ARNi utilizando uma estirpe de E. coli que produz ARNcd 0 pGN2 foi introduzido na estirpe B121 (DE3) de E. coli e a ARN-polimerase de T7 foi induzida com IPTG.
Vermes C. elegans (pha-1 (e2123)) foram inoculados com esta bactéria e cresceram à temperatura restrita de 25°C. Como este mutante é um mutante embrionário a esta temperatura, não será observada progénie. Se o gene sup-35 for inibido eficientemente pelo ARNcd presente na E. coli, será observada progénie. - 0 pGN2 foi introduzido na estirpe AB301-105 (DE3) de E. coli e a ARN-polimerase de T7 foi induzida com IPTG.
Vermes C. elegans (pha-1 (e2123)) foram inoculados com esta bactéria e cresceram à temperatura restrita de 25°C. Como este mutante é um mutante embrionário a esta temperatura, não será observada progénie. Se o gene sup-35 for inibido eficientemente pelo ARNcd presente na E. coli, será observada progénie. 3) Aperfeiçoamento da estirpe do verme para melhor captação de ARNcd.
Antes de plaquear o pha-1 de C. elegans na estirpe de E. coli que produz o ARN de cadeia dupla de sup-35, o verme foi submetido a mutação com EMS (Ácido etilmetanossulfónico) . A progénie deste verme que foi submetido à mutação é então plaqueada sobre a bactéria. 0 verme que se alimenta desta bactéria proporciona maior progénie que o que apresenta uma mutação que resulta num aperfeiçoamento da captação do ARNcd, e pode ser utilizada para outras experiências.
Integração estável do vector que produz ARNcd no genoma do verme que produz ARN-polimerase de T7.
Pode ser construído um vector de E. coli portador das seguintes características; dois promotores de T7 orientados um para o outro, com um local de restrição ou um local de clonagem múltipla entre si. Além disso, o vector pode conter ADN genómico de sup-35 de C. elegans, manipulado de tal forma que contenha vários codões de paragem em vários intervalos, de modo a que não possa ser expressa proteína de comprimento 18 ΕΡ 1 197 567/ΡΤ total a partir do fragmento de ADN genómico de sup35, conforme ilustrado na Figura 8. Qualquer ADNc ou fragmento de ADNc pode ser clonado no local de clonagem múltipla entre os dois promotores de T7. Quando esse vector é introduzido numa estirpe de C. elegans que expressa ARN-polimerase de T7, o ADNc ou fragmento de ADN clonado entre os dois promotores de T7 será transcrito, gerando ARNcd a partir do fragmento clonado. 0 vector é projectado para ser utilizado em vermes mutantes pha-1 (e2123) que expressam ARN-polimerase de T7. A expressão da ARN-polimerase de T7 pode ser constitutiva ou regulada, geral ou especifica de tecidos. Estes vermes pha-1 (e2123) não podem produzir progénie a temperaturas maiores que 25 °C, o que se deve a um problema de desenvolvimento na embriogénese. Quando o sup-35 é inibido ou anulado nesta estirpe, pode crescer progénie a esta temperatura.
Quando o vector é introduzido no verme, o vector pode-se integrar por recombinação homóloga (integração tipo Campbell). Foi demonstrado que ocorre recombinação homóloga em C. elegans, embora com baixas frequências (Plasterk e Groenen, EMBO J. 11:287-290, 1992). A recombinação homóloga no gene sup35 irá resultar numa anulação do gene pois os dois genes sup35 resultantes, irão abrigar os codões de paragem. O verme resultante, e a sua progénie, se essa recombinação acontecer nos ovos, irá possuir uma cópia do vector integrado no genoma. Isto pode ser seleccionado pois somente os vermes onde o sup35 tenha sido anulado apresentarão progénie a temperaturas maiores que 25°C. Além disso, o verme resultante irá produzir de forma estável ARN de cadeia dupla a partir do fragmento de ADN clonado entre os dois promotores de T7. Este verme pode agora ser considerado como uma estirpe de verme transgénico estável com uma redução da função do gene, e onde um fragmento foi clonado entre os dois promotores de T7. O ADN pode ser proporcionado ao verme através de várias técnicas, incluindo injecção, transformação balística, imersão na solução de ADN, alimentação com bactérias. Novos e outros métodos que aumentam as eficiências da transformação podem ser considerados A estirpe alvo de C. elegans pode adicionalmente possuir outras mutações além da mutação pha-1 (e2123) e pode expressar outros genes para além do da ARN-polimerase de T7. 19 ΕΡ 1 197 567/ΡΤ
Exemplo Β: um vector de ARNi de dois híbridos em levedura
Um vector de dois híbridos em levedura pode ser construído portador de dois promotores de T7. Os vectores podem ser projectados para replicar tanto em levedura com em E . coli . Em geral, as bibliotecas de ADNc para o sistema de dois híbridos em levedura são feitas nos vectores Gal4 ou LexA. A biblioteca é construída em vectores que apresentam o domínio de activação de um destes genes. Pode ser construído um vector que possa ainda desempenhar na levedura a pesquisa de dois híbridos, mas que também contenha dois promotores de T7 orientados um para o outro, com um local de clonagem entre si. A ordem das sequências no plasmídeo será então "esqueleto do plasmídeo, (GAL4-T7) , MCS, T7, esqueleto". Uma biblioteca de ADNc no C. elegans construída neste vector pode ser utilizada como uma biblioteca padrão de dois híbridos em levedura numa experiência para isolar proteínas com interacção com uma determinada proteína. Uma vez isolado o clone, o plasmídeo pode ser introduzido numa estirpe de E. coli expressando a ARN-polimerase de T7, e portanto irá produzir ARNcd do fragmento clonado. A bactéria que produz este ARNcd pode ser alimentada ao verme e os fenótipos podem ser classificados. Como no exemplo anterior, este procedimento de validação para um clone de dois híbridos em levedura, isolado de novo é notavelmente mias curto que o procedimento padrão, que requer etapas de PCR e/ou clonagem experiências de ARN e/ou experiências de anulação. Na maioria dos casos os clones isolados são primeiro sequenciados, e com base na sequência, é tomada uma decisão para continuar com outras experiências. Na presente invenção, cada clone isolado pode ser facilmente introduzido na E. coli apropriada e alimentado ao verme. A validação é então realizada por análise do fenótipo.
Para aplicar este procedimento, foi aplicado um sistema de dois híbridos em levedura utilizando um gene conhecido como isco e a biblioteca construída de novo como o alvo. Proteínas codificadas pelos clones no alvo que interactuam com a proteína isco, irão resultar em clones de levedura positivos expressando a molécula repórter como pode ser observado pela coloração de Lacz com X-gal. 0 plasmídeo que codifica para a proteína alvo é isolado directamente da estirpe de levedura e introduzido em E. coli. A E. coli é E. coli produtora de ARN-polimerase de T7. Neste caso, o ARN de cadeia dupla é 20 ΕΡ 1 197 567/ΡΤ produzido a partir do ADN clonado no local de clonagem múltipla do vector. Quando este ARNcd é alimentado ao verme utilizando os métodos descritos previamente, o gene foi inibido no verme, resultando num fenótipo particular. - Este vector de dois híbridos em levedura pode de forma vantajosa, ser utilizado para construir uma biblioteca ordenada e agrupada hierarquicamente conforme descrito no exemplo anterior. - Pode ser também construída uma estirpe de levedura que produz condicionalmente ARN-polimerase de T7. Após as experiências com o sistema de dois híbridos em levedura, a expressão da polimerase de T7 pode ser induzida, resultando na produção de ARNcd na célula de levedura. Consequentemente, a levedura pode ser alimentada ao verme. Estão disponíveis evidências mostrando que os vermes C. elegans se podem alimentar da levedura.
Construção de uma estirpe que produz ARN-polimerase de T7, e suas aplicações
Pode ser construída Uma estirpe de C. elegans que expressa ARN-polimerase de T7. A expressão pode ser geral e constitutiva, mas pode também ser regulada por um promotor específico do tecido, um promotor indutível, ou um promotor temporal ou um promotor que contenha uma destas características ou combinação de características. O ADN pode ser introduzido nesta estirpe de C. elegans. Isto é feito por injecção, por bombardeio de partículas, por electroporação ou como mencionado acima através de alimento. Se o ADN for um plasmídeo conforme descrito nos exemplos anteriores, isto é, um plasmídeo portador de um fragmento ADNc clonado ou um fragmento de PCR entre dois promotores de T7 flanqueadores, então o ARNcd deste ADNc ou fragmento de PCR é formado na célula ou no organismo completo resultando em infra-regulação do gene correspondente. O ADN introduzido pode ter uma infra-regulação transitória eficiente. O ADN introduzido pode formar um arranjo extracromossómico, arranjo este que pode resultar numa maior anulação catalítica ou redução do fenótipo de função. O plasmídeo pode integrar-se também no genoma do organismo, resultando a mesma anulação catalítica ou redução do fenótipo de função, mas que é transmissível de forma estável. 21 ΕΡ 1 197 567/ΡΤ - Ο ADN do plasmídeo portador de um ADNc ou de uma parte de um ADNc ou uma EST ou um fragmento de PCR de C. elegans clonado entre dois promotores de T7 conforme descrito nos exemplos A) e B) pode ser introduzido no verme de ARN- polimerase de T7 através de técnicas padrão. Os fenótipos podem ser analisados - ADN de uma biblioteca agrupada e ordenada como no exemplo A) pode ser introduzido no verme de ARN-polimerase de T7, através de técnicas padrão (injecção, bombardeio). Os fenótipos podem ser analisados. Com o agrupamento hierárquico, o clone original pode ser encontrado facilmente. - 0 mesmo procedimento pode ser realizado com um verme mutante expressando a ARN-polimerase de T7. Pesquisa de fenótipos novos, reduzidos ou melhorados. - 0 procedimento pode ser utilizado para possibilitar a pesquisa de compostos. Pesquisa com uma estirpe de tipo selvagem ou uma estirpe mutante, de fenótipos novos ou melhorados. - 0 ADN pode ser introduzido no verme através de novos métodos. Um deles é a entrega do ADN pela E. coli. Neste caso, a biblioteca agrupada hierarquicamente é alimentada ao animal. Para impedir a digestão do ADN da E. coli no intestino do nematode, será utilizado preferencialmente um C. elegans deficiente em ADNase, tal como nuc-1(e 1392). Este procedimento será um dos mais interessantes pois seria independente de eficiências de transformação de outras técnicas, e geralmente mais rápidos e menos trabalhosos. 2) Supostos melhoramentos do método - É projectado um vector de modo que abriga o ADNc de sup-35 ou uma parte deste ADNc, e é clonado entre dois promotores de T7. 0 restante do vector é conforme descrito nos Exemplos A) e B). Este vector pode ser introduzido num C. elegans mutante pha-lts. Existe neste caso um sistema de selecção de temperatura e somente os vermes que captaram o ADN e expressam o ARN de sup-35 de cadeia dupla irão sobreviver a estas temperaturas restritas. A biblioteca agrupada hierarquicamente pode ser entregue por qualquer método descrito acima. 22 ΕΡ 1 197 567/ΡΤ Ο vector pode ser utilizado para construir uma biblioteca que é introduzida numa E. coli que expressa ARN- polimerase de T7. Neste caso, temos uma pesquisa análoga à da parte A) com uma pesquisa adicional de vermes onde o ARNcd de sup-35 está activo. - 0 ADN e/ou o ARNcd de sup-35 poderia ser entregue num plasmideo diferente. Para a alimentação, tanto alimentação com ADN (exemplo C) ou alimentação com ARNcd (exemplo A e B), isto significa que os dois plasmídeos poderiam estar presentes numa bactéria, ou que o verme é alimentado com uma mistura de bactérias, um deles portador da construção de sup-35.
Exemplo da construção de um C. elegans que produz ARN de T7.
Para produzir ARN-polimerase de T7 no verme, são possíveis várias possibilidades. A polimerase de T7 pode ser expressa sob vários promotores, sejam promotores indutíveis, promotores constitutivos, promotores gerais e promotores específicos de tecidos (célula), ou suas combinações. Os exemplos destes promotores são o promotor de choque térmico hsp-16, o promotor intestinal gesl, o promotor de cet858, mas também o promotor de dpy 7 e o elemento promotor GATA1. Neste exemplo, a ARN-polimerase de T7 é expressa sob o controlo do promotor hsp-16 que está disponível no vector pPD49.78. A ARN-polimerase de T7 é isolada como um produto de PCR utilizando os iniciadores de GN3 e GN4. 0 produto de PCR resultante é digerido com Nhel e Ncol, assim como o vector no qual o queremos clonar, sendo o vector Fire pPD49.78. 0 vector resultante é pGNIOO ilustrado na
Figura 2: oGN3: CAT GGC AGG ATG AAC ACG ATT AAC ATC GC; oGN4: ATG GCC CCA TGG TTA CGG GAA CGC GAA GTC CG; pGNIOO está incluído. 0 vector é introduzido no verme utilizando técnicas padrão, tais como micro-injecção, por exemplo.
As seguintes estirpes foram então construídas: - Tipo selvagem (pGNIOO) - nuc-1 (el392) (pGNIOO) - pha-1 (e2123) (pGNIOO) 23 ΕΡ 1 197 567/ΡΤ - pha-1; nuc-1 (pGNIOO)
Todas essas estirpes são capazes de produzir ARN- polimerase de T7 quando induzidas por temperatura ou alternativamente por metais tais como aplicação de cádmio ou mercúrio pesados. 0 procedimento para indução pela temperatura consiste em elevar a temperatura do animal para 30-33°C por um mínimo de 1 hora, e depois o animal pode ser mudado de volta para as temperaturas padrão (15-25°C). A estirpe de tipo selvagem que produz ARN-polimerase de T7 pode ser utilizada para a produção de qualquer ARN no verme. Mais especificamente, os plasmídeos das bibliotecas descritas podem ser introduzidos nestes vermes, e os fenótipos podem ser classificados. O verme mutante nuc-1 será utilizado para introduzir ADN através de bactérias das quais o verme se alimenta. Como o verme nuc-1 não digere o ADN, o ADN do plasmídeo pode atravessar a parede intestinal. Se captado pelas células que produzem a ARN-polimerase de T7, o ARNcd será produzido desse modo, inibindo o gene a partir do qual o ARN foi transcrito. A estirpe mutante pha-1 que produziu ARN-polimerase de T7 pode ser utilizada para melhorar os procedimentos conforme descritos acima. O ADN pode ser introduzido por bombardeio, micro-injecção ou alimentação. Mais especificamente, esta estirpe pode ser utilizada para os vectores que produzem ARNcd a partir de sup-35 e a partir do gene de interesse, podendo o último ser um produto de PCR, um ADNc ou uma biblioteca, conforme descrito. O mutante pha-1;nuc-1 que produz ARN-polimerase de T7 pode ser utilizado para a entrega bacteriana do ADN. O ADN será preferencialmente o plasmídeo que produz ARNcd de sup-35 e do gene de interesse. A estirpe do verme irá preferencialmente produzir a ARN-polimerase de T7 no intestino. A entrega irá ocorrer preferencialmente por alimentação do verme com as bactérias portadoras do plasmídeo.
Aplicação da tecnologia de ARNi em plantas
Os nematodes são responsáveis por uma grande parte dos danos causados às plantas e mais particularmente às plantas 24 ΕΡ 1 197 567/ΡΤ utilizadas na indústria da agricultura. Os procedimentos de ARNi de acordo com a invenção podem ser aplicados às plantas para impedir que estes nematodes parasitários se alimentem por mais tempo. Numa primeira etapa, um fragmento de ADN é isolado do nematode que parasita a planta, que é critico para a sobrevivência ou crescimento dos animais, ou para a nutrição ou proliferação. Qualquer gene cuja expressão é essencial, é adequado para este propósito. É clonado uma parte deste gene, um exão ou ADNc. Este fragmento de ADN pode ser clonado sob a influência de um promotor especifico do tecido, preferencialmente um promotor especifico da raiz, mais preferencialmente entre dois promotores específicos da raiz. 0 ADN do gene clonado sob o controlo do promotor específico da raiz pode ser introduzido na planta de interesse, utilizando tecnologia transgénica de plantas. Para cada nematode parasitário, pode ser necessária uma porção de ADN diferente e, da mesma maneira, para cada espécie de planta, será necessário um promotor diferente. A raiz irá produzir ARN ou ARNcd a partir da porção de ADN introduzida quando é utilizado o promotor específico da raiz. Como o nematode se alimenta da planta, o ARN e/ou ARNcd irá ser consumido ou ingerido pelo nematode. 0 ARN e/ou ARNcd podem entrar nas células do nematode e realizar a sua acção inibitória sobre ADN alvo. Dependendo da natureza da porção de ADN clonado do verme, o nematode irá ser capaz de sobreviver, comer, proliferar, etc., em qualquer caso impedindo o animal de se alimentar por muito tempo da planta, e desta forma protegendo a planta.
Construção de um C. elegans produtor de ARN-polimerase de T7
Para produzir uma ARN-polimerase de T7 ou outras ARN-polimerases em animais, e mais particularmente em nematodes e muito mais particularmente em C. elegans, podem ser consideradas muitas possibilidades. A ARN-polimerase de T7 pode ser expressa sob vários promotores. Estes promotores podem ser promotores indutíveis, promotores constitutivos, promotores gerais, promotores específico de tecidos, ou suas combinações. 25 ΕΡ 1 197 567/ΡΤ
Exemplo 1:
Construção de um vector básico para expressão de polimerase de T7 em c. elegans. A sequência que codifica a polimerase de T7 foi amplificada por PCR a partir de XCE6 (Novagen, Madison, EUA) utilizando os iniciadores oGN26 (ATGGAATTCTTACGCGAACGCGAA GTCCG) e OGN46 (CTCACCGGTAATGAACACGATTAACATCGC), utilizando procedimentos padrão (PCR, A Practical Approach, 1993, Ed. J. McPherson e colab., IRL Press). 0 fragmento de ADN resultante que codifica a ARN-polimerase de T7 foi digerido com Agel e EcoRI e inserido no vector Fire pPD97.82, digerido com Agel e EcoRI. A construção resultante codifica para uma estrutura de leitura aberta de ARN-polimerase de T7 em fusão com o sinal de localização nuclear do antigénio T grande de SV40 (NLS) com a sequência de aminoácidos MTAPKKKRKVPV. Esta sequência de sinal de localização nuclear é necessária para translocar a ARN-polimerase de T7 do citoplasma para o núcleo, onde é capaz de se ligar aos seus promotores específicos, designados promotores de T7. A montante da sequência de codificação para a proteína de fusão de polimerase de T7 está um promotor mínimo (myo-2) precedido por um local de clonagem múltipla (MCS) no qual vários promotores de C. elegans podem ser inseridos. Este plasmídeo (pGNl05 apresentado na Figura 11) é um plasmídeo básico de ARN-polimerase de T7 que permite a expressão da polimerase de T7 em C. elegans. Derivados deste plasmídeo onde os promotores são clonados no local de clonagem múltipla, permitem a expressão indutível, constitutiva, geral e específica de tecidos da ARN-polimerase de T7 em C. elegans, pois a expressão será regulada pelo promotor clonado no local de clonagem múltipla.
Embora não restrito a estes exemplos, sabe-se que os seguintes promotores induzem a expressão nos seguintes tecidos: let-858 (expressão ubíqua), myo-2 (expressão na faringe), myo-3 (músculos da parede corporal), egl-15 (músculos vulvares), unc-119 (pan-neuron). 26 ΕΡ 1 197 567/ΡΤ
Exemplo 2:
Construção de um vector para expressão de ARN-polimerase de T7 no tecido muscular de C. elegans A sequência que codifica a ARN-polimerase de T7 foi amplificada por PCR a partir de XCE6 utilizando os iniciadores OGN43 (GCCACCGGTGCGAGCTCATGAACACGATTAACATCGC) e OGN44 (CACTAGT GGGCCCTTACGCGAACGCGAAGTCCG) digeridos com Agel/Spel e inseridos no vector pGK13 digerido com Agel/Spel. (Este vector contém o promotor forte SERCA que dirige a expressão na faringe, músculo vulvar, cauda e o músculo da parede corporal). Um sinal de localização nuclear (NLS) do antigénio T grande de SV40 foi inserido na frente da sequência de codificação da polimerase de T7 por inserção de dois oligos sobrepostos, OGN45 (CCGGATGACTGCTCCAAAGAAGAAGCGTAAGCT) e oGN46 (CTCACCGGTAATGAACACGATTAACATCGC) nos locais de restrição Sacl/Agel. A construção resultante foi denominada pGNl08 conforme apresentado na Figura 10. A introdução deste plasmídeo no C. elegans resulta na expressão de ARN-polimerase de T7 na faringe, músculo vulvar, cauda e músculos da parede corporal.
Para testar a expressão e a funcionalidade da ARN-polimerase de T7 em C. elegans sob a regulação do promotor SERCA, o pGN108, que codifica a ARN-polimerase de T7 sob o controlo do promotor SERCA, foi injectado em C. elegans. Foi co-injectado um vector de teste. Este vector de teste codifica para GFP sob o controlo de um promotor de T7 (pGN401 na Figura 13). O plasmídeo pGN401 foi construído por inserção de dois oligos sobrepostos, oGN41 (CCCGGGATTAATACGACTCACTATA) e OGN42 (CCGGTATAGTGAGTCGTATTAATCCCGGGAGCT) no vector Fire pPD97.82 aberto com Sacl/Agel gerando um promotor de T7. Além disso, um marcador de selecção foi co-injectado para seleccionar transformantes (rol6, pRF4). O último vector de selecção pRF4 é bem conhecido por peritos na especialidade. Os F1 transgénicos podem ser facilmente isolados já que apresentam o fenótipo rol6. Todos estes C. elegans transgénicos expressaram GFP na faringe, no músculo vulvar, na cauda e no músculo da parede corporal. Estes dados mostram claramente que a ARN-polimerase de T7 é funcionalmente expressa sob a regulação do promotor SERCA, e que a ARN-polimerase de T7 expressa se liga ao promotor de T7 presente 38 em pGN401 e inicia a transcrição do gene de GFP, que é então expresso funcionalmente, resultando em fluorescência nos tecidos musculares onde SERCA está a induzir a expressão doa ARN-polimerase de T7.
Exemplo 3:
Construção de um vector para expressão ubíqua da polimerase de T7 em C. elegans 0 gene de fusão NLS-ARN-polimerase de T7 foi isolado de pGN108 com Xmal/Bspl20l e clonado no vector Fire pPD103.05, digerido com Xmal/Bspl20l. Isto resulta num vector onde a ARN-polimerase de T7 é clonada sob a regulação do promotor let858. Este promotor específico permite a expressão da ARN-polimerase de T7 em todos os tecidos. 0 plasmídeo resultante foi denominado pGNUO (Figura 14).
Exemplo 4:
Construção de um vector para a expressão mediada pela ARN-polimerase de T 7 de fragmentos de ADN, genes e ADNc sob o controlo de um promotor de T7 0 vector Fire pPD97.82 foi digerido com Sacl/Agel e uma sequência do promotor de T7 foi gerada pela inserção de dois oligos sobrepostos, OGN41 (CCCGGGATTAATACGACTCACTATA) e 0GN42 (CCGGTATAGTGAGTCGTATTAATCCCGGGAGCT), nos locais das endonucleases de restrição Sacl/Agel. Esta construção (pGN400, Figura 12) contém uma estrutura de leitura aberta de GFP clonada entre os locais de endonuclease de restrição Saci e EcoRI sob a regulação do promotor de T7. Qualquer gene, ADNc ou fragmento de ADN pode ser clonado neste vector eliminando o gene de GFP na forma de um fragmento Agel/SacI e clonando o fragmento de ADN de interesse no vector. Preferencialmente, o fragmento de ADN de interesse pode ser obtido por amplificação por PCR, inserindo os locais Sacl/Agel nos iniciadores. 0 fragmento de ADN resultante após a amplificação de por PCR é digerido e o gene de GFP em pGN400 é substituído pelo fragmento de ADN amplificado.
Cada vector que contém um promotor de T7 pode ser utilizado com o propósito de expressão induzida pela ARN-polimerase de T7 em C. elegans, tais como os vectores pGEM 28 ΕΡ 1 197 567/ΡΤ comercialmente disponíveis e os vectores pBluescript. Isto é claramente mostrado pelo vector pGN401 que expressa GFP sob a regulação do promotor de T7 num C. elegans transgénico que expressa ARN-polimerase de T7. 0 uso de pGN400 possui a vantagem de que o vector inclui um fragmento 3' UTR de unc-54 que melhora a transcrição ou a estabilidade do ARN.
Geração de linhas de C. elegans ARNi "pseudo-anuladas", permanentes, específicos de tecidos.
Actualmente, anulações ("knock-out") de genes em C. elegans são obtidas após a mutagénese aleatória e em larga escala e sib-selecção à base de PCR. Este método é extenso, consome muito tempo e é tedioso. Foi descrito que a introdução do ARN de cadeia dupla numa célula resulta em interferência específica e potente da expressão de genes endógenos. Em C. elegans a expressão de genes pode ser infra-regulada pela injecção' de ARN na cavidade corporal do verme, embebendo o verme numa solução contendo ARNcd ou alimentando-o com E. coli que expressa ARNcd correspondente ao gene de interesse. As células de C. elegans possuem a capacidade de retirar o ARNcd do seu meio extracelular. Foi relatado que o ARNm é o alvo desta interferência genética mediada por ARNcd (Montgomery and Fire 1998) . Sugere-se também que o ARN alvo é degradado no núcleo antes da ocorrência da tradução. Embora a redução mediada por ARNi da expressão génica possa ser passada para as gerações seguintes, a hereditariedade é fraca e o efeito é rapidamente perdido durante uma outra geração. Isto provavelmente está deve-se a um decréscimo continuado do agrupamento de ARNcd. Propomos aqui um método para construir linhas de C. elegans com um fenótipo de ARNi permanente e hereditário. 0 método abrange a geração de linhas de C. elegans transgénicas por introdução de plasmídeos contendo fragmentos de ADNc do gene alvo na orientação com sentido e anti-sentido sob controlo de um promotor do verme ou por transcrição de uma repetição invertida do ADNc de uma única construção. Alternativamente, o ARNcd pode ser transcrito a partir de um vector portador de um ADNc flanqueado por dois promotores de T7 numa estirpe de C. elegans que expressa a polimerase de T7. 0 resultado é um verme transgénico com um fenótipo "pseudo-anulado" hereditário estável. A expressão do 29 ΕΡ 1 197 567/ΡΤ ADNc ou da polimerase de T7 pode ser geral e constitutiva mas poder ser regulada também por um promotor especifico de tecidos. Ao contrário do ARNi induzido pelo ARNcdi externo (injectado, imerso ou alimentado), este método poderia permitir a obtenção da inibição especifica do tecido, condicional da expressão do gene.
Inibição da expressão de unc-22 por interferência de ARN resulta num fenótipo de "contracção espasmódica" ADNc de unc-22 (exão 22) foi clonado numa orientação com sentido e anti-sentido em pPD103.05. (A. Fire nr L2865) contendo o promotor let858 que é capaz de expressar sequências de ARN em todos os tecidos. Os plasmídeos resultantes foram denominados pGN205 (Figura 19a) e pGN207 (Figura 19b) . Estas construções foram introduzidas em C. elegans juntamente com um marcador seleccionável (rol6;GFP). Indivíduos F1 transgénicos (expressando rol-6 ou GFP) apresentaram um fenótipo de "contracção espasmódica" indicando que o ARNi poderia ser mediado pela transcrição endógena do ARN a partir do ADN transgénico. 0 fenótipo ARNi co-segregou-se com o marcador seleccionável na progénie seguinte. Isto resultou na geração de linhas de C. elegans com fenótipo ARNi permanente.
Geração de linhas estáveis de linhas de ARN-polimerase de T7 e geração de vermes transgénico duais
Um sistema de expressão em C. elegans baseado numa ARN-polimerase exógena requer dois plasmídeos. Um é codificado para a ARN-polimerase sob o controlo de um promotor específico enquanto o outro plasmídeo codifica para o fragmento de ADN a ser expresso, sob a regulação do promotor de T7. No caso de ARNi semi-estável também designado anulação pseudo-estável, o ADN de interesse é clonado entre dois promotores de T7 para que ARNcd possa ser produzido.
Como se sabe que o sistema de expressão da ARN-polimerase de T7 é um sistema de alta expressão, isto resultará em problemas para gerar animais dualmente transgénicos. Se o gene a ser expresso no nematode C. elegans é tóxico, isto resultará em efeitos letais e portanto, na construção de um C. elegans sem expressão estável altamente regulada do gene de interesse. Se o gene de interesse for essencial para a sobrevivência do organismo, o ARNi com um fragmento de ADN deste gene irá 30 ΕΡ 1 197 567/ΡΤ resultar também em efeitos letais, de modo que não são possíveis anulações pseudo-estáveis.
Para superar este problema, os presentes inventores desenvolveram um sistema que consiste em dois animais transgénicos. O primeiro animal é transgénico para a ARN-polimerase de T7. Esta ARN-polimerase de T7 pode ser expressa em todas as células ou em células ou tecidos específicos como foi mostrado nos exemplos anteriores. O segundo animal transgénico é transgénico para o fragmento de ADN de interesse. Este pode ser um gene ou ADNc ligado a um promotor de T7, ou, quando se pretende realizar ARNi, um fragmento de ADN desse gene clonado entre dois promotores de T7.
Ambos os animais transgénicos são viáveis e não apresentam nenhum fenótipo aberrante. Isto é porque a ARN-polimerase de T7 expressa no primeiro organismo transgénico não é tóxica para o organismo, mesmo se expressa em níveis relativamente altos. No segundo organismo transgénico, o gene de interesse não é expresso ou o ARNcd não é produzido, já que estes animais transgénicos não contêm a ARN-polimerase de T7. A expressão do gene ou do ADNc de interesse ou do ARNi com um fragmento de ADN pode ser agora obtida pelo acasalamento dos dois animais transgénicos. A progénie destes animais é dualmente transgénica e expressa o gene de interesse ou expressa o ARNcd do fragmento do ADN de interesse. Para gerar machos suficientes neste acasalamento, um dos animais machos transgénicos pode ser um C. elegans mutante com um fenótipo que favorece a geração de machos. Um exemplo de tal mutante é o him-5. Preferencialmente será utilizado este mutante para tornar um C. elegans transgénico para ARN-polimerase de T7, enquanto o hermafrodita é portador do fragmento de ADN sob a regulação do promotor de T7.
Para seleccionar eficientemente a progénie transgénica dual, pode ser introduzido um segundo transgene no segundo animal transgénico. Este transgene contém um gene repórter sob a regulação do promotor de T7. O gene repórter pode ser GFP, luciferase, beta-galactosidase ou betalactamase. Um exemplo de tal transgene são os vectores pGN400 e pGN401.
Para obter expressão indutível e específica do tecido de um transgene em C. elegans, podemos preparar uma reserva de 31 ΕΡ 1 197 567/ΡΤ machos (isto é, him-5), portadores da construção de polimerase de T7 sob o controlo de diferentes promotores de C. elegans que permitem a expressão especifica do tecido. Estes machos podem ser cruzados com hermafroditas portadores do gene de interesse sob o controlo de um promotor de T7.
Além disso, os transgenes podem ser integrados no genoma do animal. Métodos para gerar a integração estável de um plasmídeo no genoma do animal foram descritos (Methods in Cell Biology, Vol. 48, 1995, ed. por Epstein and Shakes, Academic
Press) e envolvem irradiação do animal. Isto pode ser feito para ambos os animais, mas preferencialmente, são sujeitos a este tratamento os animais que expressam a ARN-polimerase de T7. Isto resulta numa colecção de nematodes C. elegans que expressam de forma estável ARN-polimerase de T7 sob o controlo de vários promotores. Exemplos de tais promotores são o myo-2 (expressão na faringe), myo-3 (músculos da parede corporal), egl-15 (músculos vulvares), unc-119 (pan-neuron), SERCA (músculos), let858 (todas as células) e ges-1 (intestino).
Construção de vectores de dois híbridos em levedura com promotor de T7 ARNi. pGAD424 com T7 / T3 directo e inverso e/ou Sp6
Na maioria das experiências de dois híbridos, uma biblioteca de ADNc é clonada no plasmídeo pGAD424 (Figura 16) que foi manipulado para ter locais de restrição adicionais no poli-ligante tais como um local Ncol (Clontech) . Esta biblioteca permite a pesquisa de proteínas de ligação numa experiência de dois híbridos em levedura. Construímos um novo vector de levedura de dois híbridos com as mesmas possibilidades de realizar dois híbridos em levedura, mas que contém dois promotores de T7 adicionais, para que o vector possa ser utilizado para anulações pseudo-estáveis induzidas pela ARN-polimerase de T7. Para isto inserimos um T7 directo utilizando um ligante T7 (consistindo nos seguintes iniciadores, aattcttaatacgactcactatagggcc e catgggccctatagtgag tcgtattaag) no local EcoRI-Ncol de pGAD424. 0 vector resultante foi designado pGAD424-without-FULL-ICE-both-T7. Foi tido o cuidado de eliminar codões de paragem e utilizar o máximo de poli-ligantes de aminoácidos compatíveis. Adoptámos a mesma estratégia para ο T7 inverso (consistindo em ambos os iniciadores gatccgtcgacagatctccctatagtgagtcgtattactgca e 32 ΕΡ 1 197 567/ΡΤ gtaatacgactcactatagggagatctgtcgacg) com BamHI e PstI. Para evitar perda de Sall, incluímos este local no iniciador. 0 local Sall é importante pois a maioria das bibliotecas são clonadas neste local, estando os adaptadores disponíveis. Isto torna o vector agora construído compatível com os vectores existentes. pAS2 com T7 / T3 directo e inverso e/ou Sp6.
Um vector de dois híbridos em levedura análogo foi construído com base no pAS2 (Clontech). Pela digestão parcial com EcoRV fomos capazes de remover uma parte significativa do gene cyh2. A construção correcta pode ser isolada e verificada por uma digestão de restrição com BglII. Este local de restrição está presente no fragmento EcoRV de PAS2 a eliminar. Isto elimina o gene cyh2 que é um gene levemente tóxico e está envolvido no atraso do crescimento. Este gene não é essencial para a realização de experiências de ARNi e de dois híbridos em levedura. Após a eliminação do fragmento EcoRV, o local de restrição EcoRI que está localizado entre a sequência de ADN que codifica para GAL4DB e HA (epítopo) torna-se único para o plasmídeo, e pode ser utilizado para substituir HA por ligante contendo um promotor de T7.
Isto assegura a persistência de todos os locais de restrição, permitindo tanto a clonagem em fase como a compatibilidade com vectores prévios e pGAD424. Utilizámos o seguinte ligante (iniciadores: aattcttaatacgactcactatagggca e tatgccctatagtgagtcgtattaag) utilizando locais de clonagem EcoRI e Ndel. Adoptámos a mesma estratégia para ο T7 inverso (iniciadores: gatccgtcgacagatctccctatagtgagtcgtattactgcacatggg ccctatagtgagtcgtattaag e gtaatacgactcactatagggagatctgtcgacg), com BamHI e PstI. Para evitar perda de Sall, incluímo-lo no iniciador. 0 vector resultante foi designado PAS2-cyh2-HA+bothT7-final.
Ter o promotor de T7 (ou alternativamente o promotor de T3 ou SP6) no pGAD424 permite ir rapidamente da proteína de interacção para o ARNi e atribuir uma função ao fragmento isolado de ADN. Uma vantagem adicional é a habilidade de preparar por transcrição "in vitro" acoplada a tradução "in vitro" (existe uma ATG na estrutura com GAL4DB ou GAL4AD) a proteína marcada que pode ser utilizada para controlos " in 33 ΕΡ 1 197 567/ΡΤ vitro" (por exemplo, ensaios de imunoprecipitação) da interacção real proteína-proteína.
As sequências dos plasmídeos produzidos e da polimerase de SP6 e T3 estão identificadas na Listagem de Sequências proporcionada abaixo: ARN-polimerase dependente de ADN SP6 SEQUÊNCIA ID NO.1 número de acesso Swissprot P06221 sequência da proteína: 1 mqdlhaiqlq leeemfnggi 61 keeyegkkgr apralaflqc 121 fskleghaak yfekvkkslk 181 qigttlleil egsvfyngep 241 yapcvipprp wrtpfnggfh 301 wqinkdvlav ieevirldlg 361 wqqfinwkge carlytaetk 421 stlspqsndl gkallrfteg 481 diucrdiaadp ltftqwakad 541 giqhysamlr devgakavnl 601 sgtelramas awdsigitrs 661 grtankvhpf eddrqdyltp 721 eglmytlptg fileqkimat 781 hdashliltv celvdkgvts 841 eehevrwmvd tgievpeqge rrfeadqqrq iaagsesdta venevaayit mkvvmdmlnt asrtksyrha hnvavvaeks vfmramrtyg gktiyylqts tekvasrirl vkgnrehvrk ygvpsfkpli dkenkpanpv rgsksaavvr mvgqarkysa rpvngvealk wfcinganlw ápyeflawcf eyaqyldlvd kpsdapqdiy gavaqvvikk ltkkpvmtlp ygstrltcre gaaynymtal iwpsisevvk emlrvrtclm gdikmslqve iavihdsfgt hadntltlrv fdlneimdse yvfa wnrrllseli datlqaiams vaekdadfdr esvgqwisaf ltqkqmpkvy pvefqhlrgr fesiyfvyam gwdkktfdvr egradefrth nalymdadda svidyivdle apivamkmir tdivdeaanun alkgqmvamy apmaegiqay vaeriedqvr weawpketql kehvaqlspa kainalqntq elkemlspeq dsrsrvyvqs vsnvideefq lpvhqdgscs ttftsgsvtl ekeaqkavae qlarfaakrn gaaapnfvhg idgnalqkll ARN-polimerase dependente de ADN de T3 SEQUÊNCIA ID NO.2 número de acesso Swissprot P07659 sequência da proteína: 1 mniieniekn dfseielaai pfntladhyg salakeqlal ehesyelger rflkmlerqa 61 kageiadnaa akpllatllp klttrivewl eeyaskkgrk psayaplqll kpeasafitl 121 kvilasltst nmttiqaaag mlgkaiedea rfgrirdlea khfkkhveeq lnkrhgqvyk 181 kafmqvvead migrgllgge awsswdkett mhvgirliem liestglvel qrhnagnags 241 dhealqlaqe yvdvlakrag alagispmfq pcvvppkpwv aitgggywan grrplalvrt 301 hskkglmrye dvympevyka vnlaqntawk inkkvlavvn eivnwkncpv adipslerqe 361 lppkpddidt neaalkewkk aaagiyrldk arvsrrisle fmleqankfa skkaiwfpyn 421 mdwrgrvyav pmfnpqgndm tkglltlakg kpigeegfyw lkihgancag vdkvpfperi 481 afiekhvddi lacakdpinn twwaeqdspf cflafcfeya gvthhglsyrv cslplafdgs 541 csgiqhfsam lrdevggrav nllpsetvqd iygivaqkvn eilkqdaing tpnemitvtd 601 kdtgeisekl klgtstlaqq wlaygvtrsv tkrsvmtlay gskefgfrqq vlddtiqpai 661 dsgkglmftq pnqaagymak liwdavsvtv vaaveamnwl ksaakllaae vkdkktkeil 721 rhrcavhwtt pdgfpvwqey rkplqkrldm iflgqfrlqp tintlkdsgi dahkqesgia 781 pnfvhsqdgs hlrmtvvyah ekygiesfal ihdsfgtipa dagklfkavr etmvityenn 841 dvladfysqf adqlhetqld kmpplpkkgn lnlqdilksd fafa 34 34 ΕΡ 1 197 567/ΡΤ SEQUÊNCIA ID NO. 3 pGN108: gttgtcgtaaagagatgtnttanttacRtacaccgggtcctctctctetgccagcacagctcagtgttggctgtgtgctcgggctcctgccaccggcgg cctcatcttcttcttcttcttctctcctgctctcgcttatcacRcttcattcattcttattccttttcatcatcaaactagcaRtcttactttatttatttttttcaattttca attttcagataaaaccaaactacttgggttacagccgtcaacagatccccgggattggccaaaggacccaaaggtatgtttcgaatgatactaacataa catagaacattttcaggaggacccttgcttggagggtaccggatgactgctccaaagaagaagcgtaagctcatgaaeacgattaacatcgctaagaa cgacttctctgacatcgaaciggctgctatcccgttcaacactctggctgaccanacggtgagcgtttagctcgcgaacagnggeccttgagcatgag tcttacgagatgggtgaagcacgcnccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctclcatcact accctactccctaagatgangcacgcatcaacgactggRtgaggaagtgaaagctaagcgcggcaagcgcccgacagccRccagttcctgcaag aaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcngcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgc aatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaag cgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggca taaggaagactciancatgtaggagtacgctgcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtagg tcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccllg cgtagttcctcctaagccgtggactggcattactggtggtggctaRgggctaacggtcgtcgtcctctggcgctggtgcgtactcacagtaagaaagc actgatgcgctacgaagacgRtacatgcctgaggtgtacaaagcgaRaacaRgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcg gtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgatgaaaccggaagacatcgac atgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaagacaaggctcgcaagtctcgccgtatcagccttgagttcatg cRgagcaagccaataagRtgctaaccataaggccatctggROCcRacaacatggactggcgcggRcgtgtttacgctgtgtcaatgRcaacccgc aaggtaacgatatgaccaaaggacgtcttacgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatecacggtgcaaactg tgcgggtgtcgataaggtttcgtttectgagcgcatcaagttcangaggaaaaccacgagaacatcatggcRgcgctaagtctccactggagaacac ttSStgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccaeggcctgagctataactgctcccttccgc tggcgntgacgggtcngctctggcatccagcacRctccgcgatgctccgagatgaggtaggtggtcgcgcggRaacRgcReclagtgaaaccgt tcaggacatctacgggattgttgctaagaaagtcaacgagattctgcaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgat gagaacactggtgaaatctclgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacggtgttactcgcagtgtgactaagc gttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtc
Igatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatccgtgagcgtgacggtggtagctgcggttgaagcaatgaac tggeRaagtctgctgctaagctgcIggctgctgaggtcaaagataagaagactggagagaRcRcgcaagcgttgcgctgtgcaRgggtaactcct gatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttacagcctaccattaacacca acaaagatagcgagangatgcacacaaacaggagtctggtatcgctcctaacntgtacacagccaagacggtagccaccRcgtaagactgtagtg tgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattocggctgacgctgcgaacetgneaaagcagtgcgc gaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagc acnccggctaaaggtaacttgaacctccgtgacatottagagtcggacttcgcgttcgcgtaagggcccactagtcggccgtacgggccctttcgtct cgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcRgtctgtaagcggatgccgggagcagacaagc ccgtcagggcgcgtcagCgggtgnggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgt gaaataccgcacagatgcgtaaggagaaaataccgcatcaggcggccttaagggcctcgtgatacgcctatttttataggttaatgtcatgataataat ggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctantgtttatnttctaaatacaRcaaatatgtatccgctcatgagacaat aaccctgataaatgcttcaataataRgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccltattcccttttttgcggcattttgccttcctgttn tgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc cngagagtmcgccccgaagaacgttftccaatgatgagcacttttaaagRctgctatgtggcgcggtattatcccgtaRgacgccgggcaagagca actcggtcgccgcatacactaRctcagaatgacttggttgagtactcaccagtcacagaaaagcatcnacggatggcatgacagtaagagaattatg cagtgctgccataaccatgagtgataacactgcggccaacRacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgg gggatcatgtaaclcgccKgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcglgacaccacgatgcctgtagcaatggcaa caacgttgcgcaaactattaáctggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccactlc tgcgctcggcccnccggctggctggtttattgctgataaatctggagccggtgagcgtgggtacgcggiatcattgcagcactggggccagatggt aagccctcccgtatcgtagnatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaa gcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctca tgaccaaaatcccttaacgtgagttTtcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctg ctgcRgcaaacaaaaaaaccaccgctaccagcggtggtttgRtgccggatcaagagctaccaactcRRtccgaaggtaactggcRcagcagagc gcagataccaaatacigtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttac cagtggctgctgccagtggcgataagtcgtgtcRaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggg gttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcRcccgaaggg agaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagettccagggggaaacgcctggtatcRtatagíc ctgtcgggtttcgccacctctgacRgagcgtcgaRtRgtgatgctcglcaggggggcggagcctatggaaaaacgccagcaacgcggccRtttac ggttcctggccttttgctggccttttgctcacalgttcRtcctgcgttatcccctgattctgtggataaccgtanaccgccmgagigagctgataccgctc gccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaecgcctctccccgcgcgRggccg aRcaRaatgcagctggcacgacaggRtcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagRagctcactcaRaggcacccca ggcRtacacRtatgcnccggctcgtatgRgtgtggaaRgtgagcggataacaatUcacacaggaaacagctatgaccatgattacgccaagctgt aagtRaaacatgatcttactaactaactaRctcaRtaaattttcagagcttaaaaatggctgaaatcactcacaacgatggatacgctaacaacnggaa atgaaataagcRgcatgcctgcagagcaaaaaaatactgcttRccRgcaaaattcggtgcRtcRcaaagagaaacRRgaagtcggcgcgagcat 35 ΕΡ 1 197 567/ΡΤ ttccttctttgacttctctctttccgccaaaaagcctagcatttttattgataatttgattacacacactcagagttcttcgacatgataaagtgtttcattggcac tcgccctaacagtacatgacaagggcggattattatcgatcgatattgaagacaaactccaaatgtgtgctcattttggagccccgtgtggggcagctg ctctcaatatattactagggagacgaggagggggaccttatcgaacgtcgcatgagccattctttcttctttatgcactctcttcactctctcacacattaat cgattcatagactcccatattccttgatgaaggtgtgggttntagctttttttcccgatRgtaaaaggaagaggctgacgatgnaggaaaaagagaacg gagccgaaaaaacatccgtagtaagtcttccttttaagccgacactttttagacagcattcgccgctagttngaagtttaaattttaaaaaataaaaattag tttcaattttttttaattactaaataggcaaaagttttttcaagaactctagaaaaactagcttaattcatgggtactagaaaaattcttgttttaaatttaatattta tcttaagatgtaattacgagaagcttttttgaaaattctcaattaaaagaatttgccgatttagaataaaagtcttcagaaatgagtaaaagctcaaattaga agtttgtttttaaaggaaaaacacgaaaaaagaacactatttatcttttcctccccgcgtaaaattagngttgtgataatagtgatccgctgtctatttgcact cggctcttcacaccgtgcttcctctcacngacccaacaggaaaaaaaaacatcacgtctgagacggtgaattgccttatcaagagcgtcgtctctttca cccagtaacaaaaaaaatnggmctttactttatatttatgtaggtcacaaaaaaaaagtgatgcagttttgtgggtcggttgtctccacaccacctccgc ctccagcagcacacaatcatcttcgtgtgttctcgacgattccttgtatgccgcggtcgtgaatgcaccacattcgacgcgcaactacacaccacactc actttcggtggtattactacacgtcatcgttgttcgtagtctcccgctctttcgtccccactcactcctcattattccccttggtgtattgattttttttaaatggla caccactcctgacgtttctaccRcngttnccgtccatttagattttatctggaaatttttttaaaattttaggccagagagttctagttcttgttctaaaagtcta ggtcagacatacattttctatttctcatcaaaaaaaaagttgataaagaaaactggttattcagaaagagtgtgtctcgttgaaattgattcaaaaaaaaatt cccacccctcgcttgtnctcaaaatatgagatcaaeggattttttccttctcgattcaattttttgctgcgctctgtctgccaaagtgtgtgtgtccgagcaaa agatgagagaatttacaaacagaaatgaaaaaaagttggccaaataatgaagttttatccgagattgatgggaaagatattaatgttctttacggtttgga ggggagagagagatagattttcgeatcaaactccgccttttacatgtcttttagaatctaaaatagatttttctcatcatttttaatagaaaatcgagaaatta cagtaatttcgcaattttcttgccaaaaatacacgaaatttgtgggtctcgccacgatctcggtcttagtggttcatttggtttaaaagtttataaaatttcaaa ttctagtgtttaatttccgcataattggacctaaaatgggtttttgtcatcattttcaacaagaaatcgtgaaaatcctgttgtttcgcaatntcttttcaaaaata cacgaaatatatggtaatttcccgaaatattgagggtctcgccacgatttcagtcacagtggccaggatttatcacgaaaaaagttcgcctagtctcacat ttccggaaaaccgaatctaaattagttttttgtcatcattttgaacaaaaaatcgagacatccctatagtttcgcaattttcgtcgcttttctctccaaaaatga cagtctagaattaaaattcgctggaactgggaccatgatatcttttctccccgtttttcattttattttttattacactggattgactaaaggtcaccaccaccg ccagtgtgtgccatatcacacacacacacacacacaatgtcgagattttatgtgttatccctgcttgatttcgttccgttgtctctctctctctattcatcttttg agccgagaagctccagagaatggagcacacaggatcccggcgcgcgatgtcgtcgggagatggcgccgcctgggaagccgccgagagatatca gggaagatcgtctgatttctcctcggatgccacctcatctctcgagtttctccgcctgttactccctgccgaacctgatatttccc 36 ΕΡ 1 197 567/ΡΤ pGNIOS: SEQUÊNCIA ID NO. 4 aagcttgcatgcctgcaggccttggtcgactctagacacttttcagctacctagatacatggatatccccgcctcccaatccacccacccagggaaaaa gaagggctcgccgaaaaatcaaagttatctccaggctcgcgcatcccaccgagcggttgacttctctccaccacttttcattttaaccctcggggtacg ggattggccaaaggacccaaaggtatgtttcgaatgatactaacataacatagaacattttcaggaggacccttgcttggagggtaccgagctcagaa aaaatgactgctccaaagaagaagcgtaaggtaccggtaatgaacacgatlaacatcgctaagaacgacttctctgacatcgaactggctgctalccc gttcaacactctggctgaccatucggtgagcgrttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaa gatgtttgagcgtcaacttaaagctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaa cgactggtttgaggaagtgaaagctaagcgcggcaagcgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatca ccattaagaccactctggcttgcctaaccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcg cttcggtcgtatccgtgaccttgaagctaagcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcattta tgcaagngtcgaggctgacatgctctctaagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgct gcatcgagatgctcattgagtcaaccggaatggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacct gaatacgctgaggctatcgcaacccgtgcaggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcatt actggtggtggctattgggctaacggtcgtcgtcctctggcgctggtgcgtacfcacagtaagaaagcactgatgcgctacgaagacgtttacatgcct gaggtgtacaaagcgattaacattgcgcaaaacaccgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcacçaagtggaagcat tgtccggtcgaggacatccctgcgattgagcgtgaagaactcccgalgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaa cgtgctgccgctgctgtgtaccgcaaggacagggctcgcaagtctcgccgtatcagccttgagttcatgcdgagcaagccaataagtttgctaaccat aaggccatctggttcccttacaacatggactggcgcggtcgtgtttacgccgtgtcaalgttcaacccgcaaggtaacgatatgaccaaaggactgctt acgctggcgaaaggtaaaccaatcggtaaggaaggttactactggctgaaaatccacggtgcaaactgtgcgggtgtegataaggttccgttccctg agcgcatcaagttcattgaggaaaaccacgagaacatcatggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctocgttct gcnccttgcgttctgctngagtacgctggggtacagcaccacggcctgagctataactgctcccttccgctggcgtttgacgggtcngctctggcatc cagcacttctccgcgatgctccgagatgaggtaggtggtcgcgcggttaacttgcttcctagtgagaccgttcaggacatctacgggattgttgctaag aaagtcaacgagattctacaagcagacgcaatcaatgggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaa gtcaagctgggcactaaggcactggctggtcaatggctggctcacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtc caaagagttcggcttccgtcaacaagtgctggaagataccattcagccagctattgattccggcaagggtccgatgttcactcagccgaatcaggctg ctggatacatggctaagctgantgggaatctgtgagcgtgacggtggtagctgcggttgaagcaatgaactggcttaagtctgcrgctaagctgctgg ctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcgctgtgcattgggtaactcctgatggtttccctgtgtggcaggaataca agaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgettacagcctaccattaacaccaacaaagatagcgagattgatgcacaca aacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccaccttcgtaagactgtagtgtgggcacacgagaagiacggaatcga atcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacctgttcaaagcagtgcgcgaaactatggttgacacatatgagtctt gtgatgtactggctgarttctacgaccagttcgctgaccagttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacct ccgtgacatcttagagtcggacttcgcgttcgcgtaagaattccaactgagcgccggtcgctaccattaccaacttgtctggtgtcaaaaataataggg gccgctgtcatcagagtaagtttaaactgagttctactaactaacgagtaatatttaaattttcagcatctcgcgcccgtgcctctgacttctaagtccaatt actcttcaacatccctacatgctctttctccctgtgctcccaccccctatttttgttattatcaaaaaaacttcttcUaatttetttgttttttagcttcttttaagtca cctetaacaatgaaattgtgiagancaaaaaiagaattaattcgtaataaaaagtcgaaaaaaattgtgctccctccccccattaataataattctatccca
aaatctacacaatgttctgtgtacact1cttatgttttttttacttctgataaattttttttgaaacatcatagaaaaaaccgcacacaaaalaccRatcatalgR acgtttcagtttatgaccgcaatttttatttcttcgcacgtctgggcctctcatgacgtcaaatcatgctcatcgtgaaaaagttnggagtatttttggaattttt caatcaagtgaaagtttatgaaattaamtcctgcttttgctttttgggggtttcccctattgtttgtcaagagtttcgaggacggcgtttttcttgctaaaatca caagtattgatgagcacgatgcaagaaagatcggaagaaggtttgggtugaggctcagtggaaggtgagugaagttgataatagaaagtggagta gtgtctatggggtttttgccttaaatgacagaatacattcccaatataccaaacataactgtttcctactagtcggccgtacgggccctttcgtctcgcgcg tttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtca ÊgScgcgtcagcgggtgnggcgggtgícggggdggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtglgaaata ccgcacagatgcgtaaggagaaaataccgcatcaggcggccttaagggcctcgtgatacgcctatttttataggttaatgtcatgataalaatggtttctt agacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttmtgcggcattttgccttcctgtttttgcica cccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgag agttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcg gtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtg ctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggat catgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacg ngcgcaaactattaac(ggcgaactacttactctagcttcccggcaacaattaatagaçtggatggaggcggataaagttgcaggaccacRctgcgct cggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccc tcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggigcctcactgattaagcattg gtaaclglcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttRgataatctcatgacca aaatcccttaacgtgagnttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccrttttttctgcgcgtaatctgctgctt gcaaacaaaaaaaccaccgc(accagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcaga taccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaalcctgttaccagtg gctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggncg tgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaa aggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcc2^ggggaaacgcctggtatctttatagtcctgtc gggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttc ctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccg cagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaaiacgcaaaccgcctctccccgcgcgttggccgattca ttaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctt tacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctgtaagttt aaacatgatcttactaactaactattctcatttaaattttcagagcttaaaaaiggctgaaatcactcacaacgatggatacgctaacaacttggaaatgaa at 37 37 ΕΡ 1 197 567/ΡΤ SEQUÊNCIA ID NO. 5 pGN400: aagcttgcatgcctgcaggccttggtcgactctagacacttttcagctacctagatacatggatatccccgcctcccaatccacccacccagggaaaaa gaagggctcgccgaaaaatcaaagttatctccaggctcgcgcatcccaccgagcggngacttctctccaccacttttcattttaaccctcggggtacg ggattggccaaaggacccaaaggtatgtttcgaatgatactaacataacatagaacattttcaggaggacccttgcttggagggtaccgagctcccgg gattaatacgactcactataccggtagaaaaaatgagtaaaggagaagaacttttcactggagttgtcccaattcngttgaattagatggtgatgttaatg ggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatngcactactggaaaactacctgttccatgg gtaagtttaaacatatatatactaaetaaeectgattatttaaattttcagccaacacttgtcactactttctgttatggtgttcaatgcttctcgagatacccag atcatatgaaacggcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacac gtaagtttaaacagttcggtactaactaaccaiacatamaaattncaggtgctgaagtcaagtttgaaggtgaiacccttgttaatagaatcgagttaaa aggtattgamtaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaat ggaatcaaagngtaagmaaacatgattttactaactaactaatctgatttaaattttcagaacttcaaaattagacacaacangaagatggaagcgRca actagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatc ccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaatagcattcgtagaattc caactgagcgccggtcgctaccattaccaacttgtctggtgtcaaaaataataggggccgctgtcatcagagtaagtttaaactgagttctactaactaa cgagtaatatttaaattttcagcatctcgcgcccgtgcctctgacttctaagtccaattactcttcaacatccctacatgctcntctccctgtgctcccaccc cctatttttgttattatcaaaaaaacttcttcttaatttctttgttttttagcttçttttaagtcacctctaacaatgaaãttgtgtagattcaaaaatagaattaattcg taataaaaagtcgaaaaaaattgtgctccctccccccattaataataattctatcccaaaatctacacaatgttctgtgtacacttcttatgttttttttacttctg ataaattttttttgaaacatcaiagaaaaaaccgcacacaaaataccttatcatatgttacgtttcagtttatgaccgcaatttttatttcttcgcacgtctgggc ctctcatgacgtcaaatcatgctcatcgtgaaaaagttttggagtatttttggaatttttcaatcaagtgaaagtttatgaaattaattttcctgcttttgcttttlg ggggtttcccctattgtngtcaagagtttcgaggacggcgtttttcttgctaaaatcacaagtattgatgagcacgatgcaagaaagatcggaagaagg tttgggtttgaggctcagtggaaggtgagtagaagttgataatRgaaagtggagtagtgtctatggggtttttgccttaaatgacagaatacattcccaat ataccaaacataactgtttcctactagtcggccgtacgggccctttegtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccg gagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggctta actatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcggc cttaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcactntcggggaaatgtgcgcggaacc cctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtatt caacatítccgtgtcgcccttattcccmtttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagtt gggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaa gttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcac cagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacnctga caacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagc cataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggc aacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccg gtgagcgtgggtctcgcggtatcattgcagcactggggecagatggtaagccctcccgtatcgtagttatctacacgacggggagteaggcaactot ggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaa acttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccg tagaaaagatcaaaggatcttctigagatccttmttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg atcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataceaaatactgtccttctagtgtagccgtagttaggccaccactt caagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactca agacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactga gatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcágggtcggaacaggag agcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtc aggggggcggagcctatggaaaaacgceageaacgcggcctttttacggttcc^ggccttttgctggccttttgctcacatgttctttcctgcgttatccc ctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaag cggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggc agtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcgg ataacaatttcacacaggaaacagctatgaccatgattacgccaagctgtaagtttaaacatgatcttactaactaactattctcatttaaattncagagctt aaaaatggctgaaatcactcacaacgatggatacgctaacaacttggaaatgaaat 38ΕΡ 1 197 567/ΡΤ pGN401 SEQUÊNCIA ID NO. 6 gatcccggcgcgcgatgtcgtcgggagatggcgccgcctgggaagccgccgagagatatcagggaagatcgtctgatttctcctcggatgccacct catctctcgagtttctccgcctgttactccctgccgaacctgatatttcccgttgtcgtaâagagatgtttttattttactttacaccgggtcctctctctctgcc agcacagctcagtgttggctgtgtgctcgggctcctgccaccggcggccicatcrtcttcttcttcttctctcctgctctcgcttatcacttcttcattcattctt attccttttcatcatcaaactagcatttcttactttatttatttttttcaattttcaatttlcagataaaaccaaactacttgggttacagccgtcaacagatccccg ggattggccaaaggacccaaaggtatgtttcgaatgatactaacataacatagaacattttcaggaggacccttgcttggagggtaccggtagaaaaa atgagtaaaggagaagaactmcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtga aggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatgggtaagtttaaacatatatatactaactaaccct gattatttaaattttcagccaacacttglcactactttctgttatggtgttcaatgcttctcgagatacccagatcatatgaaacggcatgactttttcaagagt gccatgcccgaaggttatgtacaggaaagaactatamttcaaagatgacgggaactacaagacacgtaagtttaaacagttcggtactaactaaccat acata tttaaattttcaggtgctgaagtcaagtRgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattctt ggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttgtaagtttaaacttggacttac taactaacggattatatttaaattttcagaacttcaaaattagacacaacattgaagatggaagcgticaactagcagaccattatcaacaaaatactccaa ttggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttct tgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaatagcattcgtagaattccaactgagcgccggtcgctaccattaccaac ttgtctggtgtcaaaaataataggggccgctgtcatcagagtaagtttaaactgagttctactaactaacgagtaatatttaaattttcagcatctcgcgccc gtgcctclgacttctaagtccaattactcttcaacatccctacatgctctttctccctgtgctcccaccccctatttttgttattatcaaaaaaacttcttcttaatt tctttgttttttagcttcttttaagtcacctctaacaatgaaattgtgtagattcaaaaatagaattaattcgtaataaaaagtcgaaaaaaattgtgctccctcc ccccattaataataattctatcccaaaatctacacaatgttctgtgtacacttcttatgttttttttacttctgataaattttttttgaaacatcatagaaaaaaccg cacacaaaataccttatcatatgttacgtttcagtttatgaccgcaatttttatttcttcgcacgtctgggcctctcatgacgtcaaatcatgctcatcgtgaaa aagttttggagtatttttggaatttttcaatcaagtgaaagtttatgaaattaattttcctgcttttgctttttgggggtttcccctattgtttgtcaagagtttcgag gacggcgtttttcttgctaaaatcacaagtattgatgagcacgatgcaagaaagatcggaagaaggtttgggtttgaggctcagtggaaggtgagtag aagttgataatttgaaagtggagtagtgtctatggggtttttgccttaaatgacagaatacattcccaatataccaaacataactgtttcctactagtcggcc gtacgggcccggtacccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgct cacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgc ccgctttccagtegggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcc tcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggata acgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccct gacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtg cgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcag ttcggtgtaggtcgltcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaa cccggtaagacacgacttatcgccactggcagcagccactggtaacaggatiagcagagcgaggtatgtaggcggtgctacagagttcttgaagtg gtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccgg caaacaaaccaccgctggtagcggtggtnttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacg gggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaag ttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccat agttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccg gctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgc cgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttc attcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagt aagnggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaa ccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtg ctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttc agcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaata ctcatactcttcctttttcaatanattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttc cgcgcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggc cgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtgga ctccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaa gcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaa aggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtc ccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaag gcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattg gagctccaccgcggtggcggccgctctagaactagtg 39 ΕΡ 1 197 567/ΡΤ pGNUO: SEQUÊNCIA ID NO. 7 gatcctccaaaatcgtcttccgctctgaaaaacgaaagtggacclttgacatccgaaaaaatgggcgaaaaaatgaaattgagctnttgggtcgaaaa aaatgtttttagaatgctgagaacacgttaaacacgaagatcatatttattttgagacccggatgctctgaaaatgtctgacatagatttaaaaaagcatat atatatttctcattttcaacgtgaaagtmgtgcaacttutagaacctcctattggcacattgttttttatttaactgaggcagtttttgaacaccttmgaaactt tgaatctctttgaagtatactgtcgaaaagactgacttgagcgttcgaaatgccagaagaaaactatatttgaatctcgcgctaaattgagaaatgcaacc gcgclccactggacaattggaaaaaaaatnattcggaggcgacaacggtattttcgaaattgattttctgtgtattttctcattttttataaaRcttctttgattt atcgttcgtttgtgagaaamaattgtattcaaactttmatagtaagataccggtggtaccgctagccgtacgaacccgggattggccaaaggaccca aaggtalgtttcgaaigatactaacataacatagaacaattcaggaggacccngcnggagggtaccggatgactgctccaaagaagaagcgtaagc tcatgaacacgattaacatcgctaagaacgacttctctgacatcgaactggctgctalcccgttcaacactctggctgaccattacggtgagcgtttagct cgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaagctggtgaggttgcgga taacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaagctaagcgcggcaag cgcccgacagccttccagttcctgcaagaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcngcçtaaccagtgctgaca atacaaccgttcaggctgtagcaagcgcaatcggtcgggccangaggacgaggctcgcttcggtcgtatccgtgaccttgaagctaagcacttcaa gaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacalgctctctaagggtcta ctcggtggcgaggcgtggtcttcgtggcaUaggaagaetotattcalglaggagtacgcígeatcgagatgctcattgagteaaccggaatggttag cttacaccgccaaaatgctggcgtagtaggtcaagactctgagaçtatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgcaggtgcg ciggctggcatetctccgatgttccaaccttgcgtàgttcctcetaagccgtggactggcattactggtggtggctangggctaacggtcgtcgtcctct ggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgccigaggtgtacaaagcganaacattgcgcaaaacacc gcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtgaa gaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaagacaaggctc gcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcg gttcgtgtnacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggacgtcttacgctggcgaaaggtaaaccaatcggtaaggaagg ttactactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggtncgtttcctgagcgcatcaagttcangaggaaaaccacgagaacat catggcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagc accacggectgagctataactgctcccttccgctggcgmgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtg gtcgcgcggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctgcaagcagacgcaatcaatg ggaccgataacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggct ggctlacggtgttactcgcagtgtgactaagcgttcagtcatgacgctggcrtacgggtccaaagagttcggcttccgtcaacaagtgctggaagatac cattcagccagctattgattccggcaagggtctgatgRcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatccgtgagcgt gacggíggtagctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttc gcaagcgttgcgctgtgcattgggtaactcctgatggmccctgtgtggcaggaatacaagaagcctattcagacgcgcRgaacctgatgttcctcgg tcagttccgcttacagcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagcca agacggtagccaccttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggc tgacgctgcgaacctgRcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccag ttgcacgagtctcaattggacaaaatgccagcacttccggctaaaggtaacttgaacctccglgacatcttagagtcggacttcgcgttcgcgtaaggg ccctcgicgagtcggtcacaatcacctgaaactccaaaggcagccagtgaggaacgtgaagaagaagaaaaagagtcatctgaacaggtttgatttt emctggtcaaaaagatgaaattattgattttcagccagatactcccâaaactagcagegagaagtctgcaagtcgttcacagtcgcccagagaal£gc gggaagtgagccaagaggtatglttttcaaaaatcaataactgatcataatttttattgtttggtgaatttaagaaaataatattcgaaaattcctctgaattat caagattgcagtattaamcgagaaaaangagatattcatagagctattgtaaattRcttgaRtcagactgaaacttcggaaaatcaagagaaaatcaa agaaaaggatgacggggatgatcagcctggcacaccgaacagctatagaagccgggaaacttcaceagctccaaaaagglccaaggagaccag gtttgtcaaaagcttcctgcganaattctcatttcaatttttcagagaatcagagtotcctgaaaaatccccggttcgttcaagatctcccagaaggtcttca gcacgttccccgtcacgatctcctagacggcgccgagaaagaagctcagaaagaaagcaatccgaagagccagcaccgctaccagagaaaaaga agaaagagccgctggatattctacgaacaagaaccggaggagcatatattccacccgccaaacttcgacttatgcaacaacagattagtgaiaagca aagtgaacagtatcagagaatgaattgggaaagaatgaagaaaaagattcacggattggRaacagagtcaacgcgaagaatcttgRcaaattgtca gagaacttcttcaagagaatgtgattcgttcaaagtgagtgagaaaatcgaaggaaaaggaaagaattaatttaatttttcaggggacRctctgccgtg acattattcaagctcaggctnctcaccaggatictctaacgtctatgcagctttggcggcagnatcaactcgaaattccctcatgtcggtgaacttcttctc cgtcgtctgattgtacagncaaaagaagntccgtagaaatgacagaggcgtcacggtgaacgtgatcaaaRcatcgcacainganaatcaacaag ttgctcacgaagttcRgcgctggaaatcatgattctgatgcttgaagaaccaactgatgattcagttgaagtcgccattgcgttcctgaaagagtgtgga gcaaagcttctggagattgctccagcagctcttaacagtgtctacgaccgtcRcgtgcaanetcatggaaactgaaagatcggaaaatgcactggatc gacgtaRcagtatatgattgagactgcaatgcagattcgaaaggacaaamgcggtaaggtagaatatataaatagRtaRagaaaaaaaUaattag aataatttaaattcctactagccaaicaggcgacctttttgcgcatagttctattattgaaaaamggagaatttctcatattctcgclcggaaatctggaalt cgacgagatcttctggcttctgtgcagctgcalcgctttgigctccctttctcgcttgtcttctgtgtacaccaagaaccRgttgagttcatcaactgaatct gtgactggcttgttgctcactggatgcactagacgactgattctcgagaaatcagaRgagttgcgattagggtgacctagaaattgggaataatacgaa cttttgaaaatattcaggaggattaaaaaaattaRctcgacaatcctacaaatRacttattgcaccatgttgctccaacatttttcattaaaagttaatgaaaa
aatgiagaaaatcggaaaRggcaaRRcagaccaRRtaagcaRRcaaaaaaaaattgcagctgaaataaatgtcaRRcagataaatcgagcgaR ttclgngtctgacactagtnttagtTOaaaaaatgttggaagaacatggtgcaataggtaaRtcatagaamccatgtgtnutncaattaaccaanatc caaatcttccaaactcacattttgcggagctgggctatcaagaatctgctgcagRRataagacgagcatctctgatatcactgaaaanaatttRaatca aaacflgaatatcaactaaacccacnaRaactnctcgatcnctgtcgRcggtacgatgacggtgaagaagccaattgtagtagHgatRggRcaagt 40 ΕΡ 1 197 567/ΡΤ cctttcggtgttgtacgtcagtgtcctgcaatgctamagttataacttaggectaagattcaatttaatgaagtgattaâatttgttetctgaacctcttaaga tgatcttttggattagaaacatataagacaggtttacctatctattaaaaaacagatcaaaatagatacgaccaaatcggataatccatgcctacctggcat ctaggaacgtgttcttagaagatttcttacgtaatcgtatgaagaaataacaatt^aCcgttggccagcaaaaatagggctttaagtgggatagtgtttttat tagctaaccggaaaattttatagtRRRttgcaagaaaccactgaaaaccccctaattgtatacattmtggagcagcttctggtctttttgagcaataaaat tcgataaaacagaatttaagtgtaaattgttcacatttagtttctattttatcaaattttgttgctcaaaaacattcgaagctgctctaaaaaaatgcattaaaaa aggggttttcagtgginttcacanaaaaaagctaattttaactaaaaatccBtcatatttccaactttgtcacaacaataaaatgctggtcaaaatgtgttcg aaaaaatptttttttttttaatttttataaittaaaaatagttttctttcgcteegacaeaacatttttgggcetaaattttcaettcaaatttccatttttacaaccat aatcataaagciacgtotgatctctctcgcacttacctgcgcctgattcgaaagaacaaccgtagccaaaagaacaagaagaacaagcacgtagttgt ggtagtggacgttcatcacgcaatactgaccaatggtcgtggggtctcactttccgtactattgagagaggggagactgaagatggcaattgaggaca gtgtcttcgacgcacgcatgcatccataagcataatccaggagggalggagagaaaaatcttgtttctaagcccctccctttgtaatacatacacatatct aataccgaagaatggctaattgaatggacgtcagctgttgctgtagttgccaaggcatcatcgatgaaataactgaaagaaagaattaaataattattgc aggcgtatccggcggtcattgaagacttggacttgattgaggaggaggatcagatcatccatacacttaatttggaggatgcggttgatccggaaaatg ggcttagtaagtgactgaccacacgcggggggcattaatttaataaa(tgaattccatttcagatg^ttcaaactagatccagaattcgaaaagaacga ggaggtttatgaggagatccgtaaggaaatcattggaaacgccgataracggatgaggatggtggcgacgagttggatgatgaagaagagggtagt gatgtggaagaggctccgaagaagactacagagatiattgataatactgatcagaattgactgctttcagaaggtattcattttgagttttgggccggcaa atctgiaagttgccggtlgccgaaaatttgctgaatttgccggaaaaaaaaattccggaatttatttaaaaactttttgtaaaaattaaattaaatttgcaactt
Rcagagaagtctacctgacaatgcaatcatctttggactaccaagaagctgctcacaaattgctgaaaatgaagattccagacagçatgcaggtcagc gatgttgeaaagaaaaattttcgaccaaaaaaaccaaccaatcataaaatttaaaaaaaaactccgattíltcmtttmatacgagaaaaaccaaaaaa atgtatttttgccaaattctaaaatactatccccgaaattttcaatattttctctttcagaacgaactctgcgcgatgcttgtcgattgttgtgctcaacagcgla cctacgagcgattctacggaatgctcatcgaacgtttctgccgacttcgcctcgaataccagcaatactttgaaaagctctgccaggacacgtaRscac gattcaccgaattgacatcacaaaactgcggaatRggctcgccttattgctcatRgctctcgacggatgctangactggaagattttggccgatatgaa aatgaccgaagaggacacaacRcRctggcagaatctatattaaatatatatttaatgaacRgtggaggcgatgggaatggRaaaeRcaRcgagag ttactgatccgtgagtRcctagagagagttgttttcgtattcaattttccctattttcagaactttggctcaRgctttgttggattattcccacgaactaatccg aacagcgcacgatntcgatcaacttctlcacaatgãttggattgggtggntgacgttggaacttcgtgaatggctggcaaagggtctcaagaagaaga agggaatgctggatcagngaaggccgaatcaagctcagattcatcgtcgtcttcggattcgtcagactcgtctgaRcttcggattctgacgattcatcc gactcgtcttcagattcctcatcttcttcagaatcagagccagaaccaccgaagaaaaagaagaagaagaacagtgaagagagttccaaaaagaag gaaaaagagaataRggtcgacgggatcgtggagacaagagagctgaacgtcatcgtgatcaaagtgtggagaacaaggacaaggatcgtcgacg tcgccaggattctgacgaaaatcgtcggccagaacgaggagatgaccgcaaggatcggagtaaagatcgtcgtcgtcaagactcggatgatgagg atcggaaaggtcgtgaacgtcgggaagaRcaggggaaagacgtcgcggagatcgggatcgacgtgatcgaaacaaggatcaggaggatcaccg tgaagatcgccgtgaccgaagcaaggatcgtgaggatcgacgtgatcgccgtcgtcatgactctgatgatgatcgtaaaactcgtcgggatagaagt gaagagcgaggaggacgtcgtcgtgaagtggaatcggatgatcgacgccgacgtcgttgaattncaaattttaaatactgaatatttgtttttittcctatt amaraaRctctttgtgttttttttcRgctttctaaaaaattaatteaatecaaatctaaacatgagcggtttttm«ctttccgtctcccaattcgtattccgct cctctcatctgaacacaatgtgcaagRtantatcRctcgcRtcaRtcattaggacgtggggggaattggtggaagggggaaacacacaaaaggatg atggaaatgaaataaggacacacaatatgcaacaacaRcaancagaaatatggaggaaggtttaaaagaaaacataaaaatatatagaggaggaa ggaaaaciagtaaaaaataagcaaagaaattaggcgaacgatgagaaRgtcctcgcttggcaaatgcgaatccgtatggagaggcacgtttggcga aggcaaatgttcggtatggagatctgtaaaaaRRtaagttgaaatRggtgttgctcRRâcaaaatRtccgaRRcgcRgaaaRacggtgccaggtct cgacacgtcttccaatttttcaaattcaaaagagcctnaatgggctgtagRgctaatttctcgRtttgaaaatttttcttccgtttaatcgaaatttgatgtaRt tatttatgattttcaataaatttcaaagaaactggtgaaaactcggaaaattgtgaactacagtaatccaatccttaaaggcgcacacctttlaaatgtccgc cccaatacgataRRRtaagaRcgctagagcggccgccaccgcggtggagctccaattcgccctatagtgagtcgtattacaattcactggccgtcgt
RtacaacgtcgtgactgggaaaaccctggcgRacccaacttaatcgccttgcagcacatccccccttcgccagctggcgtaatagcgaagaggccc gcaccgatcgccctreccaaçagttgcgtagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgc gcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgcRtcRcccttcctttctcgccacgttcgccggctttccccgtcaagctcta aatcgggggciccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccct gatagacggtttttcgcccttfgacgttggagtccacgttcRtaatagtggaetcttgttccaaaetggaacaacactcaaccctatctcggtctattctttt gaRtaiaagggattttgccgaRtcggcctaRggRaaaaaatgagctgaRtaacaaaaaRtaacgcgaatRtaacaaaatattaacgtRacaatttca ggtggcacttttcggggaaatgtgcgçggaacccctatttgtttatttttctaaalacattcaaatatgtatccgctcatgagacaataaccctgataaatgc
RcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgceeRaRccctRRtgcggcaRRgccttcctgRtRgctcacccagaaac gctggtgaaagtaaaagatgctgaagatcagRgggtgcacgagtgggRacatcgaactggatctcaacagcggtaagatccttgagagtntcgcc ccgaagaacgRRccaatgatgagcactRtaaagttctgctatgtggcgcggtaRatcccgtattgacgccgggcaagagcaactcggtcgccgcat acactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataag catgagtgataacactgcggccaacUacttctgacaacgatcggaggaccgaaggagctaaccgctttRRcacaacatgggggatcatgtaactcg ccttgaicgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaact attaactggcgaactacttactctagcRcccggcaacaartaatagactggatggaggcggataaagttgcaggaccacttctgcgcteggcccttcc ggctggctggmaRgctgataaatctggagccggtgagcgtgggtctcgcggtatcangcagcactggggccagatggtaagccctcccgtatcgt agRatctacacgacgggcagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgaRaagcaRggtaactgtcag accaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaa cgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcdcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaa aaaccaccgctaccagcggtggmgtngccggatcaagagctaccaactctttttccgaaggtaactggcRcagcagagcgcagataccaaatact gtccttctagtgtagccgtagttaggccaccacncaagaactctgtagcaccgcctacatacctcgctctgctaatcctgRaccagtggctgctgccag tggcgataagtcgtgtcRaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggRcgtgcacacagcc ctotgacttgagcgtcgatttttgtgatgctcgtcaggggggccgagcctatggaaaaacgccageaacgcggccttwacggttcctggccttttgct ggccttRgctcacatgttctttcctgcgnatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagecgaacga ccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctg gcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttacctcactcattaggcaccccaggctttacactttatgct ^fggf^.-ra>g>f£fgfggaattgtgagcggataacaatttcaeacageaaacaEcatgaccatRatiacgccaagctcggaattaaccctcactaa agggaacaaaagctgggggg 41 ΕΡ 1 197 567/ΡΤ pAS2-cyh2-HA+bothT7-final SEQUÊNCIA ID NO. 8 gatccgtcgacagatctccctatagtgagtcgtaRactgcagccaagctaattccgggcgaatttcttatgatttatgatttttattattaaabagttataaaaaaa ataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttgttcttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggt cgctcttattgaccacacctctaccggcatgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatac gagccggaagcataaagtgtaaagcctggggtgcctaatgagtgaggtaadcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgt cgtgccagctggatlaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcncctcgctcactgactcgctgcgctcggtcg ttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcc agcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgaogagcatcacaaaaatcgacgctcaagtcagag gtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtc cgcctttctcccttcgggaagcgtggcgctttctcatagctcacgclgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaac cccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacftatcgccactggcagcagccactggtaaca ggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggclacactagaaggacagtatttggtatctgcgctctgct gaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgc agaaaaaaaggatctcaagaagatccmgatcttRctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaa aaggatcttcacctagalcctmaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggc acctatctcagcgatctgtctatncgncatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgc aatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatcc gcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtca cgctcgtcgtRggtatggcttcattcagctccggRcccaacgatcaaggcgagttacatgatcccccatgRgtgcaaaaaagcggttagctccttcggtcct ccgatcgttgtcagaagtaagnggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgac tggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagngctcttgcccggcgtcaatacgggataataccgcgccacatagcagaac tttaaaagtgctcatcattggaaaacgRcttcggggcgaaaactctcaaggatcnaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactg atcRcagcatcttttactttcaccagcgRtctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaat actcatactcttcctttncaatanattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtoRtagaaaaataaacaaataggggttccgc gcacatRccccgaaaagtgccacctgaacgaagcatctgtgcRcaRRgtagaacaaaaatgcaacgcgagagcgctaatttttcaaacaaagaatctga gctgcatttttacagaacagaaatgcaacgcgaaagcgctattttaccaacgaagaatctgtgcttcatttttgtaaaacaaaaatgcaacgcgagagcgctaa tttttcaaacaaagaatctgagctgcatttttacagaacagaaatgcaacgcgagagcgctattttaccaacaaagaatctatacttcttttttgttctacaaaaatg catcccgagagcgctantttctaacaaagcatcRagaRacRRtttctcctngtgcgctctataatgcagtctcRgataactRRgcactgtaggtccgttaag gnagaagaaggctacRtggtgtctaRRctcRccataaaaaaagcctgactccacRcccgcgntactgaRactagcgaagctgcgggtgcaRttttcaag ataaaggcatccccgattatattctataccgatgtggaRgcgcatacRtgtgaacagaaagtgatagcgRgatgaRcttcattggtcagaaaaRatgaacg gtttcttctanttgictctatatactacgtataggaaatgRtacaRncgtaRgRRcgaRcactctatgaatagRcttactacaamRttgtctaaagagtaatac tagagataaacataaaaaatgtagaggtcgagtttagatgcaagRcaaggagcgaaaggtggatgggtaggRatatagggatatagcacagagalatata gcaaagagatacttttgagcaatgtttgtggaagcggtattcgcaataRttagtagctcgttacagtccggtgcgtttttggttttttgaaagtgcgtcttcagagc gcnttggRRcaaaagcgctctgaagncctatacRtctagagaataggaacRcggaataggaacncaaagcgRtccgaaaacgagcgcRccgaaaat gcaacgcgagctgcgcacatacagctcactgttcacgtcgcacctatatctgcgtgRgcctgtatatatatatacatgagaagaacggcatagtgcgtgRtat gcRaaatgcgtacttatatgcgtctatnatgtaggatgaaaggtagtctagtacctcctgtgataRatcccaHccatgcggggtatcgtatgcRccttcagca ctaccctttagctgttctatatgctgccactcctcaaRggattagtctcatccRcaatgctatcaRtcctttgataRggatcataRaagaaaccaRaRatcatga cattaacctataaaaataggcgiatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca cagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgRggcgggtgtcggggctggcnaactatgcggcatcag agcagattgtactgagagtgcaccalagatcaacgacattactatatatataatataggaagcatttaatagacagcatcgtaatatatgtgtactttgcagttatg acgccagatggcagtagtggaagatattcntattgaaaaatagcttgtcaccttacglacaatcttgatccggagcttttctttttttgccgattaagaanaancg gtcgaaaaaagaaaaggagagggccaagagggagggcattggtgactattgagcacgtgagtatacgtgattaagcacacaaaggcagcttggagtatg tctgRaRaatRcacaggtagnctggtccattggtgaaagRtgcggcRgcagagcacagaggccgcagaatgtgctctaganccgatgctgacRgctg ggtattatatgtgtgcccaatagaaagagaacaaRgacccggRaRgcaaggaaaaRtcaagtcRgtaaaagcatataaaaatagRcaggcactccgaa atactlggttggcgtgtttcgtaatcaacctaaggaggatgttttggctctggtcaatgattacggcattgatatcgtccaactgcatggagatgagtcgtggca agaataccaagagttcctcggtttgccagttattaaaagactcgtatttccaaaagactgcaacatactactcagtgcagçttcacagaaacctcattcgtttatt cccngtngaRcagaagcaggtgggacaggtgaacmtggaRggaactcgatRctgactgggnggaaggcaagagagccccgaaagcRacaRHat gttagctggtggactgacgccagaaaatgttggtgatgcgcttagattaaatggcgttattggtgttgatgtaagcggaggtgtggagacaaatggtgtaaaa gactctaacaaaatagcaaantcgtcaaaaatgctaagaaataggttaRactgagtaglaRtatttaagtaRgRtgtgcacRgccgatctatgcggtgtgaa ataccgcacagatgcgtaaggagaaaataccgcatcaggaaangtaaacgRaataRRgRaaaaRcgcgRaaaRRtgRaaatcagctcaRRttaacc aataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggngagtgttgttccagRtggaacaagagtccactattaaagaacgt ggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagnnnggggtcgaggtgccgtaaag cactaaatcggaaccctaaagggagcccccgaRtagagcRgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggag cgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcgcgccattcg ccaRcaggctgcgcaactgttgggaagggcgatcggtgcgggccicttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagtt 42 ΕΡ 1 197 567/ΡΤ gggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtcgtccaagctttcgcgagctcgagatcccgagctttgcaaattaaagccttc gagcgtcccaaaaccttctcaagcaaggttttcagtataatgttacatgcgtacacgcgtctgtacagaaaaaaaagaaaaatttgaaatataaataacgttctt aatactaacataactataaaaaaataaatagggacctagacttcaggttgtctaactccttccttttcggnagagcggatgtggggggagggcgtgaatgtaa gcgtgacataactaaitacatgatatccttttgttgtttccgggtgtacaatatggacttcctcttttctggcaaccaaacccatacatcgggattcctataatacctt cgttggtctccctaacatgtaggtggcggaggggagatatacaatagaacagataccagacaagacataatgggctaaacaagactacaccaattacactg cctcattgatggtggtacataacgaactaatactgtagccctagacttgatagccatcatcatatcgaagtttcactacccttntccatttgccatctattgaagta ataataggcgcatgcaacttcttttctttttttttcttttctctctcccccgttgttgtctcaccatatccgcaatgacaaaaaaaatgatggaagacactaaaggaaa aaattaacgacaaagacagcaccaacagatgtcgttgttccagagctgatgaggggtatcttcgaacacacgaaactttttccttccttcattcacgcacacta ctctctaatgagcaacggtatacggccttccttccagttacttgaatttgaaataaaaaaagtttgccgctttgctatcaagtataaatagacctgcaattattaatc ttttgtttcctcgtcattgttctcgttccctttcttccttgtttctttttctgcacaatatttcaagctataccaagcatacaatcaactccaagcttgaagcaagcctcct gaaagatgaagctactgtcttctatcgaacaagcatgcgatatttgccgacttaaaaagctcaagtgctccaaagaaaaaccgaagtgcgccaagtgtctga agaacaactgggagtgtcgctactctcccaaaaccaaaaggtctccgctgactagggcacatctgacagaagtggaatcaaggctagaaagactggaaca gctatttctactgatttttcctcgagaagaccttgacatgattttgaaaatggattctttacaggatataaaagcattgttaacaggattatttgtacaagataatgtg aataaagatgccgtcacagataganggcttcagtggagactgatatgcctctaacattgagacagcatagaataagtgcgacatcatcalcggaagagagt agtaacaaaggtcaaagacagttgactgtatcgccggaattcttaatacgactcactatagggcatatggccatggaggccccggg 43 ΕΡ 1 197 567/ΡΤ SEQUÊNCIA ID NO. 9 pGAD424-without-FULL-ICE-BOTH-T7 gatccgtcgacagatctccctatagtgagtcgtattactgcagagatctatgaatcgtagatactgaaaaaccccgcaagttcacttcaactgtgcatcgtgca ccatcteaatttctttcatttatacatcgttttgccttcttttatgtaactatactcctctaagtttcaatcttggccatgtaacctctgatctatagaattttttaaatgacta gaattaatgcccatctttttmggacetaaatiettcatgaaaatatattacgagggcttattcagaagctttggacttcttcgccagaggtttggtcaagtctccaa tcaaggHgtcggcttgtctaccttgccagaaatttacgaaaagatggaaaagggtcaaatcgttggtagatacgttgttgacacttctaaataagcgaatttctt atgatttatgamttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttgttcttgagtaactctttcc tgtaggtcaggttgctttctcaggtatageatgaggtcgctcttattgaccacacctctaccggcatgcccgaaattcccctaccctatgaacatattccattttgt aatttcgtgtcgtttctattatgaatttcatttataaagtttatgtacaaatatcataaaaaaagagaatctttttaagcaaggattttcttaacttcttcggcgacagca teaccgacttcggtggtactgttggaaccacctaaatcaccagttctgatacctgcatccaaaacctttttaactgcatcttcaatggccttaccttcttcaggcaa gttcaatgacaatttcaacatcattgcaBcagacaagatagtggCBataBggtcaaccttattctttBgcaaatctgBagcagaaccKtggcatggttcgtaca aaccaaatgcggtgRcRgtctggcaaagaggccaaggacgcagatggcaacaaacccaaggaacctgggataacggaggcRcatcggagatgatatc aecaaacatgttgctggtgattataataccaraaggtgggttgggttcttaactaggatcatggcggcagaatcaatcaattgatgttgaaccttcaatgtagga aattcgttcttgatggtttcctccacagtttttctccataatcttgaagaggccaaaacattagctttatccaaggaccaaataggcaatggtggctcatgttgtag ggccatgaaagcggccaRcRgtgattcntgcacttctggaacggtgtattgttcactatcccaagcgacaccatcaccatcgtcttcctttctcttaccaaagt aaatacctcccactaattctctgacaacaacgaagtcagtacctttagcaaattgtggcttgattggagataagtctaaaagagagtcggatgcaaagttacat ggtcttaagttggcgtacaattgaagttctttacggatttttagtaaaccttgttcaggtctaacactacctgtaccccatttaggaccacccacagcacctaacaa aacggcatcaaccttcttggaggcttccagcgcctcatctggaagtgggacacctgtagcatcgatagcagcaccaccaattaaatgattttcgaaatcgaac ttgacattggaacgaacatcagaaatagctttaagaaccttaatggcttcggctgtgatttcttgaccaacgtggtcacctggcaaaacgacgatcRcttaggg gcagacattagaatggtatatccttgaaatatatatatatattgctgaaatgtaaaaggtaagaaaagttagaaagtaagacgattgctaaccacctattggaaa aaacaataggtccttaaataatattgtcaacttcaagtattgtgatgcaagcatttagtcatgaacgcttctctattctatatgaaaagccggttecggcctctcac ctttcctttttctcccaatttttcagttgaaaaaggtatatgcgtcaggcgacctctgaaattaacaaaaaatttccagtcatcgaatttgattctgtgcgatagcgc tagctagaggatcaattcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaa agcctggggtgcctaatgagtgaggtaactcacattaaRgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctggattaatgaatc ggccaacgcgcggggagaggcggtttgcgtaRgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatc agctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgta aaaaggccgcgttgclggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggact ataaagataccaggcgtnccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggataccigtccgcctttctcccttcgggaagcg tggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctg cgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt aggcggtgctacagagRcngaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaa agagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaa gatcctttgatcttttctacggggtctgacgctcagtggaacgaaaacteacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcctttt aaanaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatt tcgttcatccatagngcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgalaccgcgagacccacg ctcaccggctccagatttatcagcaataaaceagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgt tgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttca ttcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagtt ggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcat tctgagaatagtgtatgcggcgaccgagngctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaa acgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcacc agcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatatt attgaagcatttatcagggttaTtgtctcatgagcggatacatamgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcc acctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacct ctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggt gtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccataacgcatttaagcataaacacgcactatgccgttcttctcatgtatat atatatacaggcaacacgcagatataggtgcgacgtgaacagtgagctgtalgtgcgcagctcgcgttgcattttcggaagcgctcgttttcggaaacgcttt gaagttcctattccgaagttcctattctctagctagaaagtataggaacttcagagcgcttttgaaaaccaaaagcgctctgaagacgcactttcaaaaaacca aaaacgcaccggactgtaacgagctactaaaatattgcgaataccgcttccacaaacattgctcaaaagtatctctttgctatatatctctgtgctatatccctata taacctacccatccacctttcgctccttgaacttgcatctaaactcgacctctacattttttatgtttatctctagtattactctttagacaaaaaaattgtagtaagaac tattcatagagtgaatcgaaaacaatacgaaaatgtaaacatttcctatacgtagtatatagagacaaaatagaagaaaccgttcataattttctgaccaatgaa gaatcatcaacgctatcactnctgttcacaaagtatgcgcaatccacatcgglatagaatataatcggggatgcctttatcttgaaaaaatgcacccgcagctt cgctagtaatcagtaaacgcgggaagtggagtcaggctttttttatggaagagaaaatagacaccaaagtagccttcttctaaccttaacggacctacagtgc aaaaagttatcaagagaclgcattatagagcgcacaaaggagaaaaaaagtaatctaagatgctttgttagaaaaatagcgctctcgggatgcatttttgtaga acaaaaaagaagtatagancrttgttggtaaaatagcgctctcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaaaaattagcgctctcg cgttgcatttttgttttacaaaaatgaagcacagattcttcgttggtaaaatagcgctttcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaa aaattagcgctctcgcgttgcatttttgttctacaaaatgaagcacagatgcttcgttgcttgcatgcaacttcttttctttttttttcttttctctctcccccgttgttgtct caccatatccgcaatgacaaaaaaaatgatggaagacactaaaggaaaaaattaacgacaaagacagcaccaacagatgtcgttgttccagagctgatga ggggtatcttcgaacacacgaaactttttccttccttcattcacgcacactactctctaatgagcaacggtatacggccttccttccagttacttgaatttgaaataa aaaaagtttgccgctttgctatcaagtataaatagacctgcaattattaatcttrtgtttcctcgtcattgttctcgttccctttcttccttgtttctttttctgcacaatattt taccgccgccaattttaatcaaagtgggaatattgctgatagctcattgtccttcactttcactaacagtagcaacggtccgaacctcataacaactcaaacaaa ttctcaagcgctttcacaaccaattgcctcctctaacgttcatgataacttcatgaataatgaaatcacggctagtaaaattgatgatggtaataattcaaaaccac tgtcacctggttggacggaccaaactgcgtataacgcgtttggaatcactacagggatgtttaataccactacaatggatgatgtatataactatctattcgatga tgaagataccccaccaaacccaaaaaaagagatcgaattcttaatacgactcactatagggccca(ggacgaagaatccagttcattcttatgtacctatgctg agaatcgtgccaataagaagccaatacttccttagatgatgcaataaatattaaaataaaacaaaacagaaggctg 44 ΕΡ 1 197 567/ΡΤ pGN205: SEQUÊNCIA ID NO. 10 ccggtggtaccgggccccccctcgaggtcgacggtatcgataagctttcgtcattgaaaagaaggataagaatggacgatgggaagaagctctcgtt gttecaggagatcagaaaacagcaactgttccaaatcttaaggagggagaagaatatcaattcagaatttctgctcgtaacaaggctggaactggaga tccttctgatccttctgatcgtgttgttgcgaagccaagaaaccttgctccaagaattcatcgtgaagatctttctgatacaactgtcaaggtcggagccac tctcaagttcattgttcatattgatggtgagccagcaccagatgtaacatggtcattcaatggaaaaggaatcggagagagcaaggctcaaattgaaaa tgagccatacatctcgagatttgctttgccaaaggcacttcgtaagcaaagtggaaaatataccateactgcaaccaacattaatggaactgacagtgtc actatcaatatcaaggtaaaaagcaagccaacgaaaccaaagggaccaatcgaggtaactgatgtcttcgaagatcgtgcaactcttgactggaaac caccagaggatgacggaggagagccaattgagttctatgaaattgaaaagatgaacaccaaggacggaatctgggttccatgtggacgugtggag atacccaciRaeagtcgattcacteaacaagggagateaKacaagttccgtgteaaggctgtcaacagcgaaggaccttctgatccattggaaactg aaaccgaíattttggctaaaaatccacttgatcgtccagatagaccaggtcgtccagagccaactgattgggattctgatcatgttgatctcaagtgggat ccactagttctagaagcgctgctaagggggccctcgtcgagtcggtcacaatcacctgaaactccaaaggcagccagtgaggaacgtgaagaaga agaaaaagagtcatctgaacaggtttgattttctttctggtcaaaaagatgaaattattgattttcagccagatactcccaaaactagcagcgagaagtct gcaagtcgttcacagtcgcccagagaatcgcgggaagtgagccaagaggtatgtttttcaaaaatcaataactgatcataatttttattgtttggtgaattt aagaaaataatattegaaaancctctgaattateaagattgcagtattaatttcgagaaaaattgagatatteatagagctattgtaaattttcttgatttcag actgaaacttcggaaaatcaagagaaaatcaaagaaaaggatgacggggatgatcagcctggcacaccgaacagctatagaagccgggaaacttc accagctccaaaaaggtccaaggagaccaggtttgtcaaaagcttcgtgcgattaattctcatttcaatttttcagagaatcagagtçtcctgaaaaatcc ccggttcgttcaagatctcccagaaggtcttcagcacgttccccgtcacgatctcctagacggcgccgagaaagaagctcagaaagaaagcaatccg aagagccagcaccgcuccagagaaaaagaagaaagagççgctggatattctacgaacaagaaccggaggagcatatattccacccgccaaactt cgacttatgcaacaacagattagtgataagcaaagtgaacagtatcagagaatgaattgggaaagaatgaagaaaaagattcacggattggnaacag agtcaacgcgaagaatcttgttcaaattgtcagagaacttcttcaagagaatgtgattcgttcaaagtgagtgagaaaatcgaaggaaaaggaaagaat taatttaattlttcaggggacttctctgccgtgacattattcaagctcaggcttfctcaccaggattctctaacgtctatgcagctttggcggcagttatcaac tcgaaattcccicatgtcggtgaacttcttctccgtcgtctgattgtacagttcaaaagaagtrtccgtagaaatgacagaggcgtcacggtgaacgtgat caaattcatcgcacatttgattaatcaacaagttgctcacgaagttcttgcgctggaaatcatgattctgatgcttgaagaaccaactgatgattcagttga agtcgccattgcgttcctgaaagagtgtggagcaaagcttctggagattgctccagcagctcttaacagtgtctacgaccgtcttcgtgcaattctcatg gaaactgaaagatcggaaaatgcactggatcgacgtattcagtatatgattgagactgcaatgcagattcgaaaggacaaatttgcggtaaggtagaa tatataaatagtttattagaaaaaaataaattagaataatttaaattcctactagccaatcaggcgacctttttgcgcatagttctanattgaaaaatttggag aatttctcatattctcgctcggaaatctggaattcgacgagatcttctggcttctgtgcagctgcatcgctttgtgctccctttctcgcttgtcttctgtgtaca ccaagaaccttgttgagttcatcaactgaatctgtgactggcttgttgctcactggatgcactagacgactgattctcgagaaatcagattgagttgcgatt agggtgacctagaaattgggaataatacgaacmtgaaaatattcaggaggattaaaaaaattattctcgacaatcctacaaantacttangcaccatgt tgctccaacatttttcattaaaagttaatgaaaaaatgtagaaaatcggaaattggcaattttcagaccatttttaagcattttcaaaaaaaaattgcagctga aataaatgtcattttcagataaatcgagcgattnctgttgtctgacactagtttttagttttaaaaaatgttggaagaacatggtgcaataggtaatttcatag aatttccatgtgttttttttcaattaaccaattatccaaatcttccaaactcacattttgcggagctgggctatcaagaatctgctgcagttttataagacgagc atctctgatatcactgaaaattaalttttaatcaaaacttgaatatcaactaaacccacttattaactttctcgatcttctgtcgttcggtacgatgacggtgaa gaagccaattgtagtagttgatttggttcaagtcctncggtgttgtacgtcagtgtcctgcaatgctatttagttataacttaggcctaagattcaatttaatg aagtgattaaatttgttctctgaacctcttaagatgatcttttggattagaaacatataagacaggtttacctatctattaaaaaacagatcaaaatagatacg aecaaatcggataatccatgcctacetggcatctaggaacgtgttcttagaagatttcttacgtaatcgtalgaagaaataacaatttgatcgttggccag caaaaatagggtRtaagtgggatagtgtttttattagctaaccggaaaattRatagtttttttttgcaagaaaccactgaaaaccccctaattgtatacatttt
Rggagcagcttctggtctttngagcaataaaattcgataaaacagaatttaagtgtaaattgttcacatttagtttctattnatcaaattttgttgctcaaaaa cattcgaagctgctctaaaaaaatgcattaaaaaaggggttttcagtggtttttcacattaaaaaagctaattttaactaaaaatccatcatatttccaactttg tcacaacaataaaatgctggtcaaaatgtgttcgaaaaaatgtttttttttttaatttttataatttaaaaatagttttctttcgclgggacacatacatttltgggc gtaaattttcagttcaaatttccatttnacaaccataatcataaagctacgtctgatctctctcgcacttacctgcgcctgattcgaaagaacaaccgtagc caaaagaacaagaagaacaagcacgtagttgtggtagtggacgttcatcacgcaatactgaccaatggtcgtggggtctcactttccgtactattgaga gaggggagactgaagatggcaattgaggaeagtgtcttcgacgcacgcatgcatccataagcataatccaggagggatggagagaaaaatcttgttt ctaagcccctccctttgtaatacatacacatatctaataccgaagaatggctaattgaatggacgtcagctgttgctgtagttgccaaggcatcatcgatg aaataactgaaagaaagaattaaataattattgcaggcgtatccggcggtcattgaagacttggacttgattgaggaggaggatcagatcatccataca cttaaKtggaggatgcggttgatccggaaaatgggcttagtaagtgactgaccacacgcggggggcattaatttaafaaattgaattccatttcagatgt gttcaaactagatccagaattcgaaaagaacgaggaggtttatgaggagatccgtaaggaaatcanggaaacgccgatatncggatgaggatggtg gcgacgagttggatgatgaagaagagggtagtgatgtggaagaggctccgaagaagactacagagattangataatactgatcagaangactgctt tcagaaggtaUcattttgagttttgggccggcaaatctgtaagltgccggttgccgaaaatttgcIgaatttgccggaaaaaaaaattccggaatttattta aaaactttttgtaaaaattaaattaaatttgcaacttttcagagaagtctacctgacaatgcaatcatctttggactaccaagaagctgctcacaaattgctg aaaatgaagattccagacagcatgcaggtcagcgatgttgcaaagaaaaattttcgaccaaaaaaaccaaccaatcataaaatttaaaaaaaaactcc gtttttttctttttttttatacgagaaaaaccaaaaaaatgtatttttgccaaattctaaaatactatccccgaaattttcaatattttctctttcagaacgaactctg cgcgatgcttgtcgattgttgtgctcaacagcgtaectacgagcgattctaeggaatgctcatcgaacgtttctgccgacttcgcctcgaataccagcaa tactttgaaaagctctgccaggacacgtattccacgattcaccgaatlgacatcacaaaactgcggaatttggctegccttattgctcatttgctctcgacg gatgctattgaetggaagattttggccgatatgaaaatgaccgaagaggacacaacttcttctggcagaatctatattaaatatatatttaatgaacttgtg gaggcgatgggaatggttaaacttcattcgagagttactgatccgtgagtttcctagagagagttgttttcgtattcaattttccctatttteagaactttggct cattgctttgttggattattcccacgaactaatccgaacagcgcacgattttcgatcaacttcttcacaatgattggattgggtggtttgacgttggaacttc gtgaatggctggcaaagggtctcaagaagaagaagggaatgctggatcagttgaaggccgaatcaagctcagattcatcgccgtcttcggattcgtca gactcgtctgattcncggattclgacgattcatccgactcgtcttcagattcctcatcttcttcagaatcagagocagaaccaccgaagaaaaagaagaa 45 ΕΡ 1 197 567/ΡΤ
agatcgtcgtcgtcaagactcggatgalgaggatcggaaaggtcgtgaacgtcgggaagattcaggggaaagacgtcgcggagatcgggatcgac gtgatcgaaacaaggatcaggaggatcaccgtgaagatcgccgtgaccgaagcaaggatcgtgaggatcgacgtgatcgccgtcgtcatgactctg atgatgatcgtaaaactcgtcgggatagaagtgaagagcgaggaggacgtcgtcgtgaagtggaatcggatgatcgacgccgacgtcgttgaattn caaamtaaatactgaatatttgttttttttcctattatttatttattctctttgtgttttttttcttgctttctaaaaaattaattcaatccaaatctaaacatgagcggtt ttttttctctttccgtctcccaattcgtattccgctcctctcatctgaacacaatgtgcaagttiatttatcttctcgctttcatttcattaggacgtggggggaatt ggtggaagggggaaacacacaaaaggatgatggaaatgaaataaggacacacaatatgcaacaacattcaattcagaaatatggaggaaggtttaa aagaaaacataaaaatatatagaggaggaaggaaaactagtaaaaaataagcaaagaaattaggcgaacgatgagaattgtcctcgcttggcaaatg cgaatccgtatggagaggcacgtttggcgaaggcaaatgttcggtatggagatctgtaaaaatttttaagttgaaatttggtgttgctcttttacaaaatttt ccgattttcgcttgaaattacggtgccaggtctcgacacgtcttccaatttttcaaattcaaaagagcctttaatgggctgtagttgctaatttctcgtttttga aaatttttcttccgtttaatcgaaatttgatgtattttatttatgattttcaataaatttcaaagaaactggtgaaaactcggaaaattgtgaactacagtaatcca atccttaaaggcgcacaccttttaaatgtccgccccaatacgatatttttttaagattcgctagagcggccgccaccgcggtggagctccaattcgccct atagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccc cttcgceagctggcgtaatagcgaagaggcccgcaccgatcgcecttcccaacagttgcgtagcctgaatggcgaatgggacgcgccctgtagcg gcgcattaagcgcggcgggtgtggtggttaegcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttct cgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgat tagggígatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaact ggaacaacactcaaecctatctcggtctattcttttgatttataagggamtgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaa cgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatat gtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttg cggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgga tctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtatt gacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcat gacagtaagagaattatgcagtgctgccataagcatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaacc gctttttttcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgat gcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggata aagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcag cactggggccagatggtaagccctcccgtatcgtagttatctacacgacgggcagtcaggcaactatggatgaacgaaatagacagatcgctgagat aggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaa gatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcc tttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggt aactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacct cgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagc ggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaag cgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggg aacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggccgagcctatggaaaaacg ccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcct ttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgc ctctccccgcgcgttggccgatlcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaanaatgtgagtla cctcactcattaggcaccccaggctttacactttatgcttccggctcctatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatg accatgattacgccaagctcggaattaaccctcactaaagggaacaaaagctgggggggatcctccaaaatcgtcttccgctctgaaaaacgaaagt ggacctngacatccgaaaaaatgggcgaaaaaatgaaattgagctttngggtcgaaaaaaatgtttttagaatgctgagaacacgttaaacacgaag atcatatnattttgagacccggatgctctgaaaatgtctgacatagatttaaaaaagcatatatatalttttcattttcaacgtgaaagmtgtgcaactttata gaatctectattggcacattgttttttatttaactgaggcagtttttgaacacctnttgaaactttgaatctctttgaagtatactgtcgaaaagactgacttga gcgttcgaaatgccagaagaaaactatatttgaatctcgcgctaaattgagaaatgcaaccgcgctccactggacaattggaaaaaaaatttattcgga ggcgacaacggtatrttcgaaangattttctgtgtattttctcattttttataaattcttctngatttatcgttcgtttgtgagaaatttaattgtattcaaactttttt atagtaagata 46 ΕΡ 1 197 567/ΡΤ pGN207; SEQUÊNCIA ID NO. 11 ccggtggtaccgctagccgtacgaacccgggttctagaactagtggatcccacttgagatcaacatgatcagaatcccaatcagttggctctggacga cctggtctatctggacgatcaaatggattmagccaaaatatcggmcagtttccaatggatcagaaggtccttcgctgttgacagccttgacacggaa cttgtaatgatctcccttgttgagtgaatcgactgtgaagtgggtatctccactacgtccacatggaacccagattccgtccnggtgtteatcttttcaattt catagaactcaattggctctcctccgtcatcctctggtggtttccagtcaagagttgcacgatcttcgaagacatcagttacctcgattggtccctttggttt cgttggcRgctttttaccttgatattgatagtgacactgtcagttccattaatgttggRgcagtgatggtatattttccactttgcttacgaagtgcctRggca aagcaaatctcgagatgtatggctcattttcaatttgagccttgctctctccgattccttttccattgaaigaccatgttacatctggtgctggctcaccatca atatgaacaatgaaettgagagtggctccgaccttgacagttgtatcagaaagatottcacgaigaattcttggagcaaggtítcttggcttcgeaacaac acgatcagaaggatcagaaggatctccagttccagccttgttacgagcagaaattctgaattgatattcRctccctccttaagatttggaacagttgctgt tttctgatctcctggaacaacgagagcttcttcccatcgtccattcttatccttcttttcaatgacgaaagcnatcgataccgtcgacctcgagggggggc cctcgtcgagtcggtcacaatcacctgaaactccaaaggcagccagtgaggaacgtgaagaagaagaaaaagagtcatctgaacaggtttgattttct ttctggtcaaaaagatgaaattattgattttcagccagatactcccaaaactagcagcgagaagtctgcaagtcgttcacagtcgcccagagaatcgcg ggaagtgagccaagaggtatgtttttcaaaaatcaataactgatcataatttttattgtttggtgaatttaagaaaataataltcgaaaattcctctgaaRatc aagaRgcagtattaamcgagaaaaangagatattcatagagctaRgtaaattttcttgatttcagactgaaacttcggaaaatcaagagaaaatcaaa gaaaaggatgacggggatgatcagcctggcacaccgaacagctatagaagccgggaaacttcaccagctccaaaaaggtccaaggagaccaggt ttgtcaaaagcRcctgcganaanctcatttcaatttRcagagaatcagagtctcctgaaaaatccccggttcgttcaagatctcccagaaggtcttcag cacgttccccgtcacgatctcctagacggcgccgagaaagaagctcagaaagaaagcaatccgaagagccagcaccgctaccagagaaaaagaa gaaagagccgctggataRctacgaacaagaaccggaggagcatatattccacccgccaaacttcgacnatgcaacaacagattagtgataagcaa agtgaacaglatcagagaatgaaRgggaaagaatgaagaaaaagattcacggattggttaacagagtcaacgcgaagaatcRgltcaaaRgtcag agaacttcttcaagagaatgtgattcgttcaaagtgagtgagaaaatcgaaggaaaaggaaagaattaatttaatttttcaggggacttctctgccgtgac attattcaagctcaggctttctcaccaggattctctaacgtctatgcagctRggcggcagttatcaactcgaaattccctcatgtcggtgaacRcttctcc gtcgtctgaRgtacagRcaaaagaagRtccgtagaaatgacagaggcgtcacggtgaacgtgatcaaaRcatcgcacatRgaRaatcaacaagn gctcacgaagttct1gcgctggaaatcatgaUctgatgcRgaagaaccaactgatgaRcagngaagtcgccaRgcgRcctgaaagagtgtggag caaagcRctggagaRgctccagcagetCRaacagtgtctacgaccgtcRcglgcaaRctcatggaaactgaaagatcggaaaatgcactggatcg acgtaRcagtatatgaRgagactgcaatgcagaRcgaaaggacaaaRtgcggtaaggtagaatatataaatagRtaRagaaaaaaataaaRaga ataatRaaaRcctactagccaatcaggcgaccRRtgcgcatagttctanaRgaaaaatnggagaamctcataRctcgctcggaaatctggaaRc gacgagatcRctggcnctgtgcagctgcatcgcRtgtgctcccRtctcgcttgtcRctgtgtacaccaagaaccRgttgagRcatcaaetgaatctgt gactggcngngctcactggatgcactagacgactgaRctcgagaaatcagaRgagRgcgattagggtgacctagaaaRgggaataatacgaact tttgaaaatattcaggaggattaaaaaaattattctcgacaatcctacaaatttacttattgcaccatgngctccaacattmcattaaaagttaatgaaaaa atglagaaaatcggaaanggcaattttcagaccatttRaagcattUcaaaaaaaaattgcagctgaaataaatgtcattttcagataaatcgagcgatm ctgRgtctgacactagtttttagtntaaaaaatgttggaagaacatggtgcaataggtaatttcatagaatttccatgtgtttttWcaattaaccaattatcc aaatcttccaaactcacattttgcggagctgggctatcaagaatctgctgcagtmataagacgagcatctctgatatcactgaaaattaartraaatcaa aacRgaatatcaactaaacccacttaRaacRtctcgatcRctgtcgRcggtacgatgacggtgaagaagccaattgtagtagttgaRtggRcaagtc ctttcggtgttgtacgtcagtgtcctgcaatgctatRagttataacttaggcctaagaRcaamaatgaagtgattaaatttgttctctgaacctcttaagat gatcRRggaRagaa acata taagacaggRtacctatotaRaaaaaacagatcaaaatagatacgaccaaatcggataatccatgcctacctggcat ctaggaacgtgncRagaagaRtcRaegtaatcgtatgaagaaataacaatttgatcgRggccagcaaaaatagggttRaaglgggatagtgttnta( tagctaaccggaaaattttatagtttttttttgcaagaaaccactgaaaaccccctaattgtatacatntttggagcagcRctggtcttRtgagcaataaaat tcgataaaacagaatttaagtgtaaattgRcacatttagtttctattttatcaaattttgttgctcaaaaacaRcgaagctgctctaaaaaaatgcattaaaaa aggggttttcagtggntRcacattaaaaaagctaattttaactaaaaatccatcatatttocaactttgtcacaacaataaaatgctggtcaaaatgtgttcg aaaaaatgttRtRtRtaaRtRataatttaaaaatagRRctRcgctgggacacatacaRRtgggcgtaaaRttcagRcaaaRtccaRRtacaaccat aateataaagctacgtctgatctctctcgcacRacctgcgcctgaRcgaaagaacaaccgtagccaaaagaacaagaagaacaagcacgtagRgt ggtagtggacgRcatcacgcaatactgaccaatggtcgtggggtctcactttccgtactattgagagaggggagactgaagatggcaattgaggaca gtgtcRcgacgcacgcatgCatccataagcataatccaggagggatggagagaaaaatcRgtflctaagcccctcccRtgtaatacatacacatatct aataccgaagaatggctaaRgaatggacgtcagctgttgctgtagttgccaaggcatcatcgatgaaataactgaaagaaagaattaaataattattgc aggcgtatccggcggtcattgaagacttggacRgaRgaggaggaggatcagatcatccatacacttaatRggaggatgcggttgatccggaaaatg ggcttagtaagtgactgaccacacgcggggggcattaatttaataaattgaattccatttcagatgtgttcaaactagatccagaattcgaaaagaacga ggaggtttatgaggagatccgtaaggaaatcanggaaacgccgatatttcggatgaggatggtggcgacgagRggatgatgaagaagagggtagt gatgtggaagaggctccgaagaagactacagagattaRgataatactgatcagaaRgactgctttcagaaggtattcattttgagttttgggccggcaa atctgtaagttgccggflgccgaaaatttgctgaatRgccggaaaaaaaaanccggaaRtantaaaaacntRgtaaaaattaaaRaaatngcaacn
RcagagaagtctacctgacaatgcaatcatctnggactaccaagaagctgctcacaaattgctgaaaatgaagaRccagacagcatgcaggtcagc atgtatttttgccaaaRctaaaatactatccccgaaatntcaatattRctcRtcagaacgaactctgcgcgatgcRgtcgattgttgtgctcaacagcgta cctacgagcgattctacggaatgctcatcgaacgRtctgccgacttcgcctcgaataccagcaatactttgaaaagctctgccaggacacgtattccac gattcaccgaattgacatcacaaaactgcggaatttggctcgccttaRgctcatttgctctcgacggatgctaRgactggaagaRRggccgatatgaa aatgaccgaagaggacacaacttcttctggcagaatctatattaaatatatatttaatgaacRgtggaggcgatgggaatggRaaacRcaRcgagag ttactgatccgtgagRtcctagagagagttgtmcgtaRcaattflccctattttcagaactRggctcangctngnggattancccacgaactaatccg aacagcgcacgattncgatcaacttcttcacaatgattggaRgggtggRtgacgttggaacttcgtgaatggctggcaaagggtctcaagaagaaga agggaatgctggatcagRgaaggccgaatcaagctcagartcatcgtcgtcRcggattcgtcagactcgtctgattcRcggattctgacgattcatcc gactogtcRcagaRcctcatcRcRcagaatcagagccagaaccaccgaagaaaaagaagaagaagaacagtgaagagagRccaaaaagaag 47 ΕΡ 1 197 567/ΡΤ gaaaaagagaatattggtcgacgggatcgtggagacaagagagctgaacgtcatcgtgatcaaagtgtggagaacaaggacaaggatcgtcgacg tcgccaggattctgacgaaaatcgtcggccagaacgaggagatgaccgcaaggatcggagtaaagatcgtcgtcgtcaagacteggatgatgagg atcggaaaggtcgtgaacgtcgggaagattcaggggaaagacgtcgcggagatcgggatcgacgtgatcgaaacaaggatcaggaggatcaccg tgaagatcgccgtgaccgaagcaaggatcgtgaggatcgacgtgatcgccgtcgtcatgactctgatgatgatcgtaaaactcgtcgggatagaagt gaagagcgaggaggacgtcgtcgtgaagtggaatcggatgatcgacgccgacgtcgttgaattttcaaattttaaatactgaatatttgttttttttcctatt atttatttattctctttgtgttttttttcttgctttctaaaaaattaattcaatccaaatctaaacatgagcggttttttttctctttccgtctcccaattcgtattccgct cctctcatctgaacacaatgtgcaagtttatttatcttctcgctttcatttcattaggacgtggggggaattggtggaagggggaaacacacaaaaggatg atggaaatgaaataaggacacacaatatgcaacaacattcaattcagaaatatggaggaaggtttaaaagaaaacataaaaatatatagaggaggaa ggaaaactagtaaaaaataagcaaagaaattaggcgaacgatgagaattgtcctcgcttggcaaatgcgaatccgtatggagaggcacgtttggcga aggcaaatgttcggtatggagatctglaaaaatttttaagttgaaatttggtgttgctcttttacaaaattttccgattttcgcttgaaattacggtgccaggtct cgacacgtcttccaatttttcaaattcaaaagagcctttaatgggctgtagttgctaatttctcgtttttgaaaatttttcttccgtttaatcgaaatttgatgtattt tatttatgattttcaataaatttcaaagaaactggtgaaaactcggaaaattgtgaactacagtaatccaatccttaaaggcgcacaccttttaaatgtccgc cccaatacgatatttttttaagattcgctagagcggccgccaccgcggtggagctccaattcgccctatagtgagtcgtattacaattcactggccgtcgt tttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccngcagcacatccccccttcgccagctggcgtaatagcgaagaggccc gcaccgatcgcccttcccaacagttgcgtagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgc gcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctcta aatcgggggctccctttagggnccgatttagtgctttacggcacctcgaccccaaaaaacttganagggtgatggttcacgtagtgggccatcgccct gatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattctttt gantataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttca ggtggcacttttcggggaaatgtgcgcggaacecctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgc ttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaac gctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgcc ccgaagaacgttttccaatgatgagcactmaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcat acactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataag catgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctttttttcacaacatgggggatcatgtaactcg ccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaact attaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttcc ggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgt accaagtttactcatatatactttagartgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaa cgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcRgcaaacaaaa aaaccaccgctaccagcgglggmgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatact gtccRctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccag tggcgataagtcgtgtcttaccgggRggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcc cagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcartgagaaagcgccacgcttcccgaagggagaaaggcggacagg tatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggggaacgcctggtatctttatagtcctgtcgggtttcgccac ctctgacttgagcgtcgatttttgtgatgctcgtcaggggggccgagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgct ggccttttgctcacatgttctttcctgcgnatcccctgattctgtggataaccgtattaccgcctngagtgagctgataccgctcgccgcagccgaacga ccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctg gcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttacctcactcanaggcaccccaggctttacactnatgct tccggctcctatgttgtgtggaangtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctcggaattaaccctcactaa agggaacaaaagctgggggggatcctccaaaatcgtcttccgctctgaaaaacgaaagtggacctttgacatccgaaaaaatgggcgaaaaaatga aattgagctttttgggtcgaaaaaaatgtttttagaatgctgagaacacgttaaacacgaagatcatatnattrtgagacccggatgctctgaaaatgtctg acatagatttaaaaaagcafatatatatttttcattttcaacgtgaaagttngtgcaactttatagaatctcctattggcacattgttttttatttaactgaggcag tttttgaacacctttttgaaactttgaatctctttgaagtatactgtcgaaaagactgacttgagcgttcgaaatgccagaagaaaactatatttgaatctcgc gctaaaRgagaaatgcaaccgcgctccactggacaattggaaaaaaaaRtattcggaggcgacaacggtattttcgaaattgattttctgtgtattttct cattttttataaattcttctttgatttatcgttcgtttgtgagaaatttaattgtattcaaâcttttttatagtaagata
LISTAGEM DE SEQUÊNCIAS <110> Devgen N.V. <120> Caracterização da função de genes utilizando Inibição de ARNcd <130> 50897/408 <140> 99932836.2 <141> 1999-07-02 <150> GB 9814536.0 <151> 1998-07-03 48 ΕΡ 1 197 567/ΡΤ <150> GB 9827152.1 <151> 1998-12-09 <160> 11 <170> Patentln Ver. 2.0
<210> 1 <211> 3216 <212> ADN <213> Sequência Artificial <220>
<223> Descrição de Sequência Artificial: ADN <400> 1 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa ggcgaaattg taaacgttaa tattttgtta aaattcgcgt taaatatttg tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg cccaaatcaa gttttttgcg gtcgaggtgc cgtaaagctc taaatcggaa agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattc cccggggatc ctctagagtc gaaagcttct cgccctatag tgagtcgtat gtattctata gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc plasmídico taccgcatca 60 ttaaatcagc 120 agaatagacc 180 gaacgtggac 240 tgaaccatca 300 ccctaaaggg 360 ggaagggaag 420 gcgcgtaacc 480 ttcaggctgc 540 ctggcgaaag 600 tcacgacgtt 660 gagctcggta 720 tacagcttga 780 tttcctgtgt 840 aagtgtaaag 900 ctgcccgctt 960 gcggggagag 1020 49 ΕΡ 1 197 567/ΡΤ gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 1080 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 1140 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 1200 aaaaggccgc gttgctggcg tttttcgata ggctccgccc ccctgacgag catcacaaaa 1260 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 1320 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 1380 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 1440 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 1500 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 1560 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 1620 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 1680 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tocggcaaac 1740 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 1800 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 1860 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 1920 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 1980 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 2040 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 2100 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagoaataa 2160 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 2220 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 2280 acgttgttgg cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 2340 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 2400 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 2460 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 2520 ctgtgactgg tgagtactca accaagtcat tctgagaata ccgcgcccgg cgaccgagtt 2580 gctcttgccc ggcgtcaata cgggataata gtgtatgaca tagcagaact ttaaaagtgc 2640 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 2700 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 2760 gpgi-H-n-hgg ghgagraaaa ar.aggaaggc aaaa1~gr:f^ac aaaaaaggga ataaaQQÇqa 2820 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 2880 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 2940 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga 3000 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg 3060 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg 3120 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct 3180 ggcttaacta tgcggcatca gagcagattg tactga 3216
<210> 2 <211> 6460 <212> ADN <213> Sequência Artificial <220> <223> Descrição de Sequência Artificial: ADN plasmidico <400> 2 ctagcatgaa cacgattaac atcgotaaga aogacttctc tgacatcgaa ctggctgcta 60 50 ΕΡ 1 197 567/ΡΤ tcccgttcaa cactctggct gaccattacg gtgagcgttt agctcgcgaa cagttggccc 120 ttgagcatga gtcttacgag atgggtgaag cacgcttccg caagatgttt gagcgtcaac 180 ttaaagctgg tgaggttgcg gataacgctg ccgccaagcc tctcatcact accctactcc 240 ctaagatgat tgcacgcatc aacgactggt ttgaggaagt gaaagctaag cgcggcaagc 300 gcccgacagc cttccagttc ctgcaagaaa tcaagccgga agccgtagcg tacatcacca 360 ttaagaccac tctggcttgc ctaaccagtg ctgacaatac aaccgttcag gctgtagcaa 420 gcgcaatcgg tcgggccatt gaggacgagg ctcgcttcgg tcgtatccgt gaccttgaag 480 ctaagcactt caagaaaaac gttgaggaac aactcaacaa gcgcgtaggg cacgtctaca 540 agaaagcatt tatgcaagtt gtcgaggctg acatgctctc taagggtcta ctcggtggcg 600 aggcgtggtc ttcgtggcat aaggaagact ctattcatgt aggagtacgc tgcatcgaga 660 tgctcattga gtcaaccgga atggttagct tacaccgcca aaatgctggc gtagtaggtc 720 aagactctga gactatcgaa ctcgcacctg aatacgctga ggctatcgca acccgtgcag 780 gtgcgctggc tggcatctct ccgatgttcc aaccttgcgt agttcctcct aagccgtgga 840 ctggcattac tggtggtggc tattgggcta acggtcgtcg tcctctggcg ctggtgcgta 900 ctcacagtaa gaaagcactg atgcgctacg aagacgttta catgcctgag gtgtacaaag 960 cgattaacat tgcgcaaaac accgcatgga aaatcaacaa gaaagtccta gcggtcgcca 1020 acgtaatcac caagtggaag cattgtccgg tcgaggacat ccctgcgatt gagcgtgaag 1080 aaotcccgat gaaaccggaa gacatcgaca tgaatcctga ggctctcacc gcgtggaaac 1140 qtgctqccgc tqctgtgtac cgcaaggaca gggctcgcaa gtctcgccgt atcagccttg 1200 agttcatgct tgagcaagcc aataagtttg ctaaccataa ggccatctgg ttcccttaca 1260 acatggactg gcgcggtcgt gtttacgccg tgtcaatgtt caacccgcaa ggtaacgata 1320 tgaccaaagg actgcttacg ccggcgaaag gtaaaccaat cggtaaggaa ggttactact 1380 ggctgaaaat ccacggtgca aactgtgcgg gtgtcgataa ggttccgttc cctgagcgca 1440 tcaagttcat tgaggaaaac cacgagaaca tcatggcttg cgctaagtct ccactggaga 1500 acacttggtg ggctgagcaa gattctccgt tctgcttcct tgcgttctgc tttgagtacg 1560 ctggggtaca gcaccacggc ctgagctata actgctccct tccgctggcg tttgacgggt 1620 cttgctctgg catccagcac ttctccgcga tgctccgaga tgaggtaggt ggtcgcgcgg 1680 ttaacttgct tcctagtgag accgttcagg acatctacgg gattgttgct aagaaagtca 1740 acgagattct acaaacagac gcaatcaata aaaccaataa caaaqtaatt accataacca 1800 atgagaacac tggtgaaatc tctgagaaag tcaagctggg cactaaggca ctggctggtc 1860 aatggctggc tcacggtgtt actcgcagtg tgactaagcg ttcagtcatg acgctggctt 1920 acgggtccaa agagttcggc ttccgtcaac aagtgctgga agataccatt cagccagcta 1980 ttgattccgg caagggtccg atgttcactc agccgaatca ggctgctgga tacatggcta 2040 agctgatttg ggaatctgtg agcgtgacgg tggtagctgc ggttgaagca atgaactggc 2100 ttaagtctgc tgctaagctg ctggctgctg aggtcaaaga taagaagact ggagagattc 2160 ttcgcaagcg ttgcgctgtg cattgggtaa ctcctgatgg tttccctgtg tggcaggaat 2220 acaagaagcc tattcagacg cgcttgaacc tgatgttcct cggtcagttc cgcttacagc 2280 ctaccattaa caccaacaaa gatagcgaga ttgatgcaca caaacaggag tctggtatcg 2340 cfcectaactt tgtacacagc caagacggta gccaccttcg taagactgta gtgtgggcac 2400 acgagaagta cggaatcgaa tcttttgcac tgattcacga ctccttcggt accattccgg 2460 ctgacgctgc gaacotgttc aaagcagtgc gcgaaactat ggttgacaca tatgagtctt 2520 gtgatgtact ggctgatttc tacgaccagt tcgctgacca gttgcacgag tctcaattgg 2580 acaaaatgcc agcacttccg gctaaaggta acttgaacct ccgtgacatc ttagagtcgg 2640 acttcgcgtt cgcgtaacca tggtattgat atctgagotc cgcatcggcc gctgtcatca 2700 gatcgccatc tcgcgcccgt gcctctgact tctaagtcca attactcttc aacatcccta 2760 catgctcttt ctccctgtgc tcccaccccc tatttttgtt attatcaaaa aaacttcttc 2820 ttaatttctt tgttttttag cttcttttaa gtcacctcta acaatgaaat tgtgtagatt 2880 caaaaataga attaattcgt aataaaaagt cgaaaaaaat tgtgctccct ccccccatta 2940 51 ΕΡ 1 197 567/ΡΤ ataataatto tatcccaaaa tctacacaat gttctgtgta cacttcttat gtttttttta 3000 cttctgataa attttttttg aaacatcata gaaaaaaccg cacacaaaat accttatcat 3060 atgttacgtt tcagtttatg accgcaattt ttatttcttc gcacgtctgg gcctctcatg 3120 acgtcaaatc atgctcatcg tgaaaaagtt ttggagtatt tttggaattt ttcaatcaag 3180 tgaaagttta tgaaattaat tttcctgctt ttgctttttg ggggtttccc ctattgtttg 3240 tcaagagttt cgaggacggc gtttttcttg ctaaaatcac aagtattgat gagcacgatg 3300 caagaaagat cggaagaagg tttgggtttg aggctcagtg gaaggtgagt agaagttgat 3360 aatttgaaag tggagtagtg tctatggggt ttttgcctta aatgacagaa tacattccca 3420 atataccaaa cataactgtt tcctactagt cggccgtacg ggccctttcg tctcgcgcgt 3480 ttcggtgatg acggtgaaaa octctgacac atgcagctcc cggagacggt cacagcttgt 3540 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 3600 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg 3660 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg gccttaaggg 3720 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc 3780 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 3840 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 3900 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 3960 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 4020 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 4080 ttttcgccco gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 4140 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 4200 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 4260 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 4320 gacaaogatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 4380 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 4440 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 4500 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 4560 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 4620 acataaatct cacoatatca ttacaacact crcraaccaaat ggtaaaccct cccqtatcqt 4680 aggcgeagcg gtcgggctga actgaçatac ggacaggtat gggaaacgcc atttttgtga tttacggttc tgattctgtg aacgaccgag gcctctcccc gaaagcgggc agttatctac gataggtgcc ttagattgat taatctcatg agaaaagatc aacaaaaaaa ttttccgaag gccgtagtta aatcctgtta aagacgatag gcccagcttg aagcgccacg aacaggagag cgggtttcgc cctatggaaa tgctcacatg tgagtgagct ggaagcggaa atgcagctgg acgacgggga tcactgatta ttaaaacttc accaaaatcc aaaggatctt ccaccgctac gtaactggct ggccaccact ccagtggctg ttaccggata gagcgaacga cttcccgaag cgcacgaggg cacctctgac aacgccagca ttctttcctg gataccgctc gagcgcccaa cacgacaggt gtcaggcaac agcattggta atttttaatt cttaacgtga cttgagatcc cagcggtggt tcagcagagc tcaagaactc ctgccagtgg cctacaccga ggagaaaggc agcttccagg ttgagcgtcg acgcggcctt cgttatcccc gccgcagccg tacgcaaacc ttcccgactg tatggatgaa actgtcagac taaaaggatc gttttcgttc tttttttctg ttgtttgccg gcagatacca tgtagcaccg cgataagtcg cgaaatagac caagtttact taggtgaaga cactgagcgt cgcgtaatct gatcaagagc aatactgtcc cctacatacc tgtcttaccg acgggqggtt ctacagcgtg ccggtaagcg tggtatcttt tgctcgtcag ctggcctttt gataaccgta cgcagcgagt gcgcgttggc agtgagcgca agatcgctga catatatact tcctttttga cagaccccgt gctgcttgca taccaactct ttctagtgta tcgctctgct ggttggactc agcattgaga gcagggtcgg atagtcctgt gggggcggag gctggccttt ttaccgcctt cagtgagcga cgattcatta acgcaattaa 4740 4800 4860 4920 4980 504 0 5100 5160 5220 5340 5400 5460 5520 5580 564 0 5700 5760 5820 52 ΕΡ 1 197 567/ΡΤ tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 5880 gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta 5940 cgccaagctt gcatgcctgc aggtcgactc tagaggatca agagcatttg aatcagaata 6000 tggagaacgg agcatgagca ttttcgaagt tttttagatg cactagaaca aagcgtgttç 6060 gcttcctctg agcccgcttt ccttatatac ccgcattctg cagccttaca gaatgttcta 6120 gaaggtccta gatgcattcg tttgaaaata ctcccggtgg gtgcaaagag acgcagacgg 6180-aaaatgtatc tgggtctctt tattgtgtac actacttttc catgtaccga atgtgagtcg 6240 ccctcctttt gcaacaagca gctcgaatgt tctagaaaaa ggtgçaaaat agtataaata 6300 ccgttgaaaa taaataccga acaacatttg ctctaattgt gaaattagaa atcttcaaac 6360 tataatcatc tcactggatc cccgggattg gccaaaggac ccaaaggtat gtttcgaatg 6420 atactaacat aacatagaac attttcagga ggacccttgg 6460
<210> 3 <211> 8330 <212> ADN <213> Sequência Artificial <220> <223> Descrição de Sequência Artificial: ADN plasmidico <400> 3 gttgtcgtaa agagatgttt ttattttact ttacaccggg tcctctotct ctgccagcac 60 agctcagtgt tggctgtgtg ctcçggctcc tgccaccggc ggcctcatct tcttcttctt 120 cttctctcct gctctcgctt atcacttctt cattcattct tattcctttt catcatcaaa 180 ctagcatttc ttactttatt tatttttttc aattttcaat tttcagataa aaccaaacta 240 cttgggttac agccgtcaac agatccccgg gattggccaa aggacccaaa ggtatgtttc 300 gaatgatact aacataacat agaacatttt caqqaqqacc cttgcttgga gggtaccgga 360 tgactgctcc aaagaagaag cgtaagctca tgaacacgat taacatcgct aagaacgact 420 tctctgacat cgaactggct gctatcccgt tcaacactct ggctgaccat tacggtgagc 480 gtttagctcg cgaacagttg gcccttgagc atgagtctta ogagatgggt gaagcacgct 540 tccgcaagat gtttgagcgt caacttaaag ctggtgaggt tgcggataac gctgccgcca 600 agcctctcat cactacccta ctccctaaga tgattgcacg catcaacgac tggtttgagg 660 aagtgaaagc taagcgcggc aagcgcccga cagccttcca gttcctgcaa gaaatcaagc 720 cggaagccgt agcgtacatc accattaaga ccactctggc ttgcctaacc agtgctgaca 780 atacaaccot tcaaactqta qcaaacqcaa tcqqtccrcrac cattqaqqac qaqqctcqct 840 tcggtcgtat ccgtgacctt gaagctaagc acttcaagaa aaacgttgag gaacaactca 900 acaagcgcgt agggcacgtc tacaagaaag catttatgca agttgtcgag gctgacatgc 960 tctctaaggg tctactcggt ggcgaggcgt ggtcttcgtg gcataaggaa gactctattc 1020 atgtaggagt acgctgcatc gagatgctca ttgagtcaac cggaatggtt agcttacacc 1080 gccaaaatgc tggcgtagta ggtcaagact ctgagactat cgaactcgca cctgaatacg 1140 ctgaggctat cgcaacccgt gcaggtgcgc tggctggcat ctctccgatg ttccaacctt 1200 gcgtagttcc tcctaagccg tggactggca ttactggtgg tggctattgg gctaacggtc 1260 gtcgtcctct ggcgctggtg cgtactcaca gtaagaaagc actgatgcgc tacgaagacg 1320 tttacatgcc tgaggtgtac aaagcgatta acattgcgca aaacaccgca tggaaaatca 1380 acaagaaagt cctagcggtc gccaacgtaa tcaccaagtg gaagcattgt ccggtcgagg 1440 acatccctgc gattgagcgt gaagaactcc cgatgaaacc ggaagacatc gacatgaatc 1500 ctgaggctct caccgcgtgg aaacgtgctg ccgctgctgt gtaccgcaag acaaggctcg 1560 caagtctcgc cgtatcagcc ttgagttcat gcttgagcaa gccaataagt ttgctaacca 1620 53 ΕΡ 1 197 567/ΡΤ taaggccatc tggttccctt acaacatgga ctggcgcggt tcgtgtttac gctgtgtcaa 1680 tgttcaaccc gcaaggtaac gatatgacca aaggacgtct tacgctggcg aaaggtaaac 1740 caatcggtaa ggaaggttac tactggctga aaatccacgg tgcaaactgt gcgggtgtcg 1800 ataaggtttc gtttcctgag cgcatcaagt tcattgagga aaaccacgag aacatcatgg 1860 cttgcgctaa gtctccactg gagaacactt ggtgggctga gcaagattct ccgttctgct 1920 tccttgcgtt ctgctttgag tacgctgggg tacagcacca cggcctgagc tataactgct 1980 cccttccgct ggcgtttgac gggtcttgct ctggcatcca gcacttctcc gcgatgctcc 2040 gagatgaggt aggtggtcgc goggttaact tgcttcctag tgaaaccgtt caggacatct 2100 acgggattgt tgctaagaaa gtcaacgaga ttctgcaagc agacgcaatc aatgggaccg 2160 ataacgaagt agttaccgtg accgatgaga acactggtga aatctctgag aaagtcaagc 2220 tgggcactaa ggcactggct ggtcaatggc tggcttacgg tgttactcgc agtgtgacta 2280 agcgttcagt catgacgctg gcttacgggt ccaaagagtt cggcttccgt caacaagtgc 2340 tggaagatac cattcagcca çctattgatt ccggcaaggg tctgatgttc actcagccga 2400 atcaggctgc tggatacatg gctaagctga tttgggaatc cgtgagcgtg acggtggtag 2460 ctgcggttga agcaatgaac tggcttaagt ctgctgctaa gctgctggct gctgaggtca 2520 aagataagaa gactggagag attcttcgca agcgttgcgc tgtgcattgg gtaactcctg 2580 atggtttccc tgtgtggcag gaatacaaga agcctattca gacgcgcttg aacctgatgt 2640 tcctcggtca gttccgctta cagcctacca ttaacaccaa caaagatagc gaçattgatg 2700 cacacaaaca qgagtctqqt atcgctccta actttqtaca cagccaaqac qqtaqccacc 2760 ttcgtaagac tgtagtçtgg gcacacgaga agtacggaat cgaatctttt gcactgattc 2820 acgactcctt cggtaccatt ccggctgacg ctgcgaacct gttcaaagca gtgcgcgaaa 2880 ctatggttga cacatatgag tcttgtgatg tactggctga tttctacgac cagttcgctg 2940 accagttgca cgagtctcaa ttggacaaaa tgccagcact tccggctaaa ggtaacttga 3000 acctccgtga catcttagag tcggaottog cgttcgcgta agggcccact agtcggccgt 3060 acgggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 3120 tcccggagac ggtcaoagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg 3180 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga 3240 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 3300 accocatcaq ccaaccttaa ggqcctcatq atacqcctat ttttataaat taatqtcatq 3360 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 3420 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 3480 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 3540 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 3600 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 3660 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 3720 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 3780 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 3840 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 3900 rcraacicfímrít. λ a rrgrtttt 3960 ttgcacaaca tgçgggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 4020 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 4080 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 4140 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 4200 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 4260 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 4320 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 4380 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 4440 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 4500 54 ΕΡ 1 197 567/ΡΤ ttccactgag cgtcagaccc ogtagaaaag atcaaaggat cttcttgaga tccttttttt 4560 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4620 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4680 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4740 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4800 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4860 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4920 tacctacagc gtgagcattg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4980 tatccggtaa gcggcagggt—cggaacagga gagcgcanga gggagctt~r;n agggggaaac 5040 goctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 5100 tgatgctogt caggggggcg gagcctatgg aaaaacgcca geaacgcggc ctttttacgg 5160 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 5220 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 5280 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc 5340 cccgcgcgtt ggecgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 5400 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta 5460 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 5520 ggaaacagct actattctca atgaccatga tttaaatttt ttacgccaag cagagcttaa ctgtaagttt aaatggctga aaacatgatc aatcactcac ttactaacta aacgatggat 5580 5640 acgctaacaa ttccttgcaa ccttctttga cacacactca atgacaaggg ttggagcccc cttatcgaac cattaatcga tcccgatttg cttggaaatg aattcggtgc cttctctctt gagttcttcg cggattatta gtgtggggca gtcgcatgag ttcatagact taaaaggaag aaataagctt tttcttcaaa tccgccaaaa acatgataaa tcgatcgata gctgctctca ccattctttc cccatattcc aggctgacga acatccgtag taagtcttcc ttttaagccg gcatgcctgc gagaaacttt agcctagcat gtgtttcatt ttgaagacaa atatattact ttctttatgc ttgatgaagg tgttaggaaa acacttttta agagcaaaaa tgaagtcggc tcttattgat ggcactcgcc actccaaatg agggagacga actctcttca tgtgggtttt aagagaacgg aatactgctt gcgagcattt aatccgatta ctaacagtac tgtgctcatt ggagggggac ctctctcaca tagctttttt agccgaaaaa 5700 5760 5820 5880 5940 6000 6060 6120 6180 gacagcattc gccgctagtt 6240 caccdcctcc gcctccagca gcaeacaatc atcttcgtgt gttctcgaeg attccttgta ttgaagttta caaaagtttt ttgttttaaa caattaaaag tagaagtttg gcgtaaaatt accgtgcttc ttgccttatc tttatattta tgccgcggtc ggtattacta cattattccc cttcttgttt gagttctagt aaaaaagttg aaaaaaaaat tctcgattca gagagaattt aattttaaaa ttcaagaact tttaatattt aatttgccga tttttaaagg agttgttgtg ctctcacttg aagagcgtcg tgtaggtcac gtgaatgcac cacgtcatcg cttggtgtat tccgtccatt tcttgttcta ataaagaaaa tcccacccct attttttgct acaaacagaa aataaaaatt ctagaaaaac atcttaagat tttagaataa aaaaacacga ataatagtga acccaacagg tctctttcac aaaaaaaaag cacattcgac ttgttcgtag tgattttttt tagattttat aaagtctagg ctggttatto cgcttgtttc gcgctctgtc atgaaaaaaa agtttcaatt tagcttaatt gtaattacga aagtcttcag aaaaagaaca tccgotgtct aaaaaaaaac ccagtaacaa tgatgcagtt gcgcaactac tctcccgctc taaatggtac ctggaaattt tcagacatac agaaagagtg tcaaaatatg tgccaaagtg gttggccaaa ttttttaatt catgggtact gaagcttttt aaatgagtaa ctatttatot atttgcactc atcacgtctg aaaaaatttg ttgtgggtcg acaccacact tttcgtcccc accactcctg ttttaaaatt attttctatt tgtctcgttg agatcaacgg tgtgtgtccg taatgaagtt actaaatagg agaaaaattc tgaaaattct aagctcaaat tttoctcccc ggctcttcac agacggtgaa gtttctttac gttgtctcca cactttcggt actcactcct acgtttctac ttaggccaga tctcatcaaa aaattgattc attttttcct agcaaaagat ttatccgaga 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 55 ΕΡ 1 197 567/ΡΤ ttgatgggaa agatattaat catcaaactc cgccttttac taatagaaaa togagaaatt ttgtgggtct cgccacgatc ttcaaattct agtgtttaat ttcaacaaga aatcgtgaaa atatatggta atttcccgaa gatttatcac gaaaaaagtt ttttttgtca tcattttgaa gcttttctct ccaaaaatga tcttttctcc ccgtttttca caccgccagt gtgtgccata tatccctgct tgatttcgtt gctccagaga atggagcaca ctgggaagcc gccgagagat atctctcgag tttctccgcc <210> 4 <211> 6470 <212> ADN <213> Sequência Ar gttctttacg gtttggaggg gagagagaga tagattttcg 7440 atgtctttta gaatctaaaa tagatttttc tcatcatttt 7500 acagtaattt cgcaattttc ttgccaaaaa tacacgaaat 7560 tcggtcttag tggttcattt ggtttaaaag tttataaaat 7620 ttccgcataa ttggacctaa aatgggtttt tgtcatcatt 7680 atcctgttgt ttcgcaattt tcttttcaaa aatacacgaa 7740 atattgagçg tctcgccacg atttcagtca cagtggccag 7800 cgcctagtct cacatttccg gaaaaccgaa tctaaattag 7860 caaaaaatcg agacatccct atagtttcgc aattttcgtc 7920 cagtctagaa ttaaaattcg ctggaactgg gaccatgata 7980 ttttattttt tattacactg gattgactaa aggtcaccac 8040 tcacacacac acacacacac aatgtcgaga ttttatgtgt 8100 ccgttgtctc tctctctcta ttcatctttt gagccgagaa 8160 caggatcccg gcgcgcgatg tcgtcgggag atggcgccgc 8220 atcagggaag atcgtctgat ttctcctcgg atgccacctc 8280 tgttactccc tgccgaacct gatatttccc 8330 ificial <220> <223> Descrição de Sequência Artifi ciai: ADN plasmidico <400> 4 aagcttgcat gcctgcaggc cttggtcgac tctagacact tttcagctac ctagatacat 60 ggatatcccc gcctcccaat ccacccaccc agggaaaaag aagggctcgc cgaaaaatca 120 aagttatctc caggctcgcg catcccaccg agcggttgac ttctctccac cacttttcat 180 tttaaccctc ggggtacggg attggccaaa ggacccaaag gtatgtttcg aatgatacta 240 acataacata gaacattttc aggaggaccc ttgcttggag ggtaccgagc tcagaaaaaa 300 tgactgctcc aaagaagaag cgtaaggtac cggtaatgaa cacgattaac atcgctaaga 360 acgacttctc tgacatcgaa ctggctgcta tcccgttcaa cactctggct gaccattacg 420 gtgagcgttt agctcgcgaa cagttggccc ttgagcatga gtcttacgag atgggtgaag 480 cacgcttccg caagatgttt gagcgtcaac ttaaagctgg tgaggttgcg gataacgctg 540 ccgccaagcc tctcatcact accctactcc ctaagatgat tgcacgcatc aacgactggt 600 ttgaggaagt gaaagctaag cgcggcaagc gcccgacagc cttccagttc ctgcaagaaa 660 tcaagccgga agccgtagcg tacatcacca ttaagaccac tctggcttgc ctaaccagtg 720 ctgacaatac aaccgttcag gctgtagcaa gcgcaatcgg tcgggccatt gaggacgagg 780 ctcgcttcgg tcgtatccgt gaccttgaag ctaagcactt caagaaaaac gttgaggaac 840 aactcaacaa gcgcgtaggg cacgtctaca agaaagcatt tatgcaagtt gtcgaggctg 900 acatgctctc taagggtcta ctcggtggcg aggcgtggtc ttcgtggcat aaggaagact 960 ctattcatgt aggagtacgc tgcatcgaga tgctcattga gtcaaccgga atggttagct 1020 tacaccgcca aaatgctggc gtagtaggtc aagactctga gactatcgaa ctcgcacctg 1080 aatacgctga ggctatcgca acccgtgcag gtgcgctggc tggcatctct ccgatgttcc 1140 aaccttgcgt agttcctcct aagccgtgga otggcattac tggtggtggc tattgggcta 1200 acggtcgtcg tcctctggcg ctggtgcgta ctcacagtaa gaaagcactg atgcgctacg 1260 aagacgttta catgcctgag gtgtacaaag cgattaacat tgcgcaaaac accgcatgga 1320 56 ΕΡ 1 197 567/ΡΤ tcatggcttg cgctaagtct ccactggaaa acacttaotg aaatcaacaa tcgaggacat tgaatcctga gggctcgcaa ctaaccataa tgtcaatgtt gtaaaccaat gtgtcgataa gaaagtccta ccctgcgatt ggctctcacc gtctcgccgt ggccatctgg caacccgcaa cggtaaggaa ggttccgttc gcggtcgcca gagcgtgaag gcgtggaaac atoagccttg ttcccttaca ggtaacgata ggttactact cctgagcgca acgtaatcac aactcccgat gtgctgccgc agttcatgct acatggactg tgaccaaagg ggctgaaaat tcaagttcat caagtggaag gaaaccggaa tgctgtgtac tgagcaagcc gcgcggtcgt actgcttacg ccacggtgca tgaggaaaac ggctaaacaa cattgtccgg gacatcgaca cgcaaggaca aataagtttg gtttacgccg ctggcgaaag aactgtgcgg cacgagaaca aattctccat 1380 1440 1500 1560 1620 1680 1740 1800 1860 tctgcttcct actgctccct tgctccgaga acatctacgg ggaccgataa tcaagctggg tgactaagcg aagtgctgga agccgaatca tgcgttctgc tccgctggcg tgaggtaggt gattgttgct cgaagtagtt cactaaggca ttcagtcatg agataccatt ggctgctgga tttgagtacg tttgacgggt ggtcgcgcgg aagaaagtca accgtgaccg ctggctggtc acgctggctt cagccagcta tacatggcta ctggggtaca cttgctotgg ttaacttgct acçagattct atgagaacac aatggctggc acgggtccaa ttgattccgg agctgatttg gcaccacggc catccagcac tcctagtgag acaagcagac tggtgaaatc tcacggtgtt agagttcggc caagggtccg ggaatctgtg ctgagctata ttctccgcga accgttcagg gcaatcaatg tctgagaaag actcgcagtg ttccgtcaac atgttcactc agcgtgacgg <-ggtagfi-gr qijH-gaag<--a at-gaacvhgge: tfcaagtcrtgc tgr:taagetg ctgget-çat-g 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 aggtcaaaga ctcctgatgg tgatgttcct ttgatgcaca gccaccttcg tgattoacga gcgaaactat tcgctgacca acttgaacct taagaagact tttccctgtg cggtcagttc caaacaggag taagactgta ctccttcggt ggttgacaca gttgcacgag ccgtgacatc ggagagattc tggcaggaat cgcttacagc tctggtatcg gtgtgggcacaccattccgg tatgagtctt tctcaattgg ttagagtcgg ttcgcaagcg acaagaagcc ctaccattaa ctcctaactt acgagaagtactgacgctgc gtgatgtact acaaaatgcc acttcgcgtt ttgcgctgtg tattcagacg caccaacaaa tgtacacagc cggaatcgaagaacctgttc ggctgatttc agcacttccg cgcgtaagaa gcgccggtcg ctaccattar: naacttglv cattgggtaa cgcttgaacc gatagcgaga caagacggta tcttttgcac aaagcagtgc tacgaccagt gctaaaggta ttccaactga cegetgtrat: 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 ttcctgcttt tgctttttgg ggqtttcccc tattgtttgt cagagtaagt cgcgccogtg tccctgtgct gttttttagc ttaattcgta atcccaaaat ttttttttga cagtttatga tgctcatcgt gaaattaatt ttaaactgag cctctgactt cccaccccct ttcttttaag ataaaaagtc ctacacaatg aacatcatag ccgcaatttt gaaaaagttt ttctactaae ctaagtccaa atttttgtta tcacctctaa gaaaaaaatt ttctgtgtac aaaaaaccgc tatttcttcg tggagtattt taacgagtaa ttactcttca ttatcaaaaa caatgaaatt gtgctccctc acttcttatg acacaaaata cacgtctggg ttggaatttt tatttaaatt acatccctac aacttcttct gtgtagattc cccccattaa ttttttetac ccttatcata cctctcatga tcaatcaagt ttcagcatct atgctctttc taatttcttt aaaaatagaa taataattct ttctgataaa tgttacgttt cgtcaaatca gaaagtttat naagaqt 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 gaggacggcg ggaagaaggt ggagtagtgt ataactgttt cggtgaaaac tgccgggagc gottaaotat accgcacaga gcctattttt tttttcttgc ttgggtttga ctatggggtt cctactagtc ctctgacaca agacaagccc gcggcatcag tgcgtaagga ataggttaat taaaatcaca ggctcagtgg tttgccttaa ggccgtacgg tgcagctccc gtcagggcgc agcagattgt gaaaataccg gtcatgataa agtattgatg aaggtgagta atgacagaat gccctttcgt ggagacggtc gtcagcgggt actgagagtg catcaggcgg taatggttto agcacgatgc gaagttgata acattcccaa ctcgcgcgtt acagcttgtc gttggcgggt caccatatgc ccttaagggc ttagacgtca aagaaagatc atttgaaagt tataccaaac tcggtgatga tgtaagcgga gtcggggctg ggtgtgaaat ctcgtgatac ggtggcactt 3720 3780 3840 3900 3960 4020 4080 4140 4200 57 ΕΡ 1 197 567/ΡΤ ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 4260 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 4320 tgagtattca acatttocgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 4380 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 4440 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 4500 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 4560 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 4620 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 4680 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 4740 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 4800 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 4860 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 4920 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 4980 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 5040 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 5100 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 5160 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 5220 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 5280 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 5340 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 5400 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 54 60 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 5520 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 5580 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 5640 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 5700 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gcattgagaa agcgccacgc 5760 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 5820 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 5880 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 5940 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 6000 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 6060 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 6120 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 6180 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 6240 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 6300 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctgt 6360 aagtttaaac atgatcttac taactaacta ttctcattta aattttcaga gcttaaaaat 6420 ggctgaaatc actcacaacg atggatacgc taacaacttg gaaatgaaat 6470
<210> 5 <211> 4689 <212> ADN <213> Sequência Artificial <220> <223> Descrição de Sequência Artificial: ADN plasmídico 58 ΕΡ 1 197 567/ΡΤ <400> 5 aagcttgcat gcctgcaggc cttggtcgac tctagacact tttcagctac ctagatacat 60 ggatatcccc gcctcccaat ccacccaccc agggaaaaag aagggctcgc cgaaaaatca 120 aagttatctc caggctcgcg catcccaccg agcggttgac ttctctccac cacttttcat 180 tttaaccctc ggggtacggg attggccaaa ggacccaaag gtatgtttcg aatgatacta 240 acataacata gaacattttc aggaggaccc ttgcttggag ggtaccgagc tcccgggatt 300 aatacgactc actataccgg tagaaaaaat gagtaaagga gaagaacttt tcactggagt 360 tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg 420 agagggtgaa ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg 480 aaaactacct qttccatqqq taaqtttaaa catatatata ctaactaacc ctqattattt 540 aaattttcag ccaacacttg tcactacttt ctgttatggt gttcaatgct tctcgagata 600 cccagatcat atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca 660 ggaaagaact atatttttca aagatgacgg gaactacaag acacgtaagt ttaaacagtt 720 cggtactaac taaccataca tatttaaatt ttcaggtgct gaagtcaagt ttgaaggtga 700 tacccttgtt aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct 840 tggacacaaa ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca 900 aaagaatgga atcaaagttg taagtttaaa catgatttta ctaactaact aatctgattt 960 aaattttcag aacttcaaaa ttagacacaa cattgaagat ggaagcgttc aactagcaga 1020 ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta 1080 cctatccaca caatctaccc tttcaaaaaa tcccaacaaa aaaaqaqacc acatggtcct 1140 tcttgagttt gtaacagctg ctgggattac acatggcatg gatgaactat acaaatagca 1200 ttcgtagaat tccaactgag cgccggtcgc taccattacc aacttgtctg gtgtcaaaaa 1260 taataggggc cgctgtcatc agagtaagtt taaactgagt tctactaact aacgagtaat 1320 atttaaattt tcagcatctc gcgcccgtgc ctctgacttc taagtccaat tactcttcaa 1380 catccctaca tgctctttct ccctgtgctc ccacccccta tttttgttat tatcaaaaaa 1440 acttcttctt aatttctttg ttttttagct tcttttaagt cacctctaac aatgaaattg 1500 tgtagattca aaaatagaat taattcgtaa taaaaagtcg aaaaaaattg tgctccctcc 1560 ccccattaat aataattcta tcccaaaatc tacacaatgt tctgtgtaca cttcttatgt 1620 tttttttact tctgataaat tttttttgaa acatcataga aaaaaccgca cacaaaatac 1680 cttatr.atafc gttacgtttc agtttatgac cgcaattttt atttcttcgc acgtctgagc 1740 ctctcatgac gtcaaatcat gctcatcgtg aaaaagtttt ggagtatttt tggaattttt 1800 caatcaagtg aaagtttatg aaattaattt tcctgctttt gctttttggg ggtttcccct 1860 attgtttgtc aagagtttcg aggacggcgt ttttcttgct aaaatcacaa gtattgatga 1920 gcacgatgca agaaagatcg gaagaaggtt tgggtttgag gctcagtgga aggtgagtag 1980 aagttgataa tttgaaagtg gagtagtgtc tatggggttt ttgccttaaa tgacagaata 2040 cattcccaat ataccaaaca taactgtttc ctactagtcg gccgtacggg ccctttcgtc 2100 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 2160 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 2220 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 2280 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcggc 2340 cttaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 2400 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 2460 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 2520 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 2580 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 2640 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 2700 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 2760 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 2820 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 2880 59 ΕΡ 1 197 567/ΡΤ atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 2940 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 3000 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 3060 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 3120 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 3180 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 3240 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 3300 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 3360 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 3420 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 3480 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 3540 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 3600 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 3660 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 3720 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 3780 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 3840 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 3900 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 3960 cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 4020 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 4080 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 4140 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 4200 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 4260 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 4320 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 4380 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 4440 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 4500 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 4560 catgattacg ccaagctgta agtttaaaca tgatcttact aactaactat tctcatttaa 4620 attttcagag cttaaaaatg gctgaaatca ctcacaacga tggatacgct aacaacttgg 4680 aaatgaaat 4689
<210> 6 <211> 5175 <212> ADN <213> Sequência Artificial <220> <223> Descrição de Sequência Artificial: ADN plasmidico <400> 6 gatcccggcg cgcgatgtcg tcgggagatg gcgccgcctg ggaagccgcc gagagatatc 60 agggaagatc gtctgatttc tcctcggatg ccacctcatc tctcgagttt ctccgcctgt 120 tactccctgc cgaacctgat atttcccgtt gtcgtaaaga gatgttttta ttttacttta 180 caccgggtcc tctctctctg ccagcacagc tcagtgttgg ctgtgtgctc gggctcctgc 240 caccggcggc ctcatcttct tcttcttctt ctctcctgct ctcgcttatc acttcttcat 300 tcattcttat tccttttcat catcaaacta gcatttctta ctttatttat ttttttcaat 360 tttcaatttt cagataaaac caaactactt gggttacagc cgtcaacaga tccccgggat 420 60 ΕΡ 1 197 567/ΡΤ tggccaaagg acccaaaggt atgtttcgaa tgatactaac ataacataga acattttcag 480 gaggaccctt gcttggaggg taccggtaga aaaaatgagt aaaggagaag aacttttcac 540 tggagttgtc ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt 600 cagtggagag ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac 660 tactggaaaa ctacctgttc catgggtaag tttaaacata tatatactaa ctaaccctga 720 ttatttaaat tttcagccaa cacttgtcac tactttctgt tatggtgttc aatgcttctc 780 gagataccca gatcatatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta 840 tgtacaggaa agaactatat ttttcaaaga tgacgggaac tacaagacac gtaagtttaa 900 acagttcggt actaactaac catacatatt taaattttca ggtgctgaag tcaagtttga 960 aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag aagatggaaa 1020 cattcttgga cacaaattgg aatacaacta taactcacac aatgtataca tcatggcaga 1080 caaacaaaag aatggaatca aagttgtaag tttaaacttg gacttactaa ctaacggatt 1140 atatttaaat tttcagaact tcaaaattag acacaacatt gaagatggaa gcgttcaact 1200 agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa 1260 ccattacctg tccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat 1320 ggtccttctt gagtttgtaa cagctgctgg gattacacat ggcatggatg aactatacaa 1380 atagcattcg tagaattcca actgagcgcc ggtcgctacc attaccaact tgtctggtgt 1440 caaaaataat aggggccgct gtcatcagag taagtttaaa ctgagttcta ctaactaacg 1500 _agt-aat-al-tt- aaaltUrnag catctcgcgc ccgtçcctct aacttctaao tccaattact 1560 cttcaacatc cctacatgct ctttctccct gtgctcccac cccctatttt tgttattatc 1620 aaaaaaactt cttcttaatt tctttgtttt ttagcttctt ttaagtcacc tctaacaatg 1680 aaattgtgta gattcaaaaa tagaattaat tcgtaataaa aagtcgaaaa aaattgtgct 1740 ccctcccccc attaataata attctatccc aaaatctaca caatgttctg tgtacacttc 1800 ttatgttttt tttacttctg ataaattttt tttgaaacat catagaaaaa accgcacaca 1860 aaatacctta tcatatgtta cgtttcagtt tatgaccgca atttttattt cttcgcacgt 1920 ctgggcctct catgacgtca aatcatgctc atcgtgaaaa agttttggag tatttttgga 1980 atttttcaat caagtgaaag tttatgaaat taattttcct gcttttgctt tttgggggtt 2040 tcccctattg tttgtcaaga gtttcgagga cggcgttttt cttgctaaaa tcacaagtat 2100 tgatgagcac gatgcaagaa agatcggaag aaggtttggg tttgaggctc agtggaaggt 2160 gagtagaagt tgataatttg aaagtggagt agtgtctatg gggtttttgc cttaaatgac 2220 agaatacatt cccaatatac caaacataac tgtttcctac tagtcggccg tacgggcccg 2280 gtacccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca 2340 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 2400 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 2460 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcçgc 2520 caacgcgcgg ggagaggcçg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 2580 tcgctgcgct cggtcgttcg gctgcgçcga gcggtatcag ctcactcaaa ggcggtaata 2640 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 2700 γΊέη gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 2820 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 2880 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 2940 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 3000 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 3060 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 3120 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 3180 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 3240 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 3300 61 ΕΡ 1 197 567/ΡΤ attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 3360 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 3420 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 3480 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 3540 ctatttcgtt eateeatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 3600 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 3660 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 3720 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 3780 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 3840 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 3900 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 3960 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 4020 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 4080 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 4140 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 4200 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 4260 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 4320 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 4380 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 4440 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg 4500 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat 4560 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg 4620 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc 4680 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt 4740 tggggtcgag gtgccgtaaa gcactaaato ggaaccctaa agggagcccc cgatttagag 4800 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg 4860 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc 4920 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag 4980 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 5040 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 5100 gtgagcgcgc gtaatacgac tcactatagg gcgaattgga gctccaccgc ggtggcggcc 5160 gctctagaac tagtg 5175 <210> 7 <211> 12482
<212> ADN <213> Sequência Artificial <220> <223> Descrição de Sequência Artificial: ADN plasmidico <400> 7_ gatcctccaa aatcgtcttc cgctctgaaa aacgaaagtg gacctttgac atccgaaaaa 60 atgggcgaaa aaatgaaatt gagctttttg ggtcgaaaaa aatgttttta gaatgctgag 120 aacacgttaa acacgaagat catatttatt ttgagacccg gatgctctga aaatgtctga 180 catagattta aaaaagcata tatatatttt tcattttcaa cgtgaaagtt ttgtgcaact 240 ttatagaatc tcctattggc acattgtttt ttatttaact gaggcagttt ttgaacacct 300 ttttgaaact ttgaatctct ttgaagtata ctgtcgaaaa gactgacttg agcgttcgaa 360 62 ΕΡ 1 197 567/ΡΤ atgccagaag aaaactatat ttgaatctcg cgctaaattg agaaatgcaa ccgcgctcca 420 ctggacaatt ggaaaaaaaa tttattcgga ggcgacaacg gtattttcga aattgatttt 480 ctgtgtattt tctcattttt tataaattct tctttgattt atcgttcgtt tgtgagaaat 540 ttaattgtat tcaaactttt ttatagtaag ataccggtgg taccgctagc cgtacgaacc 600 cgggattggc caaaggaccc aaaggtatgt ttogaatgat actaacataa catagaacat 660 tttcaggagg acccttgctt ggagggtacc ggatgactgc tccaaagaag aagcgtaagc 720 tcatgaacac gattaacatc gctaagaacg acttctctga catcgaactg gctgctatcc 780 cgttcaacac tctggctgac cattacggtg agcgtttagc tcgcgaacag ttggcccttg 840 agcatgagtc ttacgagatg ggtgaagcac gcttccgcaa gatgtttgag cgtcaactta 900 aagctggtga ggttgcggat aacgctgccg ccaagcctct catcactacc ctactcccta 960 agatgattgc acgcatcaac gactggtttg aggaagtgaa agctaagcgc ggcaagcgcc 1020 cgacagcctt ceagttcctg caagaaatca agccggaagc cgfcagcgtac atcaccatta 1080 agaccactct ggcttgccta accagtgctg acaatacaac cgttcaggct gtagcaagcg 1140 caatcggtcg ggccattgag gacgaggctc gcttcggtcg tatccgtgao cttgaagcta 1200 agcacttcaa gaaaaacgtt gaggaacaac tcaacaagcg cgtagggcac gtctacaaga 1260 aagcatttat gcaagttgtc gaggctgaca tgctctctaa gggtctactc ggtggcgagg 1320 cgtggtcttc gtggcataag gaagactcta ttcatgtagg agtacgctgc atcgagatgc 1380 tcattgagtc aaccggaatg gttagcttac accgccaaaa tgctggcgta gtaggtcaag 1440 actctgagac tatcgaactc gcacctgaat acgctgaggc tatcgcaacc cgtgcaggtg 1500 cgctggctgg catctctccg atgttccaac cttgcgtagt tcotoctaag ocgtggactg 1560 gcattactgg tggtggctat tgggctaacg gtcgtcgtcc tctggcgctg gtgcgtactc 1620 acagtaagaa agcactgatg cgctacgaag acgtttacat gcctgaggtg tacaaagoga 1680 ttaacattgc gcaaaacacc gcatggaaaa tcaacaagaa agtcctagcg gtcgccaacg 1740 taatcaccaa gtggaagcat tgtccggtcg aggacatccc tgcgattgag cgtgaagaac 1800 tcccgatgaa accggaagac atcgacatga atcctgaggc tctcaccgcg tggaaacgtg 1860 ctgccgctgc tgtgtaccgc aagacaaggc tcgcaagtct cgccgtatca gccttgagtt 1920 catgcttgag caagccaata agtttgctaa ccataaggcc atctggttcc cttacaacat 1980 ggactggcgc ggttcgtgtt tacgctgtgt caatgttcaa cocgcaaggt aacgatatga 2040 ccaaaggacg tcttacgctg gcgaaaggta aaccaatcgg taaggaaggt tactactggc 2100 tgaaaatcca cggtgcaaac tgtgcgggtg tcgataaggt ttcgtttcct gagcgcatca 2160 agttcattga ggaaaaccac gagaacatca tggcttgcgc taagtctcca ctggagaaca 2220 cttggtgggc tgagcaagat tctccgttct gcttccttgc gttctgcttt gagtacgctg 2280 gggtacagca ccacggcctg agctataact gctcccttce gctggegttt gaegggtett 2340 gctctggcat ccagcacttc tccgcgatgc tccgagatga ggtaggtggt cgcgcggtta 2400 acttgcttcc tagtgaaacc gttcaggaca tctacgggat tgttgctaag aaagtcaacg 2460 agattctgca agcagacgca atcaatggga ccgataacga agtagttacc gtgaccgatg 2520 agaacactgg tgaaatctct gagaaagtca agctgggcac taaggcactg gctggtcaat 2580 ggctggctta cggtgttact cgcagtgtga ctaagcgttc agtcatgacg ctggcttacg 2640 ggtccaaaga gttcggcttc cgtcaacaag tgctggaaga taccattcag ccagctattg 2700 attccggcaa gggtctgatg ttcactcagc cgaatcaggc tgctggatac atggctaagc 2760 tgatttggga atccgtgagc gtgacggtgg tagctgcggt tgaagcaatg aactggctta 2820 agtctgctgc taagctgctg gctgctgagg tcaaagataa gaagactgga gagattcttc 2880 goaagcgttg cgctgtgcat tgggtaactc ctgatggttt ccctgtgtgg caggaataca 2940 agaagcctat tcagacgcgc ttgaacctga tgttcotcgg tcagttccgc ttacagccta 3000 ccattaacac caacaaagat agcgagattg atgcacacaa acaggagtct ggtatcgctc 3060 ctaactttgt acacagccaa gacggtagcc accttcgtaa gactgtagtg tgggcacacg 3120 agaagtacgg aatcgaatct tttgcactga ttcacgactc cttcggtacc attccggctg 3180 acgctgcgaa cctgttcaaa gcagtgcgcg aaactatggt tgacacatat gagtcttgtg 3240 63 ΕΡ 1 197 567/ΡΤ atgtactggc tgatttctac gaccagttcg ctgaccagtt gcacgagtct caattggaca 3300 aaatgccagc acttccggct aaaggtaact tgaacctccg tgacatctta gagtcggact 3360 tcgcgttcgc gtaagggccc tcgtcgagtc ggtcacaatc acctgaaact ccaaaggcag 3420 ccagtgagga acgtgaagaa gaagaaaaag agtcatctga acaggtttga ttttctttct 3480 ggtcaaaaag atgaaattat tgattttcag ccagatactc ccaaaactag cagcgagaag 3540 tctgcaagto gttcacagtc gcccagagaa tcgcgggaag tgagccaaga çgtatgtttt 3600 tcaaaaatca ataactgatc ataattttta ttgtttggtg aatttaagaa aataatattc 3660 gaaaattcct ctgaattatc aagattgcag tattaatttc gagaaaaatt gagatattca 3720 tagagctatt gtaaattttc ttgatttoag actgaaactt cggaaaatca agagaaaatc 3780 aaagaaaagg atgacgggga tçatcagcct ggcacaccga acagctatag aagccgggaa 3840 acttcaccag ctccaaaaag gtccaaggag accaggtttg tcaaaagctt cctgcgatta 3900 attctcattt caatttttca gagaatcaga gtctcctgaa aaatccccgg ttcgttcaag 3960 atctcccaga aggtcttcag cacgttcccc gtcacgatct cctagacggc gccgagaaag 4020 aagctcagaa agaaagcaat ccgaagagcc agcaccgcta ccagagaaaa agaagaaaga 4080 gccgctggat attctacgaa caagaaccgg aggagcatat attccacccg ccaaacttcg 4140 acttatgcaa caacaçatta gtgataagca aagtgaacag tatcagagaa tgaattggga 4200 aagaatgaag aaaaagattc acggattggt taacagagtc aacgcgaaga atcttgttca 4260 aattgtcaga gaacttcttc aagagaatgt gattcgttca aagtgagtga gaaaatcgaa 4320 ggaaaaggaa agaattaatt taatttttca ggggacttct ctgccgtgac attattcaag 4380 ctcaggcttt ctcaccagga ttctctaacg tctatgcagc tttggcggca gttatcaact 4440 cgaaattccc tcatgtcggt gaacttcttc tccgtcgtct gattgtacag ttcaaaagaa 4500 gtttccgtag aaatgacaga ggcgtcacgg tgaacgtgat caaattcatc gcacatttga 4560 ttaatcaaca agttgctcac gaagttcttg cgctggaaat catçattctg atgcttgaag 4620 aaccaactga tgattcagtt gaagtcgcca ttgcgttcct gaaagagtgt ggagcaaagc 4680 ttctggagat tgctccagca gctcttaaca gtgtctacga ccgtcttcgt çcaattctca 4740 tggaaactga aagatcggaa aatgcactgg atcgacgtat tcaçtatatg attgagactg 4800 caatgcagat tcgaaaggac aaatttgcgg taaggtagaa tatataaata gtttattaga 4860 aaaaaataaa ttagaataat ttaaattcct actagccaat caggcgacct ttttgcgcat 4920 agttctatta ttgaaaaatt tggagaattt ctcatattct cgctcggaaa tctggaattc 4980 gacgagatct tctggcttct gtgcagctgc atcgctttgt gctccctttc tcgcttgtct 5040 tctgtgtaca ccaagaacct tgttgagttc atcaactgaa tctgtgactg gcttgttgct 5100 caetggatgc actagacgac tgattctcga gaaatcagat tgagttgcga ttagggtgac 5160 ctagaaattg ggaataatac gaacttttga aaatattcag gaggattaaa aaaattattc 5220 tcgacaatcc tacaaattta cttattgcac catgttgctc caacattttt cattaaaagt 5280 taatgaaaaa atgtagaaaa tcggaaattg gcaattttca gaccattttt aagcattttc 5340 aaaaaaaaat tgcagctgaa ataaatgtca ttttcagata aatcçagcga ttttctgttg 5400 tctgacacta gtttttagtt ttaaaaaatg ttggaagaac atggtgcaat aggtaattte 5460 atagaatttc catgtgtttt ttttcaatta accaattatc caaatcttcc aaactcacat 5520 tttgcggagc tgggctatca agaatctgct gcagttttat aagacgagca tctctgatat 5580 cactgaaaat taatttttaa tcaaaacttg aatatcaact aaacccactt attaactttc 5640 tcgatcttct gtcgttcggt acgatgacgg tgaagaagcc aattgtagta gttgatttgg 5700 ttcaagtcct ttcggtgttg tacgtcagtg tcctgcaatg ctatttagtt ataacttagg 5760 cctaagattc aatttaatga agtgattaaa tttgttctct gaacctctta agatgatctt 5820 ttggattaga aacatataag acaggtttac ctatctatta aaaaacagat caaaatagat 5880 acgaccaaat cggataatcc atgcctacct ggcatctagg aacgtgttct tagaagattt 5940 cttacgtaat cgtatgaaga aataacaatt tgatcgttgg ccagcaaaaa tagggtttta 6000 agtgggatag tgtttttatt agctaaccgg aaaattttat agtttttttt tgcaagaaac 6060 cactgaaaac cccctaattg tatacatttt ttggagcagc ttctggtctt tttgagcaat 6120 64 ΕΡ 1 197 567/ΡΤ aaaattcgat aaaacagaat ttaagtgtaa attgttcaca tttagtttct attttatcaa 6180 attttgttgc tcaaaaacat tcgaagctgc tctaaaaaaa tgcattaaaa aaggggtttt 6240 cagtggtttt tcacattaaa aaagctaatt ttaactaaaa atccatcata tttccaactt 6300 tgtcacaaca ataaaatgct ggtcaaaatg tgttcgaaaa aatgtttttt tttttaattt 6360 ttataattta aaaatagttt tctttcgctg ggacacatac atttttgggc gtaaattttc 6420 agttcaaatt tccattttta caaccataat cataaagcta cgtctgatct ctctcgcact 6480 tacctgcgcc tçattcgaaa gaacaaccgt agccaaaaga acaagaagaa caagcacgta 6540 gttgtggtag tggacgttca tcacgcaata ctgaccaatg gtcgtggggt ctcactttcc 6600 gtactattga gagaggggag actgaagatg gcaattgagg acagtgtctt cgacgcacgc 6660 atgcatccat aagcataatc caggagggat ggagagaaaa atcttgtttc taagcccctc 6720 cctttgtaat acatacacat atctaatacc gaagaatggc taattgaatg gacgtcagct 6780 gttgctgtag ttgccaaggc atcatcgatg aaataactga aagaaagaat taaataatta 6840 ttgcaggcgt atccggcggt cattgaagac ttggacttga ttgaggagga ggatcagatc 6900 atccatacac ttaatttgga ggatgcggtt gatccggaaa atgggcttag taagtgactg 6960 accacacgcg gggggcatta atttaataaa ttgaattcca tttcagatgt gttcaaacta 7020 gatccagaat tcgaaaagaa cgaggaggtt tatgaggaga tccgtaagga aatcattgga 7080 aacgccgata tttcggatga ggatggtggc gacgagttgg atgatgaaga agagggtaçt 7140 gatgtgçaag aggctccgaa gaagactaca gagattattg ataatactga tcagaattga 7200 ctgctttcacr aaggtattca ttttgagttt tgggccggca aatctgtaag ttgccggttg 7260 ccgaaaattt gctgaatttg ccggaaaaaa aaattccgga atttatttaa aaactttttg 7320 taaaaattaa attaaatttg caacttttca gagaagtcta cctgacaatg caatcatctt 7380 tggactacca agaagctgct cacaaattgc tgaaaatgaa gattccagac agcatgcagg 7440 tcagcgatgt tgcaaagaaa aattttcgac caaaaaaacc aaccaatcat aaaatttaaa 7500 aaaaaactcc gtttttttct ttttttttat acgagaaaaa ccaaaaaaat gtatttttçc 7560 caaattctaa aatactatcc ccgaaatttt caatattttc tctttcagaa cgaactctgc 7620 gcgatgcttg tcgattgttg tgctcaacag cgtacctacg agcgattcta cggaatgctc 7680 atcgaacgtt tctgccgaet tcgcctcgaa taccagcaat actttgaaaa gctctgccag 7740 gacacgtatt ccacgattca ccgaattgac atcacaaaac tgcggaattt ggctcgcctt 7800 attgctcatt tgctctcgac ggatgctatt gactggaaga ttttggccga tatgaaaatg 7860 accgaagagg acacaacttc ttctggcaga atctatatta aatatatatt taatgaactt 7920 gtggagçcga tgggaatggt taaacttcat tcgagagtta ctgatccgtg agtttcctag 7980 agagagttgt tttcgtattc aattttccct attttcagaa ctttggctca ttgctttgtt 8040 ggattattcc cacgaactaa tccgaacagc gcacgatttt cgatcaactt cttcacaatg 8100 attggattgg gtggtttgac gttggaactt cgtgaatggc tggcaaaggg tctcaagaag 8160 aagaagggaa tgctggatca gttgaaggcc gaatcaagct cagattcatc gtcgtcttcg 8220 gattcgtcag actcgtctga ttcttcggat tctgacçatt catccgactc gtcttcagat 8280 tcctcatctt cttcagaatc agaçccagaa ccaccgaaga aaaagaagaa gaagaacagt 8340 gaagagagtt ccaaaaagaa ggaaaaagag aatattggtc gacgggatcg tggagacaag 8400 aaaactoaac gtcatcqtaa tcaaaqtqtg qaqaacaaqq acaaggatcg tcgacgtcçc 8460 caggattctg acgaaaatcg tcggccagaa cgaggagatg accgcaagga tcggagtaaa 8520 gatcgtcgtc gtcaagactc ggatgatgag gatcggaaag gtcgtgaacg tcgggaagat 8580 tcagggçaaa gacgtcgcgg agatcgggat cgacgtgatc gaaacaagga tcaggaggat 8640 caccgtgaag atcgccgtga ccgaagcaag gatcgtgagg atcgacgtga tcgccgtcgt 8700 catgactctg atgatgatcg taaaactcgt cgggatagaa gtgaagagcg aggaggacgt 8760 cgtcgtgaag tggaatcgga tgatcgacgc cgacgtcgtt gaattttcaa attttaaata 8820 etgaatattt gttttttttc ctattattta tttattctct ttgtgttttt tttcttgctt 8880 tctaaaaaat taattcaatc caaatctaaa catgagcggt tttttttoto tttccgtctc 8940 coaattcgta ttccgctcct ctcatctgaa cacaatgtgc aagtttattt atcttctcgc 9000 65 ΕΡ 1 197 567/ΡΤ tttcatttca ttaggacgtg gggggaattg gtggaagggg gaaacacaca aaaggatgat 9060 ggaaatgaaa taaggacaca eaatatgcaa caacattcaa ttcagaaata tggaggaagg 9120 tttaaaagaa aacataaaaa tatatagagg aggaaggaaa actagtaaaa aataagcaaa 9180 gaaattaggc gaacgatgag aattgtcctc gcttggcaaa tgcgaatccg tatggagagg 9240 cacgtttggc gaaggcaaat gttcggtatg gagatctgta aaaattttta agttçaaatt 9300 tggtgttgct cttttacaaa attttocgat tttcgcttga aattacggtg ccaggtctcg 9360 acacgtottc caatttttca aattcaaaag agcctttaat gggctgtagt tgctaatttc 9420 tcgtttttga aaatttttct tccgtttaat cgaaatttga tgtattttat ttatgatttt 9480 caataaattt caaagaaact ggtgaaaact cggaaaattg tgaactacag taatccaatc 9540 cttaaaggcg cacacctttt aaatgtccgc cccaatacga tattttttta agattcgcta 9500 gagcggccgc caccgcggtg gagctccaat tcgccctata gtgagtcgta ttacaattca 9660 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc 9720 cttgcagcac atcccccctt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 9780 ccttcccaac agttgcgtag cctgaatggc gaatgggacg cgccctgtag cggcgcatta 9840 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 9900 cccgctcctt tcgctttctt cccttocttt ctcgccacgt tcgccggctt tccccgtcaa 9960 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 10020 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 10080 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 10140 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 10200 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta 10260 acgtttacaa tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 10320 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 10380 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 10440 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 10500 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 10560 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 10620 ctgctatgtg çcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 10680 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 10740 gatggoatga cagtaagaga attatgcagt gctgccataa gcatgagtga taacactgcg 10800 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt ttttcacaac 10860 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 10920 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 10980 actggcgaac tacttactot agcttcccgg caacaattaa tagactggat ggaggcggat 11040 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 11100 tctggageeg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 11160 ccctcccgta tcgtagttat ctacacgacg ggcagtcagg caactatgga tgaacgaaat 11220 agacagatog ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 11280 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 11340 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 11400 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 11460 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 11520 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 11580 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 11640 tacctcgctc tgctaatcct gttaccagtg gctgctgcoa gtggcgataa gtcgtgtctt 11700 accgggttgg actcaagacg atagttaccg gataaggogc agcggtcggg ctgaacgggg 11760 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 11820 cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 11880 66 ΕΡ 1 197 567/ΡΤ agcggcaggg tcggaacagg agagcgcacg agggagcttc caggggggaa cgcctggtat 11940 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 12000 tcaggggggc cgagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 12060 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 12120 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 12180 gagtcagtga gcgaggaagc ggaagagcgc ocaatacgca aaccgcctct ccccgcgcgt H-ZQU tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 12300 cgcaacgcaa ttaatgtgag ttacctcact cattaggcac cccaggcttt acactttatg 12360 cttccggctc ctatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 12420 tatgaccatg attacgccaa gctcggaatt aaccctcact aaagggaaca aaagctgggg 12480 gg 12482 <210> 8 <211> 7209 <212> ADN <213> Sequência Artificial <220> <223> Descrição de Sequência Artificial: ADN plasmidico <400> 8 gatccgtcga cagatctccc tatagtgagt cgtattactg cagccaagct aattccgggc 60 gaatttctta tgatttatga tttttattat taaataagtt ataaaaaaaa taagtgtata 120 caaattttaa agtgactctt aggttttaaa acgaaaattc ttgttcttga gtaactcttt 180 cctgtaggtc aggttgcttt ctcaggtata gcatgaggtc gctcttattg accacacctc 240 taccggcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 300 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 360 taatgagtga ggtaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 420 aacctgtcgt gccagctgga ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 480 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 540 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 600 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 660 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 720 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 780 tccctcgtgc gctctcctgt tccgaccctg cogcttacog gatacctgtc cgcctttctc 840 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 900 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 960 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 1020 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 1080 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 1140 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 1200 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 1260 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 1320 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 1380 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 1440 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 1500 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 1560 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 1620 67 ΕΡ 1 197 567/ΡΤ ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 1680 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 1740 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 1800 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 1860 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 1920 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 1980 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 2040 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 2100 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 2160 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 2220 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 2280 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 2340 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 2400 tttccccgaa aagtgccacc tgaacgaagc atctgtgctt cattttgtag aacaaaaatg 2460 caacgcgaga gcgctaattt ttcaaacaaa gaatctgagc tgcattttta cagaacagaa 2520 atgcaacgcg aaagcgctat tttaccaacg aagaatctgt gcttcatttt tgtaaaacaa 2580 aaatgcaacg cgagagcgct aatttttcaa acaaagaatc tgagctgcat ttttacagaa 2640 cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc ttttttgttc 2700 tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat tacttttttt 2760 ctcctttgtg egctctataa tgcagtctct tgataacttt ttgcactgta ggtccgttaa 2820 ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc ctgactccac 2880 ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga taaaggcatc 2940 cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa agtgatagcg 3000 ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg tctctatata 3060 ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat gaatagttct 3120 tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa tgtagaggtc 3180 gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata gggatatagc 3240 acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg tattcgcaat 3300 attttagtag ctcgttacag tccggtgcgt ttttggtttt ttgaaagtgc gtcttcagag 3360 cgcttttggt tttcaaaagc gctctgaagt tcctataott tctagagaat aggaacttcg 3420 gaataggaac ttcaaagcgt ttccgaaaac gagcgcttcc gaaaatgcaa cgcgagctgc 3480 gcacatacag ctcactgttc acgtcgcacc tatatctgcg tgttgcctgt atatatatat 3540 acatgagaag aacggcatag tgcgtgttta tgcttaaatg cgtacttata tgcgtctatt 3600 tatgtaggat gaaaggtagt ctagtacctc ctgtgatatt atcccattcc atgcggggta 3660 tcgtatgctt ccttcagcac taccctttag ctgttctata tgctgccact cctcaattgg 3720 attagtctca tccttcaatg ctatcatttc ctttgatatt ggatcatatt aagaaaccat 3780 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcçcgcg 3840 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 3900 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 3960 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatag 4020 atcaacgaca ttactatata tataatatag gaagcattta atagacagca tcgtaatata 4080 tgtgtacttt gcagttatga cgccagatgg cagtagtgga agatattctt tattgaaaaa 4140 tagcttgtca ccttacgtac aatcttgatc cggagctttt ctttttttgc cgattaagaa 4200 ttaattcggt cgaaaaaaga aaaggagagg gccaagaggg agggcattgg tgactattga 4260 gcacgtgagt atacgtgatt aagcacacaa aggcagcttg gagtatgtct gttattaatt 4320 tcacaggtag ttctggtcca ttggtgaaag tttgcggctt gcagagcaca gaggccgcag 4380 aatgtgctct agattccgat gctgacttgc tgggtattat atgtgtgccc aatagaaaga 4440 gaacaattga cccggttatt gcaaggaaaa tttcaagtct tgtaaaagca tataaaaata 4500 68 ΕΡ 1 197 567/ΡΤ gttcaggcac tccgaaatac ttggttggcg tgtttcgtaa tcaacctaag gaggatgttt 4560 tggctctggt caatgattac ggcattgata tcgtccaact gcatggagat gagtcgtggc 4620 aagaatacca agagttcctc ggtttgccag ttattaaaag actcgtattt ccaaaagact 4680 gcaacatact actcagtgca gcttcacaga aacctcattc gtttattccc ttgtttgatt 4740 cagaagcagg tgggacaggt gaacttttgg attggaactc gatttctgac tgggttggaa 4800 ggcaagagag ccccgaaagc ttacatttta tgttagctgg tggactgacg ccagaaaatg 4860 ttggtgatgc gcttagatta aatggcgtta ttggtgttga tgtaagcgga ggtgtggaga 4920 caaatggtgt aaaagactct aacaaaatag caaatttcgt caaaaatgct aagaaatagg 4980 gteçjtetf^ frtgfirgatrtt- af-gp-fjqf-gfcg 5040_ aaataccgca cagatgcgta aggagaaaat accgcatcag gaaattgtaa acgttaatat 5100 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 5160 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 5220 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac 5280 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 5340 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 5400 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag 5460 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 5520 gccgctacag ggcgcgtcgc gccattcgcc attcaggctg cgcaactgtt. gggaagggcg 5580 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg 5640 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtcg 5700 tccaagcttt cgcgagctcg agatcccgag ctttgcaaat taaagccttc gagcgtccca 5760 aaaccttctc aagcaaggtt ttcagtataa tgttacatgc gtacacgcgt ctgtacagaa 5820 aaaaaagaaa aatttgaaat ataaataacg ttcttaatac taacataact ataaaaaaat 5880 aaatagggac ctagacttca ggttgtctaa ctccttcctt ttcggttaga gcggatgtgg 5940 ggggagggcg tgaatgtaag cgtgacataa ctaattacat gatatccttt tgttgtttcc 6000 gggtgtacaa tatggacttc ctcttttctg gcaaccaaac ccatacatcg ggattcctat 6060 aataccttcg ttggtctccc taacatgtag gtggcggagg ggagatatac aatagaacag 6120 —ataccagaca agacataatg ggctaaaeaa gactacacca attacactgc ct.c.attgatg 6180_ gtggtacata acgaactaat actgtagccc tagacttgat agccatcatc atatcgaagt 6240 ttcactaccc tttttccatt tgccatctat tgaagtaata ataggcgcat gcaacttctt 6300 ttcttttttt ttcttttctc tctcccccgt tgttgtctca ccatatccgc aatgacaaaa 6360 aaaatgatgg aagacactaa aggaaaaaat taacgacaaa gacagcacca acagatgtcg 6420 ttgttccaga gctgatgagg ggtatcttcg aacacacgaa actttttcct tccttcattc 6480 acgcacacta ctctctaatg agcaacggta tacggccttc cttccagtta cttgaatttg 6540 aaataaaaaa agtttgccgc tttgctatca agtataaata gacctgcaat tattaatctt 6600 ttgtttcctc gtcattgttc tcgttccctt tcttccttgt ttctttttct gcacaatatt 6660 tcaagctata ccaagcatac aatcaactcc aagcttgaag caagcctcct gaaagatgaa 6720 gctactgtct tctatcgaac aagcatgcga tatttgccga cttaaaaagc tcaagtgctc 6780 caaagaaaaa ccgaagtgcg ccaagtgtct gaagaacaac tgggagtgtc gctactctcc 6840 caaaaccaaa aggtctccgc tgactagggc acatctgaca gaagtggaat caaggctaga 6900 aagactggaa cagctatttc tactgatttt tcctcgagaa gaccttgaca tgattttgaa 6960 aatggattct ttacaggata taaaagcatt gttaacagga ttatttgtac aagataatgt 7020 gaataaagat gccgtcacag atagattggc ttcagtggag actgatatgc ctctaacatt 7080 gagacagcat agaataagtg cgacatcatc atcggaagag agtagtaaca aaggtcaaag 7140 acagttgact gtatcgccgg aattcttaat acgactcact atagggcata tggccatgga 7200 ggccccggg 7209 <210> 9
<211> 6820 <212> ADN <213> Sequência Artificial <220> <223> Descrição de Sequência Artificial: ADN plasmidico 69 ΕΡ 1 197 567/ΡΤ <400> 9 gatccgtcga cagatctccc tatagtgagt atactgaaaa accccgcaag ttcacttcaa catttataca tcgttttgcc ttcttttatg ccatgtaacc tctgatctat agaatttttt ttggacctaa attcttcatg aaaatatatt ttcgccagag gtttggtcaa gtctccaatc atttacgaaa agatggaaaa gggtcaaatc taagcgaatt tcttatgatt tatgattttt gtatacaaat tttaaagtga ctcttaggtt tctttcctgt aggtcaggtt gctttctcag acctctaccg gcatgcccga aattccccta gtgtcgtttc tattatgaat ttcatttata gaatcttttt aagcaaggat tttcttaact gtactgttgg aaccacctaa atcaccagtt gcatcttcaa tggccttacc ttcttcaggc gcagacaaga tagtggcgat agggtcaacc tggcatggtt cgtacaaacc aaatgcggtg gatggcaaca aacccaaçga acctgggata aacatgttgc tggtgattat aataccattt gcggcagaat caatcaattg atgttgaacc tcctccacag tttttctcca taatcttgaa caaataggca atggtggctc atgttgtagg tgcacttctg gaacggtgta ttgttcacta tttctcttac caaagtaaat acctcccact ttagcaaatt gtggcttgat tggagataag ggtcttaagt tggcgtacaa ttgaagttct ctaacactac ctgtacccca tttaggacca ttcttggagg cttccagcgc ctcatctgga ccaccaatta aatgattttc gaaatcgaac ttaagaacct taatggcttc ggctgtgatt acgatcttct taggggcaga cattagaatg tgaaatgtaa aaggtaagaa aagttagaaa aaacaatagg tccttaaata atattgtcaa atgaacgctt ctctattcta tatgaaaagc cccaattttt cagttgaaaa aggtatatgc ttccagtcat cgaatttgat tctgtgcgat gaaaaaaata atggttgcta agagattcga ccacagttgg ggatctcgac tctagctaga ttcctgtgtg aaattgttat ccgctcacaa agtgtaaagc ctggggtgcc taatgagtga cgtattactg cagagatcta tgaatcgtag 60 ctgtgcatcg tgcaccatct caatttcttt 120 taactatact cctctaagtt tcaatcttgg 180 aaatgactag aattaatgcc catctttttt 240 acgagggctt attoagaagc tttggaottc 300 aaggttgtcg gcttgtctac cttgccagaa 360 gttggtagat acgttgttga cacttctaaa 420 attattaaat aagttataaa aaaaataagt 480 ttaaaacgaa aattfcttgtt cttgagtaac 540 gtatagcatg aggtcgctct tattgaccac 600 ccctatgaac atattccatt ttgtaatttc 660 aagtttatgt acaaatatca taaaaaaaga 720 tcttcggcga cagcatcacc gacttcggtg 780 ctgatacctg catccaaaac ctttttaact 840 aagttcaatg acaatttcaa catcattgca 900 ttattctttg gcaaatctgg agcagaaccg 960 ttcttgtctg gcaaagaggc caaggacgca 1020 acggaggctt catcggagat gatatcacca 1080 aggtgggttg ggttcttaac taggatcatg 1140 ttcaatgtag gaaattcgtt cttgatggtt 1200 gaggccaaaa cattagcttt atccaaggac 1260 gccatgaaag cggccattct tgtgattctt 1320 tcccaagcga caccatcacc atcgtcttcc 1380 aattctctga caacaacgaa gtcagtacct 1440 tctaaaagag agtcggatgc aaagttacat 1500 ttacggattt ttagtaaacc ttgttcaggt 1560 cccacagcac ctaacaaaac ggcatcaacc 1620 agtgggacac ctgtagcatc gatagcagca 1680 ttgacattgg aacgaacatc agaaatagct 1740 tcttgaccaa cgtggtcacc tggcaaaacg 1800 gtatatcctt gaaatatata tatatattgc 1860 gtaagacgat tgctaaccac ctattggaaa 1920 cttcaagtat tgtgatgcaa gcatttagtc 1980 cggttccggc ctctcacctt tcctttttct 2040 gtcaggcgac ctctgaaatt aacaaaaaat 2100 agcgcccctg tgtgttctcg ttatgttgag 2160 actcttgcat cttacgatac ctgagtattc 2220 ggatcaattc gtaatcatgg tcatagctgt 2280 ttccacacaa catacçagcc ggaagcataa 2340 ggtaactcac attaattgcg ttgcgctcac 2400 70 ΕΡ 1 197 567/ΡΤ tgcccgcttt ccagtcggga aacctgtcgt gccagctgga ttaatgaatc ggccaacgcg 2460 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 2520 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacçgttat 2580 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 2640 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 2700 atcacaaaaa tcgacgctca agtcagaggt ggcgaaacco gacaggacta taaagatacc 2760 aggcgtttcc ccctggaago tccctcgtgc gctctcctgt tccgaecctg ccgcttaccg 2820 gatacctgtc cçcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 2880 çgtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 2940 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 3000 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 3060 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 3120 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctottgat 3180 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 3240 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacgggçtct gacgctcagt 3300 ggaacgaaaa ctcacgttaa gggattttgg tcatçagatt atcaaaaagg atcttcacct 3360 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 3420 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 3480 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 3540 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 3600 cagcaataaa coagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 3660 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 3720 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 3780 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 3840 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 3900 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 3960 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 4020 gaccgagttg ctcttgcccg gcgtcaatao gggataatac cgcgccacat agcagaactt 4080 taaaagtgct catcattgga aaaogttctt oggggogaaa aotctcaagg atcttaccgc 4140 tgttgagatc cagttcgatg taacccaotc gtgcacccaa ctgatcttca gcatctttta 4200 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 4260 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 4320 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 4380 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 4440 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt 4500 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 4560 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 4620 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg oaccataacg 4680 catttaagca taaacacgca ctatgccgtt cttctcatgt atatatatat acaggcaaea 4740 cgcagatata ggtgcgacgt gaacagtgag ctgtatgtgc gcagctcgog ttgcattttc 4800 ggaagcgctc çttttcggaa acgctttgaa gttcctattc cgaagttcct attctctagc 4860 tagaaagtat aggaacttca gagcgctttt gaaaaccaaa agcgctctga agacgcactt 4920 tcaaaaaacc aaaaacgcac cggactgtaa cgagctacta aaatattgcg aataccgctt 4980 ccacaaacat tgctcaaaag tatctctttg ctatatatct ctgtgctata tccctatata 5040 acctacccat ccacctttcg ctccttgaac ttgcatctaa actcgacctc tacatttttt 5100 atgtttatct ctagtattac tctttagaca aaaaaattgt agtaagaact attcatagag 5160 tgaatcgaaa acaatacgaa aatgtaaaca tttcctatac gtagtatata gagacaaaat 5220 agaagaaacc gttcataatt ttctgaccaa tgaagaatca tcaacgctat cactttctgt 5280 71 ΕΡ 1 197 567/ΡΤ tcacaaagta tgcgcaatcc acatcggtat agaatataat cggggatgcc tttatcttga 5340 aaaaatgcac ccgcagcttc gctagtaatc agtaaacgcg ggaagtggag tcaggctttt 5400 tttatggaag agaaaataga caccaaagta gccttcttct aaccttaacg gacctacagt 5460 gcaaaaagtt atcaagagac tgcattatag agcgcacaaa ggagaaaaaa agtaatctaa 5520 gatgctttgt tagaaaaata gcgctctcgg gatgcatttt tgtagaacaa aaaagaagta 5580 tagattcttt gttggtaaaa tagcgctctc gcgttgcatt tctgttctgt aaaaatgcag 5640 ctcagattct ttgtttgaaa aattagcgct ctcgcgttgc atttttgttt tacaaaaatg 5700 aagcacagat tcttcgttgg taaaatagcg ctttcgcgtt gcatttctgt tctgtaaaaa 5760 tgcagctcag attctttgtt tgaaaaatta gcgctctcgc gttgcatttt tgttctacaa 5820 aatgaagcac agatgcttcg ttgcttgcat gcaacttctt ttcttttttt ttcttttctc 5880 tctcccccgt tgttgtctca ccatatccgc aatgacaaaa aaaatgatgg aagacactaa 5940 aggaaaaaat taacgacaaa gacagcacca acagatgtcg ttgttccaga gctgatgagg 6000 ggtatcttcg aacacacgaa actttttcct tccttcattc acgcacacta ctctctaatg 6060 agcaacggta tacggccttc cttccagtta cttgaatttg aaataaaaaa agtttgccgc 6120 tttgctatca agtataaata gacctgcaat tattaatctt ttgtttcctc gtcattgttc 6180 tcgttccctt tcttccttgt ttctttttct gcacaatatt tcaagctata ocaagcatac 6240 aatcaactcc aagctttgca aagatggata aagcggaatt aattcccgag cctccaaaaa 6300 agaagagaaa ggtcgaattg ggtaccgccg ccaattttaa tcaaagtggg aatattgctg 6360 atagctcatt gtccttcact ttcactaaca gtagcaacgg tccgaacctc ataacaactc 6420 aaacaaattc tcaagcgctt tcacaaccaa ttgcctcctc taacgttcat gataacttca 6480 tgaataatga aatcacggct agtaaaattg atgatggtaa taattcaaaa ccactgtcac 6540 ctggttggac ggaccaaact gcgtataacg cgtttggaat cactacaggg atgtttaata 6600 ccactacaat ggatgatgta tataactatc tattcgatga tgaagatacc ccaccaaacc 6660 caaaaaaaga gatcgaattc ttaatacgac tcactatagg gcccatggac gaagaatcca 6720 gttcattctt atgtacctat gctgagaatc gtgccaataa gaagccaata cttccttaga 6780 tgatgcaata aatattaáaa taaaacaaaa cagaaggctg 6820
<210> 10 <211> 10597 <212> ADN <213> Sequência Artificial
<220><223> Descrição de Sequência Artificial: ADN <400> 10 ccggtggtac gaaggataag aactgttcca ggctggaact tgctccaaga caagttcatt aaaaggaatc tttgccaaag tggaactgac accaatcgag ggatgacgga aatctgggtt cgggcccccc aatggacgat aatcttaagg ggagatcctt attcatcgtg gttcatattg ggagagagca gcacttcgta agtgtcacta gtaactgatg ggagagccaa ccatgtggac ctcgaggtcg gggaagaagc agggagaaga ctgatccttc aagatctttc atggtgagcc aggctcaaat agcaaagtgg tcaatatcaa tcttcgaaga ttgagttcta gtagtggaga acggtatcga tctcgttgtt atatcaattc tgatcgtgtt tgatacaact agcaccagat tgaaaatgag aaaatatacc ggtaaaaagc tcgtgcaact tgaaattgaa tacccacttc taagctttcg ccaggagatc agaatttctg gttgcgaagc gtcaaggtcg gtaacatggt ccatacatct atcactgcaa aagccaacga cttgactgga aagatgaaca acagtcgatt plasmídico tcattgaaaa 60 agaaaacagc 120 ctcgtaacaa 180 caagaaacct 240 gagccactct 300 cattcaatgg 360 cgagatttgc 420 ccaacattaa 480 aaccaaaggg 540 aaccaccaga 600 ccaaggacgg 660 cactcaacaa 720 72 ΕΡ 1 197 567/ΡΤ gggagatcat tacaagttcc gtgtcaaggc tgtcaacagc gaaggacctt ctgatccatt 780 ggaaactgaa accgatattt tggctaaaaa tccatttgat cgtccagata gaccaggtcg 840 tccagagcca actgattggg attctgatca tgttgatctc aagtgggatc caotagttct 900 agaagcgctg ctaagggggc octogtcgag tcggtcacaa tcacctgaaa ctccaaaggc 960 agccagtgag gaacgtgaag aagaagaaaa agagtcatct gaacaggttt gattttcttt 1020 ctggtcaaaa agatgaaatt attgattttc agccagatac tcccaaaact agcagcgaga 1080 agtctgcaag tcgttcacag tcgcccagag aatcgcggga agtgagccaa gaggtatgtt 1140 tttcaaaaat caataactga tcataatttt tattgtttgg tgaatttaag aaaataatat 1200 tcgaaaattc ctctgaatta tcaagattgc agtattaatt tcgagaaaaa ttgagatatt 1260 catagagcta ttgtaaattt tcttgatttc agactgaaac ttcggaaaat caagagaaaa 1320 tcaaagaaaa ggatgacggg gatgatcagc ctggcacacc gaacagctat agaagccggg 1380 aaacttcacc agctccaaaa aggtccaagg agaccaggtt tgtcaaaagc ttcctgcgat 1440 taattctcat ttcaattttt cagagaatca gagtctcctg aaaaatcccc ggttcgttca 1500 agatctccca gaaggtcttc agcacgttcc ccgtcacgat ctcctagacg gcgccgagaa 1560 agaagctcag aaagaaagca atccgaagag ccagcaccgc taccagagaa aaagaagaaa 1620 gagccgctgg atattctacg aacaagaacc ggaggagcat atattccacc cgccaaactt 1680 cgacttatgc aacaacagat tagtgataag caaagtgaac agtatcagag aatgaattgg 1740 gaaagaatga agaaaaagat tcacggattg gttaacagag tcaacgcgaa çaatcttgtt 1800 _caaattgtca aaaaar-t.t.nt·. fnaaaaaaat ahaattcatt caaaataaat aaaaaaatca 1860 aaggaaaagg aaagaattaa tttaattttt caggggactt ctctgccgtg acattattca 1920 agctcaggct ttctcaccag gattctctaa cgtctatgca gctttggcgg cagttatcaa 1980 ctcgaaattc cctcatgtcg gtgaacttct tctccgtcgt ctgattgtac agttcaaaag 2040 aagtttccgt agaaatgaca gaggcgtcac ggtgaacgtg atcaaattca tcgcacattt 2100 gattaatcaa eaagttgete acgaagttct tgcgctggaa atcatgattc tgatgcttga 2160 agaaccaact gatgattcag ttgaagtcgc cattgcgtto ctgaaagagt gtggagcaaa 2220 gcttctggag attgctccag cagctcttaa cagtgtctac gaccgtcttc gtgcaattct 2280 catggaaaot gaaagatcgg aaaatgcact ggatcgacgt attcagtata tgattgagac 2340 tgcaatgcag attcgaaagg acaaatttgc ggtaaggtag aatatataaa tagtttatta 2400 gaaaaaaata aattagaata atttaaattc ctactagcca atcaggcçac ctttttgcgc 2460 atagttctat tattgaaaaa tttggagaat ttctcatatt ctcgctcgga aatctggaat 2520 tcgacgagat cttctggctt otgtgcagct gcatcgcttt gtgctccctt tctcgcttgt 2580 cttotgtgta caccaagaac cttgttgagt tcatcaactg aatctgtgac tggcttgttg 2640 ctcactggat gcactagacg actgattctc gagaaatcag attgagttgc gattagggtg 2700 acctagaaat tgggaataat acgaactttt gaaaatattc aggaggatta aaaaaattat 2760 tctcgacaat cctacaaatt tacttattgc accatgttgc tccaacattt ttcattaaaa 2820 gttaatgaaa aaatgtagaa aateggaaat tggcaatttt cagaccattt ttaagcattt 2880 tcaaaaaaaa attgcagctg aaataaatgt cattttcaga taaatcgagc gattttctgt 2940 tgtctgacac tagtttttag ttttaaaaaa tgttggaaga acatggtgca ataggtaatt 3000 tcataaaatt tccatatatt ttttttoaat taaccaatta tccaaatctt ccaaactcac 3060 attttgcgga gctgggctat caagaatctg ctgcagtttt ataagacçag catctctgat 3120 atoactgaaa attaattttt aatcaaaact tgaatatcaa ctaaacccac ttattaactt 3180 tctcgatctt ctgtcgttcg gtacgatgac ggtgaagaag ccaattgtag tagttgattt 3240 ggttcaagtc ctttcggtgt tgtacgtcag tgtcctgcaa tgctatttag ttataactta 3300 ggcctaagat tcaatttaat gaagtgatta aatttgttct ctgaacctct taagatgatc 3360 ttttggatta gaaacatata agacaggttt acctatctat taaaaaacag atcaaaatag 3420 atacgaccaa atcggataat ccatgcctac ctggcatcta ggaacgtgtt cttagaagat 3480 ttcttacgta atcgtatgaa gaaataacaa tttgatcgtt ggccagcaaa aatagggttt 3540 taagtgggat agtgttttta ttagctaacc ggaaaatttt atagtttttt tttgcaagaa 3600 73 ΕΡ 1 197 567/ΡΤ accactgaaa accccctaat tgtatacatt ttttggagca gcttctggtc tttttgagca 3660 ataaaattcg ataaaacaga atttaagtgt aaattgttca catttagttt ctattttatc 3720 aaattttgtt gctcaaaaac attcgaagct gctctaaaaa aatgcattaa aaaaggggtt 3780 ttcagtggtt tttcacatta aaaaagctaa ttttaactaa aaatccatca tatttccaac 3840 tttgtcacaa caataaaatg ctggtcaaaa tgtgttcgaa aaaatgtttt tttttttaat 3900 ttttataatt taaaaatagt tttctttcgc tgggacacat acatttttgg gcgtaaattt 3960 tcagttcaaa tttccatttt tacaaccata atcataaagc tacgtctgat ctctctcgca 4020 cttacctgcg cctgattcga aagaacaacc gtagccaaaa gaacaagaag aacaagcacg 4080 tagttgtggt agtggacgtt catcacgcaa tactgaccaa tggtcgtggg gtctcacttt 4140 ccgtactatt gagagagggg agactgaaga tggcaattga ggacagtgtc ttcgacgcac 4200 gcatgcatcc ataagcataa tccaggaggg atggagagaa aaatcttgtt tctaagcccc 4260 tccctttgta atacatacac atatctaata ccgaagaatg gctaattgaa tggacgtcag 4320 ctgttgctgt agttgccaag gcatcatcga tgaaataact gaaagaaaga attaaataat 4380 tattgcaggc gtatccggcg gtcattgaag acttggactt gattgaggag gaggatcaga 4440 tcatccatac acttaatttg gãggatgcgg ttgatccgga aaatgggctt agtaagtgac 4500 tgaccacacg cggggggcat taatttaata aattgaattc catttcagat gtgttcaaac 4560 tagatccaga attcgaaaag aacgaggagg tttatgagga gatccgtaag gaaatcattg 4620 gaaacgccga tatttcggat gaggatggtg gcgacgagtt ggatgatgaa gaagagggta 4680 qtqatqtqqa aqaqqctccq aaqaaqacta caqaqattat tgataatact gatcagaatt 4740 gactgctttc agaaggtatt cattttçagt tttgggccgg caaatctgta agttgccggt 4800 tgccgaaaat ttgctgaatt tgccgçaaaa aaaaattccg gaatttattt aaaaactttt 4860 tgtaaaaatt aaattaaatt tgcaactttt cagagaagtc tacctgacaa tgcaatcatc 4920 tttggactac caagaagctg ctcacaaatt gctgaaaatg aagattccag acagcatgca 4980 ggtcagcgat gttgcaaaga aaaattttcg accaaaaaaa ccaaccaatc ataaaattta 5040 aaaaaaaact ccgttttttt cttttttttt atacgagaaa aaccaaaaaa atgtattttt 5100 gccaaattct aaaatactat ccccgaaatt ttcaatattt tctctttcag aacgaactct 5160 gcgcgatgct tgtcgattgt tgtgctcaac agcgtaocta cgagcgattc tacggaatgc 5220 tcatcgaacç tttctgocga cttcgcctcg aataccagca atactttgaa aagctctgcc 5280 aggacacgta ttccacgatt caccgaattg acatcacaaa actgcggaat ttggctcgcc 5340 ttattgctca tttgctctcg acggatgcta ttgactggaa gattttggcc gatatgaaaa 5400 tgaccgaaga ggacacaact tcttctggca gaatctatat taaatatata tttaatgaac 54 60 ttgtggaggc gatgggaatg gttaaacttc attcgagagt tactgatccg tgagtttcct 5520 agagagagtt gttttcgtat tcaattttcc ctattttcag aactttggct cattgctttg 5580 ttggattatt cccacgaact aatccgaaca gcgcacgatt ttcgatcaac ttcttcacaa 5640 tgattggatt gggtggtttg acgttggaac ttcgtgaatg gctggcaaag ggtctcaaga 5700 agaagaaggg aatgctggat cagttgaagg ccgaatcaag ctcagattca tcgtcgtctt 5760 cggattcgtc agactcgtct gattcttcgg attctgacga ttcatccgac tcgtcttcag 5820 attcctcatc ttcttcagaa tcagagccag aaccaccgaa gaaaaagaag aagaagaaca 5880 gtgaagagag ttccaaaaag aaggaaaaag agaatattgg tcgacgggat cgtggagaca 5940 agagagctga acgtcatcgt gatcaaagtg tggagaacaa ggacaaggat cgtcgacgtc 6000 gccaggattc tgacgaaaat cgtcggccag aacgaggaga tgaccgcaag gatcggagta 6060 aagatcgtcg tcgtcaagac tcggatgatg aggatcggaa aggtcgtgaa cgtcgggaag 6120 attcagggga aagacgtcgc ggagatcggg atcgacgtga tcgaaacaag gatcaggagg 6180 atcaccgtga agatcgccgt gaccgaagca aggatcgtga ggatcgacgt gatcgccgtc 6240 gtcatgactc tgatgatgat cçtaaaactc gtcgggatag aagtgaagag cgaggaggac 6300 gtcgtcgtga agtggaatcg gatgatcgac gccgacgtcg ttgaattttc aaattttaaa 6360 tactgaatat ttçttttttt tcctattatt tatttattct ctttgtgttt tttttcttgc 6420 tttctaaaaa attaattcaa tccaaatcta aacatgagcg gttttttttc tctttccgtc 6480 74 ΕΡ 1 197 567/ΡΤ tcccaattcg tattccgctc ctctcatctg aacacaatgt gcaagtttat ttatcttctc 6540 gctttcattt cattaggacg tggggggaat tggtggaagg gggaaacaca caaaaggatg 6600 atggaaatga aataaggaca cacaatatgc aacaacattc aattcagaaa tatggaggaa 6660 ggtttaaaag aaaacataaa aatatataga ggaggaagga aaactagtaa aaaataagca 6720 aagaaattag gcgaacgatg agaattgtcc tcgcttggca aatgcgaatc cgtatggaga 6780 ggcacgtttg gcgaaggcaa atgttcggta tggagatctg taaaaatttt taagttgaaa 6840 tttggtgttg ctcttttaca aaattttccg attttcgctt gaaattacgg tgccaggtct 6900 cgacaogtct tccaattttt caaattcaaa agagccttta atgggctgta gttgctaatt 6960 tctcgttttt gaaaattttt ettccgttta atcgaaattt gatgtatttt atttatgatt 7020 ttcaataaat ttcaaagaaa ctggtgaaaa ctcggaaaat tgtgaactac agtaatccaa 7080 tccttaaagg cgcacacctt ttaaatgtcc gccccaatac gatatttttt taagattcgc 7140 tagagcggcc gccaccgcgg tggagctcca attcgcccta tagtgagtcg tattacaatt 7200 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 7260 gccttgcagc acatcccccc ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 7320 gcccttccca acagttgcgt agcctgaatg çcçaatggga cgcgccctgt agcggcgcat 7380 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 7440 cgcccgctcc tttcgcttto ttcccttcct ttctcgccac gttcgccggc tttccccgtc 7500 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 7560 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggccc “7620- ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 7680 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 7740 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 7800 taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 7860 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 7920 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 7980 cettttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 8040 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 8100 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 8160 ttctgctatg tggcgcçgta ttatcocgta ttgacgccgg gcaagagcaa ctcggtcgcc 8220 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 8280 cggatggcat gacagtaaga gaattatgca gtgctgccat aagcatgagt gataacactg 8340 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttteaca 8400 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 8460 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 8520 taactggcga actacttact ctagcttccc ggoaaoaatt aatagactgg atggaggcgg 8580 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 8640 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 8700 agccctcccg tatcgtagtt atctacacga cgggcagtca ggcaactatg gatgaacgaa 8760 r tttactcata tataotttag attçatttaa aacttcattt ttaatttaaa aggatctagg 8880 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 8940 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 9000 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtçgtttgt ttgccggatc 9060 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 9120 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 9180 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtegtgtc 9240 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 9300 ggggttcgtg cacacagccc agcttgçagc gaacgaccta caccgaactg agatacctac 9360 75
ΕΡ 1 197 567/PT agcgtgagca ttgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 9420 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccagggggg aacgcctggt 9480 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 9540 cgtcaggggg gccgagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 9600 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 9660 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 9720 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc 9780 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg 9840 agcgcaacgc aattaatgtg agttacctca ctcattaggc accccaggct ttacacttta 9900 tgcttccggc tcctatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca 9960 gctatgacca tgattacgcc aagctcggaa ttaaccctca ctaaagggaa caaaagctgg 10020 gggggatcct ccaaaatcgt cttccgctct gaaaaacgaa agtggacctt tgacatccga 10080 aaaaatgggc gaaaaaatga aattgagctt tttgggtcga aaaaaatgtt tttagaatgc 10140 tgagaacacg ttaaacacga agatcatatt tattttgaga cccggatgct ctgaaaatgt 10200 ctgacataga tttaaaaaag catatatata tttttcattt tcaacgtgaa agttttgtgc 10260 aactttatag aatctcctat tggcacattg ttttttattt aactgaggca gtttttgaac 10320 acctttttga aactttgaat ctctttgaag tatactgtcg aaaagactga cttgagcgtt 10380 cgaaatgcca gaagaaaact atatttgaat ctcgcgctaa attgagaaat gcaaccgcgc 10440 tccactggac aattggaaaa aaaatttatt cggaggcgac aacggtattt tcgaaattga 10500 ttttctgtgt attttctcat tttttataaa ttcttctttg atttatcgtt cgtttgtgag 10560 aaatttaatt gtattcaaac ttttttatag taagata 10597 <210> 11 <211> 10599 <212> ADN <213> Sequência Artificial <220> <223> Descrição de Sequência Artificial: ADN plasmidico <400> 11 ccggtggtac cgctagccgt acgaacccgg gttctagaac tagtggatcc cacttgagat 60 caacatgatc agaatcccaa tcagttggct ctggacgacc tggtctatct ggacgatcaa 120 atggattttt agccaaaata tcggttteag tttccaatgg atcagaaggt ccttcgctgt 180 tgacagcctt gacacggaac ttgtaatgat ctcccttgtt gagtgaatcg actgtgaagt 240 gggtatctcc actacgtcca catggaaccc agattccgtc cttggtgttc atcttttcaa 300 tttcatagaa ctcaattggc tctcctccgt catcctctgg tggtttccag tcaagagttg 360 cacgatcttc gaagacatca gttacctcga ttggtccctt tggtttcgtt ggcttgcttt 420 ttaccttgat attgatagtg acactgtcag ttccattaat gttggttgca gtgatggtat 480 attttccact ttgcttacga agtgcctttg gcaaagcaaa tctcgagatg tatggctcat 540 tttcaatttg agccttgctc tctccgattc cttttccatt gaatgaccat gttacatctg 600 gtgctggctc accatcaata tgaacaatga acttgagagt ggctccgacc ttgacagttg 660 tatcagaaag atcttcacga tgaattcttg gagcaaggtt tcttggcttc gcaacaacac 720 gatcagaagg atcagaagga tctocagttc cagccttgtt acgagcagaa attctgaatt 780 gatattcttc tccctcctta agatttggaa cagttgctgt tttctgatct cctggaacaa 840 cgagagcttc ttcccatcgt ccattcttat ccttcttttc aatgacgaaa gcttatcgat 900 accgtcgacc tcgagggggg gccctcgtcg agtcggtcac aatcacctga aactccaaag 960 gcagccagtg aggaacgtga agaagaagaa aaagagtcat ctgaacaggt ttgattttct 1020 76 ΕΡ 1 197 567/ΡΤ ttctggtcaa aaagatgaaa ttattgattt tcagccagat actcccaaaa ctagcagcga 1080 gaagtctgca agtcgttcac agtcgcccag agaatcgcgg gaagtgagcc aagaggtatg 1140 tttttcaaaa atcaataact gatcataatt tttattgttt ggtgaattta agaaaataat 1200 attcgaaaat tcctctgaat tatcaagatt gcagtattaa tttcgagaaa aattgagata 1260 ttcatagagc tattgtaaat tttcttgatt tcagactgaa acttcggaaa atcaagagaa 1320 aatcaaagaa aaggatgacg gggatgatca gcctggcaca ccgaacagct atagaagccg 1380 ggaaacttca ccagctccaa aaaggtccaa ggagaccagg tttgtcaaaa gcttcctgcg 1440 attaattctc atttcaattt ttcagagaat cagagtctcc tgaaaaatcc ccggttcgtt 1500 caagatctcc cagaaggtct tcagcacgtt ccccgtcacg atctcctaga cggcgccgag 1560 aaagaagctc agaaagaaag caatccgaag agccagcacc gctaccagag aaaaagaaga 1620 aagagccgct ggatattcta cgaacaagaa ccggaggagc atatattcca cccgccaaac 1680 ttcgacttat gcaacaacag attagtgata agcaaagtga acagtatcag agaatgaatt 1740 gggaaagaat gaagaaaaag attcacggat tggttaacag agtcaacgcg aagaatcttg 1800 ttcaaattgt cagagaactt cttcaagaga atgtgattcg ttcaaagtga gtgagaaaat 1860 cgaaggaaaa ggaaagaatt aatttaattt ttcaggggac ttctctgccg tgacattatt 1920 caagctcagg ctttctcacc aggattctct aacgtctatg cagctttggc ggcagttatc 1980 aactcgaaat tccctcatgt cggtgaactt cttctccgtc gtctgattgt acagttcaaa 2040 agaagtttcc gtagaaatga cagaçgcgtc acggtgaacg tgatcaaatt catcgcacat 2100 ttgattaatc aacaagttgc tcacgaagtt cttgcgctgg aaatcatgat tctgatgctt 2160 gaagaaccaa ctgatgattc agttgaagtc gccattgcgt tcctgaaaga gtgtggagca 2220 aagcttctgg agattgctcc agcagctctt aacagtgtct acgaccgtct tcgtgcaatt 2280 ctcatggaaa ctgaaagatc ggaaaatgca ctggatcgac gtattcagta tatgattgag 2340 actgcaatgc agattcgaaa ggacaaattt gcggtaaggt açaatatata aatagtttat 2400 tagaaaaaaa taaattagaa taatttaaat tcctactagc caatcaggcg acctttttgc 2460 gcatagttct attattgaaa aatttggaga atttctcata ttctcgctcg gaaatctgga 2520 attcgacgag atcttctggc ttctgtgcag ctgcatcgct ttgtgctccc tttctcgctt 2580 gtcttctgtg tacaccaaga accttgttga gttcatcaac tgaatctgtg actggcttgt 2640 tgctcactgg atgcactaga cgactgattc tcgagaaatc agattgagtt gcgattaggg 2700 tgacctagaa attggçaata atacçaactt ttgaaaatat tcaggaggat taaaaaaatt 2760 attctcgaca atcctacaaa tttacttatt gcaccatgtt gctccaacat ttttcattaa 2820 aagttaatga aaaaatgtag aaaatcggaa attggcaatt ttcagaecat ttttaagcat 2880 tttcaaaaaa aaattgcagc tgaaataaat gtcattttca gataaatcga gcgattttct 2940 gttgtctgac actagttttt agttttaaaa aatgttggaa gaacatggtg caataggtaa 3000 tttcatagaa tttccatgtg ttttttttca attaaccaat tatccaaatc ttccaaactc 3060 acattttgcg gagctgggct atcaagaatc tgctgcagtt ttataagacg agcatctctg 3120 atatcactga aaattaattt ttaatcaaaa cttgaatatc aactaaaccc acttattaac 3180 tttctcgatc ttctgtcgtt cggtacgatg acggtgaaga agccaattgt agtagttgat 3240 ttgçttcaag tcctttcggt gttgtacgtc agtgtcctgc aatgctattt agttataact 3300 taggcctaag attcaattta atgaagtgat taaatttgtt ctctgaacct cttaagatga 3360 tcttttggat tagaaacata taagacaggt ttacctatct attaaaaaac agatcaaaat 3420 agatacgacc aaatcggata atccatgcct acctggcatc taggaaegtg ttettagaag 3480 atttcttacg taatcgtatg aagaaataac aatttgatcg ttggccagca aaaatagggt 3540 tttaagtggg atagtgtttt tattagctaa ccggaaaatt ttatagtttt tttttgcaag 3600 aaaccactga aaacccccta attgtataca ttttttggag cagcttctgg tctttttgag 3660 caataaaatt cgataaaaca gaatttaagt gtaaattgtt cacatttagt ttctatttta 3720 tcaaattttg ttgctcaaaa acattcgaag ctgctctaaa aaaatgcatt aaaaaagggg 3780 ttttcagtgg tttttcacat taaaaaagct aattttaact aaaaatccat catatttcca 3840 actttgtcac aacaataaaa tgctggtcaa aatgtgttcg aaaaaatgtt ttttttttta 3900 77 ΕΡ 1 197 567/ΡΤ atttttataa tttaaaaata gttttctttc gctgggacac atacattttt gggcgtaaat 3960 tttcagttca aatttccatt tttacaacca taatcataaa gctacgtctg atctctctcg 4020 cacttacctg cgcctgattc qaaagaacaa ccgtagccaa aagaacaaça agaacaagca 4080 cgtagttgtg gtagtggacg ttcatcacgc aatactgacc aatggtcgtg gggtctcact 4140 ttccgtaota ttgagagagg ggagactgaa çatggcaatt gaggacagtg tottcgacgc 4200 acgcatgoat ccataagcat aatccaggag ggatggagag aaaaatcttg tttctaagco 4260 cctccctttg taatacatac acatatctaa taccgaagaa tggctaattg aatggacgtc 4320 agctgttgct gtagttgcca aggcatcatc gatgaaataa ctgaaagaaa gaattaaata 4380 attattgcag gcgtatccgg cggtcattga agacttggac ttgattgagg aggaggatca 4440 gatcatccat acacttaatt tggaggatgc ggttgatccg gaaaatggçc ttagtaagtg 4500 actgaccaca cgcggggggc attaatttaa taaattgaat tccatttcag atçtgttcaa 4560 actagatcca gaattcgaaa agaacgagga ggtttatgag gagatccgta agçaaatcat 4 620 tggaaacgcc gatatttcgg atgaggatgg tggcgacgag ttggatçatg aagaagaggg 4680 tagtgatgtg gaagaggctc cgaagaagac tacagagatt attgataata ctgatcagaa 4740 ttgactgctt tcagaaggta ttcattttga gttttgggcc ggcaaatctg taagttgccg 4800 çttgccgaaa atttgctgaa tttgccggaa aaaaaaattc cggaatttat ttaaaaactt 4860 tttgtaaaaa ttaaattaaa tttgcaactt ttcagagaag tctacctgac aatgcaatca 4920 tctttggact accaagaagc tgctcacaaa ttgctgaaaa tgaagattcc agacagcatg 4980 caggtcagcg atgttgcaaa gaaaaatttt cgaccaaaaa aaccaaccaa tcataaaatt 5040 taaaaaaaaa ctccgttttt ttcttttttt ttatacgaga aaaaccaaaa aaatgtattt 5100 ttgccaaatt ctaaaatact atccccgaaa ttttcaatat tttctctttc agaacgaact 5160 ctgcgcgatg cttgtcgatt gttgtçctca acagcgtacc tacgagcgat tctacggaat 5220 gctcatcgaa cgtttctgcc gacttcgcct cgaataccag caatactttg aaaagctctg 5280 ccaggacacg tattccacga ttcaccgaat tgacatcaca aaactgcgga atttggctcg 5340 ccttattgct catttgctct cgacggatgc tattgactgg aagattttgg ccgatatgaa 5400 aatgaccgaa gaggacacaa cttcttctgg cagaatctat attaaatata tatttaatga 5460 acttgtggag gcgatgggaa tggttaaact tcattcgaga gttactgatc cgtgagtttc 5520 ctagagagag ttgttttcgt attcaatttt ccctattttc agaactttgg ctcattgctt 5580 tgttggatta ttcccacgaa ctaatccgaa cagcgcacga ttttcgatca acttcttcac 5640 aatgattgga ttgggtggtt tgacgttgga acttcgtgaa tggctggcaa agggtctcaa 5700 gaagaagaag ggaatgctgg atcagttgaa ggccgaatca agctcagatt catcgtcgtc 5760 ttcggattcg tcagactcgt ctgattcttc ggattctgac gattcatccg actcgtcttc 5820 agattcctca tcttcttcag aatcagagcc agaaccaccg aagaaaaaga agaagaagaa 5880 cagtgaagag agttccaaaa agaaggaaaa agagaatatt ggtcgacggg atcgtggaga 5940 caagagagct gaacgtcatc gtgatcaaag tgtggagaac aaggacaagg atcgtcgaog 6000 tcgccaggat tctgacgaaa atcgtcggcc agaacgagga gatgaccgca aggatcggag 6060 taaagatcgt cgtcgtcaag actcggatga tgaggatcgg aaaggtcgtg aacgtcggga 6120 agattcaggg gaaagacgtc gcggagatog gçatcgacgt gatcgaaaca aggatcagga 6180 ggateaccgt gaagatcgcc gtgaccgaag caaggatcgt gaggatcgac gtgatcgccg 6240 tcgtcatgac tctgatgatg atogtaaaao tcgtcgggat agaagtgaag agcgaggagg 6300 acgtcgtcgt gaagtggaat cggatgatcg acgccgacgt cgttgaattt tcaaatttta 6360 aatactgaat atttgttttt tttcctatta tttatttatt ctctttgtgt tttttttctt 6420 gctttctaaa aaattaattc aatccaaatc taaacatgag cggttttttt tctctttccg 6480 tctcccaatt cgtattccgc tcctctcatc tgaacacaat gtgcaagttt atttatcttc 654 0 tcgctttcat ttcattagga cgtgggggga attggtggaa gggggaaaca cacaaaagga 6600 tgatggaaat gaaataagga cacacaatat gcaacaacat tcaattcaga aatatggagg 6660 aaggtttaaa agaaaacata aaaatatata gaggaggaag gaaaactagt aaaaaataag 6720 caaagaaatt aggcgaacga tgagaattgt cctcgcttgg caaatgcgaa tccgtatgga 6780 78 ΕΡ 1 197 567/ΡΤ gaggcacgtt tggcgaaggc aaatgttcgg tatggagatc tgtaaaaatt tttaagttga 6840 aatttggtgt tgctctttta caaaattttc cgattttcgc ttgaaattac ggtgccaggt 6900 ctcgacacgt cttccaattt ttcaaattca aaagagcctt taatgggctg tagttgctaa 6960 tttctcgttt ttgaaaattt ttcttccgtt taatcgaaat ttgatgtatt ttatttatga 7020 ttttcaataa atttcaaaga aactggtgaa aactcggaaa attgtgaact acagtaatcc 7080 aatccttaaa ggcgcacacc ttttaaatgt ccgccccaat acgatatttt tttaagattc 7140 gctagagcgg ccgccaccgc ggtggagctc caattcgccc tatagtgagt ogtattacaa 7200 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa 7260 tcgccttgca gcacatcccc ccttcgccag ctggcgtaat agcgaagaçg cccgcaccga 7320 togcoottcc oaacagttgc gtagcctgaa tggcgaatgg gacgcgccct gtagcggcgc 7380 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 7440 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 7500 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 7560 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 7620 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 7680 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 7740 ggcctattgg ttaaaaaatg agotgattta acaaaaattt aaggcgaatt ttaacaaaat 7800 attaacgttt acaatttcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 7860 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 7920 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 7980 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaaçt 8040 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 8100 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 8160 agttctgcta tgtggogogg tattatcccg tattgacgcc gggcaaçagc aactcggtcg 8220 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 8280 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataagcatga gtgataacac 8340 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg ctttttttca 8400 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 8460 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact 8520 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 8580 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 8640 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 8700 taagccctcc cgtatcgtag ttatctacac gacgggcagt caggcaacta tggatgaacg 8760 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 8820 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 8880 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 8940 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 9000 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 9060 tcaagagcta ccaacfccttt ttccgaaggt aaetggettc agcagagcgc agataccaaa 9120 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagoaccgcc 9180 tacatacctc gctctgctaa tcctgttacc agtggctgct gocagtggcg ataagtcgtg 9240 tcttaccggg ttggactcaa gacgatagtt accggataag gcçcagcggt cgggctgaac 9300 ggggggttcg tgcacacagc ccagcttçga gcgaacgacc tacaccgaac tgagatacct 9360 acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 9420 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg ggaacgcctg 9480 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 9540 ctcgtcaggg gggccgagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 9600 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 9660 79 ΕΡ 1 197 567/ΡΤ taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 9720 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc 9780 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 9840 tgagcgcaac gcaattaatg tgagttacct cactcattag gcaccccagg ctttacactt 9900 tatgcttccg gctcctatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 9960 cagctatgac catgattacg ccaagctcgg aattaaccct cactaaaggg aacaaaagct 10020 gggggggatc ctccaaaatc gtcttccgct ctgaaaaacg aaagtggacc tttgacatcc 10080 gaaaaaatgg gcgaaaaaat gaaattgagc tttttgggtc gaaaaaaatg tttttagaat 10140 gctgagaaca cgttaaacac gaagatcata tttattttga gacccggatg ctctgaaaat 10200 gtctgacata gatttaaaaa agcatatata tatttttcat tttcaacgtg aaagttttgt 10260 gcaactttat agaatctcct attggcacat tgttttttat ttaactgagg cagtttttga 10320 acaccttttt gaaactttga atctctttga agtatactgt cgaaaagact gacttgagcg 10380 ttcgaaatgc cagaagaaaa ctatatttga atctcgcgct aaattgagaa atgcaaccgc 10440 gctccactgg acaattggaa aaaaaattta ttcggaggcg acaacggtat tttcgaaatt 10500 gattttctgt gtattttctc attttttata aattcttctt tgatttatcg ttcgtttgtg 10560 agaaatttaa ttgtattcaa acttttttat agtaagata 10599
Lisboa, 2009-03-30

Claims (14)

  1. ΕΡ 1 197 567/ΡΤ 1/2 REIVINDICAÇÕES 1. Método de introdução de ARNcd, ou ADN capaz de produzir ARNcd, num organismo não humano, método este que compreende a alimentação do referido organismo com um microorganismo adequado compreendendo um vector de expressão compreendendo um promotor ou promotores orientados relativamente a uma sequência de ADN tal que sejam capazes de iniciar a transcrição da referida sequência de ADN em ARN de cadeia dupla após ligação de um factor de transcrição apropriado aos referidos promotor ou promotores ou a alimentação do referido organismo directamente com este vector de expressão.
  2. 2. Método de acordo com a reivindicação 1 em que o vector de expressão compreende dois promotores idênticos flanqueando a sequência de ADN.
  3. 3. Método de acordo com a reivindicação 1 em que o vector de expressão compreende a sequência de ADN numa orientação com sentido e anti-sentido relativamente ao referido promotor.
  4. 4. Método de acordo com qualquer uma das reivindicações 1 a 3 em que o vector de expressão compreende adicionalmente uma sequência de nucleótidos que codifica um marcador seleccionável.
  5. 5. Método de acordo com a reivindicação 4 em que a referida sequência de nucleótidos que codifica o referido marcador seleccionável está orientada relativamente ao(s) promotor(es) de modo a que ocorra a transcrição da sequência de nucleótidos em ARN de cadeia dupla após ligação de um factor de transcrição apropriado ao(s) referido(s) promotor(es).
  6. 6. Método de acordo com a reivindicação 5 em que a referida sequência de nucleótidos que codifica o marcador seleccionável é proporcionada entre os promotores idênticos capazes de iniciar a transcrição a sequência de nucleótidos em ARNcd após ligação do factor de transcrição aos promotores.
  7. 7. Método de acordo com a reivindicação 5 em que a referida sequência de nucleótidos que codifica o marcador seleccionável é proporcionada numa orientação com sentido e ΕΡ 1 197 567/ΡΤ 2/2 anti-sentido relativamente ao promotor de modo a que ocorra a transcrição da sequência de nucleótidos em ARNcd após ligação do factor de transcrição ao referido promotor.
  8. 8. Método de acordo com a reivindicação 4 ou 5 em que o referido marcador seleccionável compreende uma sequência de nucleótidos que codifica sup-35, para introdução em C. elegans possuindo uma mutação pha-1.
  9. 9. Método de acordo com qualquer uma das reivindicações 1 a 8 em que o referido microorganismo ou o referido organismo estão adaptados para expressar o referido factor de transcrição.
  10. 10. Método de acordo com a reivindicação 9 em que ou o referido microorganismo ou o referido organismo compreendem um vector de expressão compreendendo uma sequência de nucleótidos que codifica o referido factor de transcrição operativamente ligado a sequências de controlo da expressão adequadas.
  11. 11. Método de acordo com qualquer uma das reivindicações 1 a 10 em que o referido organismo é C. elegans e o referido microorganismo é E. coli.
  12. 12. Método de acordo com a reivindicação 11 em que a referida estirpe de E. coli é uma estirpe negativa para ARNase III.
  13. 13. Método de acordo com qualquer uma das reivindicações 1 a 10 em que o referido organismo é um mutante nuc-1 de C. elegans.
  14. 14. Método de acordo com qualquer uma das reivindicações 1 a 13 em que o referido factor de transcrição é ARN-polimerase de T7, T3 ou SP6. Lisboa, 2009-03-30
PT01129274T 1998-07-03 1999-07-02 Caracterização da função de genes utilizando inibição de arn de cadeia dupla PT1197567E (pt)

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GBGB9827152.1A GB9827152D0 (en) 1998-07-03 1998-12-09 Characterisation of gene function using double stranded rna inhibition

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Families Citing this family (255)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
AUPP249298A0 (en) * 1998-03-20 1998-04-23 Ag-Gene Australia Limited Synthetic genes and genetic constructs comprising same I
CA2513336A1 (en) 1998-03-20 1999-09-30 Benitec Australia Ltd. Control of gene expression in a non-human eukaryotic cell, tissue or organ
GB9827152D0 (en) * 1998-07-03 1999-02-03 Devgen Nv Characterisation of gene function using double stranded rna inhibition
AU776150B2 (en) * 1999-01-28 2004-08-26 Medical College Of Georgia Research Institute, Inc. Composition and method for (in vivo) and (in vitro) attenuation of gene expression using double stranded RNA
DE19956568A1 (de) 1999-01-30 2000-08-17 Roland Kreutzer Verfahren und Medikament zur Hemmung der Expression eines vorgegebenen Gens
US7601494B2 (en) 1999-03-17 2009-10-13 The University Of North Carolina At Chapel Hill Method of screening candidate compounds for susceptibility to biliary excretion
US20040138168A1 (en) * 1999-04-21 2004-07-15 Wyeth Methods and compositions for inhibiting the function of polynucleotide sequences
CN1375004A (zh) * 1999-04-21 2002-10-16 惠氏公司 抑制多核苷酸序列的功能的方法和组合物
US6924109B2 (en) * 1999-07-30 2005-08-02 Agy Therapeutics, Inc. High-throughput transcriptome and functional validation analysis
US6423885B1 (en) 1999-08-13 2002-07-23 Commonwealth Scientific And Industrial Research Organization (Csiro) Methods for obtaining modified phenotypes in plant cells
WO2001029058A1 (en) 1999-10-15 2001-04-26 University Of Massachusetts Rna interference pathway genes as tools for targeted genetic interference
GB9927444D0 (en) * 1999-11-19 2000-01-19 Cancer Res Campaign Tech Inhibiting gene expression
US7829693B2 (en) 1999-11-24 2010-11-09 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a target gene
DE10100586C1 (de) 2001-01-09 2002-04-11 Ribopharma Ag Verfahren zur Hemmung der Expression eines Ziegens
GB9930691D0 (en) * 1999-12-24 2000-02-16 Devgen Nv Improvements relating to double-stranded RNA inhibition
US8202979B2 (en) 2002-02-20 2012-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid
US7491805B2 (en) 2001-05-18 2009-02-17 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
US8273866B2 (en) 2002-02-20 2012-09-25 Merck Sharp & Dohme Corp. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
US7833992B2 (en) 2001-05-18 2010-11-16 Merck Sharpe & Dohme Conjugates and compositions for cellular delivery
US8202846B2 (en) 2000-03-16 2012-06-19 Cold Spring Harbor Laboratory Methods and compositions for RNA interference
EP1272630A2 (en) 2000-03-16 2003-01-08 Genetica, Inc. Methods and compositions for rna interference
KR20080023768A (ko) 2000-03-30 2008-03-14 화이트헤드 인스티튜트 포 바이오메디칼 리서치 Ｒｎａ 간섭의 ｒｎａ 서열 특이적인 매개체
US7083947B2 (en) 2000-05-19 2006-08-01 Devgen Nv Assay techniques using nematode worms
GB0012229D0 (en) 2000-05-19 2000-07-12 Devgen Nv Lipid uptake assays
GB2362383B (en) * 2000-05-19 2003-12-31 Devgen Nv Gene expression system
GB0012233D0 (en) 2000-05-19 2000-07-12 Devgen Nv Vector constructs
WO2001096584A2 (en) * 2000-06-12 2001-12-20 Akkadix Corporation Materials and methods for the control of nematodes
US20030190635A1 (en) * 2002-02-20 2003-10-09 Mcswiggen James A. RNA interference mediated treatment of Alzheimer's disease using short interfering RNA
WO2002044321A2 (en) 2000-12-01 2002-06-06 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Rna interference mediating small rna molecules
US7423142B2 (en) 2001-01-09 2008-09-09 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of anti-apoptotic genes
US7767802B2 (en) 2001-01-09 2010-08-03 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of anti-apoptotic genes
US8546143B2 (en) 2001-01-09 2013-10-01 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of a target gene
EP1392857B1 (en) * 2001-01-25 2010-01-20 VIRxSYS Corporation Methods and compositions for identifying gene function
CA2429397C (en) 2001-01-26 2014-06-03 Commonwealth Scientific And Industrial Research Organisation Methods and means for producing efficient silencing construct using recombinational cloning
EP1229134A3 (en) 2001-01-31 2004-01-28 Nucleonics, Inc Use of post-transcriptional gene silencing for identifying nucleic acid sequences that modulate the function of a cell
US20040110177A1 (en) * 2001-02-02 2004-06-10 Axel Ullrich Method for identifying functional nucleic acids
US9994853B2 (en) 2001-05-18 2018-06-12 Sirna Therapeutics, Inc. Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference
EP2415486B1 (en) 2001-05-18 2017-02-22 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
US20050148530A1 (en) 2002-02-20 2005-07-07 Sirna Therapeutics, Inc. RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
WO2005078097A2 (en) 2004-02-10 2005-08-25 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING MULTIFUNCTIONAL SHORT INTERFERING NUCLEIC ACID (Multifunctional siNA)
WO2003070887A2 (en) * 2002-02-20 2003-08-28 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF POLYCOMB GROUP PROTEIN EZH2 GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
WO2003102131A2 (en) * 2002-04-22 2003-12-11 Sirna Therapeutics Inc. Nucleic acid mediated disruption of hiv fusogenic peptide interactions
WO2003070972A2 (en) * 2002-02-20 2003-08-28 Sirna Therapeutics Inc. RNA INTERFERENCE MEDIATED INHIBITION OF CHROMOSOME TRANSLOCATION GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7109165B2 (en) 2001-05-18 2006-09-19 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
BR0211111A (pt) 2001-07-12 2004-06-22 Univ Massachusetts Molécula de ácido nucleico isolada, vetor, célula hospedeira, transgene, precursor de rna engenheirado, animal transgênico não humano, e, método de induzir a interferência de ácido ribonucleico de um gene alvo em uma célula
US7612194B2 (en) * 2001-07-24 2009-11-03 Monsanto Technology Llc Nucleic acid sequences from Diabrotica virgifera virgifera LeConte and uses thereof
CA2458842C (en) * 2001-08-31 2011-01-25 Riken Plant system for comprehensive gene function analysis with the use of full-length cdnas
DE10163098B4 (de) 2001-10-12 2005-06-02 Alnylam Europe Ag Verfahren zur Hemmung der Replikation von Viren
US7745418B2 (en) 2001-10-12 2010-06-29 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting viral replication
US20030150017A1 (en) * 2001-11-07 2003-08-07 Mesa Jose Ramon Botella Method for facilitating pathogen resistance
DE10202419A1 (de) 2002-01-22 2003-08-07 Ribopharma Ag Verfahren zur Hemmung der Expression eines durch eine Chromosomen-Aberration entstandenen Zielgens
US20060009409A1 (en) 2002-02-01 2006-01-12 Woolf Tod M Double-stranded oligonucleotides
WO2003064625A2 (en) 2002-02-01 2003-08-07 Sequitur, Inc. Oligonucleotide compositions with enhanced efficiency
US20030166282A1 (en) * 2002-02-01 2003-09-04 David Brown High potency siRNAS for reducing the expression of target genes
AU2003216255A1 (en) * 2002-02-20 2003-09-09 Ribozyme Pharmaceuticals, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF MDR P-GLYCOPROTEIN GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US7667029B2 (en) 2002-02-20 2010-02-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of checkpoint kinase-1 (CHK-1) gene expression using short interfering nucleic acid (siNA)
EP1432724A4 (en) * 2002-02-20 2006-02-01 Sirna Therapeutics Inc RNA inhibition mediated inhibition of MAP KINASE GENES
US9657294B2 (en) 2002-02-20 2017-05-23 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
AU2003211058A1 (en) * 2002-02-20 2003-09-09 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED TARGET DISCOVERY AND TARGET VALIDATION USING SHORT INTERFERING NUCLEIC ACID (siNA)
AU2003213057A1 (en) * 2002-02-20 2003-09-09 Ribozyme Pharmaceuticals, Incoporated Rna interference mediated inhibition of checkpoint kinase-1 (chk-1) gene expression using short interfering nucleic acid
US9181551B2 (en) 2002-02-20 2015-11-10 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US20040180438A1 (en) 2002-04-26 2004-09-16 Pachuk Catherine J. Methods and compositions for silencing genes without inducing toxicity
AU2003239851A1 (en) * 2002-05-08 2003-11-11 Xantos Biomedicine Ag Expression constructs for producing double-stranded (ds) rna and the use thereof
AU2003239706A1 (en) * 2002-05-23 2003-12-12 Devgen Nv Method for determining the influence of a compound on cholesterol transport
WO2004001044A1 (en) * 2002-06-21 2003-12-31 Sinogenomax Company Ltd. Randomised dna libraries and double-stranded rna libraries, use and method of production thereof
AU2003247951A1 (en) * 2002-07-10 2004-01-23 Kansas State University Research Foundation Compositions and methods for controlling parasitic nematodes
US7655790B2 (en) 2002-07-12 2010-02-02 Sirna Therapeutics, Inc. Deprotection and purification of oligonucleotides and their derivatives
DK3222724T3 (en) 2002-08-05 2018-12-03 Silence Therapeutics Gmbh ADDITIONALLY UNKNOWN FORMS OF INTERFERRING RNA MOLECULES
US8729036B2 (en) 2002-08-07 2014-05-20 University Of Massachusetts Compositions for RNA interference and methods of use thereof
CA2497892A1 (en) * 2002-09-04 2004-03-18 Provost, Fellows And Scholars Of The College Of The Holy And Undivided T Rinity Of Queen Elizabeth Near Dublin Compositions and methods for tissue specific or inducible inhibition of gene expression
US7923547B2 (en) 2002-09-05 2011-04-12 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
JP4786904B2 (ja) 2002-11-27 2011-10-05 セクエノム，インコーポレイティド 配列変化検出及び発見用の断片化をベースとする方法及びシステム
WO2004056982A2 (en) * 2002-12-23 2004-07-08 Devgen Nv Kinase sequences
EP1622572B1 (en) 2003-04-30 2017-12-20 Sirna Therapeutics, Inc. Conjugates and compositions for cellular delivery
IL155783A (en) 2003-05-05 2010-11-30 Technion Res & Dev Foundation Multicellular systems of multi-potential embryonic human stem cells and cancer cells and their use
EP1633784B1 (en) 2003-05-09 2011-07-13 Diadexus, Inc. Ovr110 antibody compositions and methods of use
JP2006527593A (ja) * 2003-06-17 2006-12-07 デヴゲン エヌブイ 代謝性疾患の予防及び／又は治療のための化合物を開発するために有用なアルコールデヒドロゲナーゼ配列
US9394565B2 (en) 2003-09-05 2016-07-19 Agena Bioscience, Inc. Allele-specific sequence variation analysis
WO2005049841A1 (en) * 2003-11-17 2005-06-02 Commonwealth Scientific And Industrial Research Organisation Insect resistance using inhibition of gene expression
ES2394799T3 (es) 2003-12-31 2013-02-05 The Penn State Research Foundation Métodos para predecir y superar la resistencia a quimioterapia en cáncer de ovario
US20060019914A1 (en) 2004-02-11 2006-01-26 University Of Tennessee Research Foundation Inhibition of tumor growth and invasion by anti-matrix metalloproteinase DNAzymes
US7622301B2 (en) * 2004-02-24 2009-11-24 Basf Plant Science Gmbh Compositions and methods using RNA interference for control of nematodes
WO2006074418A2 (en) 2005-01-07 2006-07-13 Diadexus, Inc. Ovr110 antibody compositions and methods of use
CA2561381C (en) 2004-03-26 2015-05-12 Sequenom, Inc. Base specific cleavage of methylation-specific amplification products in combination with mass analysis
US7608394B2 (en) * 2004-03-26 2009-10-27 Sequenom, Inc. Methods and compositions for phenotype identification based on nucleic acid methylation
AU2005229015C1 (en) 2004-04-02 2013-01-17 The Regents Of The University Of California Methods and compositions for treating and preventing disease associated with alphaVbeta5 integrin
CA2562022C (en) 2004-04-09 2016-01-26 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
AU2005238034A1 (en) 2004-04-23 2005-11-10 The Trustees Of Columbia University In The City Of New York Inhibition of hairless protein mRNA
US10508277B2 (en) 2004-05-24 2019-12-17 Sirna Therapeutics, Inc. Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference
DE602005026811D1 (de) 2004-06-22 2011-04-21 Univ Illinois Verfahren zur inhibierung von tumorzellwachstum mit foxm1 sirns
US7968762B2 (en) 2004-07-13 2011-06-28 Van Andel Research Institute Immune-compromised transgenic mice expressing human hepatocyte growth factor (hHGF)
WO2007001324A2 (en) 2004-07-23 2007-01-04 The University Of North Carolina At Chapel Hill Methods and materials for determining pain sensitivity and predicting and treating related disorders
ATE518954T1 (de) 2004-08-18 2011-08-15 Lorus Therapeutics Inc Kleine interferierende rna-moleküle gegen ribonukleotidreduktase und ihre verwendungen
CN101389755B (zh) * 2004-09-24 2014-06-11 J.R.西姆普罗特公司 基因沉默
US20060247197A1 (en) 2004-10-04 2006-11-02 Van De Craen Marc Method for down-regulating gene expression in fungi
MX2007004310A (es) 2004-10-13 2007-06-18 Univ Georgia Res Found Plantas transgenicas resistentes a nematodos.
CA2584960A1 (en) 2004-10-21 2006-05-04 Charles L. Niblett Methods and materials for conferring resistance to pests and pathogens of plants
WO2006045591A2 (en) * 2004-10-25 2006-05-04 Devgen N.V. Method and constructs for delivering double stranded rna to pest organisms
EP1807520B1 (en) 2004-10-25 2012-07-25 Devgen NV Rna constructs
US8088976B2 (en) * 2005-02-24 2012-01-03 Monsanto Technology Llc Methods for genetic control of plant pest infestation and compositions thereof
DE202005004135U1 (de) * 2005-03-11 2005-05-19 Klocke Verpackungs-Service Gmbh Mehrkomponentenverpackung mit Applikator
CA2603730A1 (en) 2005-03-31 2006-10-05 Calando Pharmaceuticals, Inc. Inhibitors of ribonucleotide reductase subunit 2 and uses thereof
WO2006124699A2 (en) 2005-05-12 2006-11-23 Wisconsin Alumni Research Foundation Blockade of pin1 prevents cytokine production by activated immune cells
WO2006129204A2 (en) * 2005-05-31 2006-12-07 Devgen Nv Rnai for control of insects and arachnids
WO2007011702A2 (en) 2005-07-15 2007-01-25 The University Of North Carolina At Chapel Hill Use of egfr inhibitors to prevent or treat obesity
BRPI0615791B1 (pt) 2005-09-16 2018-04-03 Devgen Nv Rna de fita dupla isolado compreendendo fitas complementares aneladas, método de controle de infestação de peste e uso de uma ração artificial compreendendo a sequência de ribonucleotídeo de fita dupla para tratar infestação de plantas por insetos
JP5530632B2 (ja) 2005-09-16 2014-06-25 デブジェン エヌブイ ＲＮＡｉを使用した害虫の抑制方法
TWI390037B (zh) * 2005-09-16 2013-03-21 Monsanto Technology Llc 用於植物之昆蟲感染的基因控制方法及其組合物
EP1931789B1 (en) 2005-09-20 2016-05-04 BASF Plant Science GmbH Methods for controlling gene expression using ta-siran
US9286469B2 (en) * 2005-12-16 2016-03-15 Cisco Technology, Inc. Methods and apparatus providing computer and network security utilizing probabilistic signature generation
WO2007087153A2 (en) 2006-01-06 2007-08-02 University Of Georgia Research Foundation Cyst nematode resistant transgenic plants
WO2007080127A2 (en) 2006-01-12 2007-07-19 Devgen N.V. Dsrna as insect control agent
US8906876B2 (en) 2006-01-12 2014-12-09 Devgen Nv Methods for controlling pests using RNAi
CA2633576A1 (en) 2006-01-12 2007-07-19 Devgen N.V. Dsrna as insect control agent
US20080022423A1 (en) * 2006-02-03 2008-01-24 Monsanto Technology Llc IN PLANTA RNAi CONTROL OF FUNGI
CN101501199B (zh) 2006-02-10 2016-11-09 孟山都技术有限公司 用于控制植物寄生线虫的靶基因的鉴定和用途
CN101384721A (zh) * 2006-02-13 2009-03-11 孟山都技术有限公司 选择和稳定dsRNA构建体
US20100068172A1 (en) * 2006-03-16 2010-03-18 Devgen N.V. Nematode Control
WO2007123777A2 (en) 2006-03-30 2007-11-01 Duke University Inhibition of hif-1 activation for anti-tumor and anti-inflammatory responses
PL216037B1 (pl) * 2006-06-09 2014-02-28 Inst Biotechnologii I Antybiotykow Kaseta ekspresyjna, zastosowanie kasety ekspresyjnej, wektor, komórka gospodarza oraz sposób otrzymywania polipeptydu
EP2471809B1 (en) 2006-07-11 2015-09-02 University Of Medicine And Dentistry Of New Jersey Proteins, nucleic acids encoding the same and associated methods of use
WO2008014426A2 (en) 2006-07-28 2008-01-31 Children's Memorial Hospital Methods of inhibiting tumor cell aggressiveness using the microenvironment of human embryonic stem cells
JP5391073B2 (ja) 2006-11-27 2014-01-15 ディアデクサス インコーポレーテッド Ｏｖｒ１１０抗体組成物および使用方法
CN101195821A (zh) * 2006-12-04 2008-06-11 中国科学院上海生命科学研究院 利用RNAi技术改良植物抗虫性的方法
US20100129358A1 (en) 2006-12-22 2010-05-27 University Of Utah Research Foundation Method of detecting ocular diseases and pathologic conditions and treatment of same
CA2676143A1 (en) 2007-01-26 2008-07-31 University Of Louisville Research Foundation, Inc. Modification of exosomal components for use as a vaccine
US20080184391A1 (en) * 2007-01-29 2008-07-31 Kuppuswamy Subramaniam Pathogen resistant transgenic plants, associated nucleic acids and techniques involving the same
EP2115141A4 (en) * 2007-02-20 2010-08-04 Monsanto Technology Llc INVERTEBRA MICRO-RNA
JP5759673B2 (ja) 2007-03-21 2015-08-05 ブルックヘブン サイエンス アソシエイツ，エルエルシー 組み合わされたヘアピン−アンチセンス組成物および発現を調節するための方法
EP2152903A2 (en) 2007-04-26 2010-02-17 Ludwig Institute for Cancer Research, Ltd. Methods for diagnosing and treating astrocytomas
WO2008137115A1 (en) 2007-05-03 2008-11-13 The Brigham And Women's Hospital, Inc. Multipotent stem cells and uses thereof
US8097422B2 (en) 2007-06-20 2012-01-17 Salk Institute For Biological Studies Kir channel modulators
CN101827942A (zh) * 2007-06-29 2010-09-08 波士顿生物医药公司 促使长dsRNA可用于哺乳动物和其他所选动物细胞中的基因寻靶
US9689031B2 (en) 2007-07-14 2017-06-27 Ionian Technologies, Inc. Nicking and extension amplification reaction for the exponential amplification of nucleic acids
WO2009012263A2 (en) 2007-07-18 2009-01-22 The Trustees Of Columbia University In The City Of New York Tissue-specific micrornas and compositions and uses thereof
ES2651911T3 (es) 2007-08-14 2018-01-30 Commonwealth Scientific And Industrial Research Organisation Métodos mejorados de silenciamiento génico
KR101142209B1 (ko) 2007-09-22 2012-05-04 재단법인서울대학교산학협력재단 섭취 ＲＮＡｉ를 이용한 꼬마선충에서 두 유전자의동시발현 억제방법
US7968525B1 (en) 2007-12-03 2011-06-28 University Of Florida Research Foundation, Inc. Use of RNA interference to validate new termiticide target sites and a method of termite control
BRPI0911332A2 (pt) 2008-04-04 2019-09-24 Calando Pharmaceuticals Inc composições e uso de inibidores de epas1
CN102027121B (zh) 2008-04-10 2017-08-08 孟山都技术公司 用于根结线虫防治的方法和组合物
GB0807018D0 (en) 2008-04-17 2008-05-21 Fusion Antibodies Ltd Antibodies and treatment
WO2010054221A2 (en) 2008-11-06 2010-05-14 The Johns Hopkins University Treatment of chronic inflammatory respiratory disorders
WO2010054379A2 (en) 2008-11-10 2010-05-14 The United States Of America, As Represensted By The Secretary, Department Of Health And Human Services Gene signature for predicting prognosis of patients with solid tumors
DK2379722T3 (da) 2008-12-16 2017-01-02 C-Lecta Gmbh Ekspressionsvektor
EP2379076B1 (en) 2008-12-23 2014-11-12 The Trustees of Columbia University in the City of New York Phosphodiesterase inhibitors and uses thereof
WO2010074783A1 (en) 2008-12-23 2010-07-01 The Trustees Of Columbia University In The City Of New York Phosphodiesterase inhibitors and uses thereof
WO2010107958A1 (en) 2009-03-19 2010-09-23 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 6 (STAT6) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
EP2408915A2 (en) 2009-03-19 2012-01-25 Merck Sharp&Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF GATA BINDING PROTEIN 3 (GATA3) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
JP2012520683A (ja) 2009-03-19 2012-09-10 メルク・シャープ・エンド・ドーム・コーポレイション 低分子干渉核酸（ｓｉＮＡ）を用いた結合組織増殖因子（ＣＴＧＦ）遺伝子発現のＲＮＡ干渉媒介性阻害
JP2012520684A (ja) 2009-03-19 2012-09-10 メルク・シャープ・エンド・ドーム・コーポレイション 低分子干渉核酸（ｓｉＮＡ）を用いたＢＴＢａｎｄＣＮＣＨｏｍｏｌｏｇｙ１（塩基性ロイシンジッパー転写因子１）（Ｂａｃｈ１）遺伝子発現のＲＮＡ干渉媒介性阻害
CA2756069A1 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. Rna interference mediated inhibition of the intercellular adhesion molecule 1 (icam-1)gene expression using short interfering nucleic acid (sina)
US20120004281A1 (en) 2009-03-27 2012-01-05 Merck Sharp & Dohme Corp RNA Interference Mediated Inhibition of the Nerve Growth Factor Beta Chain (NGFB) Gene Expression Using Short Interfering Nucleic Acid (siNA)
US20120022143A1 (en) 2009-03-27 2012-01-26 Merck Sharp & Dohme Corp RNA Interference Mediated Inhibition of the Thymic Stromal Lymphopoietin (TSLP) Gene Expression Using Short Interfering Nucliec Acid (siNA)
WO2010111471A2 (en) 2009-03-27 2010-09-30 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 1 (STAT1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
EP2411516A1 (en) 2009-03-27 2012-02-01 Merck Sharp&Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF APOPTOSIS SIGNAL-REGULATING KINASE 1 (ASK1) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US20100257634A1 (en) * 2009-04-03 2010-10-07 Venganza Inc. Bioassay for gene silencing constructs
US8283332B2 (en) 2009-04-17 2012-10-09 University Of Louisville Research Foundation, Inc. PFKFB4 inhibitors and methods of using the same
EP2258858A1 (en) 2009-06-05 2010-12-08 Universitätsklinikum Freiburg Transgenic LSD1 animal model for cancer
SI2453923T1 (sl) 2009-07-14 2016-04-29 Mayo Foundation For Medical Education And Research S peptidi posredovana ne-kovalentna dostava aktivnih učinkovin preko krvno možganske bariere
AU2010275367B2 (en) 2009-07-24 2015-09-03 The Regents Of The University Of California Methods and compositions for treating and preventing disease associated with avB5 integrin
EP2470662B1 (en) * 2009-08-28 2016-08-10 E. I. du Pont de Nemours and Company Compositions and methods to control insect pests
AU2010328295B2 (en) 2009-12-07 2015-09-10 The Johns Hopkins University SR-BI as a predictor of human female infertility and responsiveness to treatment
CN106701758B (zh) 2009-12-09 2020-02-07 日东电工株式会社 Hsp47表达的调节
US10640457B2 (en) 2009-12-10 2020-05-05 The Trustees Of Columbia University In The City Of New York Histone acetyltransferase activators and uses thereof
WO2011072243A1 (en) 2009-12-10 2011-06-16 The Trustees Of Columbia University In The City Of New York Histone acetyltransferase activators and uses thereof
KR101605932B1 (ko) 2009-12-18 2016-03-24 노파르티스 아게 Ｈｓｆ1-관련 질환을 치료하기 위한 유기 조성물
BR112012015755B1 (pt) 2009-12-23 2021-06-22 Novartis Ag Lipídeo furtivo, e composição
CA3101636A1 (en) 2010-01-26 2011-08-04 National Jewish Health Diagnosis and prognosis of idiopathic interstitial pneumonia by rs35705950 snp in muc5b gene promoter
CN102770767A (zh) 2010-02-10 2012-11-07 诺瓦提斯公司 用于肌肉生长的方法和组合物
WO2011146938A1 (en) 2010-05-21 2011-11-24 NanoOncology, Inc. Reagents and methods for treating cancer
GB201009601D0 (en) 2010-06-08 2010-07-21 Devgen Private Ltd Method for down-grading gene expression in fungi
WO2011163466A1 (en) 2010-06-23 2011-12-29 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Regulation of skin pigmentation by neuregulin-1 (nrg-1)
EP3330377A1 (en) 2010-08-02 2018-06-06 Sirna Therapeutics, Inc. Rna interference mediated inhibition of catenin (cadherin-associated protein), beta 1 (ctnnb1) gene expression using short interfering nucleic acid (sina)
EP2606134B1 (en) 2010-08-17 2019-04-10 Sirna Therapeutics, Inc. RNA INTERFERENCE MEDIATED INHIBITION OF HEPATITIS B VIRUS (HBV) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
JP6106085B2 (ja) 2010-08-24 2017-03-29 サーナ・セラピューティクス・インコーポレイテッドＳｉｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． 内部非核酸スペーサーを含む一本鎖ＲＮＡｉ剤
WO2012027467A1 (en) 2010-08-26 2012-03-01 Merck Sharp & Dohme Corp. RNA INTERFERENCE MEDIATED INHIBITION OF PROLYL HYDROXYLASE DOMAIN 2 (PHD2) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)
US20140134231A1 (en) 2010-10-11 2014-05-15 Sanford-Burnham Medical Research Institute Mir-211 expression and related pathways in human melanoma
CN108404115A (zh) 2010-10-15 2018-08-17 纽约市哥伦比亚大学理事会 肥胖症-相关的基因和它们的蛋白和其用途
ES2663009T3 (es) 2010-10-29 2018-04-10 Sirna Therapeutics, Inc. Inhibición de la expresión génica mediada por interferencia por ARN utilizando ácidos nucleicos de interferencia cortos (ANic)
CN103313730B (zh) 2010-11-01 2016-06-01 佩普蒂梅德股份有限公司 用于治疗癌症的肽靶向系统的组合物
KR102027394B1 (ko) 2010-11-02 2019-10-01 더 트러스티스 오브 콜롬비아 유니버시티 인 더 시티 오브 뉴욕 탈모 질환의 치료 방법
US9198911B2 (en) 2010-11-02 2015-12-01 The Trustees Of Columbia University In The City Of New York Methods for treating hair loss disorders
WO2012078536A2 (en) 2010-12-06 2012-06-14 Quark Pharmaceuticals, Inc. Double stranded oligonucleotide compounds comprising positional modifications
US9150926B2 (en) 2010-12-06 2015-10-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Diagnosis and treatment of adrenocortical tumors using human microRNA-483
EP3323813B1 (en) 2010-12-22 2020-08-26 The Trustees of Columbia University in the City of New York Histone acetyltransferase modulators and uses thereof
CN103492572A (zh) 2011-03-03 2014-01-01 夸克医药公司 用于治疗肺疾病和损伤的组合物和方法
TWI658830B (zh) 2011-06-08 2019-05-11 日東電工股份有限公司 Ｈｓｐ47表現調控強化用類視色素脂質體
US10196637B2 (en) 2011-06-08 2019-02-05 Nitto Denko Corporation Retinoid-lipid drug carrier
WO2013033657A2 (en) 2011-09-02 2013-03-07 The Trustees Of Columbia University In The City Of New York CaMKII, IP3R, CALCINEURIN, P38 AND MK2/3 INHIBITORS TO TREAT METABOLIC DISTURBANCES OF OBESITY
US9352312B2 (en) 2011-09-23 2016-05-31 Alere Switzerland Gmbh System and apparatus for reactions
HUE039133T2 (hu) 2011-10-14 2018-12-28 Hoffmann La Roche Anti-HtrA1 antitestek és felhasználási módszerek
PT3597644T (pt) 2011-10-18 2021-11-03 Dicerna Pharmaceuticals Inc Lípidos catiónicos de amina e suas utilizações
US9422560B2 (en) 2011-11-03 2016-08-23 Quark Pharmaceuticals, Inc. Methods and compositions for neuroprotection
CN104334191A (zh) 2012-03-29 2015-02-04 纽约市哥伦比亚大学托管会 治疗毛发脱落疾病的方法
BR112014025983A2 (pt) 2012-04-20 2018-07-03 Futuragene Israel Ltd molécula de ácido ribonucleico, dsrna, vetor, célula hospedeira, tecido vegetal, ácido nucleico e métodos de produção de plantas e de inibição de infestação de pragas
BR112014026363A2 (pt) 2012-04-23 2017-06-27 Futuragene Israel Ltd molécula de ácido ribonucleico, dsrna, vetor, célula hospedeira, tecido vegetal, ácido nucleico isolado, método de produção de planta resistente a pragas e método de inibição de infestação de pragas
EP3919620A1 (en) 2012-05-02 2021-12-08 Sirna Therapeutics, Inc. Short interfering nucleic acid (sina) compositions
US20150175979A1 (en) 2012-07-23 2015-06-25 La Jolla Institute For Allergy And Immunology Ptprs and proteoglycans in autoimmune disease
BR112015007123A2 (pt) 2012-10-03 2017-08-08 Futuragene Israel Ltd molécula de ácido ribonucléico de filamento duplo (dsrna) isolada, vetor, célula hospedeira, tecido vegetal, ácido nucléico isolado, e, métodos para produzir uma planta resistente a uma praga e para inibir uma infestação de praga
ES2776029T3 (es) 2012-10-08 2020-07-28 St Jude Childrens Res Hospital Terapias basadas en el control de la estabilidad y función de las células T reguladoras por medio de un eje neuropilina-1:semaforina
US9920316B2 (en) 2013-03-14 2018-03-20 Pioneer Hi-Bred International, Inc. Compositions and methods to control insect pests
EP2968551B1 (en) 2013-03-15 2021-05-05 The Trustees of Columbia University in the City of New York Fusion proteins and methods thereof
EP2810952A1 (en) 2013-06-03 2014-12-10 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Novel pest control methods
KR20160027971A (ko) 2013-07-03 2016-03-10 시티 오브 호프 항암 병용제
US10584387B2 (en) 2013-10-09 2020-03-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Detection of hepatitis delta virus (HDV) for the diagnosis and treatment of Sjögren's syndrome and lymphoma
EP3068407A1 (en) 2013-11-11 2016-09-21 Sirna Therapeutics, Inc. Systemic delivery of myostatin short interfering nucleic acids (sina) conjugated to a lipophilic moiety
US9682123B2 (en) 2013-12-20 2017-06-20 The Trustees Of Columbia University In The City Of New York Methods of treating metabolic disease
WO2015098113A1 (ja) 2013-12-27 2015-07-02 独立行政法人医薬基盤研究所 悪性腫瘍の治療薬
US10342761B2 (en) 2014-07-16 2019-07-09 Novartis Ag Method of encapsulating a nucleic acid in a lipid nanoparticle host
WO2016059187A1 (en) * 2014-10-16 2016-04-21 Universite De Strasbourg Method of capturing and identifying novel rnas
EP3218497A1 (en) 2014-11-12 2017-09-20 NMC Inc. Transgenic plants with engineered redox sensitive modulation of photosynthetic antenna complex pigments and methods for making the same
WO2016105517A1 (en) 2014-12-23 2016-06-30 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
US10264976B2 (en) 2014-12-26 2019-04-23 The University Of Akron Biocompatible flavonoid compounds for organelle and cell imaging
CN107429251A (zh) 2015-03-09 2017-12-01 肯塔基大学研究基金会 用于治疗乳腺癌的miRNA
CN107531740B (zh) 2015-03-09 2021-03-19 肯塔基大学研究基金会 用于脑肿瘤治疗的rna纳米颗粒
WO2016145003A1 (en) 2015-03-09 2016-09-15 University Of Kentucky Research Foundation Rna nanoparticle for treatment of gastric cancer
US11279768B1 (en) 2015-04-03 2022-03-22 Precision Biologics, Inc. Anti-cancer antibodies, combination therapies, and uses thereof
WO2016168784A2 (en) 2015-04-17 2016-10-20 University Of Kentucky Research Foundation Rna nanoparticles and method of use thereof
EP3289104B1 (en) 2015-04-29 2020-11-04 New York University Method for treating high-grade gliomas
JP6980534B2 (ja) 2015-06-25 2021-12-15 ザ チルドレンズ メディカル センター コーポレーション 造血幹細胞の増大、富化、および維持に関する方法および組成物
US10072065B2 (en) 2015-08-24 2018-09-11 Mayo Foundation For Medical Education And Research Peptide-mediated delivery of immunoglobulins across the blood-brain barrier
CA2997947A1 (en) 2015-09-09 2017-03-16 The Trustees Of Columbia University In The City Of New York Reduction of er-mam-localized app-c99 and methods of treating alzheimer's disease
US11285142B2 (en) 2015-09-29 2022-03-29 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compositions and methods for identifying and treating dystonia disorders
US10421821B2 (en) 2015-10-30 2019-09-24 Genentech, Inc. Anti-HtrA1 antibodies and methods of use thereof
KR20180086260A (ko) 2015-12-13 2018-07-30 닛토덴코 가부시키가이샤 높은 활성 및 감소한 오프 타겟을 위한 sirna 구조
WO2017152073A1 (en) 2016-03-04 2017-09-08 University Of Louisville Research Foundation, Inc. Methods and compositions for ex vivo expansion of very small embryonic-like stem cells (vsels)
AU2017235461B2 (en) 2016-03-15 2023-02-23 Children's Medical Center Corporation Methods and compositions relating to hematopoietic stem cell expansion
US10883108B2 (en) 2016-03-31 2021-01-05 The Schepens Eye Research Institute, Inc. Endomucin inhibitor as an anti-angiogenic agent
EP3516062A1 (en) 2016-09-21 2019-07-31 Alnylam Pharmaceuticals, Inc. Myostatin irna compositions and methods of use thereof
CN107858405B (zh) * 2017-10-12 2021-09-24 华南农业大学 一种测定外源dsRNA对瓢虫毒性影响的方法
US20210162007A1 (en) 2018-04-09 2021-06-03 President And Fellows Of Harvard College Modulating nuclear receptors and methods of using same
CN109266677B (zh) * 2018-08-31 2021-11-23 中国烟草总公司郑州烟草研究院 一种全长转录因子酵母双杂交文库的构建方法
CN109183158B (zh) * 2018-08-31 2021-11-23 中国烟草总公司郑州烟草研究院 一种全长转录因子酵母单杂交文库的构建方法
WO2020061381A1 (en) 2018-09-19 2020-03-26 La Jolla Institute For Immunology Ptprs and proteoglycans in rheumatoid arthritis
PT3880212T (pt) 2018-11-16 2024-02-08 Nitto Denko Corp Formulação e métodos de administração de rna de interferência para tumores malignos
JP7317379B2 (ja) * 2019-01-04 2023-07-31 国立大学法人京都大学 潰瘍性大腸炎及び原発性硬化性胆管炎の検査方法
CN110229839B (zh) * 2019-06-04 2021-06-08 中国农业大学 一种提升大肠杆菌dsRNA表达产率的方法
CN110746497A (zh) * 2019-11-18 2020-02-04 维塔恩(广州)医药有限公司 肺炎衣原体相关抗原短肽及其应用
CN110804088A (zh) * 2019-11-18 2020-02-18 维塔恩(广州)医药有限公司 巨细胞病毒相关抗原短肽及其应用
EP3825408A1 (en) 2019-11-19 2021-05-26 FRAUNHOFER-GESELLSCHAFT zur Förderung der angewandten Forschung e.V. Methods of multi-species insect pest control
CR20220278A (es) 2019-12-18 2022-07-01 Novartis Ag Derivados de 3-(5-metoxi-1-oxoisoindolin-2-il)piperidin-2,6-diona y usos de los mismos
EP4077676A1 (en) 2019-12-18 2022-10-26 Novartis AG Compositions and methods for the treatment of hemoglobinopathies
EP4087652A1 (en) 2020-01-08 2022-11-16 Regeneron Pharmaceuticals, Inc. Treatment of fibrodysplasia ossificans progressiva
US20210222128A1 (en) 2020-01-22 2021-07-22 Massachusetts Institute Of Technology Inducible tissue constructs and uses thereof
US11642407B2 (en) 2020-02-28 2023-05-09 Massachusetts Institute Of Technology Identification of variable influenza residues and uses thereof
CN113638055B (zh) * 2020-05-22 2023-07-07 江苏省疾病预防控制中心(江苏省公共卫生研究院) 一种制备双链rna测序文库的方法
WO2022147481A1 (en) 2020-12-30 2022-07-07 Ansun Biopharma Inc. Combination therapy of an oncolytic virus delivering a foreign antigen and an engineered immune cell expressing a chimeric receptor targeting the foreign antigen
WO2022269518A2 (en) 2021-06-23 2022-12-29 Novartis Ag Compositions and methods for the treatment of hemoglobinopathies
EP4381069A1 (en) 2021-08-02 2024-06-12 Universite De Montpellier Compositions and methods for treating cmt1a or cmt1e diseases with rnai molecules targeting pmp22
CN113533007B (zh) * 2021-08-06 2023-11-24 青岛瑞斯凯尔生物科技有限公司 一种抗体染色标记装置及其方法
MX2024003887A (es) 2021-10-14 2024-07-09 Arsenal Biosciences Inc Células inmunitarias que tienen arnch coespresados y sistemas de compuerta lógica.
WO2024059618A2 (en) 2022-09-13 2024-03-21 Arsenal Biosciences, Inc. Immune cells having co-expressed tgfbr shrnas
WO2024059824A2 (en) 2022-09-16 2024-03-21 Arsenal Biosciences, Inc. Immune cells with combination gene perturbations
US20240342284A1 (en) 2023-03-03 2024-10-17 Arsenal Biosciences, Inc. Systems targeting psma and ca9

Family Cites Families (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US589801A (en) * 1897-09-07 woltereck
ATE201444T1 (de) 1985-03-21 2001-06-15 Johnston Stephen Ph D Vom parasit abgeleiteter widerstand
US6608241B1 (en) 1985-10-29 2003-08-19 Monsanto Technology Llc Protection of plants against viral infection
NZ219472A (en) 1986-03-28 1990-08-28 Calgene Inc Regulation of phenotype in plant cells using dsdna constructs
US5017488A (en) * 1986-04-01 1991-05-21 University Of Medicine And Dentistry Of New Jersey Highly efficient dual T7/T3 promoter vector PJKF16 and dual SP6/T3 promoter vector PJFK15
US4970168A (en) * 1989-01-27 1990-11-13 Monsanto Company Virus-resistant plants
EP0473576A4 (en) * 1989-05-19 1993-03-10 Hem Research, Inc. Short therapeutic dsrna of defined structure
HUT57265A (en) 1989-11-03 1991-11-28 Zaadunie Bv Process for producing plants of diminished infection-sensitivity
US5837848A (en) * 1990-03-16 1998-11-17 Zeneca Limited Root-specific promoter
WO1991015111A1 (en) * 1990-03-30 1991-10-17 President And Fellows Of Harvard College A binary genetic system to control expression of a transgene in a transgenic animal
US5831011A (en) * 1990-07-27 1998-11-03 Mycogen Corporation Bacillus thuringiensis genes encoding nematode-active toxins
US5459252A (en) * 1991-01-31 1995-10-17 North Carolina State University Root specific gene promoter
AU676471B2 (en) 1992-03-20 1997-03-13 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Fungus-responsive chimaeric gene
DE4234131C2 (de) * 1992-10-09 1995-08-24 Max Planck Gesellschaft Transgener pathogen-resistenter Organismus
CA2088379A1 (en) * 1993-01-29 1994-07-30 University Of British Columbia Biological systems incorporating stress-inducible genes and reporter constructs for environmental biomonitoring and toxicology
IL104830A (en) * 1993-02-23 2001-01-28 Yissum Res Dev Co A chimeric plasmid containing a non-structural gene for the protease of the potato virus and its various uses
DE4317845A1 (de) 1993-05-28 1994-12-01 Bayer Ag Desoxyribonukleinsäuren
WO1995034680A1 (en) * 1994-06-15 1995-12-21 The Trustees Of Columbia University In The City Of New York Method to identify tumor suppressor genes
US5691140A (en) * 1995-05-18 1997-11-25 New England Biolabs, Inc. Bidirectional in vitro transcription vectors utilizing a single RNA polymerase for both directions
GB9510944D0 (en) * 1995-05-31 1995-07-26 Bogaert Thierry Assays and processes for the identification of compounds which control cell behaviour,the compounds identified and their use in the control of cell behaviour
ES2151167T3 (es) * 1995-06-02 2000-12-16 M & E Biotech As S Procedimiento para la identificacion de acidos nucleicos y peptidos biologicamente activos.
US5679551A (en) * 1995-10-31 1997-10-21 Board Of Regents, The University Of Texas System Unique double-stranded RNAS associated with the Trichomonas vaginalis virus
US5898031A (en) * 1996-06-06 1999-04-27 Isis Pharmaceuticals, Inc. Oligoribonucleotides for cleaving RNA
DE19631919C2 (de) * 1996-08-07 1998-07-16 Deutsches Krebsforsch Anti-Sinn-RNA mit Sekundärstruktur
ATE279523T1 (de) * 1996-08-09 2004-10-15 Keygene Nv Resistenz gegen nematoden und/oder blattläuse
JP2001503972A (ja) * 1996-09-18 2001-03-27 ユング，クリスチャン 線虫抵抗性遺伝子
CA2267780A1 (en) 1996-10-03 1998-04-09 Hajime Kato Method for hydrocarbon steam reforming
AT409761B (de) 1996-12-18 2002-11-25 Exxon Chemical Patents Inc Unter verwendung eines multifunktionellen mittels hergestellte kompatibilisierte polymermischungen
GB9703146D0 (en) 1997-02-14 1997-04-02 Innes John Centre Innov Ltd Methods and means for gene silencing in transgenic plants
US6506559B1 (en) * 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
AUPP249298A0 (en) * 1998-03-20 1998-04-23 Ag-Gene Australia Limited Synthetic genes and genetic constructs comprising same I
EP2267138B1 (en) * 1998-04-08 2016-06-08 Commonwealth Scientific and Industrial Research Organization Methods and means for obtaining modified phenotypes
AR020078A1 (es) * 1998-05-26 2002-04-10 Syngenta Participations Ag Metodo para alterar la expresion de un gen objetivo en una celula de planta
GB9814536D0 (en) 1998-07-03 1998-09-02 Devgen Nv Characterisation of gene function using double stranded rna inhibition
GB9827152D0 (en) 1998-07-03 1999-02-03 Devgen Nv Characterisation of gene function using double stranded rna inhibition
AUPR621501A0 (en) 2001-07-06 2001-08-02 Commonwealth Scientific And Industrial Research Organisation Delivery of ds rna
TWI390037B (zh) 2005-09-16 2013-03-21 Monsanto Technology Llc 用於植物之昆蟲感染的基因控制方法及其組合物

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US20040133943A1 (en) 2004-07-08

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