CN117083071A - 人工甲病毒衍生的rna复制子表达系统 - Google Patents

人工甲病毒衍生的rna复制子表达系统 Download PDF

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CN117083071A
CN117083071A CN202280022669.3A CN202280022669A CN117083071A CN 117083071 A CN117083071 A CN 117083071A CN 202280022669 A CN202280022669 A CN 202280022669A CN 117083071 A CN117083071 A CN 117083071A
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nucleic acid
alphavirus
sequence
synthetic
acid molecule
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J·S·查哈尔
J·S·马克帕特兰
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Tiba Biotechnology Co ltd
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Abstract

描述了合成的甲病毒衍生复制子表达系统，其包含编码至少一种经过修饰的非结构蛋白的核酸序列和编码至少一种异源蛋白的合成核酸序列。提供了在细胞中产生至少一种异源蛋白的方法，或通过施用和/或表达合成的甲病毒衍生复制子表达系统在受试者中诱导免疫应答的方法。

Description

人工甲病毒衍生的RNA复制子表达系统
相关申请的交叉引用
本申请要求2021年3月19日提交的美国临时申请No.63/163,475的权益，其通过引用并入本文，如同已完整阐述。
与本申请一起电子提交的名为“SEQUENCE LISTING”的序列表创建于2022年3月18日，大小为413,759字节，其通过引用并入本文，如同已完整阐述。
发明领域
本发明涉及源自甲病毒基因组的自我复制RNA分子，以及通过突变和以其他方式编辑初级核苷酸序列来提高其安全性和基因表达能力的方法。本发明还涉及产生和使用此类工程化RNA分子来治疗和预防人类或动物疾病的方法。
背景技术
自我复制RNA，或“复制子”制子”，已被用作体外和体内转基因表达的载体。甲病毒属的病毒被用作复制子RNA设计的模板，因为它们的基因组布局在不同物种之间是一致的，并且适合分子生物学操作。源自许多不同种类的甲病毒(例如委内瑞拉马脑炎病毒、辛德比斯病毒、塞姆利基森林病毒)的基于甲病毒的复制子已被用作体外和体内转基因表达的载体。这些载体依赖于其中编码的非结构病毒蛋白的活性来介导在结构基因开放阅读框(ORF)位置编码的外源蛋白的表达，所述结构基因开放阅读框(ORF)是从复制生命周期后期合成的亚基因组(SG)mRNA翻译而来的。
复制子是先天免疫反应的强效诱导剂，使其可用于疫苗和其他免疫调节用途。甲病毒复制子RNA通常基于常见病毒种类，例如委内瑞拉马脑炎病毒(VEEV)，该病毒在世界大部分地区自然存在并流行。当复制子载体的序列与野生型基因组相同时，与野生型病毒重组的风险更大。RNA病毒的一级序列对基因组复制、翻译和包装成病毒体所需的二级结构和非蛋白质编码功能的保护很敏感。
对复制子RNA的免疫反应很难调整，因为最初的干扰素反应是在病毒生命周期的早期阶段启动的，此时只有非结构多蛋白(nsP)以有效的细胞内浓度表达。目前，将额外的调控基因产物纳入复制子中的修饰必须通过在SG ORF中插入来进行，这导致其仅在病毒复制周期的后期阶段表达，还会破坏该ORF中编码的任何其他转基因序列。如果转基因可以从nsP ORF中顺式表达，或在nsP ORF之外顺式表达，确保它们在复制生命周期的早期表达，那么RNA复制子的作用就会大得多，因为此时关键的生物过程，例如模式识别受体(PRR)的触发或蛋白质的翻译后加工，都可以通过传递其中编码的外源转基因来有意调节。此外，如果nsP区域的编码序列与天然存在的病毒基因组的序列同一性较低，那么RNA复制子在现场使用中将更安全。
发明内容
在一个方面，本发明涉及合成的甲病毒衍生的自我复制或复制子核酸(RNA)分子。合成的甲病毒衍生复制子核酸分子(synthetic alphavirus-derived replicon nucleicacid molecule)包含第一核酸，其编码甲病毒非结构蛋白nsP1、nsP2、nsP3和nsP4，并且包含在如SEQ ID NO：17所示的甲病毒基因组序列中从nt 503至nt 658、nt 658至nt 1620、nt1620至nt 2560、nt 2560至nt 3954、nt 3954至nt 4120、nt 6381至nt 7083和nt 6966至nt7526的区域内的任意位点引入的至少一个沉默突变。合成的甲病毒衍生复制子核酸分子还包含第二核酸，其包含亚基因组启动子(SGP)和经过修饰的亚基因组开放阅读框(SG ORF)。
在一个方面，本发明涉及甲病毒衍生RNA复制子表达系统，其包含任何一种合成的甲病毒衍生复制子核酸分子。在分子中，非结构蛋白4(nsP4)的C末端通过不可裂解、可白裂解或可蛋白水解裂解的肽连接序列与异源蛋白融合。合成的甲病毒衍生复制子核酸分子还编码nsP4融合蛋白下游(即3’)的功能性SGP序列，以驱动经过修饰的SG ORF的表达。在一个方面，nsP4编码序列中存在至少一个沉默突变，使其中包含的SGP同源序列丧失功能并且与nsP4融合蛋白ORF下游编码的功能性SGP不同。
在一个方面，本发明涉及甲病毒衍生RNA复制子表达系统。该甲病毒衍生RNA复制子表达系统包含本发明所述的任何一种合成的甲病毒衍生复制子核酸分子，其被封装或配制用于以病毒、病毒样颗粒、脂质体或脂质、聚合物或树枝状聚合物纳米颗粒的形式递送至生物体细胞中。
在一个方面，本发明涉及疫苗。该疫苗包含本发明所述的任意一种合成的甲病毒衍生复制子核酸分子。
在一个方面，本发明涉及治疗药物(drug)或药品(medicine)。该治疗药物或药品包含本发明所述的任意一种合成的甲病毒衍生复制子核酸分子。
在一个方面，本发明涉及在细胞中产生至少一种异源蛋白的方法。该方法包括在细胞中表达本发明所述的任意一种合成的甲病毒衍生复制子核酸分子。
在一个方面，本发明涉及预防、抑制或治疗受试者的疾病(disease)或病症(condition)的症状的方法，其包括向受试者施用治疗有效量的本发明所述的任意一种合成的甲病毒衍生复制子核酸分子。
附图说明
当结合附图阅读时，将更好地理解以下详细描述的本发明的优选实施方式。为了说明本发明，在附图中示出了特定的实施方式。然而，应当理解的是，本发明不限于附图中所示的精确布置和手段。在附图中：
图1是说明野生型甲病毒基因组非结构多蛋白(nsP1-nsP2-nsP3-nsP4)的基因表达和翻译后加工步骤的示意图。
图2是说明甲病毒基因组非结构多蛋白的基因表达步骤的示意图，该多蛋白通过与nsP4(nsP1-nsP2-nsP3-nsP4-xP)框内的附属物人工修饰以表达另外的外源蛋白(“xP")。
图3是说明克隆可修饰的nsP4开放阅读框(ORF)同时保持完整的经过延伸的亚基因组启动子(SGP、复制域(duplicated region)，以条纹框表示)的步骤的示意图。
图4是说明克隆可修饰的nsP4开放阅读框(ORF)同时保持完整的经过延伸的亚基因组启动子(SGP)的最佳步骤的示意图。在此图中，灰色框表示合成的nsP4 C末端区域和xP区域，条纹框表示原始nsP4 C末端CDS(非翻译)。
图5是编码构建体8(C08)的复制子序列模板的DNA质粒的示意图。质粒从T7启动子转录产生C08复制子RNA。C08的5'UTR、nsP1-4 ORF和SGP如SEQ ID NO：27所示。
图6是描绘了用于选择甲病毒基因组区域的示例性过程示意图，所述区域可通过应用沉默突变进行修改(alter)，从而降低与野生型病毒同源性，同时不降低复制子的复制功能。在此图中，圈出的是可以在不扰乱二级结构氨基酸编码序列的情况下修改的核酸碱基。修改此类核酸碱基以产生沉默突变被称为密码子适应过程。
图7是编码构建体2(C02)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸503-658位(灰色框)的密码子以降低与野生型病毒的同源性。质粒从T7启动子转录产生C02复制子RNA。C02的5'UTR、nsP1-4 ORF和SGP如SEQ ID NO：21所示。
图8是编码构建体6(C06)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸658-1620位(灰色框)的密码子以降低与野生型病毒的同源性。质粒从T7启动子转录产生C06复制子RNA。C06的5'UTR、nsP1-4 ORF和SGP如SEQ ID NO：25所示。
图9是编码构建体5(C05)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸1620-2560位(灰色框)的密码子以降低与野生型病毒的同源性。质粒从T7启动子转录产生C05复制子RNA。C05的5'UTR、nsP1-4 ORF和SGP如SEQ ID NO：24所示。
图10是编码构建体3(C03)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸3694-3954位(灰色框)的密码子以降低与野生型病毒的同源性。质粒从T7启动子转录产生C03复制子RNA。C03的5'UTR、nsP1-4 ORF和SGP如SEQ ID NO：22所示。
图11是编码构建体7(C07)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸2560-3954位(灰色框)的密码子以降低与野生型病毒的同源性。质粒从T7启动子转录产生C07复制子RNA。C07的5'UTR、nsP1-4 ORF和SGP如SEQ ID NO：26所示。
图12是编码构建体4(C04)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸3954-4120位(灰色框)的密码子以降低与野生型病毒的同源性。质粒从T7启动子转录产生C04复制子RNA。C04的5'UTR、nsP1-4 ORF和SGP如SEQ ID NO：23所示。
图13是编码构建体1(C01)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸6381-7083位(灰色框)的密码子以降低与野生型病毒的同源性。质粒从T7启动子转录产生C01复制子RNA。C01的5'UTR、nsP1-4 ORF和SGP如SEQ ID NO：20所示。
图14是编码构建体9(C09)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸658-1620位和2560-3954位(灰色框)的密码子以降低与野生型病毒的同源性。质粒从T7启动子转录产生C09复制子RNA。C09的5'UTR、nsP1-4 ORF和SGP如SEQ ID NO：28所示。
图15是编码构建体10(C10)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸3694-3954位和6381-7083位(灰色框)的密码子以降低与野生型病毒的同源性。质粒从T7启动子转录产生C10复制子RNA。C10的5’UTR、nsP1-4 ORF和SGP如SEQ ID NO 29所示。
图16是编码构建体11(C11)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5所示类似，不同之处仅在于修改了基因组核苷酸503-658位、658-1620位、1620-2560位、2560-3954位、3954-4120位和6381-7083位(灰色框)的密码子以降低与野生型病毒的同源性。质粒从T7启动子转录产生C11复制子RNA。C11的5’UTR、nsP1-4 ORF和SGP如SEQID NO：30所示。
图17A-17D示出了SEAP表达测试的结果，以证明存在于密码子适应复制子RNA中的编码SEAP的SG ORF的功能，并且通过比色试验定量SEAP浓度。
图17A是示出了SEAP在C01、C02、C03、C04、C05、C06、C07和C08复制子RNA上的表达测试结果与野生型(WT，类似编码SEAP的非密码子适应的复制子RNA)对比的柱状图，该测试是通过对转染所示时间的BHK细胞的条件上清液取样来进行的，转染时间为4小时(左侧柱)、16小时(中间柱)和24小时(右侧柱)。
图17B和17C是示出了SEAP表达测试结果的柱状图，该测试通过对用指定复制子RNA(图17B-C09，图17C-C10)转染16小时的BHK细胞的条件上清液取样并通过比色法在650nm波长处测量吸光度来定量SEAP表达。据观察，密码子适应构建体的表达比野生型(WT)对照更强。
图17D是示出了构建体C03、C05和C06与对照野生型RNA复制子(VEEVrep-SEAP；WT)和未处理(Untransf.)小鼠相比的通过发光试验(RLU)定量的SEAP表达结果的柱状散点图。在治疗中，每组四只小鼠注射1μg所示RNA，并于第二天收集血清。通过发光试验以相对发光单位(RLU)测量血清SEAP浓度。柱状图：带有误差条的组的平均RLU＝S.D。
图18是VEEV基因组的比例图，其中通过本发明所述的RNA折叠分析和实验确定的非结构多蛋白区域被标记为灰色框，其富集于易于沉默突变的非结构区域。
图19是示出图4所示的为实验而修改的nsP4复制子序列的一个示例的示意图，其中附加到nsP4蛋白的外源蛋白(xP)是绿色荧光蛋白(GFP；灰色框)。在此图中，SG ORF中的结构多蛋白被报告基因SEAP取代。此复制子RNA被称为VEEVrep-nsP4[GFP]-SEAP，RNA序列如SEQ ID NO：31所示。
图20A-20C示出了体外细胞中由工程化的经nsP4修饰的VEEV复制子介导的基因表达。BHK是用在非结构蛋白ORF中编码GFP和在SG ORF的SEAP编码SEAP的实验性经nsP4修饰的复制子(VEEVrep-nsP4[GFP]-SEAP)转染的细胞。
图20A示出了荧光显微镜检查的结果，证明当用nsP4(GFP)修饰的VEEV复制子RNA(VEEVrep-nsP4[GFP]-SEAP)进行转染时存在多个GFP阳性细胞，并且在未转染的对照细胞中不存在GFP表达。
图20B是用GFP特异性单克隆抗体转染的BHK细胞裂解物的免疫印迹照片，其中GAPDFI检测物用作上样对照，其确认了VEEVrep-nsP4[GFP]-SEAP的两个独立分离的克隆对GFP的表达。
图20C是示出通过对转染的BHK细胞的条件上清液取样并通过比色试验定量SEAP表达来测试编码SEAP的SG ORF的功能的柱状图。
图21A-21B示出了由工程化的经nsP4修饰的VEEV复制子介导的基因表达。此复制子RNA结合了如本发明的实施方式中所描述的密码子适应和nsP4 C末端修饰。
图21A是显示荧光显微镜检查结果的照片，示出了与明场(BF)显微镜(左图)相比，当用密码子适应的、经nsP4(GFP)-修饰的VEEV-SEAP复制子RNA(C13)进行转染时存在多个GFP阳性细胞(上图，右)，且在未转染的对照细胞(下图，右)中不存在GFP表达。
图21B是示出了发光试验(RLU)结果的图表，该发光试验与从未注射的对照小鼠收集的血清进行比较，对从注射了野生型VEEV-SEAP复制子RNA的小鼠(WT)或注射了在非结构化多蛋白基因的非结构区中携带优化密码子的经nsP4(GFP)-修饰的VEEV复制子RNA的小鼠(经修饰的)收集的血清中的SEAP浓度进行定量。血清于注射后第1天、第3天和第5天收集。
图22A-22B示出了经修饰以编码小核糖核酸病毒P1结构多蛋白(其在SG ORF中编码)所需的3Cpro蛋白酶的甲病毒复制子RNA的基因表达。
图22A是示出了对甲病毒复制子的nsP4编码序列进行修饰以编码小核糖核酸病毒P1结构多蛋白(O1 Manisa P1多蛋白)所需的3Cpro蛋白酶的构建体的示意图，称为VEEVrep-nsP4[3C]-P1。此RNA的5’UTR、nsP1-4、3Cpro和SGP区域的序列如SEQ ID NO：32所示。在SG ORF中编码的P1多蛋白的氨基酸序列如SEQ ID NO：40所示。
图22B是对用以下RNA转染的BHK细胞裂解物进行的免疫印迹的照片：泳道1，VEEVrep-nsP4[3C]-P1；泳道2，复制子RNA与泳道1相似，但携带插入突变，引入提前终止密码子并切除3Cpro多肽的表达，作为阴性对照(VEEVrep-nsP4[3Cmut]-P1)；泳道3，VEEVrep-P1(类似于VEEVrep-nsP4[3C]-P1，但缺少nsP4融合的3Cpro)；泳道4，将VEEVrep-P1与1/40质量的编码3Cpro的mRNA组合转染，作为阳性对照。参考该图，只有在VEEVrep-nsP4[3C]-P1的介导下，才能将～100kDa的P1多蛋白成功蛋白水解加工成免疫原性～35kDa的VP0衣壳片段。可以看到代表完整VP4+VP2+VP3的不完全裂解中间产物，其表观分子量为～55kDa。使用VP2特异性抗体来探测印迹。M，分子量参照标记；C，未经处理的阴性对照细胞。
图23A-23D示出了经修饰以编码人STING蛋白的甲病毒复制子RNA的基因表达数据，该甲病毒复制子RNA被突变为对IFN刺激活性具有组成型活性，并在SG ORF中编码报告基因SEAP。
图23A是构建体VEEVrep-nsP4[STING]-SEAP的示意图，该构建体是包含编码人STING蛋白的编码序列修饰的复制子RNA，其被突变为对IFN刺激活性具有组成型活性，并且在SG ORF中编码报告基因SEAP。此RNA的5'UTR、nsP1-4、STING和SGP区域的序列如SEQ IDNO：34所示。
图23B是在用以下RNA转染的HEK-LuciaTM空白细胞上进行的免疫印迹的照片：泳道1，VEEVrep-SEAP；泳道2，VEEVrep-nsP4[STING]-SEAP；泳道3，无转染对照。
图23C是示出与对照(未转染细胞)相比，在VEEVrep-SEAP和VEEVrep-nsP4[STING]-SEAP复制子RNA中的SG ORF中编码的SEAP报告基因的表达结果(通过比色试验中650nm处的吸光度测量)的柱状图。
图23D是示出与对照(未转细胞)相比由VEEVrep-SEAP SEAP和VEEVrep-nsP4[STING]-SEAP复制子RNA诱导的IFN信号传导活性的替代测量结果的柱状图。
图24A-24D示出了在B16黑色素瘤中来自经修饰以编码人STING蛋白的甲病毒复制子RNA的基因表达结果，该甲病毒复制子RNA被突变为对IFN刺激活性具有组成型活性，并且在SG ORF中编码报告基因SEAP。
图24A是示出与未转染的对照细胞相比，用1μg VEEVrep-nsP4[GFP]-SEAP复制子RNA转染后约1天，在12孔培养皿培养的B16黑色素瘤细胞中的GFP表达结果的照片。
图24B是示出与未转染的对照细胞相比，在用1μg指定复制子RNA：VEEVrep-nsP4[STING]-SEAP、VEEVrep-nsP4[GFP]-SEAP和VEEVrep-SEAP转染后，在12孔培养皿中培养的B16黑素瘤细胞中SEAP表达结果的柱状图。
图24C是示出与未转染的对照细胞相比，在用1μg指定复制子RNA：VEEVrep-nsP4[STING]-SEAP和VEEVrep-SEAP转染～1天后培养的B16黑色素瘤细胞(通过ELISA夹心法测定培养上清)的IFN-β分泌测量结果的柱状图。
图24D是与健康未转染的对照细胞(最左图)相比，用2μg VEEVrep-nsP4[GFP]-SEAP或VEEVrep-nsP4[STING]-SEAP转染1天后B16细胞的光学显微镜检查结果的照片。
图25A-25D示出了在TC-1癌细胞中来自经修饰以编码人STING蛋白的甲病毒复制子RNA的基因表达，该甲病毒复制子RNA被突变为对IFN刺激活性具有组成型活性，并在SGORF中编码报告基因SEAP。
图25A是示出与未转染的对照细胞相比，用2μg指定复制子RNA：VEEVrep-nsP4[GFP]-SEAP、VEEVrep-nsP4[STING]-SEAP转染2天后，在12孔培养皿中培养的TC-1癌细胞中SEAP表达结果的柱状图。在本实验中，通过培养基的比色试验对SEAP表达进行定量，显示在SG ORF中编码的SEAP基因的成功表达。
图25B是示出与未转染的对照细胞相比，用2μg指定复制子RNA：VEEVrep-nsP4[GFP]-SEAP或VEEVrep-nsP4[STING]-SEAP转染1天后，培养的TC-1癌细胞(通过ELISA夹心法测定培养上清)的IFN-β分泌测量结果的柱状图。
图25C是示出用1μg VEEVrep-nsP4[GFP]-SEAP转染后约3天，转染后的TC-1癌细胞团聚物中GFP表达结果的照片。参考此图，与未处理的对照细胞中没有GFP表达(下图，右)相比，通过在构建体nsP4 C末端编码荧光蛋白，在整个多细胞结构中观察到大量GFP表达。
图25D是示出与未处理的对照细胞相比，用4μg指定复制子RNA VEEVrep-nsP4[STING]-SEAP转染1天后，TC-1癌细胞的光学显微镜检查结果的照片。参考此图，观察到未处理的细胞(对照)已增殖并填满培养皿；转染VEEVrep-nsP4[STING]-SEAP后观察到生长抑制和细胞死亡。
图26A-26G示出了经修饰与野生型同源性降低的VEEV复制子RNA以及来自这些构建体的表达数据。
图26A是编码构建体12(VEEVrepHK-SEAP)的复制子序列模板的DNA质粒的示意图。参考此图，DNA质粒与图5所示的类似，不同之处仅在于修改了基因组核苷酸705-3406位(灰色框)的密码子以降低与野生型病毒的同源性，而不考虑可能存在的任何二级结构元件。
图26B是编码构建体13(VEEVrepHK(ΔU)-SEAP)的复制子序列模板的DNA质粒的示意图。参考此图，DNA质粒与图58中所示的类似，不同之处仅在于修改了基因组核苷酸658-3359位(灰色框)的密码子以降低与野生型病毒的同源性，而不考虑可能存在的任何二级结构元件，并且优先选择规避尿嘧啶碱基的密码子。
图26C是编码构建体14(VEEVrepES-SEAP)的复制子序列模板的DNA质粒的示意图。参考此图，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸4120-6965位(灰色框)的密码子以降低与野生型病毒的同源性，而不考虑可能存在的任何二级结构元件。
图26D是编码构建体15(VEEVrepES(ΔU)-SEAP)的复制子序列模板的DNA质粒的示意图。参考此图，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸4120-6965位(灰色框)的密码子以降低与野生型病毒的同源性，而不考虑可能存在的任何二级结构元件，并且优先选择规避尿嘧啶核苷碱基的密码子。
图26E是示出与未转染的对照细胞相比，在用1μgVEEVrep SEAP、VEEVrepHK SEAP或VEEVrepHK(ΔU)-SEAP RNA转染16小时后，在12孔培养皿中培养的BHK细胞中SEAP表达结果的柱状图。在本实验中，通过对培养基的比色试验来定量SEAP的表达，其显示在SG ORF中编码的SEAP基因的成功表达。
图26F是示出与未转染的对照相比，在用1μg VEEVrep-SEAP、VEEVrepES-SEAP或VEEVrepES(ΔU)-SEAP RNA转染16小时后，在12孔培养皿中培养的BHK细胞中SEAP表达结果的柱状图。
图26G是示出与对照(来自未处理的小鼠的血清)相比，注射2μg LNP配制的VEEVrep-SEAP、VEEVrepHK-SEAP或VEEVrepHK(ΔU)-SEAP后(通过化学发光试验)测量的血清中SEAP体内表达结果的柱状图。
图27A-27D示出了正常HFF细胞中的VEEV复制子RNA表达结果。
图27A是示出与未转染的对照细胞相比，在用2或4μg指定复制子RNA(VEEVrep-nsP4[STING]-SEAP、VEEVrep-SEAP)转染后1天，在12孔培养皿中培养的正常HFF细胞中SEAP表达结果的柱状图。在此实验中，通过对培养基的比色试验来定量SEAP的表达，其显示在SGORF中编码的SEAP基因的成功表达。
图27B是用4μg VEEVrep-nsP4[GFP]-SEAP转染后约4天，胰蛋白酶解离的正常人HFF细胞中GFP表达结果的照片，其显示来自nsP4 C末端编码的GFP蛋白在这些健康的非癌细胞中没有有效的基因表达。
图27C是示出来自经转染的HFF的IFN-β活性的测量结果的柱状图。在转染后1天收集来自12孔培养皿中的HFF的条件培养基，与未转染的HFF细胞对照，以及无培养基对照进行比较，转染指定RNA(VEEVrep-nsP4[STING]-SEAP、VEEVrep-nsP4[GFP]-SEAP、VEEVrep-SEAP)的条件培养基应用于HEK-Lucia空白细胞以测量其对培养基中I型IFN的应答。在本实验中，通过化学发光试验在第二天检测上清液中的IFN应答报告基因Lucia。
图27D是与未转染的对照相比，用4μg指定复制子RNA(VEEVrep-nsP4[GFP]-SEAP和VEEVrep-nsP4[STING]-SEAP)转染后4天HFF细胞的光学显微镜检查结果的照片。
具体实施方式
除非另有明确说明，否则权利要求书和说明书的相应部分中使用的词语“一种(a)”和“一个(one)”被定义为包括一个或多个所引用的项目。此术语包括上述具体提及的词语、其派生词以及类似含义的词语。短语“至少一个”后跟两个或更多个项目的列表，例如“A、B或C”，是指A、B或C中的任何单独一个及其任何组合。
甲病毒
如本发明所用，“甲病毒”是指披膜病毒科(Togaviridae)家族的包膜单链正义RNA病毒。甲病毒属包含大约30个成员，其可以感染人类和其他动物。甲病毒颗粒的直径通常为70nm，往往呈球形或略带多形性，并具有40nm等距核衣壳(isometric nucleocapsid)。
甲病毒基因组是大约12000nt长的ssRNA分子，其编码两个开放阅读框(ORF)，即非结构性(ns)和结构性(本发明也称为亚基因组或SG，ORF)。ns ORF从基因组5’端附近开始，占基因组总长度的前三分之二。它编码非结构蛋白(nsP)，即nsP1、nsP2、nsP3和nsP4，这些蛋白作为单一多蛋白前体产生，通过蛋白水解加工裂解成成熟蛋白。nsP通过细胞质中典型的帽结构依赖性翻译(cap-dependent translation)来表达。感染后，甲病毒基因组RNA被翻译成nsP多蛋白，该多蛋白被裂解为四种蛋白：nsP1、nsP2、nsP3和nsP4(图1)，它们共同作用以催化基因组复制和第二个甲病毒ORF的转录/翻译。nsP1的大小约为60kDa且可能具有甲基转移酶活性，并参与病毒加帽反应(viral capping reaction)。nsP2的大小约为90kDa且可能具有解旋酶和蛋白酶活性，而nsP3约为60kDa并包含三个结构域：宏结构域、中央(或甲病毒属独具的)结构域和高变结构域(HVD)。nsP4大小约为70kDa并包含核心RNA依赖性RNA聚合酶(RdRp)催化结构域。
结构性的SG ORF大约占基因组长度的三分之一，本质上，此ORF编码病毒体结构蛋白：核心核衣壳蛋白C，以及作为异二聚体结合的包膜蛋白P62和E1。病毒包膜锚着的表面糖蛋白负责受体识别并通过膜融合进入靶细胞。
直到nsP产物介导基因组的RNA依赖性RNA转录以产生互补的负义链(-RNA)模板后，才会发生此SG ORF的翻译。nsP产物从-RNA模板的3’端开始完成全长转录以重建原始基因组序列，从而完成基因组复制的循环。基因组复制的开始代表了从病毒复制生命周期的“早期”阶段到“晚期”阶段的转变。由于基因组复制是通过构成了病原体相关分子模式(PAMP)的dsRNA中间体的产生而发生的，因此细胞内模式识别受体(PRR)会触发强烈的先天免疫反应。从-RNA模板，nsP产物还介导来自内部RNA依赖性RNA聚合酶启动子(“亚基因组启动子”或SGP)的转录，该启动子映射到(当从原始有义链RNA基因组的角度读取时)紧邻第二个ORF上游的遗传区域以产生仅编码第二个ORF的较小的或“亚基因组的”(SG)mRNA。SGmRNA的转录比全长基因组的转录更有利，其导致感染细胞中SG mRNA拷贝数高，并因此其中编码的结构基因的产量也在高水平。
如本发明所用，RNA复制子是指从甲病毒属(披膜病毒科家族)基因组而来的序列衍生的核酸分子，其在引入合适的细胞内环境时能够自我复制。由于其自我复制，RNA复制子可以大幅放大所编码蛋白质的产生，从而导致所需蛋白质在体内持续翻译。术语“复制子RNA"和"RNA复制子”在本文中可互换使用以指代这些RNA分子。
在一种实施方式中，RNA复制子可以是源自甲病毒属物种的RNA复制子。甲病毒属物种可包括但不限于东部马脑炎病毒(Eastern equine encephalitis virus，EEEV)、委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus，VEEV)、大沼泽地病毒(Everglades virus，EVEV)、穆坎博热病毒(Mucambo virus，MUCV)、塞姆利基森林病毒(Semliki forest virus，SFV)、皮苏纳病毒(Pixuna virus，PIXV)、米德尔堡病毒(Middleburg virus，MIDV)、基孔肯雅病毒(Chikungunya virus，CHIKV)、奥尼永-尼永病毒(O′Nyong-Nyong virus，ONNV)、罗斯河病毒(Ross River virus，RRV)、巴尔马森林病毒(Barmah Forest virus，BF)、盖塔病毒(Getah virus，GET)、鹭山病毒(Sagiyama virus，SAGV)、贝巴鲁病毒(Bebaru virus，BEBV)、马亚罗病毒(Mayaro virus，MAYV)、乌纳病毒(Una virus，UNAV)、辛德比斯病毒(Sindbis virus，SINV)、奥拉病毒(Aura virus，AURAV)、沃塔罗阿病毒(Whataroa virus，WHAV)、巴班基病毒(Babanki virus，BABV)、孜拉加奇病毒(Kyzylagach virus，KYZV)、西部马脑炎病毒(Western equine encephalitis virus，WEEV)、高地J病毒(Highland J virus，HJV)、摩根堡病毒(Fort Morgan virus，FMV)、恩杜茂病毒(Ndumu，NDUV)和虫溪病毒(Buggy Creek virus)。
在一种实施方式中，RNA复制子可以是自通过自然选择产生的RNA病毒基因组衍生的复制子。
在一种实施方式中，本发明公开的RNA复制子可以是工程化的、合成的或重组的RNA复制子。RNA复制子可以在体外合成或修饰。修饰可以包括但不限于以下程序：进行化学或酶促技术处理，例如通过使用化学核酸合成、使用用于复制、聚合、核酸外切消化、核酸内切消化、连接、逆转录、转录、碱基修饰(例如甲基化)或核酸分子的重组的酶。RNA复制子可以通过使用合理的设计方法来工程化，该方法选择性地保持关键序列元件、附加功能并减少与危险的、流行的病毒的同源性。RNA复制子可以与在自然界中不结合的核苷酸序列结合。可以使用分子克隆技术来操作RNA复制子，使得其相对于天然存在的核苷酸序列具有一种或多种序列改变或重排。
基于甲病毒的RNA复制子可用作体外和体内基因表达的载体。甲病毒的结构基因在可以用所需的重组蛋白替换的单个亚基因组(SG)开放阅读框(ORF)中编码。非结构(nsP)编码序列位于单独的前一个ORF中，其携带必要的生化功能，可介导细胞内复制子RNA的复制以及SG ORF的翻译。当nsP编码序列与流行的甲病毒序列几乎相同时，会产生在现场与病原病毒基因组重组的可能性，从而存在安全风险。为了防止与病原病毒序列重组，也可以修饰nsP序列。nsP的修饰具有挑战性，因为修改一级氨基酸序列可能会影响生化功能，而且修改一级核苷酸序列可能会破坏复制子翻译和转录所需的关键二级结构。
合成的甲病毒衍生复制子核酸分子
在一种实施方式中，提供了合成的甲病毒衍生复制子核酸分子。合成的甲病毒衍生复制子核酸分子可包含第一核酸，其编码甲病毒非结构蛋白nsP1、nsP2、nsP3和nsP4。第一核酸可包含从5’非翻译区(UTR)的起点到亚基因组启动子(SGP)的末端范围内的序列。第一核酸可包含在如SEQ ID NO：17所示的甲病毒基因组序列中从nt 503至nt 658、nt 658至nt 1620、nt 1620至nt 2560、nt 2560至nt 3954、nt 3954至nt 4120、nt 6381至nt 7083和nt 6966至nt 7526的区域内的任意位置处引入的至少一个沉默突变。合成的甲病毒衍生复制子核酸分子还可以包含第二核酸序列，其包含经过修饰的亚基因组(SG)开放阅读框(ORF)。经过修饰的SG ORF可以包含编码第一异源蛋白的序列。第一核酸序列可包含与nsP4C末端融合编码的第二异源蛋白。nsP4编码序列可以含有在从nt 6966至nt 7526的区域内的任意位置处引入的至少一个沉默突变。
如本发明所用，术语“异源蛋白”是指任意在生物体中非天然存在的蛋白质。
如本发明所用，术语“沉默突变”是指不修改所编码蛋白质的氨基酸序列的核苷酸碱基序列的改变。例如，沉默突变可以是将第一密码子改变为编码相同氨基酸的第二密码子的点突变。沉默突变可包括一个或多于一个核苷酸的改变。
如本发明所用，术语“密码子优化”是指用于通过适应宿主生物体的密码子偏好来改善基因表达并增加感兴趣基因的翻译效率的过程。
如本发明所用，术语“密码子适应”是指用于通过沉默突变修改密码子的过程，其经过选择从而不会影响通过RNA折叠的生物信息学分析确定的二级结构基序(motif)。
在一种实施方式中，第一核酸可包含在甲病毒基因组中从nt 503至nt 658的区域内的任意位置处引入的至少一个沉默突变。如本发明所用，给出的所有基因组位置均参考委内瑞拉马脑炎病毒的特立尼达驴病毒株，其完整基因组参考序列L01442见NCBI GenBank数据库(SEQ ID NO：17；Kinney,R.M.，Johnson,B.J.，Brown,V.L.and Trent,D.W.1986，Nucleofide sequence of the 26S mRNA of the virulent Trinidad donkey strain ofVenezuelan equine encephalitis virus and deduced sequence of the encodedstructural proteins，Virology152(2)，400-413，其通过引用并入本发明，如同已完全阐述)。
携带此沉默突变的第一核酸可以包含编码nsP1的多核苷酸，并且包含与如SEQ IDNO：21所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98、99％或100％的同一性的序列。
在一种实施方式中，第一核酸可以包含在如SEQ ID NO：17所示的甲病毒基因组序列中从nt 658至nt 1620的区域内的任意位置处引入的至少一个沉默突变的序列。携带此沉默突变的第一核酸可以包含编码nsP1的多核苷酸，并且包含与如SEQ ID NO：25所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列。
在一种实施方式中，第一核酸可以包含在甲病毒基因组序列(SEQ ID NO：17)中从nt 1620至nt 2560的区域内的任意位置处引入的至少一个沉默突变的序列。携带沉默突变的第一核酸可以包含编码nsPl和nsP2的接合物(junction)，并且包含与如SEQ ID NO：24所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列。
在一种实施方式中，第一核酸可以包含在甲病毒基因组序列(SEQ ID NO：17)中从nt 3694至nt 3954的区域内的任意位置处引入的至少一个沉默突变的序列。包含沉默突变的第一核酸可以包含编码nsPl和nsP2的接合物的多核苷酸，并且包含与如SEQ ID NO：22所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列。
在一种实施方式中，第一核酸可以包含在甲病毒基因组序列(SEQ ID NO：17)中从nt2560到nt 3954的区域内的任意位置处引入的至少一个沉默突变的序列。包含沉默突变的核酸可包含编码nsP1和nsP2的接合物的多核苷酸，并且包含与如SEQ ID NO：26所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列。
在一种实施方式中，第一核酸可以包含在甲病毒基因组序列(SEQ ID NO：17)中从nt 3954至nt 4120的区域内的任意位置引入的至少一个沉默突变的序列。包含沉默突变的第一核酸可包含编码nsP2和nsP3的接合物的多核苷酸，并且包含与如SEQ ID NO：23所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列。
在一种实施方式中，第一核酸可以包含在甲病毒基因组序列(SEQ ID NO：17)中从nt 6381到nt 7083的区域内的任意位置处引入的至少一个沉默突变的序列。包含沉默突变的第一核酸可包含编码nsP4的多核苷酸，并且包含具有与如SEQ ID NO：20所示的参考序列至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列。
在一种实施方式中，第一核酸序列可以包含在甲病毒基因组序列(SEQ ID NO：17)中从nt 6966到nt 7526的区域内的任意位置处引入的至少一个沉默突变的序列。包含沉默突变的第一核酸可包含编码nsP4的C末端区域的多核苷酸，并且包含与如SEQ ID NO：18或19所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列。
在一种实施方式中，第一核酸可以包含在基因组的一个以上区域中具有沉默突变的序列。该核酸可包含在甲病毒基因组序列(SEQ ID NO：17)中从nt 658至nt 1620的区域内和nt 2560至nt 3954的区域内的任意位置处引入的一个以上沉默突变。包含沉默突变的第一核酸可包含编码nsP1的多核苷酸，并且包含与如SEQ ID NO：25所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列，以及，包含编码nsP2的多核苷酸，并且包含与如SEQ ID NO：26所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列。
在一种实施方式中，第一核酸可以包含在甲病毒基因组序列(SEQ ID NO：17)中从nt3694至nt 3954的区域内和nt 6381至nt 7083的区域内的任意位置处引入的一个以上沉默突变的序列。包含这些沉默突变的第一核酸可以包含编码nsP2的多核苷酸，并且包含与如SEQ ID NO：22所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列，以及，包含编码nsP4的多核苷酸，并且包含与如SEQ ID NO：20所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列。
在一种实施方式中，第一核酸可以包含在甲病毒基因组序列(SEQ ID NO：17)中，在从nt 503到nt 658的区域内、在从nt 658到nt 1620的区域内、在从nt 1620到nt 2560的区域内、在从nt 2560至nt 3954的区域内、在从nt 3954至nt 4120的区域内以及在从nt6381至nt 7083的区域内的任意位置处引入的一个以上沉默突变的序列。包含这些沉默突变的第一核酸可包含编码nsP1的多核苷酸，并且包含具有与如SEQ ID NO：21所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列；编码nsP1的多核苷酸，并且包含与如SEQ ID NO：25所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列；编码nsP1和nsP2的接合物的多核苷酸，并且包含与如SEQ ID NO：24所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列；编码nsP2的多核苷酸，并且包含与如SEQ ID NO：26所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列；编码nsP2和nsP3的接合物的多核苷酸，并且包含与如SEQ ID NO：23所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列；以及，编码nsP4的多核苷酸，并且包含与如SEQ ID NO：20所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列。包括这些沉默突变的第一核酸可以包含编码非结构蛋白的多核苷酸，其包含与如SEQ ID NO：30所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的序列。
确定两个氨基酸序列或两个核酸序列的百分比同一性可以包括调整和比较两个序列中相应位置处的氨基酸残基或核苷酸。如果两个序列中的所有位置都被相同的氨基酸残基或核苷酸占据，则称该序列100％同一。百分比同一性通过Smith Waterman算法测量(Smith TF,Waterman MS1981“Identification of Common Molecular Subsequences，”JMol Biol 147：195-197，其通过引用并入本文，如同已完全阐述)。
在一种实施方式中，第一核酸可以包含表1中列出的甲病毒之一的nsP区域的序列，其中nsP1编码序列中的一个以上的沉默突变在在第85位氨基酸之后的位置处产生，使得10-30％的核苷酸与野生型nsP1编码序列不同。
表1甲病毒及其对应的可以如本发明所述通过沉默突变进行修饰以降低与其同源的野生型基因组的同源性的nsP区域。
在一种实施方式中，第一核酸可以包含表1中列出的甲病毒之一的nsP区域的序列，其中在nsP2编码序列中产生一个以上沉默突变，使得10-30％的核苷酸不同于野生型nsP2编码序列。
在一种实施方式中，第一核酸可以包含表1中列出的甲病毒之一的nsP区域的序列，其中在nsP3编码序列中产生一个以上沉默突变，使得10-30％的核苷酸不同于野生型nsP3编码序列。
在一种实施方式中，第一核酸可以包含表1中列出的甲病毒之一的nsP区域的序列，其中在该nsP的最后30个氨基酸之前的位置产生nsP4编码序列中的一个以上沉默突变，使得10-30％的核苷酸与野生型nsP4编码序列不同。
在一种实施方式中，第一核酸可以包含表1中列出的甲病毒之一的nsP区域的序列，其中在该nsP的最后30个氨基酸内的位置处产生nsP4编码序列中的一个以上沉默突变，使得10-30％的核苷酸与野生型nsP4编码序列不同并降低与内源SGP的同源性。
在一种实施方式中，基于表1中的物种之一的甲病毒属衍生复制子核酸分子可包含第二核酸，其包含编码第一异源蛋白的多核苷酸。第一异源蛋白可以是替代由甲病毒亚基因组(SG)ORF编码的甲病毒结构蛋白的蛋白质。
在一种实施方式中，甲病毒衍生复制子核酸分子可包含编码第二异源蛋白的多核苷酸。第二异源蛋白可以是插入经修改的nsP1、nsP2、nsP3或nsP4蛋白中的蛋白质。
在一种实施方式中，经修改的nsP蛋白可以是经修改的nsP4蛋白。经修改的nsP4可以包含与其C末端区域融合的第二异源蛋白。编码异源C末端区域的多核苷酸序列可以在其3’末端包含编码甲病毒亚基因组启动子的序列。该序列可以插入nsP4编码序列区域的下游(即3’)并且可以翻译成蛋白质。或者，此插入序列可以不被翻译成蛋白质。插入此区域的非翻译序列可以是短发夹RNA(shRNA)。
在一种实施方式中，经修改的nsP4可以包含GFP蛋白作为第二异源蛋白。经修改的nsP4可包含与如SEQ ID NO：12或41所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％同一性的氨基酸序列。经修改的nsP4可以由SEQ ID NO：31所示的多核苷酸序列编码。经修改的nsP4可包含具有与如SEQ ID NO：15所示的参考序列至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％同一性的氨基酸序列。
(在SG ORF中的)第一异源蛋白或(在nsP ORF中的)第二异源蛋白可以是从病毒病原体分离或衍生的任意抗原蛋白。病毒病原体可选自腺病毒科(Adenoviridae)、乳多孔病毒科(Papovaviridae)、细小病毒科(Parvoviridae)、疱疹病毒科(Herpesviridae)、痘病毒科(Poxviridae)、指环病毒科(Anelloviridae)、多形性包膜病毒病毒科(Pleolipoviridae)、呼肠病毒科(Reoviridae)、小核糖核酸病毒科(Picomaviridae)、杯状病毒科(Caliciviridae)、披膜病毒科(Togaviridae)、沙粒病毒科(Arenaviridae)、黄病毒科(Flaviviridae)、正粘病毒科(Orthomyxoviridae)、副粘液病毒科(Paramyxoviridae)、布尼亚病毒科(Bunyaviridae)、弹状病毒科(Rhabdoviridae)、丝状病毒科(Filoviridae)、冠状病毒科(Coronaviridae)、星状病毒科(Astroviridae)、玻那病毒科(Bomaviridae)、动脉炎病毒科(Arteriviridae)、肝炎病毒科(Hepeviridae)、逆转录病毒科(Retroviridae)、花椰菜病毒科(Caulimoviridae)或肝脱氧核糖核酸病毒科(Hepadnaviridae)的家族成员。例如，病毒病原体可以为腺病毒、鼻病毒、轮状病毒、西尼罗病毒、寨卡病毒、疱疹病毒、丝状病毒或冠状病毒(CoV)。冠状病毒可以为MERS CoV、SARS-CoV和SARS-CoV-2中的一种。
如本发明所用，术语“抗原蛋白”是指包含一种或多种触发免疫应答的抗原决定簇的蛋白质。免疫反应可能涉及抗体的产生，或特定免疫活性细胞的激活，或两者兼而有之。抗原蛋白可以是病原体或癌细胞的结构成分。抗原蛋白可以在宿主中合成、重组产生，或者可以衍生自生物样品，包括但不限于组织样品、细胞或生物液体。
抗原蛋白可以是但不限于寄生虫抗原蛋白、细菌抗原蛋白、肿瘤抗原蛋白、环境抗原蛋白、治疗性抗原蛋白或过敏原。抗原蛋白可以是包含一种或多种本发明所述的病毒病原体的抗原决定簇的蛋白质。
在一种实施方式中，第一异源蛋白或第二异源蛋白可以是酶。该酶可以源自哺乳动物并可调节细胞代谢或细胞信号传导。该酶可以是细胞或病毒蛋白酶。病毒蛋白酶可以是小核糖核酸病毒3C蛋白酶(3Cpro)。如本发明所用，术语“3Cpro”、“3C蛋白酶”、“3Cpro”或"3CP"是指在小核糖核酸病毒物种中发现的半胱氨酸蛋白酶，其其活性位点内通常含有保守的半胱氨酸-组氨酸-天冬氨酸/谷氨酸催化三联体或含有半胱氨酸-组氨酸二联体。催化三联体或二联体通常形成电荷中继网络，极化并激活攻击底物的亲核体(通常为半胱氨酸)，形成共价中间体，然后水解以再生游离酶。3Cpro可在多聚蛋白的结构区和非结构区中进行成熟裂解且对谷氨酰胺-甘氨酸/丝氨酸/丙氨酸/缬氨酸/组氨酸/精氨酸和谷氨酸-丝氨酸/甘氨酸/精氨酸/甲硫氨酸中具有显著的底物偏好。3Cpro蛋白酶可源自口蹄疫病毒(FMDV)。
FMDV 3C蛋白酶是分子量约23.1-kDa并且含有213个氨基酸的半胱氨酸蛋白酶。FMDV 3C蛋白酶活性位点的半胱氨酸-组氨酸-天冬氨酸催化三联体由残基H46、D84和C163形成。
在一种实施方式中，经修改的nsP4可以包含第二异源蛋白，其为3C蛋白酶。经修改的nsP4可包含与如SEQ ID NO：40所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的氨基酸序列。编码包含3C蛋白酶的改变的nsP4多核苷酸序列可包含具有与SEQ ID NO：32中所示参考序列至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性。
在一种实施方式中，合成的甲病毒衍生复制子核酸分子可以进一步包含第一异源蛋白，其为FMDV P1前体多肽或其他感兴趣的多肽。FMDV P1可衍生自FMDV血清变型O1Manisa，并且在本发明中称为01 Manisa P1多蛋白。O1 Manisa P1多蛋白可由包含与如SEQID NO：33所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96、97％、98％、99％或100％的同一性的多核苷酸序列编码。
在一种实施方式中，第一异源蛋白可以是FMDV P1前体多肽(O1 Manisa P1多蛋白)，并且第二异源蛋白可以是3C蛋白酶。
第一或第二异源蛋白可以是病毒衍生的或哺乳动物细胞衍生的细胞代谢、免疫或信号传导功能的调节剂。这种异源蛋白的表达可能会影响细胞抗病毒先天免疫反应，从而增强甲病毒复制子RNA分子的复制。
在一种实施方式中，第二异源蛋白可以是模式识别受体(PRR)蛋白。PRR蛋白可以是STING(干扰素基因刺激因子)蛋白。STING蛋白可以是野生型STING蛋白。包含STING蛋白的经修改的nsP4可包含与如SEQ ID NO：42所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。包含STING蛋白的经修改的nsP4蛋白可以由与如SEQ ID NO：34所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的多核苷酸序列编码。第一异源蛋白可包含抗原蛋白并且第二异源蛋白可包含STING蛋白。
第一异源蛋白或第二异源蛋白可以是适合保护受试者免受疾病侵害的任何免疫原性多肽。所述疾病可以是癌症或由微生物、细菌、原生动物、寄生虫或病毒病原体引起的疾病。
第一异源蛋白可以是与第二异源蛋白相同的蛋白质。
在一种实施方式中，合成的甲病毒衍生复制子核酸分子可以包含翻译中断序列，例如2A序列。翻译中断序列允许蛋白质翻译的中断，并在不一定被蛋白酶识别的位点(例如经修改的nsP4蛋白中的位点)进行有效切割。2A序列可以在编码第二异源蛋白质的序列之前插入到编码经修改的nsP4的核酸序列中。2A序列的插入可以导致通过2A造成的翻译的中断以及不具有N端Met残基的多肽的产生。2A序列可以是源自但不限于明脉扁刺蛾病毒(Thosea asigna virus)(SEQ ID NO：43)、猪疱疹病毒-1(porcine teschovirus-1)(SEQID NO：44)、口蹄疫病毒(SEQ ID NO：45)、A型马鼻炎病毒(equine rhinitis A virus)(SEQID NO：46)或其他小核糖核酸病毒科或Permutotetraviridae科病毒的2A的氨基酸序列。
在一种实施方式中，合成的甲病毒衍生复制子核酸分子可包含指导编码第一异源蛋白和/或第二异源蛋白的序列表达的亚基因组(SGP)启动子。亚基因组启动子可以是源自甲病毒属物种(参见表1)的26S亚基因组启动子。编码异源蛋白的序列也可以处于内部核糖体进入位点(IRES)的控制之下。
本发明所述的合成的甲病毒衍生复制子核酸分子可包含一种或多种经修饰的核苷酸。经修饰的核苷酸可以是5-甲基尿苷、5-甲基胞苷、假尿苷或N6-甲基腺苷。
合成的甲病毒衍生复制子核酸分子可以具有3’poly-A尾巴。
在一种实施方式中，可以对合成的甲病毒衍生复制子核酸分子的RNA序列进行密码子优化以提高翻译效率。RNA分子可以通过本领域已知的任何方法进行修饰以增强稳定性和/或翻译(效率)。RNA分子可以通过添加例如至少30个腺苷残基的polyA尾巴来修饰。RNA分子可以通过用经修饰的核糖核苷酸在5末端进行加帽修饰(例如7-甲基鸟苷帽)，这种修饰可以在RNA合成期间掺入，也可由在RNA转录后通过酶工程的方式加入。
在一种实施方式中，提供了包含编码合成的甲病毒衍生复制子核酸分子的DNA序列的核酸。该核酸可以是例如DNA质粒或线性化DNA质粒的片段。该核酸还可以包含可操作地连接至DNA序列的5’末端的启动子，例如T7启动子。根据本发明公开的内容，核酸可用于使用本领域已知的方法生产本申请的RNA复制子。例如，合成的甲病毒衍生复制子核酸分子可通过体内或体外核酸的转录获得。
在一种实施方式中，DNA序列可以是DNA质粒。DNA质粒可包含编码nsP1并包含与如SEQ ID NO：2所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸。
DNA质粒可包含编码nsP1并且包含与如SEQ ID NO：6所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸。
DNA质粒可包含编码nsP1和nsP2接合物并且包含与如SEQ ID NO：5所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸。
DNA质粒可包含编码nsP1和nsP2接合物并且包含与如SEQ ID NO：3所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸。
DNA质粒可包含编码nsPl和nsP2接合物并且包含与如SEQ ID NO：7所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸。
DNA质粒可包含编码nsP2和nsP3接合物并且包含与如SEQ ID NO：4所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸。
DNA质粒可包含编码nsP4并包含与如SEQ ID NO：1所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸。
DNA质粒可包含编码nsP1，并且包含与如SEQ ID NO：6所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸，以及编码nsP2，并且包含与如SEQ ID NO：7所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸。
DNA质粒可包含编码nsP2，并且包含与如SEQ ID NO：3所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸，以及编码nsP4，并且包含与如SEQ ID NO：1所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸。
DNA质粒可包含编码nsP1，并且包含与如SEQ ID NO：2所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸；编码nsP1，并且包含与如SEQ ID NO：6所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸，编码nsP1和nsP2的接合物，并且包含与如SEQ ID NO：5所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸，编码nsP2，并且包含与如SEQ ID NO：7所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸；编码nsP2和nsP3接合物，并且包含与如SEQ ID NO：4所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸；以及编码nsP4，并且包含与如SEQ ID NO：1所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列的多核苷酸。包括这些沉默突变的第一核酸可以包含编码非结构蛋白的多核苷酸，其包含与如SEQ ID NO：11所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的序列。
在一种实施方式中，包含编码甲病毒衍生复制子核酸分子的DNA序列的核酸可以含有包含编码第一异源蛋白的多核苷酸的第二核酸。第一异源蛋白可以是替代由甲病毒SGORF编码的甲病毒结构蛋白的蛋白质。
在一种实施方式中，核酸可以包含编码第二异源蛋白的多核苷酸。第二异源蛋白可以是插入到经修改的nsP1、nsP2、nsP3或nsP4蛋白中的蛋白质。
在一种实施方式中，经修改的nsP蛋白可以是经修改的nsP4蛋白。经修改的nsP4可以包含第二异源蛋白。编码经修改的nsP4的多核苷酸可包含编码第一C末端区域的多核苷酸序列和编码第二C末端区域的多核苷酸序列。编码第二C末端区域的多核苷酸序列可包含编码甲病毒亚基因组启动子的序列。编码亚基因组启动子的序列可以与如SEQ ID NO：14所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性。
在一种实施方式中，经修改的nsP4可以包含GFP蛋白作为第二异源蛋白。经修改的nsP4可包含与如SEQ ID NO：12所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。经修改的nsP4可以由如SEQ ID NO：13所示的多核苷酸序列编码。经修改的nsP4可包含与如SEQ ID NO：15所示的参考序列具有至少70％、72％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性的氨基酸序列。经修改的nsP4可以由如SEQ ID NO：16所示的多核苷酸序列编码。
甲病毒衍生RNA复制子表达系统
在一种实施方式中，提供了甲病毒衍的RNA复制子表达系统。甲病毒衍生RNA复制子表达系统可包含本发明所述的任意一种合成的甲病毒衍生复制子核酸分子以及一个或多个宿主细胞(a host cell or cells)。
宿主细胞可以是但不限于仓鼠卵巢(CHO)细胞、肿瘤细胞系、BHK细胞、人细胞系，例如HEK293细胞、PER.C6细胞。宿主细胞可以为酵母、真菌、昆虫细胞、动物或植物细胞。可以使用来自人类或任意哺乳动物物种的未转化的原代正常细胞，例如成纤维细胞、干细胞或者淋巴或髓系细胞。
在一种实施方式中，在宿主细胞中第一异源蛋白和/或第二异源蛋白的生产包括合成的甲病毒衍生的复制子核酸分子的引入，所述复制子核酸分子包含编码将在宿主细胞中表达的第一异源蛋白质和/或第二异源蛋白质的核酸序列，在适合核酸分子表达的条件下培养宿主细胞并允许第一异源蛋白和/或第二异源蛋白在宿主细胞中表达。
为了在宿主细胞中表达，包含编码第一异源蛋白和/或第二异源蛋白的核酸序列的合成的甲病毒衍生复制子核酸分子可以包含在表达盒中，所述表达盒包含核酸序列的表达所需的或促进表达的调控序列。调节序列可包括启动子、增强子、启动子和/或多聚腺苷酸化信号分子等。本领域已知的各种启动子可用于在宿主细胞中表达本发明公开的核酸。启动子可以是组成型或诱导型启动子。启动子可以源自原核或真核生物，或者可以人工设计。宿主细胞可以在可商购的合适用于培养表达第一异源蛋白和/或第二异源蛋白的细胞的培养基中培养。
一种实施方式提供了含有合成的甲病毒衍生复制子核酸分子的宿主细胞，所述复制子核酸分子包含编码3C蛋白酶的核酸序列。宿主细胞还可以包含编码FMDV P1前体多肽或其他目的多肽的一种或多种多核苷酸构建体。
生产至少一种异源蛋白的方法
在一种实施方式中，提供了在细胞中生产至少一种异源蛋白的方法。该方法可以包括在细胞中表达本发明所述的任意一种合成的甲病毒衍生复制子核酸分子。
本发明描述了如何成功编辑甲病毒复制子的nsP基因区域，以降低与野生型病毒的同源性，并将新的生物学功能纳入此类人工复制子的自我扩增的早期阶段，该早期阶段在亚基因组编码的转基因翻译之前。这可以通过识别nsP区域中可以在不破坏保守的结构元件的情况下安全编辑的关键核苷酸片段，并且将额外的序列附加到nsP的天然末端来实现。本发明展示了如何可以在不损害复制子的自扩增功能的情况下进行此类修饰。因此，本发明提供了一种产生更安全的复制子的方法，所述复制子配备有额外的功能以修饰宿主细胞的细胞和免疫学参数。
在一种实施方式中，该方法可以包括使用合适的培养基培养宿主细胞，并在宿主细胞中表达3C蛋白酶。该方法还可以包括将FMVD P1前体多肽(或其他目标多肽)表达和/或加工成FMDV病毒蛋白。该方法可包括培养宿主细胞并回收病毒蛋白VP0、VP1、VP2、VP3或VP4，或3C蛋白酶的其他切割产物。
在一种实施方式中，该方法可以包括培养宿主细胞并表达含有合成的甲病毒衍生复制子核酸分子，该核酸分子包含编码作为第一异源蛋白的O1 Manisa P1多蛋白和作为第二异源蛋白的3C蛋白酶的核酸序列。
在一种实施方式中，该方法可以包括培养含有翻译中断序列(例如2A序列)的合成的甲病毒衍生复制子核酸分子。翻译中断序列允许蛋白质翻译的中断，并在不一定被3C蛋白酶识别的位点(例如经修改的nsP4蛋白质中的位点)进行有效切割。2A序列可以在编码第二异源蛋白的序列之前插入到编码经修改的nsP4的核酸序列中。2A序列的插入可以导致由2A造成的翻译的中断以及不具有N末端Met残基的多肽的产生。
组合物
在一种实施方式中，本发明描述的合成的甲病毒衍生复制子核酸分子或异源蛋白可以在组合物中施用。
在一种实施方式中，组合物可以用于治疗自身免疫性疾病或过敏性疾病。该治疗可包括任意一种包含如本发明所述的异源蛋白的甲病毒衍生的复制子核酸分子，其表达抑制或以其他方式减少针对所需靶向抗原的适应性免疫应答的因子。
在一种实施方式中，组合物可以包含用于表达靶向特异性抗原的合成的甲病毒衍生复制子核酸分子。靶向特异性抗原可以是肿瘤抗原。癌症特异性抗原可以衍生自以下任一种：自靶细胞分离的总mRNA、一种或多种特异性靶向mRNA分子、靶细胞的蛋白质裂解物、来自靶细胞的特异性蛋白，或合成的靶向特异性肽或蛋白质以及编码靶向特异性抗原或其衍生肽的合成mRNA或DNA。非限制性实例为以下癌症或肿瘤特异性抗原：WT1、MUC1、LMP2、HPV E6 E7、EGFRvIII、HER-2/neu、独特型(Idiotype)、MAGE A3、p53(非突变体)、NY-ESO-1、PSMA、GD2、CEA、MelanA/MART 1、Ras突变体、gp100、p53突变体、蛋白酶3(PR1)、Bcr-abl、酪氨酸酶、生存素、PSA、hTERT、肉瘤易位断点、EphA2、PAP、ML-IAP、AFP、EpCAM、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、ALK、雄激素受体、细胞周期蛋白B1、聚唾液酸、MYCN、RhoC、TRP-2、GD3、岩藻糖基GM1、间皮素、PSCA、MAGEA1、sLe(a)、CYP1B1、PLAC1、GM3、BORIS、Tn、GloboH、ETV6-AML、NY-BR-1、RGS5、SART3、STn、碳酸酐酶IX、PAX5、OY-TES1、精子蛋白17、LCK、HMWMAA、AKAP-4、SSX2、XAGE 1、B7H3、豆荚蛋白(Legumain)、Tie 2、Page4、VEGFR2、MAD-CT-1、FAP，PDGFR--、MAD-CT-2、Fos相关抗原1。
在一种实施方式中，组合物可以用于治疗癌症。该治疗可包括任意一种包含本发明所述的异源蛋白质的甲病毒衍生复制子核酸分子，其表达抑制或以其他方式减少人或动物体内肿瘤细胞生长或增殖的因子。
在一种实施方式中，组合物可包含3C蛋白酶。此类组合物可用于诱导或检测针对3C蛋白酶的免疫应答，例如直接针对3C蛋白酶的体液或细胞免疫应答。该组合物还可以包含前体多肽，例如FMDV P1前体，其含有被经修饰的FMDV 3C蛋白酶识别的位点。该组合物可以是可用于体外加工前体多肽的形式。
在一种实施方式中，组合物可以是疫苗。疫苗可包含任意一种包含本发明所述的抗原蛋白的甲病毒衍生复制子核酸分子。术语“疫苗”是指含有活性成分的试剂或组合物，能够有效诱导受试者针对某种病原体或疾病产生一定程度的免疫力，从而至少降低与病原体或疾病感染相关的症状的严重程度、持续时间、其他表现的降低或消除症状。
在一种实施方式中，本发明任一实施方式中公开的合成的甲病毒衍生复制子核酸分子可以封装在载体系统中用于体内施用。载体可以是但不限于阴离子脂质体、阳离子脂质体或树枝状聚合物(dendrimer)。术语“阴离子脂质体”是指包括含有阴离子基团的脂质的脂质体。阴离子脂质体可以由阴离子磷脂形成。术语“阳离子脂质体”是指全部或部分由带正电荷的脂质，或更具体地，由包含阳离子基团和亲脂部分的脂质制成的脂质体。用于阳离子脂质体的阳离子脂质的带正电的分子提供了有利的结构特征。例如，阳离子脂质的亲脂性部分是疏水性的，因此可以引导其自身远离脂质体的水性内部并与其他非极性和疏水性物质缔合，或者相反，阳离子部分可以与极性分子和物质缔合，从而在阳离子脂质体的水性内部与之络合。带正电荷的脂质体可以与带负电荷的核酸分子相互作用以形成稳定的复合物。
脂质体可以包括两性离子脂质。如本发明所用，术语“两性离子”是指同时含有正电荷和负电荷但具有净中性电荷的分子。
脂质体可以由单一脂质或脂质混合物形成。脂质的亲水部分可以被聚乙二醇化，即通过聚乙二醇的共价连接进行修饰，以增加稳定性并防止脂质体的非特异性吸附(Heyeset al.(2005)J Controlled Release 107：276-87，其通过引用并入本文，如同已完全阐述)。
术语“树枝状聚合物”是指具有球体形状的高度分支化的大分子。树枝状聚合物分子的表面可以通过多种方式进行修饰，并且所得构建体的许多性质可以由其表面决定。树枝状聚合物可以被修饰以具有正表面电荷，即成为阳离子树枝状聚合物。阳离子树枝状聚合物可以与核酸形成暂时缔合。到达其目的地后，树枝状聚合物-核酸复合物可通过胞吞作用被摄入细胞中。
单个树枝状聚合物-核酸复合物(本文也称为经修饰的树枝状聚合物纳米粒子(MDN Ps))的示例性尺寸在最长尺寸上可以在30nm至1,000nm的范围内。MDNP可具有30nm至450nm(含端值)、50nm至300nm(含端值)、或60nm至250nm(含端值)的平均尺寸。MDNPS可以是烷基改性的树枝状聚合物纳米颗粒。纳米颗粒的尺寸可能受到取代核心树枝状聚合物的烷基链的长度的影响。制备和配制改性树枝状聚合物纳米颗粒的方法可见WO2021 207020(公开日为2021年10月14日)；US20210330600(公开日为2021年10月28日)；以及US20210338789(公开日为2021年11月4日)中的描述；所有这些内容均通过引用并入本文，如同已完整阐述。
在一种实施方式中，组合物还可包含一种或多种佐剂。术语“佐剂”是指引起免疫系统刺激的一种或多种物质。佐剂可用于增强对用于使免疫受试者抵抗疾病的异源蛋白的免疫应答。一种或多种佐剂可以包括铝，例如氢氧化铝和/或磷酸铝；油乳液组合物或水包油组合物，例如角鲨烯-水乳液，例如MF59；皂苷制剂，例如QS21和免疫刺激复合物(ISCOMS)；细菌或微生物衍生物，例如单磷酰脂质A(MPL)、3-O-脱酰化MPL(3dMPL)、含有寡核苷酸的CpG基序、ADP-核糖基化细菌毒素或其突变体，例如大肠杆菌热不稳定肠毒素LT，霍乱毒素CT、核酸和其他TLR激动剂，例如聚(I：C)；等等；真核蛋白，例如抗体或其片段，以及受体的配体，其在与受体细胞相互作用时刺激免疫反应。
在一种实施方式中，本发明描述的组合物可以在没有佐剂的情况下使用。
在一种实施方式中，组合物可以在药学上可接受的载体或赋形剂中施用。本发明所用的术语“药学上可接受的载体”是指药学上可接受的材料、组合物或媒介物，例如液体或固体填充剂、稀释剂、赋形剂、制造助剂(例如润滑剂、滑石粉镁、硬脂酸镁、硬脂酸钙或硬脂酸锌，或硬脂酸)或溶剂封装材料，其参与将主体化合物从一个器官或身体的一部分携带或运输至另一器官或身体的一部分。每种载体必须是“可接受的”，即与制剂中的其他成分相容并且不会对患者造成伤害。可用作药学上可接受的载体的材料的一些实例包括：(1)糖类，如乳糖、葡萄糖和蔗糖；(2)淀粉类，如玉米淀粉和马铃薯淀粉；(3)纤维素及其衍生物，如羧甲基纤维素钠、甲基纤维素、乙基纤维素、微晶纤维素和醋酸纤维素；(4)黄芪胶粉；(5)麦芽；(6)明胶；(7)润滑剂，如硬脂酸镁、十二烷基硫酸钠和滑石粉；(8)赋形剂，例如可可脂和栓剂蜡；(9)油类，如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和大豆油；(10)二醇类，例如丙二醇；(11)多元醇，例如甘油、山梨醇、甘露醇和聚乙二醇(PEG)；(12)酯类，例如油酸乙酯和月桂酸乙酯；(13)琼脂；(14)缓冲剂，例如氢氧化镁和氢氧化铝；(15)海藻酸；(16)无热原水(pyrogen-free water)；(17)等渗盐水；(18)林格氏溶液；(19)乙醇；(20)pH缓冲溶液；(21)聚酯、聚碳酸酯和/或聚酐；(22)增容剂，例如多肽和氨基酸(23)血清成分，例如血清白蛋白、HDL和LDL；(22)C2-C12醇类，例如乙醇；以及(23)用于药物制剂的其他无毒相容物质。润湿剂、着色剂、脱模剂、包衣剂、甜味剂、矫味剂、芳香剂、防腐剂和抗氧化剂也可以存在于制剂中。诸如“赋形剂”、“载体”、“药学上可接受的载体”或类似的术语在本发明中可互换使用。药学上可接受的载体和赋形剂是本领域已知的(Remington′sPharmaceutical Sciences，18th edition，A.R.Gennaro，Ed.，Mack Publishing Company，1990；Pharmaceutical Formulation Development of Peptides and Proteins，S.Frokjaer and L.Hovgaard，Eds.，Taylor&Francis，2000；Handbook of PharmaceuticalExcipients，3rd edition，A.Kibbe，Ed.，Pharmaceutical Press，2000)。本发明所述的合成的甲病毒衍生复制子核酸分子可以配制为无菌和/或冻干溶液并施用。无菌溶液可以通过无菌过滤或任何其他已知的方法来制备。然后可以将溶液冻干或填充到药物剂量容器中。溶液的pH通常可以在pH 3.0至9.5的范围内，例如pH 5.0至7.5。在一种实施方式中，可以添加稳定剂，例如白蛋白或另外的糖(例如蔗糖)。在一种实施方式中，可以添加去垢剂(detergent)。在一种实施方式中，包含本发明所述的任意复制子RNA的核酸混合物可以配制为可注射制剂。
一种实施方式提供了通过本发明公开的方法产生的复制子RNA制剂在制备能够在有需要的患者中引发针对癌症的免疫应答的疫苗中的用途。
在一种实施方式中，提供了在受试者中诱导免疫应答的方法。该方法可以包括向受试者施用治疗有效量的疫苗，该疫苗包含本发明所述的任意一种合成的甲病毒衍生复制子核酸分子。
在一种实施方式中，提供了用于预防和/或治疗受试者的疾病或病症的方法。该方法可以包括利用本发明描述的任意一种组合物和疫苗。该方法可以包括向有需要的受试者施用治疗有效量的包含本发明如上所述的合成的甲病毒衍生复制子核酸分子的组合物或疫苗。
本发明描述的组合物或疫苗的施用可以使用已知的施用途径进行。组合物或疫苗可以通过使用肠胃外给药途径来施用，例如皮内、肌内、皮下、经皮或粘膜给药途径，例如鼻内、口服等。在一种实施方式中，通过肌内注射施用组合物。如本发明所用，术语“施用”是指通过使所述组合物至少部分定位在期望部位处从而产生期望效果的方法或途径将组合物置入受试者体内。本发明所述的化合物或组合物可以通过本领域已知的任何适当途径施用，包括但不限于口服或肠胃外途径，包括静脉内、肌内、皮下、经皮、气道(气雾剂)、肺、鼻、直肠或局部(包括口腔和舌下)给药。
示例性的施用模式包括但不限于注射、输注、滴注、吸入或吞食。“注射”包括但不限于静脉注射、肌肉注射、动脉注射、鞘内注射、心室内注射、囊内注射、眼眶内注射、心内注射、皮内注射、腹腔注射、经气管注射、皮下注射、表皮下注射、关节内注射、囊下注射、蛛网膜下注射、椎管内注射、脑脊液内注射和皮内注射(intrastemal injection)以及输液。在一种实施方式中，组合物可以通过静脉内输注或注射来施用。
本发明描述的疫苗可以通过任何已知途径施用，以诱导针对疫苗中的抗原的免疫应答。
在一种实施方式中，施用或递送的方法可以不限于上述方法，并且可以使用用于细胞内递送的任何手段。
治疗有效量是指有效预防、改善和/或治疗由病原体感染引起的疾病或病症的蛋白质或核酸分子的量。预防包括抑制或减少病原体的传播或者抑制或减少与病原体感染相关的一种或多种症状的发作、发展或进展。如本发明所用，改善是指可见或可感知的疾病症状、病毒血症或由病原体诱导的感染的任何其他可测量的表现的减少。
就治疗癌症而言，“治疗有效量”是有效防止癌症进一步发展或转化生长、甚至实现癌症或实体瘤消退的量。
治疗有效量的确定通常完全在本领域技术人员的能力范围之内。一般来说，治疗有效量可以根据受试者的病史、年龄、状况、性别以及受试者的严重性和医疗状况的类型，以及其他药剂的施用减轻了待治疗的疾病或病症的情况而变化。
毒性和治疗功效可以通过细胞培养物或实验动物中的标准制药程序来确定，例如，用于确定LD50(半数致死剂量)和ED50(半数治疗有效剂量)。毒性作用与治疗作用之间的剂量比是治疗指数，可以用LD50/ED50的比值表示。优选表现出大治疗指数的组合物。如本发明所用，术语ED表示有效剂量并且与动物模型结合使用。术语EC表示有效浓度并且与体外模型结合使用。
从细胞培养试验和动物研究中获得的数据可用于制定用于人类的一系列剂量。此类化合物的剂量优选位于包括具有很小或无毒性的ED50的循环浓度范围内。剂量可以在此范围内变化，这取决于所采用的剂型和所采用的施用途径。
治疗有效剂量最初可以通过细胞培养试验进行估计。可以在动物模型中配制剂量，以达到包括在细胞培养中确定的IC50(即实现症状半最大抑制的治疗药物浓度)的循环血浆浓度范围。血浆中的水平可以通过例如高效液相色谱法来测量。任何特定剂量的效果可以通过合适的生物试验来监测。
剂量可由医生确定并根据需要进行调整以适应所观察到的治疗效果。一般而言，可以施用组合物使得活性剂以1μg/kg至150mg/kg、1μg/kg至100mg/kg、1μg/kg至50mg/kg、1μg/kg至20mg/kg、1μg/kg至10mg/kg、1μg/kg至1mg/kg、100μg/kg至100mg/kg、100μg/kg至50mg/kg、100μg/kg至20mg/kg、100μg/kg至10mg/kg、100μg/kg至1mg/kg、1mg/kg至100mg/kg、1mg/kg至50mg/kg、1mg/kg至20mg/kg、1mg/kg至10mg/kg、10mg/kg至100mg/kg、10mg/kg至50mg/kg或10mg/kg至20mg/kg的计量给药。应理解的是，本发明给出的范围包括所有中间范围，例如，范围1mg/kg至10mg/kg包括1mg/kg至2mg/kg、1mg/kg至3mg/kg、1mg/kg至4mg/kg、1mg/kg至5mg/kg、1mg/kg至6mg/kg、1mg/kg至7mg/kg、1mg/kg至8mg/kg、1mg/kg至9mg/kg、2mg/kg至10mg/kg、3mg/kg至10mg/kg、4mg/kg至10mg/kg、5mg/kg至10mg/kg、6mg/kg至10mg/kg、7mg/kg至10mg/kg、8mg/kg至10mg/kg、9mg/kg至10mg/kg等。还应当理解，上述给出的中间范围也在本发明的范围内，例如，在1mg/kg至10mg/kg的范围内，剂量范围例如2mg/kg至8mg/kg、3mg/kg至7mg/kg、4mg/kg至6mg/kg等。
在一种实施方式中，可以以剂量施用该组合物，以使该活性剂的体内浓度在给药15分钟、30分钟、1小时、1.5小时、2小时、2.5小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时或更多的给药时间后，小于500nM、小于400nM、小于300nM、小于250nM、小于200nM、小于150nM、小于100nM、小于50nM、小于25nM、小于20nM、小于10nM、小于5nM、小于1nM、小于0.5nM、小于0.1nM、小于0.05、小于0.01nM、小于0.005nM、小于0.001nM。
关于治疗的持续时间和频率，通常由熟练的临床医生监测受试者以确定治疗何时提供治疗益处，并确定是否增加或减少剂量、增加或减少给药频率、停止治疗、恢复治疗或对治疗方案进行其他修改。给药方式可以从每周一次到每天一次，具体取决于许多临床因素，例如受试者对多肽的敏感性。可以每天或每第三、第四、第五或第六天施用所需剂量。所需剂量可以一次施用或分成亚剂量，例如2-4个亚剂量，并在一段时间内施用，例如在一天中以适当的间隔或其他适当的方案施用。此类亚剂量可以作为单位剂型施用。在一种实施方式中，施用可以是长期的，例如一次或在数周或数月内每天施用一剂量更多剂量。给药方案的实例可包括在1周、2周、3周、4周、1个月、2个月、3个月、4个月、5个月或6个月或更长时间的时间内每天施用、每天两次、每天三次或每天四次或更多次施用。
在一种实施方式中，提供了制备针对疾病或病症的疫苗或治疗组合物的方法。该方法可包括提供本发明所述的疫苗或治疗组合物并将其配制为药学上可接受的组合物。疫苗组合物可包含可引起针对疾病的免疫应答的，有效量的异源免疫原性蛋白和/或编码该蛋白的核酸分子。
在一种实施方式中，疾病可以由病毒病原体引起。病原体可以是病毒病原体。病毒病原体可以是腺病毒、鼻病毒、轮状病毒、西尼罗病毒、寨卡病毒、疱疹病毒或冠状病毒(CoV)。冠状病毒可以为MERS CoV、SARS-CoV和SARS-CoV-2病毒中的一种。
在一种实施方式中，疾病可以是口蹄疫(FMD)。FMD是家养和野生偶蹄动物(包括家养和野生牛和猪)的急性且高度传染性的病毒性疾病。示例性的易感动物包括牛、水牛、绵羊、山羊、猪、羚羊、鹿和野牛、刺猬和大象；美洲驼和羊驼。在实验室实验中，小鼠、大鼠和鸡已通过人工手段成功感染，但据信它们在自然条件下不会感染该疾病。人类很少被感染。FMD的症状包括持续大约两到六天的高烧，然后口腔内和脚上出现水泡，这些水泡可能会破裂并导致跛行。口蹄疫对畜牧业具有严重影响，因为它具有高度传染性，受感染的动物可以通过气溶胶、通过接触受污染的农业设备、车辆、织物或饲料以及家养和野生捕食者传播。特别是由口蹄疫病毒感染引起的牛和猪的症状。口蹄疫爆发造成的经济损失是所有牲畜疾病中最高的。病原体口蹄疫病毒(FMDV)是小核糖核酸病毒科的家族成员，因此，病毒病原体可以是小核糖核酸病毒。如本发明所用，术语“小核糖核酸病毒”是指一种非包膜病毒，代表一个具有二十面体衣壳的小细胞质正链RNA(7.0-8.5kb)的病毒家族。它是一个大型病毒家族，包括多个属，含有重要的人类和动物病原体，例如肠道病毒(人类脊髓灰质炎病毒)、肝病毒(甲型肝炎病毒)和以口蹄疫为原型成员的口疮病毒(aphthoviruses)。FMDV存在七种不同的血清型(欧亚血清型A、0、C以及亚洲和南非领土[SAT]血清型SAT1、SAT2和SAT3)和反映显着遗传变异性的多个亚型。该科内的属包括口疮病毒属(Aphthovims)、水病毒属(Aquamavirus)、禽肝病毒属(Aquamavirus)、心脏病毒属(Cardiovirus)、科萨维病毒属(Cosavirus)、二分病毒属(Dicipivirus)、肠道病毒属(Enterovirus)、埃博病毒属(Erbovirus)、肝病毒属(Hepatovirus)、柯布病毒属(Kobuvirus)、巨型病毒属(Megrivirus)、帕雷奇病毒属(Parechovirus)、鱼病毒属(Piscevirus)、唾液病毒属(Salivirus)、沙佩洛病毒属(Sapelovirus)、塞内卡病毒属(Senecavirus)、特沙病毒属(Teschovirus)和特雷莫病毒属(Tremovirus)。小核糖核酸病毒的病毒基因组通常含有一个开放阅读框，其编码包含结构蛋白区域P1和非结构蛋白区域P2和P3的单一多蛋白。成熟和功能性蛋白质从多蛋白中的释放主要由包括3C蛋白酶在内的病毒蛋白酶介导。
提供疫苗组合物和免疫方案
本发明描述的“疫苗”可以作为药物组合物提供。组合物可以包含药学上可接受的稀释剂、载体或赋形剂。该组合物可以包含进一步的的活性成分。可以以单剂量给药或在初免-加强组合给药(prime-boost setting)中提供施用。初免-加强组合给药可以涉及“初免”和“加强”免疫方案，其中初免疫苗诱导的免疫反应可以通过加强疫苗加强。例如，在用编码第一异源蛋白的任意一种多核苷酸引发(至少一次)后，可以施用包含有效量的第二异源抗原蛋白或其亚基的加强疫苗以加强初免的免疫应答。
本发明描述的组合物可以施用于受试者，例如人类受试者。用于单次施用的组合物中的异源免疫原性蛋白的总剂量可以是例如约0.01μg至约10mg，例如1μg-1mg，例如10μg-100μg。确定推荐剂量可以通过实验进行，并且对于本领域技术人员来说是常规的。
术语“受试者”和“个体”在本文中可互换使用，并且是指人类或动物。通常，动物是脊椎动物，例如灵长类动物、啮齿类动物、家养或猎用动物。灵长类动物包括黑猩猩、食蟹猴(cynomologous monkeys)、蜘蛛猴和猕猴，例如恒河猴。啮齿动物包括小鼠、大鼠、土拨鼠、雪貂、兔子和仓鼠。家养和猎用动物包括牛、马、猪、鹿、野牛、水牛、猫科动物，例如家猫，犬科动物，例如狗、狐狸、狼、禽类，例如鸡、鸸鹋、鸵鸟和鱼，例如鳟鱼、鲶鱼和鲑鱼。患者或受试者包括上述的任何子集，例如上述所有，但不包括一个或多个群体或物种，例如人类、灵长类动物或啮齿类动物。在一种实施方式中，受试者可以是哺乳动物，例如灵长类动物，例如人类。术语“患者”和“受试者”在本发明中可互换使用。术语“患者”和“受试者”在本文中可互换使用。优选地，受试者是哺乳动物。哺乳动物可以是人、非人灵长类动物、小鼠、大鼠、狗、猫、马或牛，但不限于这些实例。
下面的清单包括本发明的特定实施方式。但该清单不是限制性的并且不排除替代实施方式或本文另外描述的实施方式。以下实施方式清单中描述的百分比同一性是指所列举的序列沿着参考序列的全长的同一性。
治疗癌症的方法
一种实施方式提供了治疗癌症的方法。该方法可以包括向有需要的受试者施用治疗有效量的包含本发明描述的任何一种免疫原性蛋白的组合物。免疫原性蛋白可包含刺激先天免疫应答的模式识别受体或效应蛋白。免疫原性蛋白可以是细胞外信号分子，例如细胞因子、膜表面受体、膜通道、或整联蛋白或影响免疫细胞识别的其他糖蛋白。免疫原性蛋白质可包含STING蛋白。干扰素基因刺激剂(STING)在感染和癌症期间的先天免疫中发挥着核心作用。STING被2′，3′-环-GMP-AMP(cGAMP)内源激活，cGAMP是一种由cGAMP合酶(cGAS)响应胞质DNA作为危险信号而合成的环状二核苷酸。STING的激活介导多方面的I型干扰素(IFN-I)反应，其促进树突状细胞的成熟和迁移，并启动细胞毒性T淋巴细胞和自然杀伤细胞进行自发免疫反应。
在一种实施方式中，用于治疗癌症的方法可以包括向有需要的受试者施用治疗有效量的本发明所述的任意一种免疫原性蛋白。
在一种实施方式中，该方法还可以包括分析肿瘤生长的抑制。分析步骤可以包括观察受试者中肿瘤生长的超过约60％、70％、80％或约90％的抑制。
多种已知的控释或缓释剂型、制剂和装置可适合与本发明公开的免疫原性组合物一起使用。实例包括但不限于美国专利Nos.3,845,770；3,916,899；3,536,809；3,598,123；4,008,719；5674,533；5,059,595；5,591,767；5,120,548；5,073,543；5,639,476；5,354,556；5,733,566和6,365,185 B1中描述的那些，所有这些都通过引用并入本文，就如已完全阐述。这些剂型可用于通过使用例如羟丙基甲基纤维素、其他聚合物基质、凝胶、渗透膜、渗透系统(例如(Alza Corporation，Mountain View，Calif.USA))，或其组合，以提供一种或多种活性成分的缓慢或受控释放，
如本发明所用，术语“癌症”是指可能干扰身体器官和系统的正常功能的细胞不受控制的生长。癌症可以是原发性癌症，或转移性癌症，或两者兼有。从原来的位置迁移并播散到重要器官的癌症最终会因受影响器官的功能恶化而导致受试者死亡。转移是指与原发肿瘤位置不同的癌细胞或癌细胞群，是由于癌细胞从原发肿瘤扩散到身体其他部位而产生的结果。在诊断出原发性肿瘤块时，可以监测受试者是否存在转移灶，例如扩散过程中的癌细胞。
如本发明所用，术语“癌症”还包括但不限于实体瘤和血源性肿瘤。术语癌症是指皮肤、组织、器官、骨、软骨、血液和血管的疾病。术语“癌症”包括恶性上皮肿瘤(来自肺、胃、十二指肠、结肠、直肠、乳腺、子宫、前列腺、膀胱等)。
术语“癌症”进一步涵盖原发性癌症和转移性癌症。可以用本发明的方法治疗的癌症的实例包括但不限于实体瘤；黑色素瘤、脑癌，包括但不限于胶质瘤、胶质母细胞瘤、多形性胶质母细胞瘤(GBM)、少突胶质细胞瘤、原始神经外胚层肿瘤、低、中和高级别星形细胞瘤、室管膜瘤(例如粘液乳头状室管膜瘤、乳头状室管膜瘤、室管膜下瘤、间变性室管膜瘤)、少突胶质细胞瘤、髓母细胞瘤、脑膜瘤、垂体腺瘤、神经母细胞瘤和颅咽管瘤；乳腺癌，包括但不限于导管原位癌、浸润(invasive)(或浸润性(infiltrating))导管癌、浸润(或浸润性)小叶癌、腺样囊性(或腺囊性)癌、低度腺鳞癌、髓样癌、粘液性(或胶质)癌、乳头状癌、小管癌、炎性乳腺癌、乳头佩吉特病、叶状瘤、三阴性乳腺癌、转移性乳腺癌；恶性上皮癌(carcinoma)，包括在膀胱、乳腺、结肠、肾脏、肺、卵巢、胰腺、胃、子宫颈、甲状腺的恶性上皮癌，以及皮肤，包括鳞状细胞癌；其他肿瘤包括精原细胞瘤、四子细胞癌(tetratocarcinoma)；中枢和周围神经系统肿瘤；以及其他肿瘤，包括但不限于异皮瘤、色素瘤、角化棘皮瘤、甲状腺滤泡癌和畸胎瘤。
本发明公开的方法可用于治疗先前已接受过癌症治疗的患者以及先前未接受过癌症治疗的患者。事实上，本发明描述的方法和组合物可用于一线和二线癌症治疗。
如本发明所用，术语“癌前病症”具有其普通含义，即无转移的不受调节的生长，并且包括各种形式的增生和良性肥大。因此，“癌前病症”是一种疾病、综合征或如果不治疗，可能导致癌症的发现物。这是一种与癌症风险显着增加相关的全身状态。恶性肿瘤前病变是一种形态发生改变的组织，其中比表面正常的组织更容易发生癌症。恶性肿瘤前病变的实例包括但不限于口腔白斑、光化性角化病(日光性角化病)、巴雷特食管、萎缩性胃炎、前列腺良性增生、结肠或直肠癌前息肉、胃上皮发育不良、腺瘤性发育不良、遗传性非息肉病结肠癌综合征(HNPCC)、巴雷特食管、膀胱发育不良、宫颈癌前病变和宫颈发育不良。
在一种实施方式中，癌症可以选自由以下组成的组中：乳腺癌；卵巢癌；脑癌；胃肠道癌；前列腺癌；恶性上皮癌、肺恶性上皮癌、肝细胞恶性上皮癌、睾丸癌；宫颈癌；子宫内膜癌；膀胱癌；头颈癌；肺癌；胃食管癌和妇科癌症。
在一种实施方式中，癌症可以是乳腺癌，包括但不限于导管原位癌、浸润(或浸润性)导管癌、浸润(或浸润性)小叶癌、腺样囊性(或腺囊性)癌、低度腺鳞癌、髓样癌、粘液性(或胶质)癌、乳头状癌、小管癌、炎性乳腺癌、乳头佩吉特病、叶状瘤、三阴性乳腺癌、转移性乳腺癌。
在一种实施方式中，本发明描述的方法可以涉及治疗患有或诊断为患有癌症的受试者。患有癌症的受试者可以由医生使用当前诊断癌症的方法来鉴定。表征这些病症并有助于诊断的癌症症状和/或并发症是本领域众所周知的，并且可以是但不限于肿瘤生长、含有癌细胞的器官或组织的功能受损等。可能有助于诊断的测试，例如癌症包括但不限于组织活检和组织学检查。癌症家族史或接触癌症危险因素(例如烟草产品、辐射等)也可能有助于确定受试者是否可能患有癌症或做出癌症诊断。
下面的清单包括本发明的特定实施方式。但该清单不是限制性的并且不排除替代实施方式或本文另外描述的实施方式。以下实施方式清单中描述的百分比同一性是指所列举的序列沿着参考序列的全长的同一性。
实施方式
1、一种合成的甲病毒衍生复制子核酸分子，其包含：(i)第一核酸，其编码甲病毒非结构蛋白nsP1、nsP2、nsP3和nsP4，并且包含在如SEQ ID NO：17所示的甲病毒基因组序列中从nt 503至nt 658、nt 658至nt 1620、nt 1620至nt 2560、nt 2560至nt 3954、nt 3954至nt 4120、nt 6381至nt 7083和nt 6966至nt 7526的区域内的任意位置处引入的至少一个沉默突变；以及(ii)第二核酸，其包含经过修饰的亚基因组开放阅读框(ORF)。
2、实施方式1所述的合成的甲病毒衍生复制子核酸分子，其中，经过修饰的亚基因组ORF包含编码第一异源蛋白的序列。
3、实施方式1和2中任意一个或两个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 503至nt 658的区域内的任意位置处引入的至少一个沉默突变。
4、实施方式1-3中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：21所示的参考序列具有至少90％同一性的序列的多核苷酸。
5、实施方式1-4中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 658至nt 1620的区域内的任意位置处引入的至少一个沉默突变。
6、实施方式1-5中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：25所示的参考序列具有至少90％同一性的序列的多核苷酸。
7、实施方式1-6中任意一个或多个所述的合成的甲病毒衍生的复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 1620至nt 2560的区域内的任意位置处引入的至少一个沉默突变。
8、实施方式1-7中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：24所示的参考序列具有至少90％同一性的序列的多核苷酸。
9、实施方式1-8中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 3694至nt 3954的区域内的任意位置处引入的至少一个沉默突变。
10、实施方式1-9中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：22所示的参考序列具有至少90％同一性的序列的多核苷酸。
11、实施方式1-10中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 2560至nt 3954的区域内的任意位置处引入的至少一个沉默突变。
12、实施方式1-11中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：26所示的参考序列具有至少90％同一性的序列的多核苷酸。
13、实施方式1-12中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 3954至nt 4120的区域内的任意位置处引入的至少一个沉默突变。
14、实施方式1-13中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP2和nsP3的接合物并且含有与如SEQ ID NO：23所示的参考序列具有至少90％同一性的序列的多核苷酸。
15、实施方式1-15中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 6381至nt 7083的区域内的任意位置处引入的至少一个沉默突变。
16、实施方式1-15中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP4并且含有与如SEQ ID NO：20所示的参考序列具有至少90％同一性的序列的多核苷酸。
17、实施方式1-16中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 658至nt 1620和nt 2560至nt 3954的区域内的任意位点处引入的至少一个沉默突变。
18、实施方式1-17中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：25所示的参考序列具有至少90％同一性的序列的多核苷酸，以及编码nsP2并且含有与如SEQ ID NO：26所示的参考序列具有至少90％同一性的序列的多核苷酸。
19、实施方式1-18中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 3694至nt 3954和nt 6381至nt 7083的区域内的任意位置处引入的至少一个沉默突变。
20、实施方式1-19中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP2并且含有与如SEQ ID NO：22所示的参考序列具有至少90％同一性的序列的多核苷酸，以及编码nsP4并且含有与如SEQ ID NO：20所示的参考序列具有至少90％同一性的序列的多核苷酸。
21、实施方式1-20中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：21所示的参考序列具有至少90％同一性的序列的多核苷酸；编码nsPl并且含有与如SEQ ID NO：25所示的参考序列具有至少90％同一性的序列的多核苷酸，编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：24所示的参考序列具有至少90％同一性的序列的多核苷酸；编码nsP2并且含有与如SEQ ID NO：26所示的参考序列具有至少90％同一性的序列的多核苷酸；编码nsP2和nsP3的接合物并且含有与如SEQ ID NO：23所示的参考序列具有至少90％同一性的序列的多核苷酸；以及编码nsP4并且含有与如SEQ ID NO：20所示的参考序列具有至少90％同一性的序列的多核苷酸。
22、实施方式1-21中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码所述非结构蛋白并且含有与如SEQ ID NO：30所示的参考序列具有至少90％同一性的多核苷酸序列的多核苷酸。
23、实施方式1-22中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，第一核酸分子包含在甲病毒基因组的从nt 6966到nt 7523的区域内的任何位置处引入的至少一个沉默突变。
24实施方式1-23中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，第一核酸分子包含与如SEQ ID NO：18所示的参考序列具有至少90％同一性的多核苷酸序列。
25、实施方式1-24中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含引入编码nsP4蛋白的多核苷酸的至少一个沉默突变。
26、实施方式1-25中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，第一核酸分子包含与如SEQ ID NO：19所示参考序列具有至少90％同一性的多核苷酸序列。
27、实施方式1-26中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码经过修改的nsP4的多核苷酸，所述经过修改的nsP4包含第二异源蛋白。
28、实施方式1-27中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，编码经过修改的nsP4的多核苷酸包含编码第一C末端区域的多核苷酸序列和编码第二C末端区域的多核苷酸序列。
29、实施方式1-28中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第二C末端区域可翻译成蛋白质。
30、实施方式1-28中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第二C末端区域不可翻译成蛋白质。
31、实施方式1-28中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，编码第一C末端区域的多核苷酸序列不同于编码第二C末端区域的多核苷酸序列。
32、实施方式1-31中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第二异源蛋白包含GFP蛋白，并且经过修改的nsP4包含与如SEQ ID NO：41所示的参考序列具有至少90％同一性的氨基酸序列。
33、实施方式1-32中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，经过修改的nsP4由包含与如SEQ ID NO：31所示的参考序列具有至少90％同一性的核苷酸序列编码。
34、实施方式1-33中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白或第二异源蛋白包含由病毒衍生的或由哺乳动物细胞衍生的细胞代谢、免疫或信号传导功能的调节剂。
35、实施方式1-34中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白或第二异源蛋白影响细胞抗病毒先天免疫应答并增强甲病毒复制子RNA分子的复制。
36、实施方式1-35中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白或第二异源蛋白包含自病毒病原体分离或衍生的抗原蛋白。
37、实施方式1-36中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，病毒病原体选自由：小核糖核酸病毒、腺病毒、鼻病毒、轮状病毒、西尼罗病毒、寨卡病毒、疱疹病毒或冠状病毒(CoV)组成的组中。
38、实施方式1-37中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，病毒病原体为小核糖核酸病毒。
39、实施例1-35中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第二异源蛋白包含3C蛋白酶蛋白，并且经过修改的nsP4包含与如SEQ ID NO：40所示的参考序列具有至少90％同一性的氨基酸序列。
40、实施方式1-35和39中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，经过修改的nsP4由与如SEQ ID NO：32所示的参考序列具有至少90％同一性的多核苷酸序列编码。
41、实施方式1-35和39-40中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白包含O1 Manisa P1蛋白。
42、实施方式1-35和39-41中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白包含O1 Manisa P1蛋白并且第二异源蛋白包含3C蛋白酶。
43、实施方式1-35中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第二异源蛋白包含STING蛋白，并且所述经过修改的nsP4包含与如SEQ ID NO：42所示的参考序列具有至少90％同一性的氨基酸序列。
44、实施方式1-35和44中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白包含抗原蛋白并且第二异源蛋白包含STING蛋白。
45、一种核酸，其包含编码合成的甲病毒衍生复制子核酸分子的DNA序列，其包含：(i)第一核酸，其编码甲病毒非结构蛋白nsP1、nsP2、nsP3和nsP4，并且包含在如SEQ ID NO：17所示的甲病毒基因组序列中从nt 503至nt 658、nt 658至nt 1620、nt 1620至nt 2560、nt 2560至nt 3954、nt 3954至nt 4120、nt 6381至nt 7083和nt 6966至nt 7526的区域内的任意位置处引入的至少一个沉默突变；以及(ii)第二核酸，其包含经过修饰的亚基因组开放阅读框(ORF)。
46、实施方式45所述的核酸，其中，经过修饰的亚基因组ORF包含编码第一异源蛋白的序列。
47、实施方式45和46中任意一个或多个所述的核酸，其中，第一核酸包含在甲病毒基因组中从nt 503至nt 658的区域内的任意位置处引入的至少一个沉默突变。
48、实施方式45-47中任意一个或多个所述的核酸，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：2所示的参考序列具有至少90％同一性的序列的多核苷酸。
49、实施方式45-48中任意一个或多个所述的核酸，其中，第一核酸包含在甲病毒基因组中从nt 658至nt 1620的区域内的任意位置处引入的至少一个沉默突变。
50、实施方式45-49中任意一个或多个所述的核酸，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：6所示的参考序列具有至少90％同一性的序列的多核苷酸。
51、实施方式45-50中任意一个或多个所述的核酸，其中，第一核酸包含在甲病毒基因组中从nt 1620至nt 2560的区域内的任意位置处引入的至少一个沉默突变。
52、实施方式45-51中任意一个或多个所述的核酸，其中，第一核酸包含编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：5所示的参考序列具有至少90％同一性的序列的多核苷酸。
53、实施方式45-52中任意一个或多个所述的核酸，其中，第一核酸包含在甲病毒基因组中从nt 3694至nt 3954的区域内的任意位置处引入的至少一个沉默突变。
54、实施方式45-53中任意一个或多个所述的核酸，其中，第一核酸包含编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：3所示的参考序列具有至少90％同一性的序列的多核苷酸。
55、实施方式45-54中任意一个或多个所述的核酸，其中，第一核酸包含在甲病毒基因组中从nt 2560至nt 3954的区域内的任意位置处引入的至少一个沉默突变。
56、实施方式45-55中任意一个或多个所述的核酸，其中，第一核酸包含编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：7所示的参考序列具有至少90％同一性的序列的多核苷酸。
57、实施方式45-57中任意一个或多个所述的核酸，其中，第一核酸包含在甲病毒基因组中从nt 3954至nt 4120的区域内的任意位置处引入的至少一个沉默突变。
58、实施方式45-57中任意一个或多个所述的核酸，其中，第一核酸包含编码nsP2和nsP3的接合物并且含有与如SEQ ID NO：4所示的参考序列具有至少90％同一性的序列的多核苷酸。
59、实施方式45-58中任意一个或多个所述的核酸，其中，第一核酸包含在甲病毒基因组中从nt 6381至nt 7083的区域内的任意位置处引入的至少一个沉默突变。
60、实施方式45-59中任意一个或多个所述的核酸，其中，第一核酸包含编码nsP4并且含有与如SEQ ID NO：1所示的参考序列具有至少90％同一性的序列的多核苷酸。
61、实施方式45-60中任意一个或多个所述的核酸，其中，第一核酸包含在甲病毒基因组中从nt 658至nt 1620和nt 2560至nt 3954的区域内的任意位点处引入的至少一个沉默突变。
62、实施方式45-61中任意一个或多个所述的核酸，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：6所示的参考序列具有至少90％同一性的序列的多核苷酸，以及编码nsP2并且含有与如SEQ ID NO：7所示的参考序列具有至少90％同一性的序列的多核苷酸。
63、实施方式45-63中任意一个或多个所述的核酸，其中，第一核酸包含在甲病毒基因组中从nt 3694至nt 3954和nt 6381至nt 7083的区域内的任意位置处引入的至少一个沉默突变。
64、实施方式45-63中任意一个或多个所述的核酸，其中，第一核酸包含编码nsP2并且含有与如SEQ ID NO：3所示的参考序列具有至少90％同一性的序列的多核苷酸，以及编码nsP4并且含有与如SEQ ID NO：1所示的参考序列具有至少90％同一性的序列的多核苷酸。
65、实施方式45-64中任意一个或多个所述的核酸，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：2所示的参考序列具有至少90％同一性的序列的多核苷酸；编码nsP1并且含有与如SEQ ID NO：6所示的参考序列具有至少90％同一性的序列的多核苷酸，编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：5所示的参考序列具有至少90％同一性的序列的多核苷酸；编码nsP2并且含有与如SEQ ID NO：7所示的参考序列具有至少90％同一性的序列的多核苷酸；编码nsP2和nsP3的接合物并且含有与如SEQ ID NO：4所示的参考序列具有至少90％同一性的序列的多核苷酸；以及编码nsP4并且含有与如SEQ ID NO：1所示的参考序列具有至少90％同一性的序列的多核苷酸。
66、实施方式45-65中任意一个或多个所述的核酸，其中，第一核酸包含编码非结构蛋白并且含有与如SEQ ID NO：11所示的参考序列具有至少90％同一性的多核苷酸序列的多核苷酸。
67、实施方式45-66中任意一个或多个所述的核酸，其中，第一核酸包含编码经过修改的nsP4的多核苷酸，所述经过修改的nsP4包含第二异源蛋白。
68、实施方式45-67中任意一个或多个所述的核酸，其中，编码经修改的nsP4的多核苷酸包含编码第一C末端区域的多核苷酸序列和编码第二C末端区域的多核苷酸序列。
69、实施方式45-68中任意一个或多个所述的核酸，其中，第二C末端区域包含亚基因组启动子。
70、实施方式45-69中任意一个或多个所述的核酸，其中，亚基因组启动子由与如SEQ ID NO：14所示的参考序列具有至少90％同一性的序列编码。
71、实施方式45-70中任意一个或多个所述的核酸，其中，第二C末端区域可翻译成蛋白质。
72、实施方式45-70中任意一个或多个所述的核酸，其中，第二C末端区域不可翻译成蛋白质。
73、实施方式45-70中任意一个或多个所述的核酸，其中，编码第一C末端区域的多核苷酸序列不同于编码第二C末端区域的多核苷酸序列。
74、实施方式45-73中任意一个或多个所述的核酸，其中，第二异源蛋白包含GFP蛋白，并且经过修改的nsP4包含与如SEQ ID NO：12所示的参考序列具有至少90％同一性的氨基酸序列。
75、实施方式45-74中任意一个或多个所述的核酸，其中，经过修改的nsP4由如SEQID NO：13所示的多核苷酸序列编码。
76、实施方式45-75中任意一个或多个所述的核酸，其中，第二异源蛋白包含GFP蛋白，并且经过修改的nsP4包含与如SEQ ID NO：15所示的参考序列具有至少90％同一性的氨基酸序列。
77、实施方式45-76中任意一个或多个所述的核酸，其中，经过修改的nsP4由如SEQID NO：16所示的多核苷酸序列编码。
78、实施方式45-73中任意一个或多个所述的核酸，其中，第一异源蛋白或第二异源蛋白包含由病毒衍生的或由哺乳动物细胞衍生的细胞代谢、免疫或信号传导功能的调节剂。
79、实施方式45X73中任意一个或多个所述的核酸，其中，第一异源蛋白或第二异源蛋白影响细胞抗病毒先天免疫应答并增强甲病毒复制子RNA分子的复制。
80、实施方式45-73中任意一个或多个所述的核酸，其中，第一异源蛋白或第二异源蛋白包含自病毒病原体分离或衍生的抗原蛋白。
81、实施方式45-73和80中任意一个或多个所述的核酸，其中，病毒病原体选自由：小核糖核酸病毒、腺病毒、鼻病毒、轮状病毒、西尼罗病毒、寨卡病毒、疱疹病毒或冠状病毒(CoV)组成的组中。
82、实施方式45-73和80-81中任意一个或多个所述的核酸，其中，病毒病原体为小核糖核酸病毒。
83、实施方式45-73和80-82中任意一个或多个所述的核酸，其中，第一异源蛋白包含O1 ManisaP1蛋白，和/或第二异源蛋白包含3C蛋白酶。
84、实施方式45-73中任意一个或多个所述的核酸，其中，第一异源蛋白和/或第二异源蛋白包含STING蛋白并且第一异源蛋白质包含抗原蛋白。
85、一种甲病毒衍生RNA复制子表达系统，其包含实施方式1-44中任意一个或多个所述的合成的甲病毒衍生复制子核酸分子和宿主细胞。
86、一种药学上可接受的组合物，其包含实施方式1-44中任意一个或多个所述的甲病毒衍生复制子和药学上可接受的载体或赋形剂。
87、一种在细胞中产生至少一种异源蛋白的方法，包括在宿主细胞中表达实施方式1-44中任意一个或多个所述的合成甲病毒衍生的复制子核酸分子。
88、一种预防、抑制或治疗受试者的疾病或病症的症状的方法，包括：提供合成的甲病毒属衍生的复制子核酸分子，其包含：
(i)第一核酸，其编码甲病毒非结构蛋白nsP1、nsP2、nsP3和nsP4，并且包含在如SEQ ID NO：17所示的甲病毒基因组序列中从nt 503至nt 658、nt 658至nt 1620、m 1620至nt 2560、nt 2560至nt 3954、nt 3954至nt 4120、nt 6381至nt 7083和nt 6966至nt 7526的区域内的任意位置处引入的至少一个沉默突变；以及
(ii)第二核酸，其包含经过修饰的亚基因组开放阅读框(ORF)；以及
施用治疗有效量的合成的甲病毒衍生复制子核酸分子予以受试者。
89、实施方式88所述的方法，其中，经过修饰的亚基因组ORF包含编码第一异源蛋白的序列。
90、实施方式88-89中任意一个或两个所述的方法，其中，第一核酸包含与选自由SEQ ID NOS：20-30组成的组中的参考序列具有至少90％同一性的至少一个序列。
91、实施方式88-90中任意一个或多个所述的方法，其中，第一核酸包含编码经过修改的nsP4的多核苷酸，所述经过修改的nsP4包含第二异源蛋白。
92、实施方式88-91中任意一个或多个所述的方法，其中，编码经修改的nsP4的多核苷酸包含编码第一C末端区域的多核苷酸序列和编码第二C末端区域的多核苷酸序列。
93、实施方式88-92中任意一个或多个所述的方法，其中，第二C末端区域可翻译成蛋白质。
94、实施方式88-92中任意一个或多个所述的方法，其中，第二C末端区域不可翻译成蛋白质。
95、实施方式88-92中任意一个或多个所述的方法，其中，编码第一C末端区域的多核苷酸序列不同于编码第二C末端区域的多核苷酸序列。
96、实施方式88-95中任意一个或多个所述的方法，其中，第一异源蛋白或第二异源蛋白包含由病毒衍生的或由哺乳动物细胞衍生的细胞代谢、免疫或信号传导功能的调节剂。
97、实施方式88-95中任意一个或多个所述的方法，其中，第一异源蛋白或第二异源蛋白影响细胞抗病毒先天免疫应答并增强甲病毒复制子RNA分子的复制。
98、实施方式88-95中任意一个或多个所述的方法，其中，第一异源蛋白或第二异源蛋白包含自病毒病原体分离或衍生的抗原蛋白。
99、实施方式88-95和98中任意一个或多个所述的方法，其中，病毒病原体选自由：小核糖核酸病毒、腺病毒、鼻病毒、轮状病毒、西尼罗病毒、寨卡病毒、疱疹病毒或冠状病毒(CoV)组成的组中。
100、实施方式88-95和98-99中任意一个或多个所述的方法，其中，病毒病原体为小核糖核酸病毒。
101、实施方式88-95和98-100中任意一个或多个所述的方法，其中，第一异源蛋白包含O1 ManisaP1蛋白并且第二异源蛋白包含3C蛋白酶。
102、实施方式88-95中任意一个或多个所述的方法，其中，第一异源蛋白或第二异源蛋白包含STING蛋白。
103、实施方式88-95和102中任意一个或多个所述的方法，其中，第二异源蛋白包含STING蛋白并且第一异源蛋白包含抗原蛋白。
104、实施方式88-104中任意一个或多个所述的方法，其中，施用步骤使得受试者的疾病得到预防、治疗、严重性减轻或病程发展减缓。
105、实施方式88-95和103-104中任意一个或多个所述的方法，其中，疾病为癌症。
106、实施方式88-95和103-105中任意一个或多个所述的方法，其中，癌症为黑色素瘤或上皮性肿瘤。
107、实施方式88-95和103-106中任意一个或多个所述的方法，其中，该方法进一步包括向受试者共同施用一种或多种另外的抗癌疗法。
108、实施方式88-95和103-107中任意一个或多个所述的方法，其中，另外的抗癌疗法选自由外科手术、化学疗法、放射疗法、温热疗法、免疫疗法、激素疗法、激光疗法、抗血管生成疗法及其任意组合组成的组中。
109、实施方式88-95和98-101中任意一个或多个所述的方法，其中，疾病为FMD。
110、实施方式88-109中任意一个或多个所述的方法，其中，受试者为哺乳动物。
111、实施方式88-110中任意一个或多个所述的方法，其中，哺乳动物为偶蹄动物或人。
112、实施方式88-111中任意一个或多个所述的方法，其中，偶蹄动物选自：牛、水牛、绵羊、山羊、猪、羚羊、鹿、野牛、刺猬、大象、美洲驼和羊驼组成的组中。
本发明的进一步的实施方式可以通过用来自本发明的任何一个或多个其他实施方式中的一种或多种要素补充实施方式和/或用来自一个或多个其他实施方式中的一种或多种要素替换来自一个实施方式的一种或多种要素来形成。
实施例
提供以下非限制性实施方式来说明特定的实施方式。贯穿全文的实施方式可以用来自下面的一个或多个实施例中的一个或多个细节来补充，和/或来自一个实施方式中的一个或多个元件可以用来自下面的一个或多个实施例中的一个或多个细节替代。
实施例1 nsP4修饰复制子的设计
图1说明了甲病毒基因组非结构多蛋白(nsP；nsP1-nsP2-nsP3-nsP4)的基因表达步骤。此图表明，非结构多蛋白的表达是基因组合成和随后亚基因组ORF(SG ORF)表达的先决条件，在天然病毒中SG ORF编码结构多蛋白。在图中，显示出nsP包括野生型VEEV中存在的早期终止密码子，该密码子导致nsP-1-2-3多蛋白的翻译终止；nsP4通过基因组5682位处的琥珀终止密码子(UGA)的低频翻译通读进行翻译，其结合了精氨酸，半胱氨酸或色氨酸。随后对多蛋白进行蛋白水解，产生分离的nsP1、nsP2、nsP3和nsP4蛋白。
病毒感染的“早期阶段”是指在发生基因组复制之前，主要以nsP多蛋白的表达为特征的感染后时间。“晚期”是由基因组复制发生后的基因表达模式定义的，亚基组多蛋白的表达是主要的病毒基因产物。
本发明报道了成功编辑甲病毒复制子的nsP基因区域，以降低与野生型病毒的同源性，并将新的生物学功能纳入此类人工复制子的自我扩增(在亚基因组编码的转基因翻译之前)的早期阶段。这是通过识别nsP区域中的关键核苷酸片段来实现的，这些核苷酸片段可以安全地进行编辑而不破坏保守的结构元件，并将额外的序列附加到nsP的天然末端。本发明显示了如何在不损害复制子的自扩增功能的情况下进行此类修饰。因此，本发明提供了一种产生更安全的复制子的新方法，所述复制子配备有额外的功能以修饰宿主细胞的细胞和免疫学参数。
甲病毒复制子目前主要用作疫苗载体，其中目标抗原在亚基因组ORF中编码，从而取代通常组成病毒颗粒的天然甲病毒结构蛋白。目前，如果需要以额外多肽(例如报告基因、免疫调节因子或其他宿主细胞功能调节因子)的形式提供辅助生物学功能，则辅助因子必须(i)通过编码序列的融合在与第一个转基因相同的亚基因组ORF中编码，(ii)在单独的RNA转录/翻译启动子(例如复制的亚基因组启动子(SGP)或内部核糖体进入位点(IRES)的控制下编码，或(iii)通过将额外的核酸或蛋白质种类与复制子RNA产物混合来从复制子中分离。方法(i)需要对功能性多肽进行工程设计，而对于某些抗原而言，在不改变其主要序列的情况下这并不总是可能的。方法(ii)除了所需的辅助ORF之外，还需要添加大量额外的核苷酸序列以产生SGP或IRES。方法(i)和使用额外的SGP作为(ii)中的方法仅允许辅助因子在病毒复制周期的后期与亚基因组ORF同时表达，从而使其无法在早期影响复制子功能。方法(iii)需要生产完全额外的生物制品，并且必须通过某种方式解决与复制子RNA的共同递送。目前，使用的复制子nsP序列与流行的地方性甲病毒基本相同，在与另一个密切相关的甲病毒(例如流行的致病性野生型病毒菌株)基因组共同感染的情况下，会增加重组的风险。这种重组有可能产生有生命力的复制病毒，从而造成环境安全问题。与野生型天然病毒株同源性降低的复制子将具有安全性提高的优点。
通过特异性编辑甲病毒基因组的天然编码序列，故意保留关键的一级和二级结构元件完整，解决了(a)与天然病毒的序列同源性和(b)包括辅助早期生物功能的方法的挑战。通过在编辑之前分析区域以检测预计携带特定功能的保守一级和二级序列元件(例如，如图6所示)，可以修改nsP编码区的天然密码子以产生沉默突变，从而产生相同的蛋白质产物并且不损害复制子的功能。扩展该技术，可以编辑nsP多蛋白的C末端，以通过融合到nsP编码序列中允许附加新的转基因x，而不损害复制子的功能。对nsP ORF 3’区域的必要非编码元件进行复制，以重新创建SGP并确保SG ORF表达，并对上游副本进行沉默密码子修改，以创建新的nsP-x融合蛋白并消除重复位点之间的假性同源性，可以抑制标准细菌生产菌株中此类构建体的DNA模板的增殖。
图2示出了甲病毒基因组非结构多蛋白的基因表达步骤，该多蛋白通过与nsP4(nsP1-nsP2-nsP3-nsP4-xP)框内的附属物人工修饰以表达另外的外源蛋白(“xP")。该图显示，外源蛋白xP被引入编码nsP多蛋白的nsP4片段的基因，导致nsP4和xP蛋白通过接合点连接在一起进行翻译和蛋白水解。nsP4-xP接合点可能包含不可裂解的、可自我裂解的(例如，2A肽)或蛋白裂解的(例如，nsP2蛋白酶或弗林蛋白酶识别位点)肽接头序列。
图3示出了可用于对甲病毒复制子RNA序列进行遗传修饰以产生如图2中所描述的构建体的步骤的示意图。如图3所示，所需的插入位置在亚基因组启动子(SGP)区域内，步骤1涉及SGP区域的复制。步骤2涉及在重复区域插入编码xP蛋白的序列。由于SGP的复制，该方法导致近端相同序列，从而使构建体在遗传上不稳定。这个问题的解决方式是在上游重复元件中实施沉默密码子修改，从而消除有问题的同源性。
图4示出了克隆可修饰的nsP4开放阅读框(ORF)，同时保持完整的延伸亚基因组启动子的最佳步骤。如图4所示，步骤1涉及使用不同的密码子优化选择，将合成的nsP4末端区域插入SGP区域(灰色框)，并加入末端限制位点。步骤2涉及在合成的nsP4末端区域和原始nsP4 C末端编码序列(非翻译编码序列CDS，条纹框)之间插入编码xP蛋白的序列。该方法不会产生任何近端相同的序列延伸，并产生遗传稳定的构建体。这种方法消除了有问题的重复序列元素，从而DNA模板可以很容易地在常见菌株中繁殖和制造。对nsP4的这种附加转基因x的修饰可以与本发明所述的nsP区域中的其他沉默突变结合进行，以产生进一步改进的复制子RNA。
实施例2基因组45-260位核苷酸内nsP1同源性降低的VEEV复制子RNA修饰
为了证明甲病毒nsP对任意核苷酸序列修改的敏感性，在对应于基因组核苷酸45-260位的nsP1蛋白的起始处替换了一系列沉默突变，如图5所示。图5是编码构建体8(C08)的复制子序列模板的DNA质粒的示意图。
在此图中，复制子序列源自甲病毒中的委内瑞拉马脑炎病毒(VEEV)。该DNA编码氨苄青霉素抗性基因(AmpR)，以促进在细菌细胞培养物中的特异性生产，编码复制起点(ori)以允许在细菌细胞中繁殖，工程化VEEV复制子的DNA模板(包含病毒的基因组5′UTR、nsPs、亚基因组启动子和5′UTR、亚基因组ORF中的目的基因、基因组3’UTR和poly(A)尾巴)可通过从T7启动子开始的流式转录将其转录为RNA。修改基因组核苷酸45-260位(灰色框)的密码子以减少与野生型病毒的同源性。这个45-260nt编码区包含细胞内RNA复制所需的甲病毒保守序列元件(CSE)。已知复制子功能所需的CSE已通过该构建体中的沉默突变(密码子适应)在二级结构水平上被破坏，因此它作为阴性对照。总的来说，沉默突变使nsP1 CDS与野生型的序列同一性降低了19.04％。质粒从T7启动子转录产生C08复制子RNA。C08的5’UTR、nsP1-4ORF和SGP如SEQ ID NO：27所示。
C08区包括公认的甲病毒保守序列元件(CSE)，这是一个含有51nt的核苷酸跨度，折叠成有序的茎环。该二级结构基序是基因组复制所必需的，从而从亚基因组ORF转基因表达。BHK细胞的体外转染证实，该RNA(C08)无法介导分泌胚胎碱性磷酸酶的亚基因组编码的报告基因的表达(如图17A中所示)。
实施例3基因组503-658位核苷酸内nsP1同源性降低的VEEV复制子RNA修饰
与实施例1中描述的构建体C08相反，在CSE下游的区域中进行不同的nsP1 ORF修饰，其中如图6所示，基于热力学参数对局部RNA折叠进行生物信息学检查用于选择区域非结构化核苷酸，因此一系列沉默突变在整个区域被取代以产生命名为C02的构建体，如图7所示。
图6是描绘用于选择甲病毒基因组区域的示意图，该区域可通过使用降低与野生型病毒同源性的沉默突变修改，而不降低复制子的复制功能。在此图中示出了野生型VEEV基因组(NCBI GenBank数据库中的参考序列L01442)中横跨BsiWI至EcoNI限制片段的区域的RNA二级结构，该结构是通过计算77计时的最小杂交自由能预测的。图中框出的是涉及局部(即，在大约100nt窗口内)发夹结构的多核苷酸延伸，这些结构是完整且未发生突变的，以保存基因组的总体二级结构。图中圈出了可以修改以产生沉默突变同时保留局部序列的无序性质的核酸位置的例子。
图7是编码构建体2(C02)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸503-658(灰色框)的密码子以降低与野生型病毒的同源性。总的来说，沉默突变使nsP1 CDS与野生型的序列同一性降低了14.09％。质粒从T7启动子转录产生C02复制子RNA。C02的5’UTR、nsP1-4ORF和SGP如SEQID NO：21所示。
据观察，BHK细胞的体外转染证实了C02构建体的RNA能够介导亚基因组ORF编码的报告基因(分泌型胚胎碱性磷酸酶)的表达(图17A)。
实施例4基因组658-1620位核苷酸内nsP1同源性降低的VEEV复制子RNA修饰
如实施例2中描述的构建体C02一样，基于热力学参数对局部RNA折叠进行生物信息学检查用于选择非结构化核苷酸区域，并且相应地在该区域中替换一系列沉默突变，以创建命名为C06的构建体。图8是编码构建体6(C06)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸658-1620(灰色框)中的密码子以降低与野生型病毒的同源性。总的来说，沉默突变使nsP1 CDS与野生型的序列同一性降低了8.96％。质粒从T7启动子转录产生C06复制子RNA。C06的5’UTR、nsP1-4ORF和SGP如SEQ ID NO：25所示。
据观察，BHK细胞的体外转染证实了该RNA(构建体C06)能够介导亚基因组ORF编码的报告基因(分泌型胚胎碱性磷酸酶)的表达(图17A)。注射该构建体的小鼠表现出与注射野生型VEEV-SEAP的小鼠同等或更高的平均SEAP血清表达(图17D)。
实施例5基因组1620-2560位核苷酸内nsP1-nsP2接合物区域中同源性降低的VEEV 复制子RNA修饰
如实施例2中描述的构建体C02一样，基于热力学参数对局部RNA折叠进行生物信息学检查用于选择nsP1和nsP2接合处的非结构化核苷酸区域，并且相应地在该区域中替换一系列沉默突变，以创建命名为C05的构建体。图9是编码构建体5(C05)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸1620-2560(灰色框)中的密码子以降低与野生型病毒的同源性。总的来说，沉默突变使nsP1 CDS与野生型的序列同一性降低了15.94％，与nsP2CDS的序列同一性降低了0.8％。
质粒从T7启动子转录产生C05复制子RNA。C05的5’UTR、nsP1-4 ORF和SGP如SEQ IDNO：24所示。
BHK细胞的体外转染证实了该RNA(构建体C05)能够介导亚基因组ORF编码的报告基因(分泌型胚胎碱性磷酸酶)的表达(图17A)。注射该构建体的小鼠表现出与注射野生型VEEV-SEAP的小鼠同等或更高的平均SEAP血清表达(图17D)。
实施例6基因组3694-3954位核苷酸内nsP2同源性降低的VEEV复制子RNA修饰[211]如实施例2中描述的构建体C02一样，基于热力学参数对局部RNA折叠进行生物信息学检查用于选择非结构化核苷酸区域，并且相应地在该区域中替换一系列沉默突变，以创建命名为C03的构建体。图10是编码构建体3(C03)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸3694-3954(灰色框)中的密码子以降低与野生型病毒的同源性。总的来说，沉默突变使nsP2 CDS与野生型的序列同一性降低了3.19％。
质粒从T7启动子转录产生C03复制子RNA。C03的5’UTR、nsP1-4 ORF和SGP如SEQ IDNO：22所示。
BHK细胞的体外转染证实了RNA能够介导亚基因组ORF编码的报告基因(分泌型胚胎碱性磷酸酶)的表达(图17A)。据观察，注射构建体C03的小鼠表现出与注射野生型VEEV-SEAP的小鼠同等或更高的平均SEAP血清表达(图17D)。
实施例7基因组2560-3954位核苷酸内nsP2同源性降低的VEEV复制/子RNA修饰
鉴于实施例5中观察到的成功，同源性降低的区域在5’方向进一步延伸。再次基于基因组核苷酸2560-3954位的热力学参数对局部RNA折叠进行生物信息学检查，并且相应地用沉默突变取代非结构化核苷酸区域，以创建命名为C07的构建体。图11是编码构建体7(C07)的复制子序列模板的DNA质粒的示意图。在该图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸2560-3954(灰色框)中的密码子以降低与野生型病毒的同源性。总的来说，沉默突变使nsP2 CDS与野生型的序列同一性降低了15.97％。质粒从T7启动子转录产生C07复制子RNA。C07的5’UTR、nsP1-4 ORF和SGP如SEQ ID NO：26所示。
BHK细胞的体外转染证实了该RNA(构建体C07)能够介导亚基因组ORF编码的报告基因(分泌型胚胎碱性磷酸酶)的表达(图17A)。
实施例8基因组3954-4120位核苷酸内nsP1-nsP2接合物区域中同源性降低的VEEV 复制子RNA修饰
如实施例2中描述的构建体C02一样，基于热力学参数对局部RNA折叠进行生物信息学检查用于选择nsP1和nsP2接合处的非结构化核苷酸区域，并且相应地在该区域中替换一系列沉默突变，以创建命名为C04的构建体。图12是编码构建体4(C04)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸3954-4120(灰色框)中的密码子以降低与野生型病毒的同源性。总的来说，沉默突变使nsP2 CDS与野生型的序列同一性降低了4.61％、与nsP3 CDS的序列同一性降低了0.12％。质粒从T7启动子转录产生C04复制子RNA。C04的5’UTR、nsP1-4 ORF和SGP如SEQ IDNO：23所示。
BHK细胞的体外转染证实了该RNA能够介导亚基因组ORF编码的报告基因(分泌型胚胎碱性磷酸酶)的表达(图17A)。
实施例9基因组6381-7083位核苷酸内nsP4同源性降低的VEEV复制子RNA修饰
如实施例2中描述的构建体C02一样，基于热力学参数对局部RNA折叠进行生物信息学检查用于选择非结构化核苷酸区域，并且相应地在该区域中替换一系列沉默突变，以创建命名为C01的构建体。图13是编码构建体1(C01)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸6381-7083(灰色框)中的密码子以降低与野生型病毒的同源性。总的来说，沉默突变使nsP4 CDS与野生型的序列同一性降低了4.69％。质粒从T7启动子转录产生C01复制子RNA。C01的5’UTR、nsP1-4ORF和SGP如SEQ ID NO：20所示。
BHK细胞的体外转染证实了该RNA(C01)能够介导亚基因组ORF编码的报告基因(分泌型胚胎碱性磷酸酶)的表达(图17A)。
实施例10由C06的nsP1同源性降低和C07的nsP2同源性降低修饰的单个VEEV复制 子RNA
鉴于在实施例4和7中观察到的成功，将同源性降低的区域组合在单个克隆中，以创建命名为C09的构建体。图14是编码构建体9(C09)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸658-1620和2560-3954(灰色框)中的密码子以降低与野生型病毒的同源性。总的来说，沉默突变使nsP1 CDS与野生型的序列同一性降低了8.96％，nsP2 CDS的序列同一性降低了15.97％。质粒从T7启动子转录产生C09复制子RNA。C09的5’UTR、nsP1-4ORF和SGP如SEQ ID NO：28所示。
图17B的柱状图示出了SEAP表达测试的结果，该测试通过对用指定复制子RNA转染16小时的BHK细胞的条件上清液取样并通过比色法在650nm波长处吸光度来定量SEAP表达。
如图17B所示，BHK细胞的体外转染证实该RNA(C09)能够介导亚基因组ORF编码的报告基因(分泌型胚胎碱性磷酸酶)的表达。
实施例11由C03的nsP2同源性降低和C01的nsP4同源性降低修饰的单个VEEV复制 子RNA
鉴于在实施例6和9中观察到的成功，将同源性降低的区域组合在单个克隆中，以创建命名为C10的构建体。图15是编码构建体10(C10)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸3694-3954位和6381-7083位(灰色框)中的密码子以降低与野生型病毒的同源性。总的来说，沉默突变使nsP2 CDS与野生型的序列同一性降低了3.19％，与nsP4 CDS的序列同一性降低了4.69％。质粒从T7启动子转录产生C10复制子RNA。C10的5’UTR、nsP1-4 ORF和SGP如SEQ IDNO 29所示。
图17C是示出了SEAP表达测试的结果的柱状图，该测试通过对用指定复制子RNA转染16小时的BHK细胞的条件上清液取样并通过比色法在650nm波长处吸光度来定量SEAP表达。
如图17C所示，BHK细胞的体外转染证实该RNA(C09)能够介导亚基因组ORF编码的报告基因(分泌型胚胎碱性磷酸酶)的表达。
实施例12在所有nsP的区域中用同源性降低修饰单个VEEV复制子RNA
考虑到先前实施例中描述的方法的普遍成功，先前被分析且同源性降低以创建构建体C01、C02、C04、C05、C06和C07的区域被重新集中分析，并在确定的非结构化核苷酸延伸中替换沉默突变，以创建命名为C11的构建体。图16是编码构建体11(C11)的复制子序列模板的DNA质粒的示意图。在此图中，DNA质粒与图5中所示的类似，不同之处仅在于修改了基因组核苷酸503-658、658-1620、1620-2560、2560-3954、3954-4120和6381-7083(灰色框)中的密码子以降低与野生型病毒的同源性。在此实施例中，与野生型病毒相比，所使用的沉默突变导致nsP CDS的同源性总体降低，降低程度相当于所有先前突变区域的总和。质粒从T7启动子转录产生C11复制子RNA。C11的5’UTR、nsP1-4 ORF和SGP如SEQ ID NO：30所示。
图17A-17D示出了SEAP表达测试的结果，以证明密码子适应复制子RNA中存在的编码SEAP的亚基因组ORF的功能。图17A是示出了SEAP表达测试的结果的柱状图，该测试是通过与对照、野生型(WT)和未转染(无转染)细胞相比，对转染了4小时(左测柱)、16小时(中间柱)和24小时(右侧柱)的BHK细胞的条件上清液取样进行检测的。图17B和17C是显示SEAP表达测试的结果的柱状图，其通过对用指定复制子RNA(图17B-C09，以及图17C-C10)转染16小时的BHK细胞的条件上清液进行取样，并通过在比色试验中测量650nm处的吸光度来量化SEAP表达。正如该数据所证明的，生物信息学分析确定的区域中nsP密码子的修饰不会对亚基因组ORF中编码的转基因的体外表达产生负面影响。图17D示出了小鼠中SEAP表达的结果。在这些实验中，每组四只小鼠通过肌内注射1μg指定的密码子适应复制子进行给药。收集血清并通过发光试验定量SEAP的相对表达。图17D显示了每只动物的平均RLU、误差条、每组的标准差。两只未注射的小鼠(标记为“误差条、每组的标.”)用作阴性对照以指示背景信号。如该数据所证明的，通过生物信息学分析鉴定的区域中nsP密码子的修饰不会对体内亚基因组ORF中编码的转基因的表达产生负面影响。
总的来说，通过该分析揭示了nsP的广泛区域包含非结构化核苷酸片段，这些片段易于发生不影响基因表达的沉默突变。图18是VEEV基因组的比例图，其中通过RNA折叠分析和本发明所述的实验确定的非结构多蛋白的区域富集于易于沉默突变的非结构区域，被标记为灰色框。本发明的实施方式公开了跨越这些区域的沉默突变，使得通过基本局部比对搜索(BLAST)得到的序列与天然存在的VEEV型基因组nsP编码区域相差高达10％的非同一性。
实施例13复制具有nsP4 C末端编码区的VEEV复制子RNA以引入C末端融合位点并 恢复亚基因组启动子(SGP)
通过常规克隆直接插入任意附加蛋白(“过常规)以融合nsP4和xP ORF会破坏组成SGP的核苷酸序列元件。由于介导亚基因组RNA的转录，从而介导亚基因组ORF的后期翻译需要SGP，因此这种将xP融合到nsP4 C末端的方法将破坏复制子介导亚基因组基因座转基因表达的能力。图3示出了通过复制SGP区域(包括nsP4 C末端的编码区域)来规避该问题的过程。通过掺入产生常规克隆的限制性位点的核苷酸，然后，上游重复区域可以用作xP框内插入的靶标。图3示出了可遵循的简单步骤的实例，以克隆可修饰的nsP4开放阅读框(ORF)，同时保持完整的延伸亚基因组启动子(SGP，条纹框)。然而，这种简单化的方法留下了两个具有相同序列的区域，当在制造所需的细菌菌株中繁殖该DNA模板序列时，该序列会导致遗传不稳定。
当该构建体在实践中被克隆为DNA模板时，代表编码nsP4 C末端的密码子的相同序列使得标准细菌菌株(DH5-a)无法保持重复序列完整。从用图3中概述的方法构建的DNA模板中分离的所有克隆都携带消除nsP4 C-末端重复的缺失。
实施例14具有nsP4 C末端编码区重复和同源性降低以引入C末端融合位点，从而 恢复亚基因组启动子(SGP)，并消除了重复序列元件的VEEV复制子RNA
图4示出了在保持完整的延伸亚基因组启动子(SGP，条纹框)的同时克隆可修饰的nsP4开放阅读框(ORF)所遵循的最佳步骤的实例。这种方法消除了有问题的重复序列元素，DNA模板可以很容易地在常见菌株中繁殖和制造。图4示出了将xP融合到nsP4C末端，同时保持完整的SGP区域的最佳过程。上游重复序列的密码子首先被修改，以消除与下游重复序列的同源性，下游重复序列保持野生型序列以确保亚基因组ORF附近的完整SGP。位于任意xP的3’的下游SGP的序列如SEQ ID NO：14所示。然后可以通过常规分子克隆将上游同源性降低的重复区域(以SEQ ID NO：18所示的序列为例)融合在nsP4末端框内插入的任意xP。最终nsP4编码RNA序列的实例如SEQ ID NO：19所示。图19是说明为了实验而修饰的nsP4复制子序列的示意图，其中附加到nsP4蛋白的外源蛋白(xP)是绿色荧光蛋白(GFP)。在此图中，SGORF中的结构多蛋白被报告基因SEAP取代。在此图中，nsP包括编码GFP的基因，其前面是作为xP的自裂解2A肽，亚基因组ORF包括分泌型胚胎碱性磷酸酶(SEAP)报告基因。由这些修改产生的nsP1-nsP2-nsP3-nsP4-2A融合多蛋白氨基酸序列可以进一步与目标xP(例如GFP)融合，如SEQ ID NO：12所示。编码该工程化的nP-2A ORF的示例性DNA序列如SEQ ID NO：13所示。nsP1-nsP2-nsP3-nsP4-2A-GFP氨基酸序列如SEQ ID NO：15所示，编码该工程化的nsP-2A-GFP ORF的示例性DNA序列如SEQ ID NO：16所示。经过总的nsP4-2A-GFP修饰的复制子RNA构建体被命名为C16。该复制子RNA被称为VEEVrep-nsP4[GFP]-SEAP，其5′UTR、nsP1-4-GFP融合ORF和SGP的相应RNA序列如SEQ ID NO：31所示。
图20A-20C示出了体外细胞中由工程化的nsP4修饰的VEEV复制子介导的基因表达。BHK细胞用实验性nsP4修饰的复制子转染，该复制子(VEEVrep-nsP4[GFP]-SEAP)编码非结构蛋白ORF中的GFP和亚基因组ORF中的SEAP。图20A示出了荧光显微镜检查的结果，证明当用nsP4(GFP)修饰的VEEV-SEAP复制子RNA进行转染时存在多个GFP阳性细胞(右上)，并且在未转染的对照细胞中不存在GFP表达(右下)。图20B是用GFP特异性单克隆抗体转染的BHK细胞裂解物的免疫印迹(顶部条带)，以GAPDH检测用作负载对照(下方条带)，其显示与未转染对照(NoTx)相比，通过两个独立分离的nsP4(GFP)修饰的VEEV复制子RNA克隆(克隆1和克隆2)确认了GFP表达。图20C是显示编码SEAP的亚基因组ORF的功能的柱状图，该实验通过对转染的BHK细胞的条件上清液取样并通过比色试验定量SEAP表达。参考此图，与未转染对照(其中仅观察到试验背景吸光度)相比，克隆1和克隆2都显示出高SEAP表达。如图20A-20C所示，该RNA在转染至BHK细胞中时同时驱动GFP表达(图20A和29B)和SEAP表达(图20C)。这证实了nsP4介导的基因组复制转录和SGP介导的翻译都得到了维持，同时，从独立于亚基因组ORF的早期翻译位点成功产生了额外的辅助功能蛋白(GFP)。
实施例15同时包含nsP4C末端xP融合和同源性降低的nsP的单个VEEV复制子RNA
鉴于实施例9和12中的成功，生成了组合了C09的同源性降低的区域和C16的nsP4-2A-GFP修饰的VEEV复制子，以创建命名为C13的新构建体。图21A-21B示出了由该工程化的nsP4修饰的VEEV复制子介导的基因表达。该复制子RNA结合了如本发明中所述的密码子适应和nsP4 C末端修饰。
图21A是示出了荧光显微镜检查结果的照片，显示了当用密码子适应的nsP4(GFP)修饰的VEEV-SEAP复制子RNA(C13)进行转染时的多个GFP阳性细胞(上图，右)，以及在未转染的对照细胞中不存在GFP表达(下图，右)。
图21B是显示发光试验(RLU)结果的图表，该发光试验与从未注射的对照小鼠收集的血清相比，对从注射了野生型VEEVrep-SEAP复制子RNA(WT)或注射了在非结构多蛋白基因的非结构化区域中携带优化的密码子的经nsP4(GFP)修饰的VEEV(经修饰的)复制子RNA(类似于VEEVrep-nsP4[GFP]-SEAP)的小鼠收集的血清中的SEAP浓度进行定量。于注射后第1天、第3天和第5天收集血清。在这些实验中，小鼠(每组三只)通过肌肉注射标准VEEV-SEAP复制子RNA或等剂量在非结构多蛋白基因的非结构区域中携带优化的密码子的经nsP4(GFP)修饰的VEEV复制子RNA。在指定时间收集血清并通过发光试验定量SEAP的浓度。示出了每组小鼠的平均RLU、误差条、标准差。两只未注射的小鼠用作阴性对照以指示背景信号。如该数据所证明的，通过此处描述的方法对nsP4基因的修饰不会对体内亚基因组ORF中编码的转基因的表达产生负面影响。
如图21A所示，观察到该RNA能够在体外转染的细胞中表达GFP。如图21B所示，还观察到在小鼠中注射C13导致与亲代VEEV-SEAP复制子RNA相等的SEAP血清浓度，表明与野生型病毒的同源性降低，以及通过与nsP4融合而结合的额外转基因有效载荷并不会妨碍体内基因表达功效。
实施例16编码与nsP4 C末端融合的功能性酶，并在SG ORF中分离转基因的复制子 RNA
按照实施例14中描述的设计，可以将酶安装在nsP4 C末端(即，xP可以是功能性酶)。如上文概述和SEQ ID NOS：18和SEQ ID NO：14所示，SGP可以通过复制来维持，将野生型SGP序列置于xP融合体的下游(在本实施例中为酶的下游)，并修改nsP4 C末端编码区的上游重复序列，以保持正确的氨基酸序列，同时降低与野生型SGP的同源性。这确保了构建体的遗传稳定性并避免了同源驱动的重组，否则会导致xP的删除(如实施例13中所例示的，如果不进行密码子改变。就会发生这种情况)。
图22A-22B示出了经修饰以编码小核糖核酸病毒P1结构多蛋白(其在SG ORF中编码)的蛋白水解加工所需的3Cpro蛋白酶的甲病毒复制子RNA的基因表达。
图22A示出了对甲病毒复制子的nsP4编码序列进行修饰以编码小核糖核酸病毒P1结构多蛋白(O1 Manisa P1多蛋白)所需的3Cpro蛋白酶的构建体的示意图，称为VEEVrep-nsP4[3C]-P1。该RNA的5’UTR、nsPl-4、3Cpro和SGP区域的序列如SEQ ID NO：32所示。编码SGORF中的P1结构多蛋白的RNA序列如SEQ ID NO：33所示。nsPORF中编码的nsP4-2A-3Cpro融合多蛋白的氨基酸序列如SEQ ID NO：40所示。
该图说明了示例性VEEV复制子RNA的设计，其中使用本发明描述的方法通过2A自裂解肽将C末端融合至病毒蛋白酶。本实施例中的病毒蛋白酶是小核糖核酸病毒(口蹄疫病毒(FMDV))Al2株的3Cpro。3Cpro酶是插入复制子NPS4修饰区域的良好候选酶的一个例子。3Cpro是一种高度加工酶，当外源递送至细胞时，与其底物相比，仅需要相对较低的分子拷贝数。3Cpro活性是小核糖核酸病毒衣壳多蛋白(称为P1多蛋白)的翻译后加工所必需的。除了在低表达水平下发挥全部功能外，由于蛋白酶固有的毒性，3Cpro的低拷贝数也是理想的选择。当3Cpro在体外高水平表达时，细胞代谢停滞且活力下降。人们已尝试创建FMDV疫苗，其中编码P1多蛋白的复制子RNA和编码3Cpro的mRNA共同配制，目的是将两种分子递送至同一细胞并允许P1的加工(反式互补)。在这种反式互补疫苗设计中，P1-2A与3Cpro的比例需要为～30-40：1，以尽量减少3Cpro的毒性。虽然存在允许将多个RNA分子共封装和递送到同一细胞的递送平台，但由于这些比率要求，不可能100％的纳米颗粒斗含有至少一个编码3Cpro的mRNA拷贝。因此，反式互补方法最大限度地降低了疫苗产品的潜在功效，因为单个纳米颗粒如果不具有至少一个拷贝的两种RNA分子，将无法有效生成经过加工的衣壳，从而无法促进体液免疫反应。
鉴于3Cpro酶具有高度细胞毒性，并且必须维持在低稳态浓度，以避免通过其蛋白水解活性最终破坏细胞过程，因此它在nsP4 C末端区域编码，作为FMDV疫苗构建体的一部分来介导表达。P1多蛋白包含VP0、VP2和VP3亚基，这些亚基必须被3Cpro切割成单独的多肽链，以便组装成衣壳结构。为了制造功能性小核糖核酸病毒疫苗，这些衣壳结构必须组装起来才能被免疫系统识别，从而驱动中和抗体的产生。P1多蛋白的VP0片段还包含VP4和VP2蛋白，它们通过3Cpro独立过程进行裂解，并且不是衣壳组装所必需的。为了制造有用的FMDV疫苗，FMDV O1 Manisa株的P1多蛋白被编码在VEEV的复制子的SG ORF中。如图22A中所示，称为VEEVrep-nsP4[3C]-P1的复制子通过在nsP4 C末端进行编码而提供顺式3Cpro活性。另一个复制子VEEVrep-nsP4[3Cmut]-P1包含一个会中止3Cpro序列的翻译的插入突变，以用作平行对照。作为额外的对照，制备了具有野生型nsP4和SGP区域的复制子，其仅编码SG ORF中的P1多蛋白(VEEVrep-P1)。这些RNA通过体外转录和转录后加帽合成。用每孔1μg的每种P1编码复制子转染12孔培养皿中的BHK细胞，并于约1天后制备细胞裂解物用于通过免疫印迹进行分析。
图22B是对用以下RNA转染的BHK细胞裂解物进行的免疫印迹的照片：泳道1，VEEVrep-nsP4[3C]-P1；泳道2，复制子RNA与泳道1相似，但携带插入突变，引入提前终止密码子并切除3Cpro多肽的表达，作为阴性对照(VEEVrep-nsP4[3Cmut]-P1)；泳道3，VEEVrep-P1(类似于VEEVrep-nsP4[3C]-P1，但缺少nsP4融合的3Cpro)；泳道4，将VEEVrep-P与1/40质量的编码3Cpro的mRNA组合转染，作为阳性对照。参考该图，只有在VEEVrep-nsP4[3C]-P1(泳道1)的介导下，才能将～100kDa的P1多蛋白成功蛋白水解加工成～35kDa的免疫原性VP0衣壳片段。可以看到代表完整VP4+VP2+vP3的不完全裂解中间产物，其表观分子量为～55kDa。使用VP2特异性抗体来探测印迹。M，分子量参照标记；C，未经处理的阴性对照细胞。该图显示了用FMDV VP2特异性单克隆抗体探测后样品的免疫印迹结果。缺乏3Cpro活性的VEEVrep-nsP4[3Cmut]-P1和VEEVrep-P1，仅产生全长完整P1多肽，视为～100kDa带。VEEVrep-nsP4[3C]-P1成功产生了所需的完全加工的VP0片段，表明衣壳加工按预期进行。值得注意的是，处理效果优于阳性对照(泳道4)，其中VEEVrep-P1与常规mRNA分子中反式编码的单独3Cpro以40∶1的质量比(已知当作为疫苗施用时在猪中产生保护性抗体滴度的比例)组合转染。由于缺乏加工中间片段VP4+2+3，因此卓越的加工效率是显而易见的。这证明了使用本发明中解释的方法在nsP4 C末端区域编码功能性蛋白(在本特定实施例中为酶)以引发所需的生物化学结果的实用性。
实施例17编码与nsP4 C末端融合的免疫调节因子，并在SG ORF中分离转基因的复 制子RNA
为了产生期望的结果，增加或减少细胞中复制子RNA活性引发的免疫反应是有用的。例如，可以通过在癌细胞中诱导强烈的先天免疫反应来治疗癌症。
图23A-23D示出了来自经修饰以编码人STING蛋白的甲病毒复制子RNA的基因表达数据，所述甲病毒复制子RNA被突变为对于IFN刺激活性具有组成型活性，并且在SG ORF中编码报道基因SEAP。
图23A是构建体VEEVrep-nsP4[STING]-SEAP的示意图，其为包括甲病毒复制子的nsP4编码序列的修饰，以编码人STING蛋白的复制子RNA，其被突变为对于IFN刺激活性具有组成型活性，并且报道基因SEAP在SG ORF中编码。该RNA的5'UTR、nsP1-4、STING和SGP区域的序列如SEQ ID NO：34所示。产生的经翻译的nsP4-2A-STING融合多肽的氨基酸序列如SEQID NO：42所示。该图示出了复制子RNA构建体，其中通过与nsP4 C末端融合来编码有效的免疫调节蛋白STING(突变为组成型活性)。在癌症治疗的情况下，SG ORF中可以编码肿瘤相关抗原或其他免疫调节因子，以驱动针对所需癌症靶点的特异性细胞免疫。为了说明STING修饰的nsP4的功能，在此实施例中，SG ORF编码报告基因SEAP，其可以轻松地从肿瘤细胞培养物中进行定量。为了证明该设计的STING和SEAP编码复制子(称为VEEVrep-nsP4[STING]-SEAP)在人类细胞中可以发挥作用，使用商业转染试剂(TransIT-mRNA，Mirus)与1μgVEEVrep-nsP4[STING]-SEAP或作为对照的非nsP4修饰的VEEVrep-SEAP转染HEK-LuciaTM空白细胞。约1天后，对上清液培养基进行取样并裂解细胞以通过免疫印迹分析STING含量。如图。图23B是在用以下RNA转染的HEK-LuciaTM空白细胞上进行的免疫印迹的照片：泳道1，VEEVrep-SEAP；泳道2，VEEVrep-nsP4[STING]-SEAP；泳道3，无转染对照。在此图中，常见的背景条带用星号标记(*，可能是内源野生型STING)，并且使用人STING特异性抗体来探测印迹。据观察，只有VEEVrep-nsP4[STING]-SEAP表现出一条额外的条带，代表nsP4 C末端区域编码的组成型活性STING蛋白。
图23C是示出与对照(未转染细胞)相比，在VEEVrep-SEAP和VEEVrep-nsP4[STING]-SEAP复制子RNA中的SG ORF中编码的SEAP报道基因的表达结果(通过比色试验中650nm处的吸光度测量)的柱状图。据观察，尽管用免疫调节多肽对nsP4 C末端进行了新的修饰，但VEEVrep-nsP4[STING]-SEAP亚基因组启动子活性得以保留，并且转基因表达完整。参见该图，在比色试验中对培养物上清液中的SEAP进行定量，该基因被证明由两种构建体表达，证明VEEVrep-nsP4[STING]-SEAP构建体成功地进行了复制和SGP驱动的转基因表达。SEAP表达较低可能是由于STING蛋白触发的强烈先天免疫反应诱导HEK细胞活力降低。I型IFN信号传导活性可以通过HEK-LuciaTM空白细胞中分泌的荧光素酶的上清液浓度来测量，在这些细胞中，荧光素酶是在包含IFN刺激基因(ISG)54启动子的IFN诱导型启动子的控制下进行编码的，IFN刺激基因(ISG)54启动子通过多聚体干扰素刺激反应元件(ISRE)增强。如图23D示出与对照(未转染细胞)相比，由VEEVrep-SEAP和VEEVrep-nsP4[STING]-SEAP复制子RNA诱导的IFN信号传导活性的替代测量结果的柱状图。HEK-LuciaTM空白细胞编码受IFN诱导型启动子控制的分泌型荧光素酶报告基因，IFN诱导型启动子包含由多聚体IFN刺激反应元件(ISRE)增强的IFN刺激基因(ISG)54启动子。据观察，VEEVrep-nsP4[STING]-SEAP RNA触发有效的IFN信号传导，而未修饰的VEEVrep-SEAP RNA则不会。
参考图23D，在分泌型荧光素酶的发光试验中，仅VEEVrep-nsP4[STING]-SEAP显示出I型IFN信号激活，证明活性STING蛋白是有功能的并且对转染的人细胞赋予期望的效果。STING是一种免疫调节蛋白，可通过IFN信号级联具有免疫刺激和免疫抑制两种特性，具体取决于施用的组织和细胞类型。因此，这种嵌入STING或其他免疫调节蛋白的方法是一种引发或减弱适应性免疫反应的方法，其使得工程复制子不仅可用于治疗传染病和癌症，还可通过在需要时触发免疫耐受来治疗自身免疫性疾病和过敏。
实施例18黑色素瘤的治疗
使用本发明描述的方法工程化的表达来自nsP4编码区的辅助因子的复制子可用于治疗例如黑色素瘤等癌症。图24A-24D示出了在B16黑色素瘤中来自甲病毒复制子RNA的基因表达和生物学效应，所述甲病毒复制基RNA经修饰以在nsP ORF中编码GFP或人STING蛋白(突变为对IFN刺激活性具有组成型活性)，并在SG ORF中编码报告基因SEAP。
图24A是示出与未转染的对照细胞相比，用1μg VEEVrep-nsP4[GFP]-SEAP转染后约1天，在12孔培养皿中培养的B16黑色素瘤细胞中的GFP表达结果的照片。由本发明所述构建体的nsP4 C末端编码荧光蛋白，观察到大量的GFP表达。图24A描绘了B16黑色素瘤细胞中，由RNA VEEVrep-nsP4[GFP]-SEAP介导的GFP成功表达，其证明了nsP4-融合蛋白在肿瘤细胞系中具有有效表达的能力。
图24B是示出与未转染的对照细胞相比，在用1μg指定复制子RNA：VEEVrep-nsP4[STING]-SEAP、VEEVrep-nsP4[GFP]-SEAP和VEEVrep-SEAP转染后，在12孔培养皿中培养的B16黑素瘤细胞中SEAP表达结果的柱状图。在实验中，SEAP表达定量在转染后1天，通过对培养基的比色试验进行，其显示SG ORF中编码的SEAP基因成功表达。参考图24B，无论nsP4融合蛋白的性质如何，SG ORF(SEAP)(代表抗原或其他癌症治疗的基因序列的转基因位置)中编码的报告基因序列都能在相同细胞中有效表达。
图24C是示出与未转染的对照细胞相比，在用1μg指定复制子RNA：VEEVrep-nsP4[STING]-SEAP和VEEVrep-SEAP转染～1天后培养的B16黑色素瘤细胞(通过ELISA夹心法测定培养上清)的IFN-β分泌测量结果的柱状图。参考该图，观察到VEEVrep-nsP4[STING]-SEAP RNA成功诱导这些癌细胞中的IFN-β分泌，其水平比VEEVrep-SEAP高约73％。这表明与nsP4融合表达的STING蛋白成功诱导了癌细胞的先天免疫反应，这与临床环境中肿瘤的成功清除相关。
图24D是与健康未转染的对照细胞(对照；最左图)相比，用2μg VEEVrep-nsP4[GFP]-SEAP或VEEVrep-nsP4[STING]-SEAP转染1天后B16细胞的光学显微镜检查的照片。参考该图，未转染的对照细胞已增殖并填满培养皿；转染VEEVrep-nsP4[GFP]-SEAP后观察到中度生长失败和细胞病理变化；转染VEEVrep-nsP4[STING]-SEAP后观察到最大的生长抑制和细胞死亡。
参考图24D，当转染1天后通过明场显微镜检查B16细胞时，未处理的细胞(对照)已如预期那样增殖并充满培养皿；相反，在转染VEEVrep-nsP4[GFP]-SEAP后观察到中度生长失败和细胞病理变化，并且在转染VEEVrep-nsP4[STING]-SEAP后观察到更大程度的生长抑制和细胞死亡。总的来说，数据表明nsP4修饰的复制子可以在黑色素瘤细胞中复制，驱动来自nsP4 C末端编码ORF和SG ORF的基因表达，当免疫调节蛋白与nsP4融合时激活更高的先天免疫反应，并且抑制黑色素瘤细胞的生长和存活。在本实施例中描述的所有B16黑色素瘤细胞实验中，细胞在12孔培养皿中，在DMEM+10％灭活胎牛血清(IFS)中生长。为了分析基因表达(GFP、SEAP)或免疫激活(IFN-激)，用1μg指定RNA转染细胞，并在1天后进行分析。为了通过显微镜测量细胞增殖和活力，用2μgRNA转染细胞，并在1天后捕获图像。
实施例19HPV诱发的上皮肿瘤的治疗
为了证明本发明中公开的工程化复制子的普遍适用性，在另一种肿瘤细胞类型中证明了nsP4修饰的复制子RNA的功能：TC-1上皮肿瘤细胞，该细胞表现出由HPV E6和E7癌基因转化驱动的恶性生长。
图25A-25D示出了在TC-1癌细胞中来自经修饰以编码人STING蛋白的甲病毒复制子RNA的基因表达，该甲病毒复制子RNA被突变为对IFN刺激活性具有组成型活性，并在SGORF中编码报告基因SEAP。
图25A示出与未转染的对照细胞相比，用2μg指定复制子RNA：VEEVrep-nsP4[GFP]-SEAP、VEEVrep-nsP4[STING]-SEAP转染2天后，在12孔培养皿中培养的TC-1癌细胞中SEAP表达结果的柱状图。在本实验中，通过培养基的比色试验对SEAP表达进行定量，显示在SG ORF中编码的SEAP基因的成功表达。参考图25A，如在B16黑素瘤(实施例18)中所见，SEAP的表达证实了SG ORF对于VEEVrep-nsP4[STING]-SEAP和VEEVrep-nsP4[GFP]-SEAP RNA在这种不相关癌症类型中的活性。
图25B是示出与未转染的对照细胞相比，用2μg指定复制子RNA：VEEVrep-nsP4[GFP]-SEAP或VEEVrep-nsP4[STING]-SEAP转染1天后，培养的TC-1癌细胞(通过ELISA夹心法测定培养上清)的IFN-β分泌测量结果的柱状图。参考图25B，nsP4编码的组成型活性STING融合蛋白比不含STING的复制子诱导更多的IFN-β分泌。
图25C是示出用1μg VEEVrep-nsP4[GFP]-SEAP转染后约3天，转染后的TC-1癌细胞团聚物中GFP表达结果的照片。参考此图，与未处理的对照细胞中没有GFP表达(下图，右)相比，通过在构建体nsP4 C末端编码荧光蛋白，在整个多细胞结构中观察到大量GFP表达(上图，右)。参考图25C，在从TC-1培养物分离的细胞团块中，证实VEEVrep-nsP4[GFP]-SEAP的GFP表达在整个结构中广泛存在。
图25D是示出与未处理的对照细胞相比，用4μg指定复制子RNAVEEVrep-nsP4[STING]-SEAP转染1天后，TC-1癌细胞的光学显微镜检查结果的照片。参考此图，观察到未处理的细胞(对照)已增殖并填满培养皿；转染VEEVrep-nsP4[STING]-SEAP后观察到生长抑制和细胞死亡。
如图25D所示，正如对B16黑色素瘤细胞所观察到的一样，用VEEVrep-nsP4[STING]-SEAP治疗严重抑制了增殖和活力。
这些数据证实经nsP4修饰的复制子在不同类型的癌症治疗中具有广泛的适用性。在本实施例中描述的所有TC-1癌细胞实验中，细胞在12孔培养皿中，在DMEM+10％IFS中生长。为了通过显微镜分析GFP表达，用1μg RNA转染细胞，用刮刀刮擦细胞以释放团聚物，并在3天后成像。为了通过IFN-过为了通过镜分测量免疫激活，用2μg指定的RNA转染细胞，并在1天后分析培养基。为了通过显微镜测量细胞增殖和活力，用4μg RNA转染细胞，并在1天后捕获图像。用2μg指定的RNA转染2天后，通过对培养基进行比色试验来测量SEAP表达结果。
实施例20在未经生物信息举检查的情况下，经同源性降低的修饰的VEEV复制子 RNA
生成了另外四个同源性降低的复制子RNA。26A-26G示出了经修饰与野生型同源性降低的VEEV复制子RNA以及来自这些构建体的表达数据。VEEVrepHK-SEAP是通过在酸658和3359位核苷之间的nsP1和nsP2接合处使用一系列沉默突变而产生的(图26A)；核苷酸位置参考SEQ ID NO：17所示的VEEV基因组序列。图26A是编码构建体12(C12；VEEVrepHK-SEAP)的复制子序列模板的DNA质粒的示意图。参考此图，DNA质粒与图5所示的类似，不同之处仅在于修改了基因组核苷酸658-3359位(灰色框)的密码子以降低与野生型病毒的同源性，而不考虑可能存在的任何二级结构元件。C12质粒转录产生的RNA的5’UTR、nsP1-4编码区和SGP如SEQ ID NO：35所示。
VEEVrepHK(ΔU)-SEAP在相同区域内进行类似突变，但通过仅选择尽可能省略尿嘧啶核苷碱基的密码子(图26B)。图26B是构建体13(C13；VEEVrepHK(ΔU)-SEAP)的示意图。参考该图，DNA质粒与图5所示的类似，不同之处仅在于修改了基因组核苷酸658-3359位(灰色框)的密码子以降低与野生型病毒的同源性，而不考虑任何二级结构元件的可能存在，并且优先选择避开尿嘧啶碱基的密码子。C13质粒转录产生的RNA的5’UTR、nsP1-4编码区和SGP如SEQ ID NO：36所示。
VEEVrepES-SEAP是通过在核苷酸4120和6965位之间的nsP3和nsP4接合处应用一系列沉默突变而生成的。图26C是构建体14(C14；VEEVrepES-SEAP)的示意图。参考该图，DNA质粒与图5所示的类似，不同之处仅在于修改了基因组核苷酸4120-6965位(灰色框)中的密码子以降低与野生型病毒的同源性，而不考虑任何二级结构元件的可能存在。C14质粒转录产生的RNA的5’UTR、nsP1-4编码区和SGP如SEQ ID NO：37所示。
VEEVrepES(ΔU)-SEAP在相同区域进行类似突变，但是通过仅选择尽可能省略尿嘧啶核苷碱基的密码子(图26D)。图26D是构建体15(C15；VEEVrepES(ΔU)-SEAP)的示意图。参考此图，DNA质粒与图5所示的相似，不同之处仅在于修改了基因组核苷酸4120-6965位(灰色框)的密码子以降低与野生型病毒的同源性，不考虑任何二级结构元件的可能存在，并且优先选择避开尿嘧啶碱基的密码子。C15质粒转录产生的RNA的5’UTR、nsP1-4编码区和SGP如SEQ ID NO：38所示。
这些构建体在没有考虑序列中存在的二级结构元件的情况下生成，以评估减少与野生型病毒同源性的典型非生物信息学方法的性能。
图26E示出与未转染的对照细胞相比，在用1μg VEEVrep-SEAP、VEEVrepHK-SEAP或VEEVrepHK(p相比，在用典型非生物信转染16小时后，在12孔培养皿中培养的BHK细胞中SEAP表达结果的柱状图。在本实验中，通过对培养基的比色试验来定量SEAP的表达，其显示在SG ORF中编码的SEAP基因的成功表达。
图26F示出与未转染的对照相比，在用1μg VEEVrep-SEAP、VEEVrepES-SEAP或VEEVrepES(SEAPAP过对培养基的转染16小时后，在12孔培养皿中培养的BHK细胞中SEAP表达结果的柱状图。在此实验中，通过对培养基的比色试验定量SEAP表达，其显示VEEVrepES-SEAP或VEEVrepES(SEAP，P过对构建体的SG ORF中编码的SEAP基因没有表达。
BHK细胞的体外转染证实VEEVrepHK-SEAP和VEEVrepHK(SEAP，P过对能够介导亚基因组ORF编码的报道基因(分泌型胚胎碱性磷酸酶)的表达(图26E)，但VEEVrepES-SEAP和VEEVrepES(SEAP，P过对不能介导(图26F)。这表明如果不检查一级RNA序列来识别二级结构元件，就无法通过对任意编码片段的沉默突变来可靠地获得功能性复制子。VEEVrepFIK-SEAP和VEEVrepHK(-SEAP获得功似乎代表了一个偶然的发现，表明原始方法可以自发地产生工作复制子，然而进一步的表征表明，这两种构建体都失去了在体内的SEAP表达活性，该表达活性如通过在小鼠(n＝5)肌内注射2μg LNP配制的RNA 1天后，通过化学发光试验测定血清SEAP浓度来测量。图26G是示出与对照(来自未处理的小鼠的血清)相比，注射2μg LNP配制的VEEVrep-SEAP、VEEVrepHK-SEAP或VEEVrepHK(SEAP作复制子后(通过化学发光试验)测量的血清中SEAP体内表达结果的柱状图。在此实验中，小鼠(每组5只)通过肌内(i.m.)注射施用指定的RNA，并在第二天采集血清样本，并通过化学发光试验对SEAP进行定量。绘制每组小鼠的平均RLU；误差线，标准差。来自两只未经治疗的小鼠(n＝2)的血清用作阴性对照组。
虽然体内性能得到证实，但表达低于标准水平，并且凸显了本发明内容中描述的生物信息学方法的优越性，与野生型复制子相比，其在体内产生了等同或更好的基因表达(参见实施例15)。
实施例21工程复制子是安全的，不会伤害健康的人体细胞
为了证明本发明中公开的工程化复制子的安全性，确认了nsP4修饰的复制子RNA的功能在非癌性人类细胞中较低。人包皮成纤维细胞(HFF)是一种通常用于研究的正常细胞类型，代表健康的非转化细胞生物学特性，同时还是多种核酸和病毒物种的有效转染和感染宿主。图27A-27D示出了正常HFF细胞中的VEEV复制子RNA表达结果和影响。图27A示出了与未转染的对照细胞相比，在用2μg或4μg指定复制子RNA(VEEVrep-nsP4[STING]-SEAP，或VEEVrep-SEAP)转染后1天，在12孔培养皿中培养的正常HFF细胞中SEAP表达结果的柱状图。在此实验中，通过对培养基的比色试验来定量SEAP的表达。
参考该图，用2μg或4μg在SG ORF中编码SEAP报道基因的非工程复制子(VEEVrep-SEAP)转染12孔培养皿中的HFF，导致转染后1天在通过对培养基进行的比色试验测得的SEAP表达水平低；相反，含有nsP4编码的组成型活性STING融合蛋白的复制子(VEEVrep-nsP4[STING]-SEAP)不会诱导SEAP分泌，表明工程复制子在健康细胞中的复制和转基因表达受到抑制。
图27B是用47B表明工程复制子在健康细胞中的复制和转基因表达受转染后约4天，胰蛋白酶解离的正常人HFF细胞中GFP表达结果的照片，其显示来自nsP4 C末端编码的GFP蛋白在这些健康的非癌细胞中没有有效的基因表达。参考该图，与上面测试的癌细胞类型(参见实施例18和19)不同，在用VEEVrep-nsP4[GFP]-SEAP转染的HFF细胞中没有观察到GFP表达。
图27C是示出来自经转染的HFF的IFN-经活性的测量结果的柱状图。在以1胞对、2胞对和4胞对剂量转染指定RNA(VEEVrep-nsP4[STING]-SEAP、VEEVrep-nsP4[GFP]-SEAP、VEEVrep-SEAP)1天后收集来自12孔培养皿中的HFF的条件培养基。从未转染的细胞收集培养基作为阴性对照(对照HFF)。收集的培养基用于HEK-Lucia空白细胞培养，以测量其对于培养基中的I型IFN的相应。未处理的HEK-Lucia空白细胞作为额外的阴性对照(无培养基对照)包含在内。在本实验中，通过化学发光试验在第二天检测HEK-Lucia空白细胞上清液中的IFN应答报告基因Lucia。如本发明中所述的工程化的复制子VEEVrep-nsP4[GFP]-SEAP和VEEVrep-nsP4[STING]-SEAP在健康HFF型细胞中不刺激I型IFN先天免疫应答。参见图27C，基于用条件HFF培养基处理IFN-β敏感性HEK-Lucia空白细胞的结果，工程化复制子VEEVrep-nsP4[STING]-SEAP和VEEVrep-nsP4[GFP]-SEAP也不会在这些正常细胞中驱动I型IFN信号传导的急剧增加。
图27D是与未转染的对照相比，用4μg指定复制子RNA(VEEVrep-nsP4[GFP]-SEAP和VEEVrep-nsP4[STING]-SEAP)转染后4天HFF细胞的光学显微镜检查结果的照片。经观察，未处理的细胞(对照)已增殖填满培养皿并显示出健康的纤维形态。RNA转染的细胞表现出相同的增殖和健康的形态。如图27D所示，没有观察到对细胞生长、形态或活力的有害影响。为了通过显微镜测量细胞增殖并评估健康状况，用2μg或4μg RNA转染细胞，并在3天后捕获图像。即使在用4μg的高剂量RNA转染时，所有条件下的细胞都增殖并保持完整，具有预期的纤维形态(图27D)，这表明这些RNA在健康细胞中的施用是安全的，并且表现出针对非癌旁观细胞的非靶向免疫原性可忽略不计。这与它们对癌细胞的作用形成对比，其中nsP4融合蛋白和SG ORF均强烈表达并且能够阻碍癌细胞生长并触发先天免疫应答(参见实施例18和19)。
参考文献
US20140079734A1——对甲病毒复制子进行修饰，其为了影响复制效果而将基因组5’末端的一个组分复制到亚基因组区域。
WO2014170493A2——对复制子的不同的修饰方法，其提及为了调节SGP而在nsP4的3’区域的突变。
J Virol.2006Oct；80(20)：9962-9969——描述了nsP4的功能和蛋白的C末端标签，表明通过修饰这些端点，结构可能会得到保留。
SEQUENCE LISTING
<110> 蒂巴生物技术有限公司
<120> 人工甲病毒衍生的RNA复制子表达系统
<130> TIB-PT012WO
<150> 63/163,475
<151> 2021-03-19
<160> 46
<170> PatentIn version 3.5
<210> 1
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C01 nsP coding sequence DNA
<400> 1
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc tcgtaacata ggcctatgca gctctgacgt tatggagcgg 660
tcacgtagag ggatgtccat tcttagaaag aagtatttga aaccatccaa caatgttcta 720
ttctctgttg gctcgaccat ctaccacgag aagagggact tactgaggag ctggcacctg 780
ccgtctgtat ttcacttacg tggcaagcaa aattacacat gtcggtgtga gactatagtt 840
agttgcgacg ggtacgtcgt taaaagaata gctatcagtc caggcctgta tgggaagcct 900
tcaggctatg ctgctacgat gcaccgcgag ggattcttgt gctgcaaagt gacagacaca 960
ttgaacgggg agagggtctc ttttcccgtg tgcacgtatg tgccagctac attgtgtgac 1020
caaatgactg gcatactggc aacagatgtc agtgcggacg acgcgcaaaa actgctggtt 1080
gggctcaacc agcgtatagt cgtcaacggt cgcacccaga gaaacaccaa taccatgaaa 1140
aattaccttt tgcccgtagt ggcccaggca tttgctaggt gggcaaagga atataaggaa 1200
gatcaagaag atgaaaggcc actaggacta cgagatagac agttagtcat ggggtgttgt 1260
tgggctttta gaaggcacaa gataacatct atttataagc gcccggatac ccaaaccatc 1320
atcaaagtga acagcgattt ccactcattc gtgctgccca ggataggcag taacacattg 1380
gagatcgggc tgagaacaag aatcaggaaa atgttagagg agcacaagga gccgtcacct 1440
ctcattaccg ccgaggacgt acaagaagct aagtgcgcag ccgatgaggc taaggaggtg 1500
cgtgaagccg aggagttgcg cgcagctcta ccacctttgg cagctgatgt tgaggagccc 1560
actctggaag ccgatgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aaaagcatct ctcgccgttg cactaaatct gtgacttcgg tcgtctcaac cttgttttac 2580
gacaaaaaaa tgagaacgac gaatccgaaa gagactaaga ttgtgattga cactaccggc 2640
agtaccaaac ctaagcagga cgatctcatt ctcacttgtt tcagagggtg ggtgaagcag 2700
ttgcaaatag attacaaagg caacgaaata atgacggcag ctgcctctca agggctgacc 2760
cgtaaaggtg tgtatgccgt tcggtacaag gtgaatgaaa atcctctgta cgcacccacc 2820
tcagaacatg tgaacgtcct actgacccgc acggaggacc gcatcgtgtg gaaaacacta 2880
gccggcgacc catggataaa aacactgact gccaagtacc ctgggaattt cactgccacg 2940
atagaggagt ggcaagcaga gcatgatgcc atcatgaggc acatcttgga gagaccggac 3000
cctaccgacg tcttccagaa taaggcaaac gtgtgttggg ccaaggcttt agtgccggtg 3060
ctgaagaccg ctggcataga catgaccact gaacaatgga acactgtgga ttattttgaa 3120
acggacaaag ctcactcagc agagatagta ttgaaccaac tatgcgtgag gttctttgga 3180
ctcgatctgg actccggtct attttctgca cccactgttc cgttatccat taggaataat 3240
cactgggata actccccgtc gcctaacatg tacgggctga ataaagaagt ggtccgtcag 3300
ctctctcgca ggtacccaca actgcctcgg gcagttgcca ctggaagagt ctatgacatg 3360
aacactggta cactgcgcaa ttatgatccg cgcataaacc tagtacctgt aaacagaaga 3420
ctgcctcatg ctttagtcct ccaccataat gaacacccac agagtgactt ttcttcattc 3480
gtcagcaaat tgaagggcag aactgtcctg gtggtcgggg aaaagttgtc cgtcccaggc 3540
aaaatggttg actggttgtc agaccggcct gaggctacct tcagagctcg gctggattta 3600
ggcatcccag gtgatgtgcc caaatatgac ataatatttg ttaatgtgag gaccccatat 3660
aaataccatc actatcagca gtgtgaagac catgccatta agcttagcat gttgaccaag 3720
aaagcttgtc tgcatctgaa tcccggcgga acctgtgtca gcataggtta tggttacgct 3780
gacagggcca gcgaaagcat cattggtgct atagcgcggc agttcaagtt ttcccgggta 3840
tgcaaaccga aatcctcact tgaagagacg gaagttctgt ttgtattcat tgggtacgat 3900
cgcaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaagtacgc ctgcaacaac 6360
gagtactggg agacattcaa agagaacccc atccggctga ccgaggaaaa cgtggtcaac 6420
tacatcacca agctgaaggg ccccaaagcc gccgctctgt ttgccaagac acacaacctg 6480
aacatgctgc aggacatccc catggacaga ttcgtgatgg acctgaagcg ggacgtgaaa 6540
gtgacccctg gcaccaagca caccgaggaa cggcctaagg tgcaagtgat ccaggccgct 6600
gatcctctgg ccacagccta tctgtgtggc atccacagag aactcgtgcg gagactgaat 6660
gccgtgctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgctga tgattctgga agatctcgga 6840
gtggacgccg agctgctgac actgattgaa gccgcctttg gcgagatcag cagcatccat 6900
ctgcctacca agaccaagtt caagttcggc gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
acaggcagcc cttgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 2
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C02 nsP coding sequence DNA
<400> 2
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat atgccgtgga tggccctaca 480
agcctgtacc accaggccaa caagggcgtc agagtggcct actggatcgg cttcgacacc 540
acacctttca tgttcaagaa cctggctggc gcttacccca gctacagcac aaactgggcc 600
gacgaaaccg tgttaacggc tcgtaacata ggcctatgca gctctgacgt tatggagcgg 660
tcacgtagag ggatgtccat tcttagaaag aagtatttga aaccatccaa caatgttcta 720
ttctctgttg gctcgaccat ctaccacgag aagagggact tactgaggag ctggcacctg 780
ccgtctgtat ttcacttacg tggcaagcaa aattacacat gtcggtgtga gactatagtt 840
agttgcgacg ggtacgtcgt taaaagaata gctatcagtc caggcctgta tgggaagcct 900
tcaggctatg ctgctacgat gcaccgcgag ggattcttgt gctgcaaagt gacagacaca 960
ttgaacgggg agagggtctc ttttcccgtg tgcacgtatg tgccagctac attgtgtgac 1020
caaatgactg gcatactggc aacagatgtc agtgcggacg acgcgcaaaa actgctggtt 1080
gggctcaacc agcgtatagt cgtcaacggt cgcacccaga gaaacaccaa taccatgaaa 1140
aattaccttt tgcccgtagt ggcccaggca tttgctaggt gggcaaagga atataaggaa 1200
gatcaagaag atgaaaggcc actaggacta cgagatagac agttagtcat ggggtgttgt 1260
tgggctttta gaaggcacaa gataacatct atttataagc gcccggatac ccaaaccatc 1320
atcaaagtga acagcgattt ccactcattc gtgctgccca ggataggcag taacacattg 1380
gagatcgggc tgagaacaag aatcaggaaa atgttagagg agcacaagga gccgtcacct 1440
ctcattaccg ccgaggacgt acaagaagct aagtgcgcag ccgatgaggc taaggaggtg 1500
cgtgaagccg aggagttgcg cgcagctcta ccacctttgg cagctgatgt tgaggagccc 1560
actctggaag ccgatgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aaaagcatct ctcgccgttg cactaaatct gtgacttcgg tcgtctcaac cttgttttac 2580
gacaaaaaaa tgagaacgac gaatccgaaa gagactaaga ttgtgattga cactaccggc 2640
agtaccaaac ctaagcagga cgatctcatt ctcacttgtt tcagagggtg ggtgaagcag 2700
ttgcaaatag attacaaagg caacgaaata atgacggcag ctgcctctca agggctgacc 2760
cgtaaaggtg tgtatgccgt tcggtacaag gtgaatgaaa atcctctgta cgcacccacc 2820
tcagaacatg tgaacgtcct actgacccgc acggaggacc gcatcgtgtg gaaaacacta 2880
gccggcgacc catggataaa aacactgact gccaagtacc ctgggaattt cactgccacg 2940
atagaggagt ggcaagcaga gcatgatgcc atcatgaggc acatcttgga gagaccggac 3000
cctaccgacg tcttccagaa taaggcaaac gtgtgttggg ccaaggcttt agtgccggtg 3060
ctgaagaccg ctggcataga catgaccact gaacaatgga acactgtgga ttattttgaa 3120
acggacaaag ctcactcagc agagatagta ttgaaccaac tatgcgtgag gttctttgga 3180
ctcgatctgg actccggtct attttctgca cccactgttc cgttatccat taggaataat 3240
cactgggata actccccgtc gcctaacatg tacgggctga ataaagaagt ggtccgtcag 3300
ctctctcgca ggtacccaca actgcctcgg gcagttgcca ctggaagagt ctatgacatg 3360
aacactggta cactgcgcaa ttatgatccg cgcataaacc tagtacctgt aaacagaaga 3420
ctgcctcatg ctttagtcct ccaccataat gaacacccac agagtgactt ttcttcattc 3480
gtcagcaaat tgaagggcag aactgtcctg gtggtcgggg aaaagttgtc cgtcccaggc 3540
aaaatggttg actggttgtc agaccggcct gaggctacct tcagagctcg gctggattta 3600
ggcatcccag gtgatgtgcc caaatatgac ataatatttg ttaatgtgag gaccccatat 3660
aaataccatc actatcagca gtgtgaagac catgccatta agcttagcat gttgaccaag 3720
aaagcttgtc tgcatctgaa tcccggcgga acctgtgtca gcataggtta tggttacgct 3780
gacagggcca gcgaaagcat cattggtgct atagcgcggc agttcaagtt ttcccgggta 3840
tgcaaaccga aatcctcact tgaagagacg gaagttctgt ttgtattcat tgggtacgat 3900
cgcaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaaatatgc gtgtaataat 6360
gaatattggg aaacgtttaa agaaaacccc atcaggctta ctgaagaaaa cgtggtaaat 6420
tacattacca aattaaaagg accaaaagct gctgctcttt ttgcgaagac acataatttg 6480
aatatgttgc aggacatacc aatggacagg tttgtaatgg acttaaagag agacgtgaaa 6540
gtgactccag gaacaaaaca tactgaagaa cggcccaagg tacaggtgat ccaggctgcc 6600
gatccgctag caacagcgta tctgtgcgga atccaccgag agctggttag gagattaaat 6660
gcggtcctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgttaa tgattctgga agacttaggt 6840
gtggacgcag agctgttgac gctgattgag gcggctttcg gcgaaatttc atcaatacat 6900
ttgcccacta aaactaaatt taaattcgga gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
accggatcac catgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 3
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C03 nsP coding sequence DNA
<400> 3
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc tcgtaacata ggcctatgca gctctgacgt tatggagcgg 660
tcacgtagag ggatgtccat tcttagaaag aagtatttga aaccatccaa caatgttcta 720
ttctctgttg gctcgaccat ctaccacgag aagagggact tactgaggag ctggcacctg 780
ccgtctgtat ttcacttacg tggcaagcaa aattacacat gtcggtgtga gactatagtt 840
agttgcgacg ggtacgtcgt taaaagaata gctatcagtc caggcctgta tgggaagcct 900
tcaggctatg ctgctacgat gcaccgcgag ggattcttgt gctgcaaagt gacagacaca 960
ttgaacgggg agagggtctc ttttcccgtg tgcacgtatg tgccagctac attgtgtgac 1020
caaatgactg gcatactggc aacagatgtc agtgcggacg acgcgcaaaa actgctggtt 1080
gggctcaacc agcgtatagt cgtcaacggt cgcacccaga gaaacaccaa taccatgaaa 1140
aattaccttt tgcccgtagt ggcccaggca tttgctaggt gggcaaagga atataaggaa 1200
gatcaagaag atgaaaggcc actaggacta cgagatagac agttagtcat ggggtgttgt 1260
tgggctttta gaaggcacaa gataacatct atttataagc gcccggatac ccaaaccatc 1320
atcaaagtga acagcgattt ccactcattc gtgctgccca ggataggcag taacacattg 1380
gagatcgggc tgagaacaag aatcaggaaa atgttagagg agcacaagga gccgtcacct 1440
ctcattaccg ccgaggacgt acaagaagct aagtgcgcag ccgatgaggc taaggaggtg 1500
cgtgaagccg aggagttgcg cgcagctcta ccacctttgg cagctgatgt tgaggagccc 1560
actctggaag ccgatgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aaaagcatct ctcgccgttg cactaaatct gtgacttcgg tcgtctcaac cttgttttac 2580
gacaaaaaaa tgagaacgac gaatccgaaa gagactaaga ttgtgattga cactaccggc 2640
agtaccaaac ctaagcagga cgatctcatt ctcacttgtt tcagagggtg ggtgaagcag 2700
ttgcaaatag attacaaagg caacgaaata atgacggcag ctgcctctca agggctgacc 2760
cgtaaaggtg tgtatgccgt tcggtacaag gtgaatgaaa atcctctgta cgcacccacc 2820
tcagaacatg tgaacgtcct actgacccgc acggaggacc gcatcgtgtg gaaaacacta 2880
gccggcgacc catggataaa aacactgact gccaagtacc ctgggaattt cactgccacg 2940
atagaggagt ggcaagcaga gcatgatgcc atcatgaggc acatcttgga gagaccggac 3000
cctaccgacg tcttccagaa taaggcaaac gtgtgttggg ccaaggcttt agtgccggtg 3060
ctgaagaccg ctggcataga catgaccact gaacaatgga acactgtgga ttattttgaa 3120
acggacaaag ctcactcagc agagatagta ttgaaccaac tatgcgtgag gttctttgga 3180
ctcgatctgg actccggtct attttctgca cccactgttc cgttatccat taggaataat 3240
cactgggata actccccgtc gcctaacatg tacgggctga ataaagaagt ggtccgtcag 3300
ctctctcgca ggtacccaca actgcctcgg gcagttgcca ctggaagagt ctatgacatg 3360
aacactggta cactgcgcaa ttatgatccg cgcataaacc tagtacctgt aaacagaaga 3420
ctgcctcatg ctttagtcct ccaccataat gaacacccac agagtgactt ttcttcattc 3480
gtcagcaaat tgaagggcag aactgtcctg gtggtcgggg aaaagttgtc cgtcccaggc 3540
aaaatggttg actggttgtc agaccggcct gaggctacct tcagagctcg gctggattta 3600
ggcatcccag gtgatgtgcc caaatatgac ataatatttg ttaatgtgag gaccccctac 3660
aagtaccacc actaccagca gtgcgaggac cacgccatca agctgagcat gctgaccaag 3720
aaggcctgcc tgcatctgaa tcccggcgga acctgtgtca gcataggtta tggttacgct 3780
gacagggcca gcgaaagcat cattggtgct atagcgcggc agttcaagtt ttcccgggta 3840
tgcaaaccga aatcctcact tgaagagacg gaagttctgt ttgtattcat cggctacgac 3900
agaaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaaatatgc gtgtaataat 6360
gaatattggg aaacgtttaa agaaaacccc atcaggctta ctgaagaaaa cgtggtaaat 6420
tacattacca aattaaaagg accaaaagct gctgctcttt ttgcgaagac acataatttg 6480
aatatgttgc aggacatacc aatggacagg tttgtaatgg acttaaagag agacgtgaaa 6540
gtgactccag gaacaaaaca tactgaagaa cggcccaagg tacaggtgat ccaggctgcc 6600
gatccgctag caacagcgta tctgtgcgga atccaccgag agctggttag gagattaaat 6660
gcggtcctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgttaa tgattctgga agacttaggt 6840
gtggacgcag agctgttgac gctgattgag gcggctttcg gcgaaatttc atcaatacat 6900
ttgcccacta aaactaaatt taaattcgga gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
accggatcac catgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 4
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C04 nsP coding sequence DNA
<400> 4
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc tcgtaacata ggcctatgca gctctgacgt tatggagcgg 660
tcacgtagag ggatgtccat tcttagaaag aagtatttga aaccatccaa caatgttcta 720
ttctctgttg gctcgaccat ctaccacgag aagagggact tactgaggag ctggcacctg 780
ccgtctgtat ttcacttacg tggcaagcaa aattacacat gtcggtgtga gactatagtt 840
agttgcgacg ggtacgtcgt taaaagaata gctatcagtc caggcctgta tgggaagcct 900
tcaggctatg ctgctacgat gcaccgcgag ggattcttgt gctgcaaagt gacagacaca 960
ttgaacgggg agagggtctc ttttcccgtg tgcacgtatg tgccagctac attgtgtgac 1020
caaatgactg gcatactggc aacagatgtc agtgcggacg acgcgcaaaa actgctggtt 1080
gggctcaacc agcgtatagt cgtcaacggt cgcacccaga gaaacaccaa taccatgaaa 1140
aattaccttt tgcccgtagt ggcccaggca tttgctaggt gggcaaagga atataaggaa 1200
gatcaagaag atgaaaggcc actaggacta cgagatagac agttagtcat ggggtgttgt 1260
tgggctttta gaaggcacaa gataacatct atttataagc gcccggatac ccaaaccatc 1320
atcaaagtga acagcgattt ccactcattc gtgctgccca ggataggcag taacacattg 1380
gagatcgggc tgagaacaag aatcaggaaa atgttagagg agcacaagga gccgtcacct 1440
ctcattaccg ccgaggacgt acaagaagct aagtgcgcag ccgatgaggc taaggaggtg 1500
cgtgaagccg aggagttgcg cgcagctcta ccacctttgg cagctgatgt tgaggagccc 1560
actctggaag ccgatgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aaaagcatct ctcgccgttg cactaaatct gtgacttcgg tcgtctcaac cttgttttac 2580
gacaaaaaaa tgagaacgac gaatccgaaa gagactaaga ttgtgattga cactaccggc 2640
agtaccaaac ctaagcagga cgatctcatt ctcacttgtt tcagagggtg ggtgaagcag 2700
ttgcaaatag attacaaagg caacgaaata atgacggcag ctgcctctca agggctgacc 2760
cgtaaaggtg tgtatgccgt tcggtacaag gtgaatgaaa atcctctgta cgcacccacc 2820
tcagaacatg tgaacgtcct actgacccgc acggaggacc gcatcgtgtg gaaaacacta 2880
gccggcgacc catggataaa aacactgact gccaagtacc ctgggaattt cactgccacg 2940
atagaggagt ggcaagcaga gcatgatgcc atcatgaggc acatcttgga gagaccggac 3000
cctaccgacg tcttccagaa taaggcaaac gtgtgttggg ccaaggcttt agtgccggtg 3060
ctgaagaccg ctggcataga catgaccact gaacaatgga acactgtgga ttattttgaa 3120
acggacaaag ctcactcagc agagatagta ttgaaccaac tatgcgtgag gttctttgga 3180
ctcgatctgg actccggtct attttctgca cccactgttc cgttatccat taggaataat 3240
cactgggata actccccgtc gcctaacatg tacgggctga ataaagaagt ggtccgtcag 3300
ctctctcgca ggtacccaca actgcctcgg gcagttgcca ctggaagagt ctatgacatg 3360
aacactggta cactgcgcaa ttatgatccg cgcataaacc tagtacctgt aaacagaaga 3420
ctgcctcatg ctttagtcct ccaccataat gaacacccac agagtgactt ttcttcattc 3480
gtcagcaaat tgaagggcag aactgtcctg gtggtcgggg aaaagttgtc cgtcccaggc 3540
aaaatggttg actggttgtc agaccggcct gaggctacct tcagagctcg gctggattta 3600
ggcatcccag gtgatgtgcc caaatatgac ataatatttg ttaatgtgag gaccccatat 3660
aaataccatc actatcagca gtgtgaagac catgccatta agcttagcat gttgaccaag 3720
aaagcttgtc tgcatctgaa tcccggcgga acctgtgtca gcataggtta tggttacgct 3780
gacagggcca gcgaaagcat cattggtgct atagcgcggc agttcaagtt ttcccgggta 3840
tgcaaaccga aatcctcact tgaagagacg gaagttctgt ttgtattcat tgggtacgat 3900
cgcaaggccc gtacgcacaa cccctacaag ctgagcagca ccctgaccaa catctacacc 3960
ggcagcagac tgcacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacagcca cagaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaaatatgc gtgtaataat 6360
gaatattggg aaacgtttaa agaaaacccc atcaggctta ctgaagaaaa cgtggtaaat 6420
tacattacca aattaaaagg accaaaagct gctgctcttt ttgcgaagac acataatttg 6480
aatatgttgc aggacatacc aatggacagg tttgtaatgg acttaaagag agacgtgaaa 6540
gtgactccag gaacaaaaca tactgaagaa cggcccaagg tacaggtgat ccaggctgcc 6600
gatccgctag caacagcgta tctgtgcgga atccaccgag agctggttag gagattaaat 6660
gcggtcctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgttaa tgattctgga agacttaggt 6840
gtggacgcag agctgttgac gctgattgag gcggctttcg gcgaaatttc atcaatacat 6900
ttgcccacta aaactaaatt taaattcgga gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
accggatcac catgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 5
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C05 nsP coding sequence DNA
<400> 5
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc tcgtaacata ggcctatgca gctctgacgt tatggagcgg 660
tcacgtagag ggatgtccat tcttagaaag aagtatttga aaccatccaa caatgttcta 720
ttctctgttg gctcgaccat ctaccacgag aagagggact tactgaggag ctggcacctg 780
ccgtctgtat ttcacttacg tggcaagcaa aattacacat gtcggtgtga gactatagtt 840
agttgcgacg ggtacgtcgt taaaagaata gctatcagtc caggcctgta tgggaagcct 900
tcaggctatg ctgctacgat gcaccgcgag ggattcttgt gctgcaaagt gacagacaca 960
ttgaacgggg agagggtctc ttttcccgtg tgcacgtatg tgccagctac attgtgtgac 1020
caaatgactg gcatactggc aacagatgtc agtgcggacg acgcgcaaaa actgctggtt 1080
gggctcaacc agcgtatagt cgtcaacggt cgcacccaga gaaacaccaa taccatgaaa 1140
aattaccttt tgcccgtagt ggcccaggca tttgctaggt gggcaaagga atataaggaa 1200
gatcaagaag atgaaaggcc actaggacta cgagatagac agttagtcat ggggtgttgt 1260
tgggctttta gaaggcacaa gataacatct atttataagc gcccggatac ccaaaccatc 1320
atcaaagtga acagcgattt ccactcattc gtgctgccca ggataggcag taacacattg 1380
gagatcgggc tgagaacaag aatcaggaaa atgttagagg agcacaagga gccgtcacct 1440
ctcattaccg ccgaggacgt acaagaagct aagtgcgcag ccgatgaggc taaggaggtg 1500
cgtgaagccg aggagttgcg cgcagctcta ccacctttgg cagctgatgt tgaggagccc 1560
actctggaag ccgatgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagc gagaagctga gctgcattca ccctctggcc 1740
gagcaagtga tcgtgatcac acacagcggc cggaagggca gatatgccgt ggaaccttat 1800
cacggcaagg tggtggtgcc tgagggacac gctattccag tgcaggactt tcaggccctg 1860
agcgagtctg ccaccatcgt gtacaacgag cgcgagttcg tgaacagata cctgcaccac 1920
attgccacac acggcggagc cctgaacacc gacgaagagt actacaagac cgtgaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggat agcgtgctgc tgaacggctg caagcacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcctaagca gtgcggcttc 2460
ttcaacatga tgtgcctgaa ggtgcacttc aaccacgaga tctgcaccca ggtgttccac 2520
aaaagcatct ctcgccgttg cactaaatct gtgacttcgg tcgtctcaac cttgttttac 2580
gacaaaaaaa tgagaacgac gaatccgaaa gagactaaga ttgtgattga cactaccggc 2640
agtaccaaac ctaagcagga cgatctcatt ctcacttgtt tcagagggtg ggtgaagcag 2700
ttgcaaatag attacaaagg caacgaaata atgacggcag ctgcctctca agggctgacc 2760
cgtaaaggtg tgtatgccgt tcggtacaag gtgaatgaaa atcctctgta cgcacccacc 2820
tcagaacatg tgaacgtcct actgacccgc acggaggacc gcatcgtgtg gaaaacacta 2880
gccggcgacc catggataaa aacactgact gccaagtacc ctgggaattt cactgccacg 2940
atagaggagt ggcaagcaga gcatgatgcc atcatgaggc acatcttgga gagaccggac 3000
cctaccgacg tcttccagaa taaggcaaac gtgtgttggg ccaaggcttt agtgccggtg 3060
ctgaagaccg ctggcataga catgaccact gaacaatgga acactgtgga ttattttgaa 3120
acggacaaag ctcactcagc agagatagta ttgaaccaac tatgcgtgag gttctttgga 3180
ctcgatctgg actccggtct attttctgca cccactgttc cgttatccat taggaataat 3240
cactgggata actccccgtc gcctaacatg tacgggctga ataaagaagt ggtccgtcag 3300
ctctctcgca ggtacccaca actgcctcgg gcagttgcca ctggaagagt ctatgacatg 3360
aacactggta cactgcgcaa ttatgatccg cgcataaacc tagtacctgt aaacagaaga 3420
ctgcctcatg ctttagtcct ccaccataat gaacacccac agagtgactt ttcttcattc 3480
gtcagcaaat tgaagggcag aactgtcctg gtggtcgggg aaaagttgtc cgtcccaggc 3540
aaaatggttg actggttgtc agaccggcct gaggctacct tcagagctcg gctggattta 3600
ggcatcccag gtgatgtgcc caaatatgac ataatatttg ttaatgtgag gaccccatat 3660
aaataccatc actatcagca gtgtgaagac catgccatta agcttagcat gttgaccaag 3720
aaagcttgtc tgcatctgaa tcccggcgga acctgtgtca gcataggtta tggttacgct 3780
gacagggcca gcgaaagcat cattggtgct atagcgcggc agttcaagtt ttcccgggta 3840
tgcaaaccga aatcctcact tgaagagacg gaagttctgt ttgtattcat tgggtacgat 3900
cgcaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaaatatgc gtgtaataat 6360
gaatattggg aaacgtttaa agaaaacccc atcaggctta ctgaagaaaa cgtggtaaat 6420
tacattacca aattaaaagg accaaaagct gctgctcttt ttgcgaagac acataatttg 6480
aatatgttgc aggacatacc aatggacagg tttgtaatgg acttaaagag agacgtgaaa 6540
gtgactccag gaacaaaaca tactgaagaa cggcccaagg tacaggtgat ccaggctgcc 6600
gatccgctag caacagcgta tctgtgcgga atccaccgag agctggttag gagattaaat 6660
gcggtcctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgttaa tgattctgga agacttaggt 6840
gtggacgcag agctgttgac gctgattgag gcggctttcg gcgaaatttc atcaatacat 6900
ttgcccacta aaactaaatt taaattcgga gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
accggatcac catgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 6
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C06 nsP coding sequence DNA
<400> 6
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc cagaaatatc ggcctgtgta gcagcgacgt gatggaaaga 660
tccagacggg gcatgagcat cctgcggaag aagtacctga agcctagcaa caacgtgctg 720
ttcagcgtgg gcagcaccat ctaccacgag aagagggacc tgctgcggag ctggcatctg 780
ccttccgtgt ttcacctgag aggcaagcag aactacacct gtagatgcga gacaatcgtg 840
tcctgcgacg gctacgtggt caagcggatc gccatttctc ctggcctgta cggcaagcct 900
tctggctatg ccgccaccat gcacagagaa ggctttctgt gttgcaaagt gaccgacaca 960
ctgaacggcg agcgggtgtc ctttcctgtg tgtacctatg tgcccgccac actgtgcgat 1020
cagatgacag gcattctggc caccgacgtg tcagccgacg atgcccagaa actgctcgtg 1080
ggcctgaacc agagaatcgt ggtcaacggc agaacccagc ggaacaccaa caccatgaag 1140
aactacctgc tgcctgtggt ggcccaggcc tttgccagat gggccaaaga gtacaaagag 1200
gatcaagagg acgagcggcc cctgggcctg agagatagac aactggtcat gggctgctgc 1260
tgggccttca gaaggcacaa gatcaccagc atctacaagc ggcccgacac acagaccatc 1320
atcaaagtga acagcgactt ccacagcttc gtgctgcctc ggatcggcag caacacactg 1380
gaaatcggcc tgcggacccg gatcagaaag atgctggaag aacacaaaga gccctctcca 1440
ctgatcaccg ccgaggatgt gcaagaggcc aaatgtgccg ccgacgaggc taaagaagtg 1500
cgcgaagccg aggaactgag agccgcactt cctcctctgg ccgccgatgt tgaagaaccc 1560
actctggaag ccgacgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aaaagcatct ctcgccgttg cactaaatct gtgacttcgg tcgtctcaac cttgttttac 2580
gacaaaaaaa tgagaacgac gaatccgaaa gagactaaga ttgtgattga cactaccggc 2640
agtaccaaac ctaagcagga cgatctcatt ctcacttgtt tcagagggtg ggtgaagcag 2700
ttgcaaatag attacaaagg caacgaaata atgacggcag ctgcctctca agggctgacc 2760
cgtaaaggtg tgtatgccgt tcggtacaag gtgaatgaaa atcctctgta cgcacccacc 2820
tcagaacatg tgaacgtcct actgacccgc acggaggacc gcatcgtgtg gaaaacacta 2880
gccggcgacc catggataaa aacactgact gccaagtacc ctgggaattt cactgccacg 2940
atagaggagt ggcaagcaga gcatgatgcc atcatgaggc acatcttgga gagaccggac 3000
cctaccgacg tcttccagaa taaggcaaac gtgtgttggg ccaaggcttt agtgccggtg 3060
ctgaagaccg ctggcataga catgaccact gaacaatgga acactgtgga ttattttgaa 3120
acggacaaag ctcactcagc agagatagta ttgaaccaac tatgcgtgag gttctttgga 3180
ctcgatctgg actccggtct attttctgca cccactgttc cgttatccat taggaataat 3240
cactgggata actccccgtc gcctaacatg tacgggctga ataaagaagt ggtccgtcag 3300
ctctctcgca ggtacccaca actgcctcgg gcagttgcca ctggaagagt ctatgacatg 3360
aacactggta cactgcgcaa ttatgatccg cgcataaacc tagtacctgt aaacagaaga 3420
ctgcctcatg ctttagtcct ccaccataat gaacacccac agagtgactt ttcttcattc 3480
gtcagcaaat tgaagggcag aactgtcctg gtggtcgggg aaaagttgtc cgtcccaggc 3540
aaaatggttg actggttgtc agaccggcct gaggctacct tcagagctcg gctggattta 3600
ggcatcccag gtgatgtgcc caaatatgac ataatatttg ttaatgtgag gaccccatat 3660
aaataccatc actatcagca gtgtgaagac catgccatta agcttagcat gttgaccaag 3720
aaagcttgtc tgcatctgaa tcccggcgga acctgtgtca gcataggtta tggttacgct 3780
gacagggcca gcgaaagcat cattggtgct atagcgcggc agttcaagtt ttcccgggta 3840
tgcaaaccga aatcctcact tgaagagacg gaagttctgt ttgtattcat tgggtacgat 3900
cgcaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaaatatgc gtgtaataat 6360
gaatattggg aaacgtttaa agaaaacccc atcaggctta ctgaagaaaa cgtggtaaat 6420
tacattacca aattaaaagg accaaaagct gctgctcttt ttgcgaagac acataatttg 6480
aatatgttgc aggacatacc aatggacagg tttgtaatgg acttaaagag agacgtgaaa 6540
gtgactccag gaacaaaaca tactgaagaa cggcccaagg tacaggtgat ccaggctgcc 6600
gatccgctag caacagcgta tctgtgcgga atccaccgag agctggttag gagattaaat 6660
gcggtcctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgttaa tgattctgga agacttaggt 6840
gtggacgcag agctgttgac gctgattgag gcggctttcg gcgaaatttc atcaatacat 6900
ttgcccacta aaactaaatt taaattcgga gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
accggatcac catgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 7
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C07 nsP coding sequence DNA
<400> 7
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc tcgtaacata ggcctatgca gctctgacgt tatggagcgg 660
tcacgtagag ggatgtccat tcttagaaag aagtatttga aaccatccaa caatgttcta 720
ttctctgttg gctcgaccat ctaccacgag aagagggact tactgaggag ctggcacctg 780
ccgtctgtat ttcacttacg tggcaagcaa aattacacat gtcggtgtga gactatagtt 840
agttgcgacg ggtacgtcgt taaaagaata gctatcagtc caggcctgta tgggaagcct 900
tcaggctatg ctgctacgat gcaccgcgag ggattcttgt gctgcaaagt gacagacaca 960
ttgaacgggg agagggtctc ttttcccgtg tgcacgtatg tgccagctac attgtgtgac 1020
caaatgactg gcatactggc aacagatgtc agtgcggacg acgcgcaaaa actgctggtt 1080
gggctcaacc agcgtatagt cgtcaacggt cgcacccaga gaaacaccaa taccatgaaa 1140
aattaccttt tgcccgtagt ggcccaggca tttgctaggt gggcaaagga atataaggaa 1200
gatcaagaag atgaaaggcc actaggacta cgagatagac agttagtcat ggggtgttgt 1260
tgggctttta gaaggcacaa gataacatct atttataagc gcccggatac ccaaaccatc 1320
atcaaagtga acagcgattt ccactcattc gtgctgccca ggataggcag taacacattg 1380
gagatcgggc tgagaacaag aatcaggaaa atgttagagg agcacaagga gccgtcacct 1440
ctcattaccg ccgaggacgt acaagaagct aagtgcgcag ccgatgaggc taaggaggtg 1500
cgtgaagccg aggagttgcg cgcagctcta ccacctttgg cagctgatgt tgaggagccc 1560
actctggaag ccgatgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aagagcatca gcagacggtg caccaagagc gtgaccagcg tggtgtctac cctgttctac 2580
gacaagaaga tgcggacgac aaaccccaaa gagacaaaga tcgtcatcga caccaccggc 2640
agcaccaagc ctaagcagga cgatctgatc ctgacctgct tcagaggctg ggtcaagcag 2700
ctgcagatcg actacaaggg caacgagatc atgaccgccg ctgcctctca gggcctgaca 2760
agaaaaggcg tgtacgccgt gcggtacaaa gtgaacgaga accctctgta cgcccctacc 2820
agcgagcatg tgaatgtgct gctgacccgg accgaggacc ggatcgtttg gaaaacactg 2880
gccggcgatc cctggatcaa gaccctgaca gccaagtatc ccggcaactt caccgccacc 2940
atcgaggaat ggcaggccga gcacgatgcc atcatgcggc acatcctgga aagacccgat 3000
cctaccgacg tgttccagaa caaggccaac gtgtgctggg ccaaagctct ggtgcctgtg 3060
ctgaaaaccg ccggcatcga tatgaccacc gagcagtgga acaccgtgga ctacttcgag 3120
acagacaagg cccacagcgc cgagatcgtg ctgaatcagc tgtgcgtgcg gttcttcggc 3180
ctggatctgg atagcggcct gttctctgct cctaccgtgc ctctgagcat ccggaacaac 3240
cactgggaca acagcccctc tcctaatatg tacggcctga acaaagaagt cgtgcggcag 3300
ctgagcagaa gatacccaca gctgcctaga gccgtggcca caggcagagt gtacgacatg 3360
aataccggca cactgcggaa ctacgacccc agaatcaatc tggtgcccgt gaacagaagg 3420
ctgccccacg ctctggttct gcaccacaat gagcaccctc agagcgactt cagcagcttc 3480
gtgtccaagc tgaagggcag aaccgtgctg gttgtgggcg agaagctgtc tgtgcctggc 3540
aagatggtgg actggctgag cgatagaccc gaggccacct ttagagccag actggacctt 3600
ggaatccctg gcgacgtgcc caaatacgac atcatcttcg tgaacgtgcg gacgccctac 3660
aagtaccacc actaccagca gtgcgaggac cacgccatca agctgagcat gctgaccaag 3720
aaggcctgcc tgcacctgaa tcctggcggc acctgtgtgt ctatcggcta cggctatgcc 3780
gacagagcca gcgagtctat catcggcgcc attgccagac agttcaagtt cagcagagtg 3840
tgcaagccca agagcagcct ggaagagaca gaggtgctgt tcgtgttcat cggctatgac 3900
cggaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaaatatgc gtgtaataat 6360
gaatattggg aaacgtttaa agaaaacccc atcaggctta ctgaagaaaa cgtggtaaat 6420
tacattacca aattaaaagg accaaaagct gctgctcttt ttgcgaagac acataatttg 6480
aatatgttgc aggacatacc aatggacagg tttgtaatgg acttaaagag agacgtgaaa 6540
gtgactccag gaacaaaaca tactgaagaa cggcccaagg tacaggtgat ccaggctgcc 6600
gatccgctag caacagcgta tctgtgcgga atccaccgag agctggttag gagattaaat 6660
gcggtcctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgttaa tgattctgga agacttaggt 6840
gtggacgcag agctgttgac gctgattgag gcggctttcg gcgaaatttc atcaatacat 6900
ttgcccacta aaactaaatt taaattcgga gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
accggatcac catgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 8
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C08 nsP coding sequence DNA
<400> 8
atggaaaagg tgcacgtgga catcgaagag gacagcccat tcctgagagc cctgcagaga 60
agcttccctc agttcgaggt ggaagccaaa caagtgaccg acaacgatca cgccaacgcg 120
agggccttct ctcacctcgc aagcaagctg atcgagacag aggtggaccc cagcgacacc 180
atcctggata ttggatctgc ccctgccaga agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc tcgtaacata ggcctatgca gctctgacgt tatggagcgg 660
tcacgtagag ggatgtccat tcttagaaag aagtatttga aaccatccaa caatgttcta 720
ttctctgttg gctcgaccat ctaccacgag aagagggact tactgaggag ctggcacctg 780
ccgtctgtat ttcacttacg tggcaagcaa aattacacat gtcggtgtga gactatagtt 840
agttgcgacg ggtacgtcgt taaaagaata gctatcagtc caggcctgta tgggaagcct 900
tcaggctatg ctgctacgat gcaccgcgag ggattcttgt gctgcaaagt gacagacaca 960
ttgaacgggg agagggtctc ttttcccgtg tgcacgtatg tgccagctac attgtgtgac 1020
caaatgactg gcatactggc aacagatgtc agtgcggacg acgcgcaaaa actgctggtt 1080
gggctcaacc agcgtatagt cgtcaacggt cgcacccaga gaaacaccaa taccatgaaa 1140
aattaccttt tgcccgtagt ggcccaggca tttgctaggt gggcaaagga atataaggaa 1200
gatcaagaag atgaaaggcc actaggacta cgagatagac agttagtcat ggggtgttgt 1260
tgggctttta gaaggcacaa gataacatct atttataagc gcccggatac ccaaaccatc 1320
atcaaagtga acagcgattt ccactcattc gtgctgccca ggataggcag taacacattg 1380
gagatcgggc tgagaacaag aatcaggaaa atgttagagg agcacaagga gccgtcacct 1440
ctcattaccg ccgaggacgt acaagaagct aagtgcgcag ccgatgaggc taaggaggtg 1500
cgtgaagccg aggagttgcg cgcagctcta ccacctttgg cagctgatgt tgaggagccc 1560
actctggaag ccgatgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aaaagcatct ctcgccgttg cactaaatct gtgacttcgg tcgtctcaac cttgttttac 2580
gacaaaaaaa tgagaacgac gaatccgaaa gagactaaga ttgtgattga cactaccggc 2640
agtaccaaac ctaagcagga cgatctcatt ctcacttgtt tcagagggtg ggtgaagcag 2700
ttgcaaatag attacaaagg caacgaaata atgacggcag ctgcctctca agggctgacc 2760
cgtaaaggtg tgtatgccgt tcggtacaag gtgaatgaaa atcctctgta cgcacccacc 2820
tcagaacatg tgaacgtcct actgacccgc acggaggacc gcatcgtgtg gaaaacacta 2880
gccggcgacc catggataaa aacactgact gccaagtacc ctgggaattt cactgccacg 2940
atagaggagt ggcaagcaga gcatgatgcc atcatgaggc acatcttgga gagaccggac 3000
cctaccgacg tcttccagaa taaggcaaac gtgtgttggg ccaaggcttt agtgccggtg 3060
ctgaagaccg ctggcataga catgaccact gaacaatgga acactgtgga ttattttgaa 3120
acggacaaag ctcactcagc agagatagta ttgaaccaac tatgcgtgag gttctttgga 3180
ctcgatctgg actccggtct attttctgca cccactgttc cgttatccat taggaataat 3240
cactgggata actccccgtc gcctaacatg tacgggctga ataaagaagt ggtccgtcag 3300
ctctctcgca ggtacccaca actgcctcgg gcagttgcca ctggaagagt ctatgacatg 3360
aacactggta cactgcgcaa ttatgatccg cgcataaacc tagtacctgt aaacagaaga 3420
ctgcctcatg ctttagtcct ccaccataat gaacacccac agagtgactt ttcttcattc 3480
gtcagcaaat tgaagggcag aactgtcctg gtggtcgggg aaaagttgtc cgtcccaggc 3540
aaaatggttg actggttgtc agaccggcct gaggctacct tcagagctcg gctggattta 3600
ggcatcccag gtgatgtgcc caaatatgac ataatatttg ttaatgtgag gaccccatat 3660
aaataccatc actatcagca gtgtgaagac catgccatta agcttagcat gttgaccaag 3720
aaagcttgtc tgcatctgaa tcccggcgga acctgtgtca gcataggtta tggttacgct 3780
gacagggcca gcgaaagcat cattggtgct atagcgcggc agttcaagtt ttcccgggta 3840
tgcaaaccga aatcctcact tgaagagacg gaagttctgt ttgtattcat tgggtacgat 3900
cgcaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaaatatgc gtgtaataat 6360
gaatattggg aaacgtttaa agaaaacccc atcaggctta ctgaagaaaa cgtggtaaat 6420
tacattacca aattaaaagg accaaaagct gctgctcttt ttgcgaagac acataatttg 6480
aatatgttgc aggacatacc aatggacagg tttgtaatgg acttaaagag agacgtgaaa 6540
gtgactccag gaacaaaaca tactgaagaa cggcccaagg tacaggtgat ccaggctgcc 6600
gatccgctag caacagcgta tctgtgcgga atccaccgag agctggttag gagattaaat 6660
gcggtcctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgttaa tgattctgga agacttaggt 6840
gtggacgcag agctgttgac gctgattgag gcggctttcg gcgaaatttc atcaatacat 6900
ttgcccacta aaactaaatt taaattcgga gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
accggatcac catgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 9
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C09 nsP coding sequence DNA
<400> 9
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc cagaaatatc ggcctgtgta gcagcgacgt gatggaaaga 660
tccagacggg gcatgagcat cctgcggaag aagtacctga agcctagcaa caacgtgctg 720
ttcagcgtgg gcagcaccat ctaccacgag aagagggacc tgctgcggag ctggcatctg 780
ccttccgtgt ttcacctgag aggcaagcag aactacacct gtagatgcga gacaatcgtg 840
tcctgcgacg gctacgtggt caagcggatc gccatttctc ctggcctgta cggcaagcct 900
tctggctatg ccgccaccat gcacagagaa ggctttctgt gttgcaaagt gaccgacaca 960
ctgaacggcg agcgggtgtc ctttcctgtg tgtacctatg tgcccgccac actgtgcgat 1020
cagatgacag gcattctggc caccgacgtg tcagccgacg atgcccagaa actgctcgtg 1080
ggcctgaacc agagaatcgt ggtcaacggc agaacccagc ggaacaccaa caccatgaag 1140
aactacctgc tgcctgtggt ggcccaggcc tttgccagat gggccaaaga gtacaaagag 1200
gatcaagagg acgagcggcc cctgggcctg agagatagac aactggtcat gggctgctgc 1260
tgggccttca gaaggcacaa gatcaccagc atctacaagc ggcccgacac acagaccatc 1320
atcaaagtga acagcgactt ccacagcttc gtgctgcctc ggatcggcag caacacactg 1380
gaaatcggcc tgcggacccg gatcagaaag atgctggaag aacacaaaga gccctctcca 1440
ctgatcaccg ccgaggatgt gcaagaggcc aaatgtgccg ccgacgaggc taaagaagtg 1500
cgcgaagccg aggaactgag agccgcactt cctcctctgg ccgccgatgt tgaagaaccc 1560
actctggaag ccgacgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aagagcatca gcagacggtg caccaagagc gtgaccagcg tggtgtctac cctgttctac 2580
gacaagaaga tgcggacgac aaaccccaaa gagacaaaga tcgtcatcga caccaccggc 2640
agcaccaagc ctaagcagga cgatctgatc ctgacctgct tcagaggctg ggtcaagcag 2700
ctgcagatcg actacaaggg caacgagatc atgaccgccg ctgcctctca gggcctgaca 2760
agaaaaggcg tgtacgccgt gcggtacaaa gtgaacgaga accctctgta cgcccctacc 2820
agcgagcatg tgaatgtgct gctgacccgg accgaggacc ggatcgtttg gaaaacactg 2880
gccggcgatc cctggatcaa gaccctgaca gccaagtatc ccggcaactt caccgccacc 2940
atcgaggaat ggcaggccga gcacgatgcc atcatgcggc acatcctgga aagacccgat 3000
cctaccgacg tgttccagaa caaggccaac gtgtgctggg ccaaagctct ggtgcctgtg 3060
ctgaaaaccg ccggcatcga tatgaccacc gagcagtgga acaccgtgga ctacttcgag 3120
acagacaagg cccacagcgc cgagatcgtg ctgaatcagc tgtgcgtgcg gttcttcggc 3180
ctggatctgg atagcggcct gttctctgct cctaccgtgc ctctgagcat ccggaacaac 3240
cactgggaca acagcccctc tcctaatatg tacggcctga acaaagaagt cgtgcggcag 3300
ctgagcagaa gatacccaca gctgcctaga gccgtggcca caggcagagt gtacgacatg 3360
aataccggca cactgcggaa ctacgacccc agaatcaatc tggtgcccgt gaacagaagg 3420
ctgccccacg ctctggttct gcaccacaat gagcaccctc agagcgactt cagcagcttc 3480
gtgtccaagc tgaagggcag aaccgtgctg gttgtgggcg agaagctgtc tgtgcctggc 3540
aagatggtgg actggctgag cgatagaccc gaggccacct ttagagccag actggacctt 3600
ggaatccctg gcgacgtgcc caaatacgac atcatcttcg tgaacgtgcg gacgccctac 3660
aagtaccacc actaccagca gtgcgaggac cacgccatca agctgagcat gctgaccaag 3720
aaggcctgcc tgcacctgaa tcctggcggc acctgtgtgt ctatcggcta cggctatgcc 3780
gacagagcca gcgagtctat catcggcgcc attgccagac agttcaagtt cagcagagtg 3840
tgcaagccca agagcagcct ggaagagaca gaggtgctgt tcgtgttcat cggctatgac 3900
cggaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaaatatgc gtgtaataat 6360
gaatattggg aaacgtttaa agaaaacccc atcaggctta ctgaagaaaa cgtggtaaat 6420
tacattacca aattaaaagg accaaaagct gctgctcttt ttgcgaagac acataatttg 6480
aatatgttgc aggacatacc aatggacagg tttgtaatgg acttaaagag agacgtgaaa 6540
gtgactccag gaacaaaaca tactgaagaa cggcccaagg tacaggtgat ccaggctgcc 6600
gatccgctag caacagcgta tctgtgcgga atccaccgag agctggttag gagattaaat 6660
gcggtcctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgttaa tgattctgga agacttaggt 6840
gtggacgcag agctgttgac gctgattgag gcggctttcg gcgaaatttc atcaatacat 6900
ttgcccacta aaactaaatt taaattcgga gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
accggatcac catgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 10
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C10 nsP coding sequence DNA
<400> 10
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc tcgtaacata ggcctatgca gctctgacgt tatggagcgg 660
tcacgtagag ggatgtccat tcttagaaag aagtatttga aaccatccaa caatgttcta 720
ttctctgttg gctcgaccat ctaccacgag aagagggact tactgaggag ctggcacctg 780
ccgtctgtat ttcacttacg tggcaagcaa aattacacat gtcggtgtga gactatagtt 840
agttgcgacg ggtacgtcgt taaaagaata gctatcagtc caggcctgta tgggaagcct 900
tcaggctatg ctgctacgat gcaccgcgag ggattcttgt gctgcaaagt gacagacaca 960
ttgaacgggg agagggtctc ttttcccgtg tgcacgtatg tgccagctac attgtgtgac 1020
caaatgactg gcatactggc aacagatgtc agtgcggacg acgcgcaaaa actgctggtt 1080
gggctcaacc agcgtatagt cgtcaacggt cgcacccaga gaaacaccaa taccatgaaa 1140
aattaccttt tgcccgtagt ggcccaggca tttgctaggt gggcaaagga atataaggaa 1200
gatcaagaag atgaaaggcc actaggacta cgagatagac agttagtcat ggggtgttgt 1260
tgggctttta gaaggcacaa gataacatct atttataagc gcccggatac ccaaaccatc 1320
atcaaagtga acagcgattt ccactcattc gtgctgccca ggataggcag taacacattg 1380
gagatcgggc tgagaacaag aatcaggaaa atgttagagg agcacaagga gccgtcacct 1440
ctcattaccg ccgaggacgt acaagaagct aagtgcgcag ccgatgaggc taaggaggtg 1500
cgtgaagccg aggagttgcg cgcagctcta ccacctttgg cagctgatgt tgaggagccc 1560
actctggaag ccgatgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aaaagcatct ctcgccgttg cactaaatct gtgacttcgg tcgtctcaac cttgttttac 2580
gacaaaaaaa tgagaacgac gaatccgaaa gagactaaga ttgtgattga cactaccggc 2640
agtaccaaac ctaagcagga cgatctcatt ctcacttgtt tcagagggtg ggtgaagcag 2700
ttgcaaatag attacaaagg caacgaaata atgacggcag ctgcctctca agggctgacc 2760
cgtaaaggtg tgtatgccgt tcggtacaag gtgaatgaaa atcctctgta cgcacccacc 2820
tcagaacatg tgaacgtcct actgacccgc acggaggacc gcatcgtgtg gaaaacacta 2880
gccggcgacc catggataaa aacactgact gccaagtacc ctgggaattt cactgccacg 2940
atagaggagt ggcaagcaga gcatgatgcc atcatgaggc acatcttgga gagaccggac 3000
cctaccgacg tcttccagaa taaggcaaac gtgtgttggg ccaaggcttt agtgccggtg 3060
ctgaagaccg ctggcataga catgaccact gaacaatgga acactgtgga ttattttgaa 3120
acggacaaag ctcactcagc agagatagta ttgaaccaac tatgcgtgag gttctttgga 3180
ctcgatctgg actccggtct attttctgca cccactgttc cgttatccat taggaataat 3240
cactgggata actccccgtc gcctaacatg tacgggctga ataaagaagt ggtccgtcag 3300
ctctctcgca ggtacccaca actgcctcgg gcagttgcca ctggaagagt ctatgacatg 3360
aacactggta cactgcgcaa ttatgatccg cgcataaacc tagtacctgt aaacagaaga 3420
ctgcctcatg ctttagtcct ccaccataat gaacacccac agagtgactt ttcttcattc 3480
gtcagcaaat tgaagggcag aactgtcctg gtggtcgggg aaaagttgtc cgtcccaggc 3540
aaaatggttg actggttgtc agaccggcct gaggctacct tcagagctcg gctggattta 3600
ggcatcccag gtgatgtgcc caaatatgac ataatatttg ttaatgtgag gaccccctac 3660
aagtaccacc actaccagca gtgcgaggac cacgccatca agctgagcat gctgaccaag 3720
aaggcctgcc tgcatctgaa tcccggcgga acctgtgtca gcataggtta tggttacgct 3780
gacagggcca gcgaaagcat cattggtgct atagcgcggc agttcaagtt ttcccgggta 3840
tgcaaaccga aatcctcact tgaagagacg gaagttctgt ttgtattcat cggctacgac 3900
agaaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaagtacgc ctgcaacaac 6360
gagtactggg agacattcaa agagaacccc atccggctga ccgaggaaaa cgtggtcaac 6420
tacatcacca agctgaaggg ccccaaagcc gccgctctgt ttgccaagac acacaacctg 6480
aacatgctgc aggacatccc catggacaga ttcgtgatgg acctgaagcg ggacgtgaaa 6540
gtgacccctg gcaccaagca caccgaggaa cggcctaagg tgcaagtgat ccaggccgct 6600
gatcctctgg ccacagccta tctgtgtggc atccacagag aactcgtgcg gagactgaat 6660
gccgtgctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgctga tgattctgga agatctcgga 6840
gtggacgccg agctgctgac actgattgaa gccgcctttg gcgagatcag cagcatccat 6900
ctgcctacca agaccaagtt caagttcggc gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
acaggcagcc cttgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 11
<211> 7482
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C11 nsP coding sequence DNA
<400> 11
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat atgccgtgga tggccctaca 480
agcctgtacc accaggccaa caagggcgtc agagtggcct actggatcgg cttcgacacc 540
acacctttca tgttcaagaa cctggctggc gcttacccca gctacagcac aaactgggcc 600
gacgaaaccg tgttaacggc cagaaatatc ggcctgtgta gcagcgacgt gatggaaaga 660
tccagacggg gcatgagcat cctgcggaag aagtacctga agcctagcaa caacgtgctg 720
ttcagcgtgg gcagcaccat ctaccacgag aagagggacc tgctgcggag ctggcatctg 780
ccttccgtgt ttcacctgag aggcaagcag aactacacct gtagatgcga gacaatcgtg 840
tcctgcgacg gctacgtggt caagcggatc gccatttctc ctggcctgta cggcaagcct 900
tctggctatg ccgccaccat gcacagagaa ggctttctgt gttgcaaagt gaccgacaca 960
ctgaacggcg agcgggtgtc ctttcctgtg tgtacctatg tgcccgccac actgtgcgat 1020
cagatgacag gcattctggc caccgacgtg tcagccgacg atgcccagaa actgctcgtg 1080
ggcctgaacc agagaatcgt ggtcaacggc agaacccagc ggaacaccaa caccatgaag 1140
aactacctgc tgcctgtggt ggcccaggcc tttgccagat gggccaaaga gtacaaagag 1200
gatcaagagg acgagcggcc cctgggcctg agagatagac aactggtcat gggctgctgc 1260
tgggccttca gaaggcacaa gatcaccagc atctacaagc ggcccgacac acagaccatc 1320
atcaaagtga acagcgactt ccacagcttc gtgctgcctc ggatcggcag caacacactg 1380
gaaatcggcc tgcggacccg gatcagaaag atgctggaag aacacaaaga gccctctcca 1440
ctgatcaccg ccgaggatgt gcaagaggcc aaatgtgccg ccgacgaggc taaagaagtg 1500
cgcgaagccg aggaactgag agccgcactt cctcctctgg ccgccgatgt tgaagaaccc 1560
actctggaag ccgacgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagc gagaagctga gctgcattca ccctctggcc 1740
gagcaagtga tcgtgatcac acacagcggc cggaagggca gatatgccgt ggaaccttat 1800
cacggcaagg tggtggtgcc tgagggacac gctattccag tgcaggactt tcaggccctg 1860
agcgagtctg ccaccatcgt gtacaacgag cgcgagttcg tgaacagata cctgcaccac 1920
attgccacac acggcggagc cctgaacacc gacgaagagt actacaagac cgtgaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggat agcgtgctgc tgaacggctg caagcacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcctaagca gtgcggcttc 2460
ttcaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aagagcatca gcagacggtg caccaagagc gtgaccagcg tggtgtctac cctgttctac 2580
gacaagaaga tgcggacgac aaaccccaaa gagacaaaga tcgtcatcga caccaccggc 2640
agcaccaagc ctaagcagga cgatctgatc ctgacctgct tcagaggctg ggtcaagcag 2700
ctgcagatcg actacaaggg caacgagatc atgaccgccg ctgcctctca gggcctgaca 2760
agaaaaggcg tgtacgccgt gcggtacaaa gtgaacgaga accctctgta cgcccctacc 2820
agcgagcatg tgaatgtgct gctgacccgg accgaggacc ggatcgtttg gaaaacactg 2880
gccggcgatc cctggatcaa gaccctgaca gccaagtatc ccggcaactt caccgccacc 2940
atcgaggaat ggcaggccga gcacgatgcc atcatgcggc acatcctgga aagacccgat 3000
cctaccgacg tgttccagaa caaggccaac gtgtgctggg ccaaagctct ggtgcctgtg 3060
ctgaaaaccg ccggcatcga tatgaccacc gagcagtgga acaccgtgga ctacttcgag 3120
acagacaagg cccacagcgc cgagatcgtg ctgaatcagc tgtgcgtgcg gttcttcggc 3180
ctggatctgg atagcggcct gttctctgct cctaccgtgc ctctgagcat ccggaacaac 3240
cactgggaca acagcccctc tcctaatatg tacggcctga acaaagaagt cgtgcggcag 3300
ctgagcagaa gatacccaca gctgcctaga gccgtggcca caggcagagt gtacgacatg 3360
aataccggca cactgcggaa ctacgacccc agaatcaatc tggtgcccgt gaacagaagg 3420
ctgccccacg ctctggttct gcaccacaat gagcaccctc agagcgactt cagcagcttc 3480
gtgtccaagc tgaagggcag aaccgtgctg gttgtgggcg agaagctgtc tgtgcctggc 3540
aagatggtgg actggctgag cgatagaccc gaggccacct ttagagccag actggacctt 3600
ggaatccctg gcgacgtgcc caaatacgac atcatcttcg tgaacgtgcg gacgccctac 3660
aagtaccacc actaccagca gtgcgaggac cacgccatca agctgagcat gctgaccaag 3720
aaggcctgcc tgcacctgaa tcctggcggc acctgtgtgt ctatcggcta cggctatgcc 3780
gacagagcca gcgagtctat catcggcgcc attgccagac agttcaagtt cagcagagtg 3840
tgcaagccca agagcagcct ggaagagaca gaggtgctgt tcgtgttcat cggctatgac 3900
cggaaggccc gtacgcacaa cccctacaag ctgagcagca ccctgaccaa catctacacc 3960
ggcagcagac tgcacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacagcca cagaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaagtacgc ctgcaacaac 6360
gagtactggg agacattcaa agagaacccc atccggctga ccgaggaaaa cgtggtcaac 6420
tacatcacca agctgaaggg ccccaaagcc gccgctctgt ttgccaagac acacaacctg 6480
aacatgctgc aggacatccc catggacaga ttcgtgatgg acctgaagcg ggacgtgaaa 6540
gtgacccctg gcaccaagca caccgaggaa cggcctaagg tgcaagtgat ccaggccgct 6600
gatcctctgg ccacagccta tctgtgtggc atccacagag aactcgtgcg gagactgaat 6660
gccgtgctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgctga tgattctgga agatctcgga 6840
gtggacgccg agctgctgac actgattgaa gccgcctttg gcgagatcag cagcatccat 6900
ctgcctacca agaccaagtt caagttcggc gccatgatga aatctggaat gttcctcaca 6960
ctgtttgtga acacagtcat taacattgta atcgcaagca gagtgttgag agaacggcta 7020
acaggcagcc cttgtgcagc attcattgga gatgacaata tcgtgaaagg agtcaaatcg 7080
gacaaattaa tggcagacag gtgcgccacc tggttgaata tggaagtcaa gattatagat 7140
gctgtggtgg gcgagaaagc gccttatttc tgtggagggt ttattttgtg tgactccgtg 7200
accggcacag cgtgccgtgt ggcagacccc ctaaaaaggc tgtttaagct tggcaaacct 7260
ctggcagcag acgatgaaca tgatgatgac aggagaaggg cattgcatga agagtcaaca 7320
cgctggaacc gagtgggtat tctttcagag ctgtgcaagg cagtagaatc aaggtatgaa 7380
accgtaggaa cttccatcat agttatggcc atgactactc tagctagcag tgttaaatca 7440
ttcagctacc tgagaggggc ccctataact ctctacggct aa 7482
<210> 12
<211> 2513
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic construct, C16 nsP-2A protein amino acid
<220>
<221> misc_feature
<222> (1880)..(1880)
<223> Xaa can be any naturally occurring amino acid
<400> 12
Met Glu Lys Val His Val Asp Ile Glu Glu Asp Ser Pro Phe Leu Arg
1 5 10 15
Ala Leu Gln Arg Ser Phe Pro Gln Phe Glu Val Glu Ala Lys Gln Val
20 25 30
Thr Asp Asn Asp His Ala Asn Ala Arg Ala Phe Ser His Leu Ala Ser
35 40 45
Lys Leu Ile Glu Thr Glu Val Asp Pro Ser Asp Thr Ile Leu Asp Ile
50 55 60
Gly Ser Ala Pro Ala Arg Arg Met Tyr Ser Lys His Lys Tyr His Cys
65 70 75 80
Ile Cys Pro Met Arg Cys Ala Glu Asp Pro Asp Arg Leu Tyr Lys Tyr
85 90 95
Ala Thr Lys Leu Lys Lys Asn Cys Lys Glu Ile Thr Asp Lys Glu Leu
100 105 110
Asp Lys Lys Met Lys Glu Leu Ala Ala Val Met Ser Asp Pro Asp Leu
115 120 125
Glu Thr Glu Thr Met Cys Leu His Asp Asp Glu Ser Cys Arg Tyr Glu
130 135 140
Gly Gln Val Ala Val Tyr Gln Asp Val Tyr Ala Val Asp Gly Pro Thr
145 150 155 160
Ser Leu Tyr His Gln Ala Asn Lys Gly Val Arg Val Ala Tyr Trp Ile
165 170 175
Gly Phe Asp Thr Thr Pro Phe Met Phe Lys Asn Leu Ala Gly Ala Tyr
180 185 190
Pro Ser Tyr Ser Thr Asn Trp Ala Asp Glu Thr Val Leu Thr Ala Arg
195 200 205
Asn Ile Gly Leu Cys Ser Ser Asp Val Met Glu Arg Ser Arg Arg Gly
210 215 220
Met Ser Ile Leu Arg Lys Lys Tyr Leu Lys Pro Ser Asn Asn Val Leu
225 230 235 240
Phe Ser Val Gly Ser Thr Ile Tyr His Glu Lys Arg Asp Leu Leu Arg
245 250 255
Ser Trp His Leu Pro Ser Val Phe His Leu Arg Gly Lys Gln Asn Tyr
260 265 270
Thr Cys Arg Cys Glu Thr Ile Val Ser Cys Asp Gly Tyr Val Val Lys
275 280 285
Arg Ile Ala Ile Ser Pro Gly Leu Tyr Gly Lys Pro Ser Gly Tyr Ala
290 295 300
Ala Thr Met His Arg Glu Gly Phe Leu Cys Cys Lys Val Thr Asp Thr
305 310 315 320
Leu Asn Gly Glu Arg Val Ser Phe Pro Val Cys Thr Tyr Val Pro Ala
325 330 335
Thr Leu Cys Asp Gln Met Thr Gly Ile Leu Ala Thr Asp Val Ser Ala
340 345 350
Asp Asp Ala Gln Lys Leu Leu Val Gly Leu Asn Gln Arg Ile Val Val
355 360 365
Asn Gly Arg Thr Gln Arg Asn Thr Asn Thr Met Lys Asn Tyr Leu Leu
370 375 380
Pro Val Val Ala Gln Ala Phe Ala Arg Trp Ala Lys Glu Tyr Lys Glu
385 390 395 400
Asp Gln Glu Asp Glu Arg Pro Leu Gly Leu Arg Asp Arg Gln Leu Val
405 410 415
Met Gly Cys Cys Trp Ala Phe Arg Arg His Lys Ile Thr Ser Ile Tyr
420 425 430
Lys Arg Pro Asp Thr Gln Thr Ile Ile Lys Val Asn Ser Asp Phe His
435 440 445
Ser Phe Val Leu Pro Arg Ile Gly Ser Asn Thr Leu Glu Ile Gly Leu
450 455 460
Arg Thr Arg Ile Arg Lys Met Leu Glu Glu His Lys Glu Pro Ser Pro
465 470 475 480
Leu Ile Thr Ala Glu Asp Val Gln Glu Ala Lys Cys Ala Ala Asp Glu
485 490 495
Ala Lys Glu Val Arg Glu Ala Glu Glu Leu Arg Ala Ala Leu Pro Pro
500 505 510
Leu Ala Ala Asp Val Glu Glu Pro Thr Leu Glu Ala Asp Val Asp Leu
515 520 525
Met Leu Gln Glu Ala Gly Ala Gly Ser Val Glu Thr Pro Arg Gly Leu
530 535 540
Ile Lys Val Thr Ser Tyr Ala Gly Glu Asp Lys Ile Gly Ser Tyr Ala
545 550 555 560
Val Leu Ser Pro Gln Ala Val Leu Lys Ser Glu Lys Leu Ser Cys Ile
565 570 575
His Pro Leu Ala Glu Gln Val Ile Val Ile Thr His Ser Gly Arg Lys
580 585 590
Gly Arg Tyr Ala Val Glu Pro Tyr His Gly Lys Val Val Val Pro Glu
595 600 605
Gly His Ala Ile Pro Val Gln Asp Phe Gln Ala Leu Ser Glu Ser Ala
610 615 620
Thr Ile Val Tyr Asn Glu Arg Glu Phe Val Asn Arg Tyr Leu His His
625 630 635 640
Ile Ala Thr His Gly Gly Ala Leu Asn Thr Asp Glu Glu Tyr Tyr Lys
645 650 655
Thr Val Lys Pro Ser Glu His Asp Gly Glu Tyr Leu Tyr Asp Ile Asp
660 665 670
Arg Lys Gln Cys Val Lys Lys Glu Leu Val Thr Gly Leu Gly Leu Thr
675 680 685
Gly Glu Leu Val Asp Pro Pro Phe His Glu Phe Ala Tyr Glu Ser Leu
690 695 700
Arg Thr Arg Pro Ala Ala Pro Tyr Gln Val Pro Thr Ile Gly Val Tyr
705 710 715 720
Gly Val Pro Gly Ser Gly Lys Ser Gly Ile Ile Lys Ser Ala Val Thr
725 730 735
Lys Lys Asp Leu Val Val Ser Ala Lys Lys Glu Asn Cys Ala Glu Ile
740 745 750
Ile Arg Asp Val Lys Lys Met Lys Gly Leu Asp Val Asn Ala Arg Thr
755 760 765
Val Asp Ser Val Leu Leu Asn Gly Cys Lys His Pro Val Glu Thr Leu
770 775 780
Tyr Ile Asp Glu Ala Phe Ala Cys His Ala Gly Thr Leu Arg Ala Leu
785 790 795 800
Ile Ala Ile Ile Arg Pro Lys Lys Ala Val Leu Cys Gly Asp Pro Lys
805 810 815
Gln Cys Gly Phe Phe Asn Met Met Cys Leu Lys Val His Phe Asn His
820 825 830
Glu Ile Cys Thr Gln Val Phe His Lys Ser Ile Ser Arg Arg Cys Thr
835 840 845
Lys Ser Val Thr Ser Val Val Ser Thr Leu Phe Tyr Asp Lys Lys Met
850 855 860
Arg Thr Thr Asn Pro Lys Glu Thr Lys Ile Val Ile Asp Thr Thr Gly
865 870 875 880
Ser Thr Lys Pro Lys Gln Asp Asp Leu Ile Leu Thr Cys Phe Arg Gly
885 890 895
Trp Val Lys Gln Leu Gln Ile Asp Tyr Lys Gly Asn Glu Ile Met Thr
900 905 910
Ala Ala Ala Ser Gln Gly Leu Thr Arg Lys Gly Val Tyr Ala Val Arg
915 920 925
Tyr Lys Val Asn Glu Asn Pro Leu Tyr Ala Pro Thr Ser Glu His Val
930 935 940
Asn Val Leu Leu Thr Arg Thr Glu Asp Arg Ile Val Trp Lys Thr Leu
945 950 955 960
Ala Gly Asp Pro Trp Ile Lys Thr Leu Thr Ala Lys Tyr Pro Gly Asn
965 970 975
Phe Thr Ala Thr Ile Glu Glu Trp Gln Ala Glu His Asp Ala Ile Met
980 985 990
Arg His Ile Leu Glu Arg Pro Asp Pro Thr Asp Val Phe Gln Asn Lys
995 1000 1005
Ala Asn Val Cys Trp Ala Lys Ala Leu Val Pro Val Leu Lys Thr
1010 1015 1020
Ala Gly Ile Asp Met Thr Thr Glu Gln Trp Asn Thr Val Asp Tyr
1025 1030 1035
Phe Glu Thr Asp Lys Ala His Ser Ala Glu Ile Val Leu Asn Gln
1040 1045 1050
Leu Cys Val Arg Phe Phe Gly Leu Asp Leu Asp Ser Gly Leu Phe
1055 1060 1065
Ser Ala Pro Thr Val Pro Leu Ser Ile Arg Asn Asn His Trp Asp
1070 1075 1080
Asn Ser Pro Ser Pro Asn Met Tyr Gly Leu Asn Lys Glu Val Val
1085 1090 1095
Arg Gln Leu Ser Arg Arg Tyr Pro Gln Leu Pro Arg Ala Val Ala
1100 1105 1110
Thr Gly Arg Val Tyr Asp Met Asn Thr Gly Thr Leu Arg Asn Tyr
1115 1120 1125
Asp Pro Arg Ile Asn Leu Val Pro Val Asn Arg Arg Leu Pro His
1130 1135 1140
Ala Leu Val Leu His His Asn Glu His Pro Gln Ser Asp Phe Ser
1145 1150 1155
Ser Phe Val Ser Lys Leu Lys Gly Arg Thr Val Leu Val Val Gly
1160 1165 1170
Glu Lys Leu Ser Val Pro Gly Lys Met Val Asp Trp Leu Ser Asp
1175 1180 1185
Arg Pro Glu Ala Thr Phe Arg Ala Arg Leu Asp Leu Gly Ile Pro
1190 1195 1200
Gly Asp Val Pro Lys Tyr Asp Ile Ile Phe Val Asn Val Arg Thr
1205 1210 1215
Pro Tyr Lys Tyr His His Tyr Gln Gln Cys Glu Asp His Ala Ile
1220 1225 1230
Lys Leu Ser Met Leu Thr Lys Lys Ala Cys Leu His Leu Asn Pro
1235 1240 1245
Gly Gly Thr Cys Val Ser Ile Gly Tyr Gly Tyr Ala Asp Arg Ala
1250 1255 1260
Ser Glu Ser Ile Ile Gly Ala Ile Ala Arg Gln Phe Lys Phe Ser
1265 1270 1275
Arg Val Cys Lys Pro Lys Ser Ser Leu Glu Glu Thr Glu Val Leu
1280 1285 1290
Phe Val Phe Ile Gly Tyr Asp Arg Lys Ala Arg Thr His Asn Pro
1295 1300 1305
Tyr Lys Leu Ser Ser Thr Leu Thr Asn Ile Tyr Thr Gly Ser Arg
1310 1315 1320
Leu His Glu Ala Gly Cys Ala Pro Ser Tyr His Val Val Arg Gly
1325 1330 1335
Asp Ile Ala Thr Ala Thr Glu Gly Val Ile Ile Asn Ala Ala Asn
1340 1345 1350
Ser Lys Gly Gln Pro Gly Gly Gly Val Cys Gly Ala Leu Tyr Lys
1355 1360 1365
Lys Phe Pro Glu Ser Phe Asp Leu Gln Pro Ile Glu Val Gly Lys
1370 1375 1380
Ala Arg Leu Val Lys Gly Ala Ala Lys His Ile Ile His Ala Val
1385 1390 1395
Gly Pro Asn Phe Asn Lys Val Ser Glu Val Glu Gly Asp Lys Gln
1400 1405 1410
Leu Ala Glu Ala Tyr Glu Ser Ile Ala Lys Ile Val Asn Asp Asn
1415 1420 1425
Asn Tyr Lys Ser Val Ala Ile Pro Leu Leu Ser Thr Gly Ile Phe
1430 1435 1440
Ser Gly Asn Lys Asp Arg Leu Thr Gln Ser Leu Asn His Leu Leu
1445 1450 1455
Thr Ala Leu Asp Thr Thr Asp Ala Asp Val Ala Ile Tyr Cys Arg
1460 1465 1470
Asp Lys Lys Trp Glu Met Thr Leu Lys Glu Ala Val Ala Arg Arg
1475 1480 1485
Glu Ala Val Glu Glu Ile Cys Ile Ser Asp Asp Ser Ser Val Thr
1490 1495 1500
Glu Pro Asp Ala Glu Leu Val Arg Val His Pro Lys Ser Ser Leu
1505 1510 1515
Ala Gly Arg Lys Gly Tyr Ser Thr Ser Asp Gly Lys Thr Phe Ser
1520 1525 1530
Tyr Leu Glu Gly Thr Lys Phe His Gln Ala Ala Lys Asp Ile Ala
1535 1540 1545
Glu Ile Asn Ala Met Trp Pro Val Ala Thr Glu Ala Asn Glu Gln
1550 1555 1560
Val Cys Met Tyr Ile Leu Gly Glu Ser Met Ser Ser Ile Arg Ser
1565 1570 1575
Lys Cys Pro Val Glu Glu Ser Glu Ala Ser Thr Pro Pro Ser Thr
1580 1585 1590
Leu Pro Cys Leu Cys Ile His Ala Met Thr Pro Glu Arg Val Gln
1595 1600 1605
Arg Leu Lys Ala Ser Arg Pro Glu Gln Ile Thr Val Cys Ser Ser
1610 1615 1620
Phe Pro Leu Pro Lys Tyr Arg Ile Thr Gly Val Gln Lys Ile Gln
1625 1630 1635
Cys Ser Gln Pro Ile Leu Phe Ser Pro Lys Val Pro Ala Tyr Ile
1640 1645 1650
His Pro Arg Lys Tyr Leu Val Glu Thr Pro Pro Val Asp Glu Thr
1655 1660 1665
Pro Glu Pro Ser Ala Glu Asn Gln Ser Thr Glu Gly Thr Pro Glu
1670 1675 1680
Gln Pro Pro Leu Ile Thr Glu Asp Glu Thr Arg Thr Arg Thr Pro
1685 1690 1695
Glu Pro Ile Ile Ile Glu Glu Glu Glu Glu Asp Ser Ile Ser Leu
1700 1705 1710
Leu Ser Asp Gly Pro Thr His Gln Val Leu Gln Val Glu Ala Asp
1715 1720 1725
Ile His Gly Pro Pro Ser Val Ser Ser Ser Ser Trp Ser Ile Pro
1730 1735 1740
His Ala Ser Asp Phe Asp Val Asp Ser Leu Ser Ile Leu Asp Thr
1745 1750 1755
Leu Glu Gly Ala Ser Val Thr Ser Gly Ala Thr Ser Ala Glu Thr
1760 1765 1770
Asn Ser Tyr Phe Ala Lys Ser Met Glu Phe Leu Ala Arg Pro Val
1775 1780 1785
Pro Ala Pro Arg Thr Val Phe Arg Asn Pro Pro His Pro Ala Pro
1790 1795 1800
Arg Thr Arg Thr Pro Ser Leu Ala Pro Ser Arg Ala Cys Ser Arg
1805 1810 1815
Thr Ser Leu Val Ser Thr Pro Pro Gly Val Asn Arg Val Ile Thr
1820 1825 1830
Arg Glu Glu Leu Glu Ala Leu Thr Pro Ser Arg Thr Pro Ser Arg
1835 1840 1845
Ser Val Ser Arg Thr Ser Leu Val Ser Asn Pro Pro Gly Val Asn
1850 1855 1860
Arg Val Ile Thr Arg Glu Glu Phe Glu Ala Phe Val Ala Gln Gln
1865 1870 1875
Gln Xaa Arg Phe Asp Ala Gly Ala Tyr Ile Phe Ser Ser Asp Thr
1880 1885 1890
Gly Gln Gly His Leu Gln Gln Lys Ser Val Arg Gln Thr Val Leu
1895 1900 1905
Ser Glu Val Val Leu Glu Arg Thr Glu Leu Glu Ile Ser Tyr Ala
1910 1915 1920
Pro Arg Leu Asp Gln Glu Lys Glu Glu Leu Leu Arg Lys Lys Leu
1925 1930 1935
Gln Leu Asn Pro Thr Pro Ala Asn Arg Ser Arg Tyr Gln Ser Arg
1940 1945 1950
Lys Val Glu Asn Met Lys Ala Ile Thr Ala Arg Arg Ile Leu Gln
1955 1960 1965
Gly Leu Gly His Tyr Leu Lys Ala Glu Gly Lys Val Glu Cys Tyr
1970 1975 1980
Arg Thr Leu His Pro Val Pro Leu Tyr Ser Ser Ser Val Asn Arg
1985 1990 1995
Ala Phe Ser Ser Pro Lys Val Ala Val Glu Ala Cys Asn Ala Met
2000 2005 2010
Leu Lys Glu Asn Phe Pro Thr Val Ala Ser Tyr Cys Ile Ile Pro
2015 2020 2025
Glu Tyr Asp Ala Tyr Leu Asp Met Val Asp Gly Ala Ser Cys Cys
2030 2035 2040
Leu Asp Thr Ala Ser Phe Cys Pro Ala Lys Leu Arg Ser Phe Pro
2045 2050 2055
Lys Lys His Ser Tyr Leu Glu Pro Thr Ile Arg Ser Ala Val Pro
2060 2065 2070
Ser Ala Ile Gln Asn Thr Leu Gln Asn Val Leu Ala Ala Ala Thr
2075 2080 2085
Lys Arg Asn Cys Asn Val Thr Gln Met Arg Glu Leu Pro Val Leu
2090 2095 2100
Asp Ser Ala Ala Phe Asn Val Glu Cys Phe Lys Lys Tyr Ala Cys
2105 2110 2115
Asn Asn Glu Tyr Trp Glu Thr Phe Lys Glu Asn Pro Ile Arg Leu
2120 2125 2130
Thr Glu Glu Asn Val Val Asn Tyr Ile Thr Lys Leu Lys Gly Pro
2135 2140 2145
Lys Ala Ala Ala Leu Phe Ala Lys Thr His Asn Leu Asn Met Leu
2150 2155 2160
Gln Asp Ile Pro Met Asp Arg Phe Val Met Asp Leu Lys Arg Asp
2165 2170 2175
Val Lys Val Thr Pro Gly Thr Lys His Thr Glu Glu Arg Pro Lys
2180 2185 2190
Val Gln Val Ile Gln Ala Ala Asp Pro Leu Ala Thr Ala Tyr Leu
2195 2200 2205
Cys Gly Ile His Arg Glu Leu Val Arg Arg Leu Asn Ala Val Leu
2210 2215 2220
Leu Pro Asn Ile His Thr Leu Phe Asp Met Ser Ala Glu Asp Phe
2225 2230 2235
Asp Ala Ile Ile Ala Glu His Phe Gln Pro Gly Asp Cys Val Leu
2240 2245 2250
Glu Thr Asp Ile Ala Ser Phe Asp Lys Ser Glu Asp Asp Ala Met
2255 2260 2265
Ala Leu Thr Ala Leu Met Ile Leu Glu Asp Leu Gly Val Asp Ala
2270 2275 2280
Glu Leu Leu Thr Leu Ile Glu Ala Ala Phe Gly Glu Ile Ser Ser
2285 2290 2295
Ile His Leu Pro Thr Lys Thr Lys Phe Lys Phe Gly Ala Met Met
2300 2305 2310
Lys Ser Gly Met Phe Leu Thr Leu Phe Val Asn Thr Val Ile Asn
2315 2320 2325
Ile Val Ile Ala Ser Arg Val Leu Arg Glu Arg Leu Thr Gly Ser
2330 2335 2340
Pro Cys Ala Ala Phe Ile Gly Asp Asp Asn Ile Val Lys Gly Val
2345 2350 2355
Lys Ser Asp Lys Leu Met Ala Asp Arg Cys Ala Thr Trp Leu Asn
2360 2365 2370
Met Glu Val Lys Ile Ile Asp Ala Val Val Gly Glu Lys Ala Pro
2375 2380 2385
Tyr Phe Cys Gly Gly Phe Ile Leu Cys Asp Ser Val Thr Gly Thr
2390 2395 2400
Ala Cys Arg Val Ala Asp Pro Leu Lys Arg Leu Phe Lys Leu Gly
2405 2410 2415
Lys Pro Leu Ala Ala Asp Asp Glu His Asp Asp Asp Arg Arg Arg
2420 2425 2430
Ala Leu His Glu Glu Ser Thr Arg Trp Asn Arg Val Gly Ile Leu
2435 2440 2445
Ser Glu Leu Cys Lys Ala Val Glu Ser Arg Tyr Glu Thr Val Gly
2450 2455 2460
Thr Ser Ile Ile Val Met Ala Met Thr Thr Leu Ala Ser Ser Val
2465 2470 2475
Lys Ser Phe Ser Tyr Leu Arg Gly Ala Pro Ile Thr Leu Tyr Gly
2480 2485 2490
Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
2495 2500 2505
Glu Asn Pro Gly Pro
2510
<210> 13
<211> 7539
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C16 nsP-2A coding sequence DNA
<400> 13
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc tcgtaacata ggcctatgca gctctgacgt tatggagcgg 660
tcacgtagag ggatgtccat tcttagaaag aagtatttga aaccatccaa caatgttcta 720
ttctctgttg gctcgaccat ctaccacgag aagagggact tactgaggag ctggcacctg 780
ccgtctgtat ttcacttacg tggcaagcaa aattacacat gtcggtgtga gactatagtt 840
agttgcgacg ggtacgtcgt taaaagaata gctatcagtc caggcctgta tgggaagcct 900
tcaggctatg ctgctacgat gcaccgcgag ggattcttgt gctgcaaagt gacagacaca 960
ttgaacgggg agagggtctc ttttcccgtg tgcacgtatg tgccagctac attgtgtgac 1020
caaatgactg gcatactggc aacagatgtc agtgcggacg acgcgcaaaa actgctggtt 1080
gggctcaacc agcgtatagt cgtcaacggt cgcacccaga gaaacaccaa taccatgaaa 1140
aattaccttt tgcccgtagt ggcccaggca tttgctaggt gggcaaagga atataaggaa 1200
gatcaagaag atgaaaggcc actaggacta cgagatagac agttagtcat ggggtgttgt 1260
tgggctttta gaaggcacaa gataacatct atttataagc gcccggatac ccaaaccatc 1320
atcaaagtga acagcgattt ccactcattc gtgctgccca ggataggcag taacacattg 1380
gagatcgggc tgagaacaag aatcaggaaa atgttagagg agcacaagga gccgtcacct 1440
ctcattaccg ccgaggacgt acaagaagct aagtgcgcag ccgatgaggc taaggaggtg 1500
cgtgaagccg aggagttgcg cgcagctcta ccacctttgg cagctgatgt tgaggagccc 1560
actctggaag ccgatgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aaaagcatct ctcgccgttg cactaaatct gtgacttcgg tcgtctcaac cttgttttac 2580
gacaaaaaaa tgagaacgac gaatccgaaa gagactaaga ttgtgattga cactaccggc 2640
agtaccaaac ctaagcagga cgatctcatt ctcacttgtt tcagagggtg ggtgaagcag 2700
ttgcaaatag attacaaagg caacgaaata atgacggcag ctgcctctca agggctgacc 2760
cgtaaaggtg tgtatgccgt tcggtacaag gtgaatgaaa atcctctgta cgcacccacc 2820
tcagaacatg tgaacgtcct actgacccgc acggaggacc gcatcgtgtg gaaaacacta 2880
gccggcgacc catggataaa aacactgact gccaagtacc ctgggaattt cactgccacg 2940
atagaggagt ggcaagcaga gcatgatgcc atcatgaggc acatcttgga gagaccggac 3000
cctaccgacg tcttccagaa taaggcaaac gtgtgttggg ccaaggcttt agtgccggtg 3060
ctgaagaccg ctggcataga catgaccact gaacaatgga acactgtgga ttattttgaa 3120
acggacaaag ctcactcagc agagatagta ttgaaccaac tatgcgtgag gttctttgga 3180
ctcgatctgg actccggtct attttctgca cccactgttc cgttatccat taggaataat 3240
cactgggata actccccgtc gcctaacatg tacgggctga ataaagaagt ggtccgtcag 3300
ctctctcgca ggtacccaca actgcctcgg gcagttgcca ctggaagagt ctatgacatg 3360
aacactggta cactgcgcaa ttatgatccg cgcataaacc tagtacctgt aaacagaaga 3420
ctgcctcatg ctttagtcct ccaccataat gaacacccac agagtgactt ttcttcattc 3480
gtcagcaaat tgaagggcag aactgtcctg gtggtcgggg aaaagttgtc cgtcccaggc 3540
aaaatggttg actggttgtc agaccggcct gaggctacct tcagagctcg gctggattta 3600
ggcatcccag gtgatgtgcc caaatatgac ataatatttg ttaatgtgag gaccccatat 3660
aaataccatc actatcagca gtgtgaagac catgccatta agcttagcat gttgaccaag 3720
aaagcttgtc tgcatctgaa tcccggcgga acctgtgtca gcataggtta tggttacgct 3780
gacagggcca gcgaaagcat cattggtgct atagcgcggc agttcaagtt ttcccgggta 3840
tgcaaaccga aatcctcact tgaagagacg gaagttctgt ttgtattcat tgggtacgat 3900
cgcaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaaatatgc gtgtaataat 6360
gaatattggg aaacgtttaa agaaaacccc atcaggctta ctgaagaaaa cgtggtaaat 6420
tacattacca aattaaaagg accaaaagct gctgctcttt ttgcgaagac acataatttg 6480
aatatgttgc aggacatacc aatggacagg tttgtaatgg acttaaagag agacgtgaaa 6540
gtgactccag gaacaaaaca tactgaagaa cggcccaagg tacaggtgat ccaggctgcc 6600
gatccgctag caacagcgta tctgtgcgga atccaccgag agctggttag gagattaaat 6660
gcggtcctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgttaa tgattctgga agacttaggt 6840
gtggacgcag agctgttgac gctgattgag gcggctttcg gcgaaatttc atcaatacat 6900
ttgcccacta aaactaaatt taagttcggc gccatgatga agtccggcat gtttctgacc 6960
ctgttcgtga acaccgtgat caacatcgtg atcgccagcc gggtgctgag agagagactg 7020
acaggatctc cttgcgccgc cttcatcggc gacgacaata tcgtgaaggg cgtgaagtcc 7080
gacaagctga tggccgatag atgcgccacc tggctgaaca tggaagtgaa gatcatcgac 7140
gccgtcgtgg gcgagaaggc cccttatttt tgcggcggct tcatcctgtg cgacagcgtg 7200
acaggcacag cctgcagagt tgccgatcct ctgaagcggc tgttcaagct gggaaaacct 7260
ctggccgccg acgacgagca cgacgacgat agacgtagag ccctgcacga ggaatccacc 7320
agatggaaca gagtgggcat cctgagcgag ctgtgcaagg ccgtggaaag cagatacgag 7380
acagtgggca ccagcatcat tgtgatggca atgaccacac tggccagcag cgtgaaaagc 7440
ttcagctacc taaggggcgc ccctatcaca ctgtacggct ctggcgaagg cagaggcagc 7500
cttctgacat gtggcgacgt ggaagagaac cccggacct 7539
<210> 14
<211> 128
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C16 subgenomic promoter DNA
<400> 14
acttccatca tagttatggc catgactact ctagctagca gtgttaaatc attcagctac 60
ctgagagggg cccctataac tctctacggc taacctgaat ggactacgac atagtctagt 120
ccgccaag 128
<210> 15
<211> 2751
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic construct, C16 nsP-2A-eGFP protein
<220>
<221> misc_feature
<222> (1880)..(1880)
<223> Xaa can be any naturally occurring amino acid
<400> 15
Met Glu Lys Val His Val Asp Ile Glu Glu Asp Ser Pro Phe Leu Arg
1 5 10 15
Ala Leu Gln Arg Ser Phe Pro Gln Phe Glu Val Glu Ala Lys Gln Val
20 25 30
Thr Asp Asn Asp His Ala Asn Ala Arg Ala Phe Ser His Leu Ala Ser
35 40 45
Lys Leu Ile Glu Thr Glu Val Asp Pro Ser Asp Thr Ile Leu Asp Ile
50 55 60
Gly Ser Ala Pro Ala Arg Arg Met Tyr Ser Lys His Lys Tyr His Cys
65 70 75 80
Ile Cys Pro Met Arg Cys Ala Glu Asp Pro Asp Arg Leu Tyr Lys Tyr
85 90 95
Ala Thr Lys Leu Lys Lys Asn Cys Lys Glu Ile Thr Asp Lys Glu Leu
100 105 110
Asp Lys Lys Met Lys Glu Leu Ala Ala Val Met Ser Asp Pro Asp Leu
115 120 125
Glu Thr Glu Thr Met Cys Leu His Asp Asp Glu Ser Cys Arg Tyr Glu
130 135 140
Gly Gln Val Ala Val Tyr Gln Asp Val Tyr Ala Val Asp Gly Pro Thr
145 150 155 160
Ser Leu Tyr His Gln Ala Asn Lys Gly Val Arg Val Ala Tyr Trp Ile
165 170 175
Gly Phe Asp Thr Thr Pro Phe Met Phe Lys Asn Leu Ala Gly Ala Tyr
180 185 190
Pro Ser Tyr Ser Thr Asn Trp Ala Asp Glu Thr Val Leu Thr Ala Arg
195 200 205
Asn Ile Gly Leu Cys Ser Ser Asp Val Met Glu Arg Ser Arg Arg Gly
210 215 220
Met Ser Ile Leu Arg Lys Lys Tyr Leu Lys Pro Ser Asn Asn Val Leu
225 230 235 240
Phe Ser Val Gly Ser Thr Ile Tyr His Glu Lys Arg Asp Leu Leu Arg
245 250 255
Ser Trp His Leu Pro Ser Val Phe His Leu Arg Gly Lys Gln Asn Tyr
260 265 270
Thr Cys Arg Cys Glu Thr Ile Val Ser Cys Asp Gly Tyr Val Val Lys
275 280 285
Arg Ile Ala Ile Ser Pro Gly Leu Tyr Gly Lys Pro Ser Gly Tyr Ala
290 295 300
Ala Thr Met His Arg Glu Gly Phe Leu Cys Cys Lys Val Thr Asp Thr
305 310 315 320
Leu Asn Gly Glu Arg Val Ser Phe Pro Val Cys Thr Tyr Val Pro Ala
325 330 335
Thr Leu Cys Asp Gln Met Thr Gly Ile Leu Ala Thr Asp Val Ser Ala
340 345 350
Asp Asp Ala Gln Lys Leu Leu Val Gly Leu Asn Gln Arg Ile Val Val
355 360 365
Asn Gly Arg Thr Gln Arg Asn Thr Asn Thr Met Lys Asn Tyr Leu Leu
370 375 380
Pro Val Val Ala Gln Ala Phe Ala Arg Trp Ala Lys Glu Tyr Lys Glu
385 390 395 400
Asp Gln Glu Asp Glu Arg Pro Leu Gly Leu Arg Asp Arg Gln Leu Val
405 410 415
Met Gly Cys Cys Trp Ala Phe Arg Arg His Lys Ile Thr Ser Ile Tyr
420 425 430
Lys Arg Pro Asp Thr Gln Thr Ile Ile Lys Val Asn Ser Asp Phe His
435 440 445
Ser Phe Val Leu Pro Arg Ile Gly Ser Asn Thr Leu Glu Ile Gly Leu
450 455 460
Arg Thr Arg Ile Arg Lys Met Leu Glu Glu His Lys Glu Pro Ser Pro
465 470 475 480
Leu Ile Thr Ala Glu Asp Val Gln Glu Ala Lys Cys Ala Ala Asp Glu
485 490 495
Ala Lys Glu Val Arg Glu Ala Glu Glu Leu Arg Ala Ala Leu Pro Pro
500 505 510
Leu Ala Ala Asp Val Glu Glu Pro Thr Leu Glu Ala Asp Val Asp Leu
515 520 525
Met Leu Gln Glu Ala Gly Ala Gly Ser Val Glu Thr Pro Arg Gly Leu
530 535 540
Ile Lys Val Thr Ser Tyr Ala Gly Glu Asp Lys Ile Gly Ser Tyr Ala
545 550 555 560
Val Leu Ser Pro Gln Ala Val Leu Lys Ser Glu Lys Leu Ser Cys Ile
565 570 575
His Pro Leu Ala Glu Gln Val Ile Val Ile Thr His Ser Gly Arg Lys
580 585 590
Gly Arg Tyr Ala Val Glu Pro Tyr His Gly Lys Val Val Val Pro Glu
595 600 605
Gly His Ala Ile Pro Val Gln Asp Phe Gln Ala Leu Ser Glu Ser Ala
610 615 620
Thr Ile Val Tyr Asn Glu Arg Glu Phe Val Asn Arg Tyr Leu His His
625 630 635 640
Ile Ala Thr His Gly Gly Ala Leu Asn Thr Asp Glu Glu Tyr Tyr Lys
645 650 655
Thr Val Lys Pro Ser Glu His Asp Gly Glu Tyr Leu Tyr Asp Ile Asp
660 665 670
Arg Lys Gln Cys Val Lys Lys Glu Leu Val Thr Gly Leu Gly Leu Thr
675 680 685
Gly Glu Leu Val Asp Pro Pro Phe His Glu Phe Ala Tyr Glu Ser Leu
690 695 700
Arg Thr Arg Pro Ala Ala Pro Tyr Gln Val Pro Thr Ile Gly Val Tyr
705 710 715 720
Gly Val Pro Gly Ser Gly Lys Ser Gly Ile Ile Lys Ser Ala Val Thr
725 730 735
Lys Lys Asp Leu Val Val Ser Ala Lys Lys Glu Asn Cys Ala Glu Ile
740 745 750
Ile Arg Asp Val Lys Lys Met Lys Gly Leu Asp Val Asn Ala Arg Thr
755 760 765
Val Asp Ser Val Leu Leu Asn Gly Cys Lys His Pro Val Glu Thr Leu
770 775 780
Tyr Ile Asp Glu Ala Phe Ala Cys His Ala Gly Thr Leu Arg Ala Leu
785 790 795 800
Ile Ala Ile Ile Arg Pro Lys Lys Ala Val Leu Cys Gly Asp Pro Lys
805 810 815
Gln Cys Gly Phe Phe Asn Met Met Cys Leu Lys Val His Phe Asn His
820 825 830
Glu Ile Cys Thr Gln Val Phe His Lys Ser Ile Ser Arg Arg Cys Thr
835 840 845
Lys Ser Val Thr Ser Val Val Ser Thr Leu Phe Tyr Asp Lys Lys Met
850 855 860
Arg Thr Thr Asn Pro Lys Glu Thr Lys Ile Val Ile Asp Thr Thr Gly
865 870 875 880
Ser Thr Lys Pro Lys Gln Asp Asp Leu Ile Leu Thr Cys Phe Arg Gly
885 890 895
Trp Val Lys Gln Leu Gln Ile Asp Tyr Lys Gly Asn Glu Ile Met Thr
900 905 910
Ala Ala Ala Ser Gln Gly Leu Thr Arg Lys Gly Val Tyr Ala Val Arg
915 920 925
Tyr Lys Val Asn Glu Asn Pro Leu Tyr Ala Pro Thr Ser Glu His Val
930 935 940
Asn Val Leu Leu Thr Arg Thr Glu Asp Arg Ile Val Trp Lys Thr Leu
945 950 955 960
Ala Gly Asp Pro Trp Ile Lys Thr Leu Thr Ala Lys Tyr Pro Gly Asn
965 970 975
Phe Thr Ala Thr Ile Glu Glu Trp Gln Ala Glu His Asp Ala Ile Met
980 985 990
Arg His Ile Leu Glu Arg Pro Asp Pro Thr Asp Val Phe Gln Asn Lys
995 1000 1005
Ala Asn Val Cys Trp Ala Lys Ala Leu Val Pro Val Leu Lys Thr
1010 1015 1020
Ala Gly Ile Asp Met Thr Thr Glu Gln Trp Asn Thr Val Asp Tyr
1025 1030 1035
Phe Glu Thr Asp Lys Ala His Ser Ala Glu Ile Val Leu Asn Gln
1040 1045 1050
Leu Cys Val Arg Phe Phe Gly Leu Asp Leu Asp Ser Gly Leu Phe
1055 1060 1065
Ser Ala Pro Thr Val Pro Leu Ser Ile Arg Asn Asn His Trp Asp
1070 1075 1080
Asn Ser Pro Ser Pro Asn Met Tyr Gly Leu Asn Lys Glu Val Val
1085 1090 1095
Arg Gln Leu Ser Arg Arg Tyr Pro Gln Leu Pro Arg Ala Val Ala
1100 1105 1110
Thr Gly Arg Val Tyr Asp Met Asn Thr Gly Thr Leu Arg Asn Tyr
1115 1120 1125
Asp Pro Arg Ile Asn Leu Val Pro Val Asn Arg Arg Leu Pro His
1130 1135 1140
Ala Leu Val Leu His His Asn Glu His Pro Gln Ser Asp Phe Ser
1145 1150 1155
Ser Phe Val Ser Lys Leu Lys Gly Arg Thr Val Leu Val Val Gly
1160 1165 1170
Glu Lys Leu Ser Val Pro Gly Lys Met Val Asp Trp Leu Ser Asp
1175 1180 1185
Arg Pro Glu Ala Thr Phe Arg Ala Arg Leu Asp Leu Gly Ile Pro
1190 1195 1200
Gly Asp Val Pro Lys Tyr Asp Ile Ile Phe Val Asn Val Arg Thr
1205 1210 1215
Pro Tyr Lys Tyr His His Tyr Gln Gln Cys Glu Asp His Ala Ile
1220 1225 1230
Lys Leu Ser Met Leu Thr Lys Lys Ala Cys Leu His Leu Asn Pro
1235 1240 1245
Gly Gly Thr Cys Val Ser Ile Gly Tyr Gly Tyr Ala Asp Arg Ala
1250 1255 1260
Ser Glu Ser Ile Ile Gly Ala Ile Ala Arg Gln Phe Lys Phe Ser
1265 1270 1275
Arg Val Cys Lys Pro Lys Ser Ser Leu Glu Glu Thr Glu Val Leu
1280 1285 1290
Phe Val Phe Ile Gly Tyr Asp Arg Lys Ala Arg Thr His Asn Pro
1295 1300 1305
Tyr Lys Leu Ser Ser Thr Leu Thr Asn Ile Tyr Thr Gly Ser Arg
1310 1315 1320
Leu His Glu Ala Gly Cys Ala Pro Ser Tyr His Val Val Arg Gly
1325 1330 1335
Asp Ile Ala Thr Ala Thr Glu Gly Val Ile Ile Asn Ala Ala Asn
1340 1345 1350
Ser Lys Gly Gln Pro Gly Gly Gly Val Cys Gly Ala Leu Tyr Lys
1355 1360 1365
Lys Phe Pro Glu Ser Phe Asp Leu Gln Pro Ile Glu Val Gly Lys
1370 1375 1380
Ala Arg Leu Val Lys Gly Ala Ala Lys His Ile Ile His Ala Val
1385 1390 1395
Gly Pro Asn Phe Asn Lys Val Ser Glu Val Glu Gly Asp Lys Gln
1400 1405 1410
Leu Ala Glu Ala Tyr Glu Ser Ile Ala Lys Ile Val Asn Asp Asn
1415 1420 1425
Asn Tyr Lys Ser Val Ala Ile Pro Leu Leu Ser Thr Gly Ile Phe
1430 1435 1440
Ser Gly Asn Lys Asp Arg Leu Thr Gln Ser Leu Asn His Leu Leu
1445 1450 1455
Thr Ala Leu Asp Thr Thr Asp Ala Asp Val Ala Ile Tyr Cys Arg
1460 1465 1470
Asp Lys Lys Trp Glu Met Thr Leu Lys Glu Ala Val Ala Arg Arg
1475 1480 1485
Glu Ala Val Glu Glu Ile Cys Ile Ser Asp Asp Ser Ser Val Thr
1490 1495 1500
Glu Pro Asp Ala Glu Leu Val Arg Val His Pro Lys Ser Ser Leu
1505 1510 1515
Ala Gly Arg Lys Gly Tyr Ser Thr Ser Asp Gly Lys Thr Phe Ser
1520 1525 1530
Tyr Leu Glu Gly Thr Lys Phe His Gln Ala Ala Lys Asp Ile Ala
1535 1540 1545
Glu Ile Asn Ala Met Trp Pro Val Ala Thr Glu Ala Asn Glu Gln
1550 1555 1560
Val Cys Met Tyr Ile Leu Gly Glu Ser Met Ser Ser Ile Arg Ser
1565 1570 1575
Lys Cys Pro Val Glu Glu Ser Glu Ala Ser Thr Pro Pro Ser Thr
1580 1585 1590
Leu Pro Cys Leu Cys Ile His Ala Met Thr Pro Glu Arg Val Gln
1595 1600 1605
Arg Leu Lys Ala Ser Arg Pro Glu Gln Ile Thr Val Cys Ser Ser
1610 1615 1620
Phe Pro Leu Pro Lys Tyr Arg Ile Thr Gly Val Gln Lys Ile Gln
1625 1630 1635
Cys Ser Gln Pro Ile Leu Phe Ser Pro Lys Val Pro Ala Tyr Ile
1640 1645 1650
His Pro Arg Lys Tyr Leu Val Glu Thr Pro Pro Val Asp Glu Thr
1655 1660 1665
Pro Glu Pro Ser Ala Glu Asn Gln Ser Thr Glu Gly Thr Pro Glu
1670 1675 1680
Gln Pro Pro Leu Ile Thr Glu Asp Glu Thr Arg Thr Arg Thr Pro
1685 1690 1695
Glu Pro Ile Ile Ile Glu Glu Glu Glu Glu Asp Ser Ile Ser Leu
1700 1705 1710
Leu Ser Asp Gly Pro Thr His Gln Val Leu Gln Val Glu Ala Asp
1715 1720 1725
Ile His Gly Pro Pro Ser Val Ser Ser Ser Ser Trp Ser Ile Pro
1730 1735 1740
His Ala Ser Asp Phe Asp Val Asp Ser Leu Ser Ile Leu Asp Thr
1745 1750 1755
Leu Glu Gly Ala Ser Val Thr Ser Gly Ala Thr Ser Ala Glu Thr
1760 1765 1770
Asn Ser Tyr Phe Ala Lys Ser Met Glu Phe Leu Ala Arg Pro Val
1775 1780 1785
Pro Ala Pro Arg Thr Val Phe Arg Asn Pro Pro His Pro Ala Pro
1790 1795 1800
Arg Thr Arg Thr Pro Ser Leu Ala Pro Ser Arg Ala Cys Ser Arg
1805 1810 1815
Thr Ser Leu Val Ser Thr Pro Pro Gly Val Asn Arg Val Ile Thr
1820 1825 1830
Arg Glu Glu Leu Glu Ala Leu Thr Pro Ser Arg Thr Pro Ser Arg
1835 1840 1845
Ser Val Ser Arg Thr Ser Leu Val Ser Asn Pro Pro Gly Val Asn
1850 1855 1860
Arg Val Ile Thr Arg Glu Glu Phe Glu Ala Phe Val Ala Gln Gln
1865 1870 1875
Gln Xaa Arg Phe Asp Ala Gly Ala Tyr Ile Phe Ser Ser Asp Thr
1880 1885 1890
Gly Gln Gly His Leu Gln Gln Lys Ser Val Arg Gln Thr Val Leu
1895 1900 1905
Ser Glu Val Val Leu Glu Arg Thr Glu Leu Glu Ile Ser Tyr Ala
1910 1915 1920
Pro Arg Leu Asp Gln Glu Lys Glu Glu Leu Leu Arg Lys Lys Leu
1925 1930 1935
Gln Leu Asn Pro Thr Pro Ala Asn Arg Ser Arg Tyr Gln Ser Arg
1940 1945 1950
Lys Val Glu Asn Met Lys Ala Ile Thr Ala Arg Arg Ile Leu Gln
1955 1960 1965
Gly Leu Gly His Tyr Leu Lys Ala Glu Gly Lys Val Glu Cys Tyr
1970 1975 1980
Arg Thr Leu His Pro Val Pro Leu Tyr Ser Ser Ser Val Asn Arg
1985 1990 1995
Ala Phe Ser Ser Pro Lys Val Ala Val Glu Ala Cys Asn Ala Met
2000 2005 2010
Leu Lys Glu Asn Phe Pro Thr Val Ala Ser Tyr Cys Ile Ile Pro
2015 2020 2025
Glu Tyr Asp Ala Tyr Leu Asp Met Val Asp Gly Ala Ser Cys Cys
2030 2035 2040
Leu Asp Thr Ala Ser Phe Cys Pro Ala Lys Leu Arg Ser Phe Pro
2045 2050 2055
Lys Lys His Ser Tyr Leu Glu Pro Thr Ile Arg Ser Ala Val Pro
2060 2065 2070
Ser Ala Ile Gln Asn Thr Leu Gln Asn Val Leu Ala Ala Ala Thr
2075 2080 2085
Lys Arg Asn Cys Asn Val Thr Gln Met Arg Glu Leu Pro Val Leu
2090 2095 2100
Asp Ser Ala Ala Phe Asn Val Glu Cys Phe Lys Lys Tyr Ala Cys
2105 2110 2115
Asn Asn Glu Tyr Trp Glu Thr Phe Lys Glu Asn Pro Ile Arg Leu
2120 2125 2130
Thr Glu Glu Asn Val Val Asn Tyr Ile Thr Lys Leu Lys Gly Pro
2135 2140 2145
Lys Ala Ala Ala Leu Phe Ala Lys Thr His Asn Leu Asn Met Leu
2150 2155 2160
Gln Asp Ile Pro Met Asp Arg Phe Val Met Asp Leu Lys Arg Asp
2165 2170 2175
Val Lys Val Thr Pro Gly Thr Lys His Thr Glu Glu Arg Pro Lys
2180 2185 2190
Val Gln Val Ile Gln Ala Ala Asp Pro Leu Ala Thr Ala Tyr Leu
2195 2200 2205
Cys Gly Ile His Arg Glu Leu Val Arg Arg Leu Asn Ala Val Leu
2210 2215 2220
Leu Pro Asn Ile His Thr Leu Phe Asp Met Ser Ala Glu Asp Phe
2225 2230 2235
Asp Ala Ile Ile Ala Glu His Phe Gln Pro Gly Asp Cys Val Leu
2240 2245 2250
Glu Thr Asp Ile Ala Ser Phe Asp Lys Ser Glu Asp Asp Ala Met
2255 2260 2265
Ala Leu Thr Ala Leu Met Ile Leu Glu Asp Leu Gly Val Asp Ala
2270 2275 2280
Glu Leu Leu Thr Leu Ile Glu Ala Ala Phe Gly Glu Ile Ser Ser
2285 2290 2295
Ile His Leu Pro Thr Lys Thr Lys Phe Lys Phe Gly Ala Met Met
2300 2305 2310
Lys Ser Gly Met Phe Leu Thr Leu Phe Val Asn Thr Val Ile Asn
2315 2320 2325
Ile Val Ile Ala Ser Arg Val Leu Arg Glu Arg Leu Thr Gly Ser
2330 2335 2340
Pro Cys Ala Ala Phe Ile Gly Asp Asp Asn Ile Val Lys Gly Val
2345 2350 2355
Lys Ser Asp Lys Leu Met Ala Asp Arg Cys Ala Thr Trp Leu Asn
2360 2365 2370
Met Glu Val Lys Ile Ile Asp Ala Val Val Gly Glu Lys Ala Pro
2375 2380 2385
Tyr Phe Cys Gly Gly Phe Ile Leu Cys Asp Ser Val Thr Gly Thr
2390 2395 2400
Ala Cys Arg Val Ala Asp Pro Leu Lys Arg Leu Phe Lys Leu Gly
2405 2410 2415
Lys Pro Leu Ala Ala Asp Asp Glu His Asp Asp Asp Arg Arg Arg
2420 2425 2430
Ala Leu His Glu Glu Ser Thr Arg Trp Asn Arg Val Gly Ile Leu
2435 2440 2445
Ser Glu Leu Cys Lys Ala Val Glu Ser Arg Tyr Glu Thr Val Gly
2450 2455 2460
Thr Ser Ile Ile Val Met Ala Met Thr Thr Leu Ala Ser Ser Val
2465 2470 2475
Lys Ser Phe Ser Tyr Leu Arg Gly Ala Pro Ile Thr Leu Tyr Gly
2480 2485 2490
Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
2495 2500 2505
Glu Asn Pro Gly Pro Val Ser Lys Gly Glu Glu Leu Phe Thr Gly
2510 2515 2520
Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
2525 2530 2535
Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly
2540 2545 2550
Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val
2555 2560 2565
Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys
2570 2575 2580
Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
2585 2590 2595
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe
2600 2605 2610
Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu
2615 2620 2625
Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe
2630 2635 2640
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr
2645 2650 2655
Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly
2660 2665 2670
Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
2675 2680 2685
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
2690 2695 2700
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser
2705 2710 2715
Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
2720 2725 2730
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
2735 2740 2745
Leu Tyr Lys
2750
<210> 16
<211> 8256
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C16 nsP-2A-eGFP coding sequence DNA
<400> 16
atggagaaag ttcacgttga catcgaggaa gacagcccat tcctcagagc tttgcagcgg 60
agcttcccgc agtttgaggt agaagccaag caggtcactg ataatgacca tgctaatgcc 120
agagcgtttt cgcatctggc ttcaaaactg atcgaaacgg aggtggaccc atccgacacg 180
atccttgaca ttggaagtgc gcccgcccgc agaatgtatt ctaagcacaa gtatcattgt 240
atctgtccga tgagatgtgc ggaagatccg gacagattgt ataagtatgc aactaagctg 300
aagaaaaact gtaaggaaat aactgataag gaattggaca agaaaatgaa ggagctcgcc 360
gccgtcatga gcgaccctga cctggaaact gagactatgt gcctccacga cgacgagtcg 420
tgtcgctacg aagggcaagt cgctgtttac caggatgtat acgcggttga cggaccgaca 480
agtctctatc accaagccaa taagggagtt agagtcgcct actggatagg ctttgacacc 540
acccctttta tgtttaagaa cttggctgga gcatatccat catactctac caactgggcc 600
gacgaaaccg tgttaacggc tcgtaacata ggcctatgca gctctgacgt tatggagcgg 660
tcacgtagag ggatgtccat tcttagaaag aagtatttga aaccatccaa caatgttcta 720
ttctctgttg gctcgaccat ctaccacgag aagagggact tactgaggag ctggcacctg 780
ccgtctgtat ttcacttacg tggcaagcaa aattacacat gtcggtgtga gactatagtt 840
agttgcgacg ggtacgtcgt taaaagaata gctatcagtc caggcctgta tgggaagcct 900
tcaggctatg ctgctacgat gcaccgcgag ggattcttgt gctgcaaagt gacagacaca 960
ttgaacgggg agagggtctc ttttcccgtg tgcacgtatg tgccagctac attgtgtgac 1020
caaatgactg gcatactggc aacagatgtc agtgcggacg acgcgcaaaa actgctggtt 1080
gggctcaacc agcgtatagt cgtcaacggt cgcacccaga gaaacaccaa taccatgaaa 1140
aattaccttt tgcccgtagt ggcccaggca tttgctaggt gggcaaagga atataaggaa 1200
gatcaagaag atgaaaggcc actaggacta cgagatagac agttagtcat ggggtgttgt 1260
tgggctttta gaaggcacaa gataacatct atttataagc gcccggatac ccaaaccatc 1320
atcaaagtga acagcgattt ccactcattc gtgctgccca ggataggcag taacacattg 1380
gagatcgggc tgagaacaag aatcaggaaa atgttagagg agcacaagga gccgtcacct 1440
ctcattaccg ccgaggacgt acaagaagct aagtgcgcag ccgatgaggc taaggaggtg 1500
cgtgaagccg aggagttgcg cgcagctcta ccacctttgg cagctgatgt tgaggagccc 1560
actctggaag ccgatgtcga cttgatgtta caagaggctg gggccggctc agtggagaca 1620
cctcgtggct tgataaaggt taccagctac gctggcgagg acaagatcgg ctcttacgct 1680
gtgctttctc cgcaggctgt actcaagagt gaaaaattat cttgcatcca ccctctcgct 1740
gaacaagtca tagtgataac acactctggc cgaaaagggc gttatgccgt ggaaccatac 1800
catggtaaag tagtggtgcc agagggacat gcaatacccg tccaggactt tcaagctctg 1860
agtgaaagtg ccaccattgt gtacaacgaa cgtgagttcg taaacaggta cctgcaccat 1920
attgccacac atggaggagc gctgaacact gatgaagaat attacaaaac tgtcaagccc 1980
agcgagcacg acggcgaata cctgtacgac atcgacagga aacagtgcgt caagaaagaa 2040
ctagtcactg ggctagggct cacaggcgag ctggtggatc ctcccttcca tgaattcgcc 2100
tacgagagtc tgagaacacg accagccgct ccttaccaag taccaaccat aggggtgtat 2160
ggcgtgccag gatcaggcaa gtctggcatc attaaaagcg cagtcaccaa aaaagatcta 2220
gtggtgagcg ccaagaaaga aaactgtgca gaaattataa gggacgtcaa gaaaatgaaa 2280
gggctggacg tcaatgccag aactgtggac tcagtgctct tgaatggatg caaacacccc 2340
gtagagaccc tgtatattga cgaagctttt gcttgtcatg caggtactct cagagcgctc 2400
atagccatta taagacctaa aaaggcagtg ctctgcgggg atcccaaaca gtgcggtttt 2460
tttaacatga tgtgcctgaa agtgcatttt aaccacgaga tttgcacaca agtcttccac 2520
aaaagcatct ctcgccgttg cactaaatct gtgacttcgg tcgtctcaac cttgttttac 2580
gacaaaaaaa tgagaacgac gaatccgaaa gagactaaga ttgtgattga cactaccggc 2640
agtaccaaac ctaagcagga cgatctcatt ctcacttgtt tcagagggtg ggtgaagcag 2700
ttgcaaatag attacaaagg caacgaaata atgacggcag ctgcctctca agggctgacc 2760
cgtaaaggtg tgtatgccgt tcggtacaag gtgaatgaaa atcctctgta cgcacccacc 2820
tcagaacatg tgaacgtcct actgacccgc acggaggacc gcatcgtgtg gaaaacacta 2880
gccggcgacc catggataaa aacactgact gccaagtacc ctgggaattt cactgccacg 2940
atagaggagt ggcaagcaga gcatgatgcc atcatgaggc acatcttgga gagaccggac 3000
cctaccgacg tcttccagaa taaggcaaac gtgtgttggg ccaaggcttt agtgccggtg 3060
ctgaagaccg ctggcataga catgaccact gaacaatgga acactgtgga ttattttgaa 3120
acggacaaag ctcactcagc agagatagta ttgaaccaac tatgcgtgag gttctttgga 3180
ctcgatctgg actccggtct attttctgca cccactgttc cgttatccat taggaataat 3240
cactgggata actccccgtc gcctaacatg tacgggctga ataaagaagt ggtccgtcag 3300
ctctctcgca ggtacccaca actgcctcgg gcagttgcca ctggaagagt ctatgacatg 3360
aacactggta cactgcgcaa ttatgatccg cgcataaacc tagtacctgt aaacagaaga 3420
ctgcctcatg ctttagtcct ccaccataat gaacacccac agagtgactt ttcttcattc 3480
gtcagcaaat tgaagggcag aactgtcctg gtggtcgggg aaaagttgtc cgtcccaggc 3540
aaaatggttg actggttgtc agaccggcct gaggctacct tcagagctcg gctggattta 3600
ggcatcccag gtgatgtgcc caaatatgac ataatatttg ttaatgtgag gaccccatat 3660
aaataccatc actatcagca gtgtgaagac catgccatta agcttagcat gttgaccaag 3720
aaagcttgtc tgcatctgaa tcccggcgga acctgtgtca gcataggtta tggttacgct 3780
gacagggcca gcgaaagcat cattggtgct atagcgcggc agttcaagtt ttcccgggta 3840
tgcaaaccga aatcctcact tgaagagacg gaagttctgt ttgtattcat tgggtacgat 3900
cgcaaggccc gtacgcacaa tccttacaag ctttcatcaa ccttgaccaa catttataca 3960
ggttccagac tccacgaagc cggatgtgca ccctcatatc atgtggtgcg aggggatatt 4020
gccacggcca ccgaaggagt gattataaat gctgctaaca gcaaaggaca acctggcgga 4080
ggggtgtgcg gagcgctgta taagaaattc ccggaaagct tcgatttaca gccgatcgaa 4140
gtaggaaaag cgcgactggt caaaggtgca gctaaacata tcattcatgc cgtaggacca 4200
aacttcaaca aagtttcgga ggttgaaggt gacaaacagt tggcagaggc ttatgagtcc 4260
atcgctaaga ttgtcaacga taacaattac aagtcagtag cgattccact gttgtccacc 4320
ggcatctttt ccgggaacaa agatcgacta acccaatcat tgaaccattt gctgacagct 4380
ttagacacca ctgatgcaga tgtagccata tactgcaggg acaagaaatg ggaaatgact 4440
ctcaaggaag cagtggctag gagagaagca gtggaggaga tatgcatatc cgacgactct 4500
tcagtgacag aacctgatgc agagctggtg agggtgcatc cgaagagttc tttggctgga 4560
aggaagggct acagcacaag cgatggcaaa actttctcat atttggaagg gaccaagttt 4620
caccaggcgg ccaaggatat agcagaaatt aatgccatgt ggcccgttgc aacggaggcc 4680
aatgagcagg tatgcatgta tatcctcgga gaaagcatga gcagtattag gtcgaaatgc 4740
cccgtcgaag agtcggaagc ctccacacca cctagcacgc tgccttgctt gtgcatccat 4800
gccatgactc cagaaagagt acagcgccta aaagcctcac gtccagaaca aattactgtg 4860
tgctcatcct ttccattgcc gaagtataga atcactggtg tgcagaagat ccaatgctcc 4920
cagcctatat tgttctcacc gaaagtgcct gcgtatattc atccaaggaa gtatctcgtg 4980
gaaacaccac cggtagacga gactccggag ccatcggcag agaaccaatc cacagagggg 5040
acacctgaac aaccaccact tataaccgag gatgagacca ggactagaac gcctgagccg 5100
atcatcatcg aagaggaaga agaggatagc ataagtttgc tgtcagatgg cccgacccac 5160
caggtgctgc aagtcgaggc agacattcac gggccgccct ctgtatctag ctcatcctgg 5220
tccattcctc atgcatccga ctttgatgtg gacagtttat ccatacttga caccctggag 5280
ggagctagcg tgaccagcgg ggcaacgtca gccgagacta actcttactt cgcaaagagt 5340
atggagtttc tggcgcgacc ggtgcctgcg cctcgaacag tattcaggaa ccctccacat 5400
cccgctccgc gcacaagaac accgtcactt gcacccagca gggcctgctc gagaaccagc 5460
ctagtttcca ccccgccagg cgtgaatagg gtgatcacta gagaggagct cgaggcgctt 5520
accccgtcac gcactcctag caggtcggtc tcgagaacca gcctggtctc caacccgcca 5580
ggcgtaaata gggtgattac aagagaggag tttgaggcgt tcgtagcaca acaacaatga 5640
cggtttgatg cgggtgcata catcttttcc tccgacaccg gtcaagggca tttacaacaa 5700
aaatcagtaa ggcaaacggt gctatccgaa gtggtgttgg agaggaccga attggagatt 5760
tcgtatgccc cgcgcctcga ccaagaaaaa gaagaattac tacgcaagaa attacagtta 5820
aatcccacac ctgctaacag aagcagatac cagtccagga aggtggagaa catgaaagcc 5880
ataacagcta gacgtattct gcaaggccta gggcattatt tgaaggcaga aggaaaagtg 5940
gagtgctacc gaaccctgca tcctgttcct ttgtattcat ctagtgtgaa ccgtgccttt 6000
tcaagcccca aggtcgcagt ggaagcctgt aacgccatgt tgaaagagaa ctttccgact 6060
gtggcttctt actgtattat tccagagtac gatgcctatt tggacatggt tgacggagct 6120
tcatgctgct tagacactgc cagtttttgc cctgcaaagc tgcgcagctt tccaaagaaa 6180
cactcctatt tggaacccac aatacgatcg gcagtgcctt cagcgatcca gaacacgctc 6240
cagaacgtcc tggcagctgc cacaaaaaga aattgcaatg tcacgcaaat gagagaattg 6300
cccgtattgg attcggcggc ctttaatgtg gaatgcttca agaaatatgc gtgtaataat 6360
gaatattggg aaacgtttaa agaaaacccc atcaggctta ctgaagaaaa cgtggtaaat 6420
tacattacca aattaaaagg accaaaagct gctgctcttt ttgcgaagac acataatttg 6480
aatatgttgc aggacatacc aatggacagg tttgtaatgg acttaaagag agacgtgaaa 6540
gtgactccag gaacaaaaca tactgaagaa cggcccaagg tacaggtgat ccaggctgcc 6600
gatccgctag caacagcgta tctgtgcgga atccaccgag agctggttag gagattaaat 6660
gcggtcctgc ttccgaacat tcatacactg tttgatatgt cggctgaaga ctttgacgct 6720
attatagccg agcacttcca gcctggggat tgtgttctgg aaactgacat cgcgtcgttt 6780
gataaaagtg aggacgacgc catggctctg accgcgttaa tgattctgga agacttaggt 6840
gtggacgcag agctgttgac gctgattgag gcggctttcg gcgaaatttc atcaatacat 6900
ttgcccacta aaactaaatt taagttcggc gccatgatga agtccggcat gtttctgacc 6960
ctgttcgtga acaccgtgat caacatcgtg atcgccagcc gggtgctgag agagagactg 7020
acaggatctc cttgcgccgc cttcatcggc gacgacaata tcgtgaaggg cgtgaagtcc 7080
gacaagctga tggccgatag atgcgccacc tggctgaaca tggaagtgaa gatcatcgac 7140
gccgtcgtgg gcgagaaggc cccttatttt tgcggcggct tcatcctgtg cgacagcgtg 7200
acaggcacag cctgcagagt tgccgatcct ctgaagcggc tgttcaagct gggaaaacct 7260
ctggccgccg acgacgagca cgacgacgat agacgtagag ccctgcacga ggaatccacc 7320
agatggaaca gagtgggcat cctgagcgag ctgtgcaagg ccgtggaaag cagatacgag 7380
acagtgggca ccagcatcat tgtgatggca atgaccacac tggccagcag cgtgaaaagc 7440
ttcagctacc taaggggcgc ccctatcaca ctgtacggct ctggcgaagg cagaggcagc 7500
cttctgacat gtggcgacgt ggaagagaac cccggacctg tgtctaaggg cgaagaactg 7560
tttaccggcg tggtgcccat cctggtggaa ctggatgggg atgtgaacgg ccacaagttc 7620
agcgttagcg gagaaggcga aggcgacgcc acatacggaa agctgaccct gaagttcatc 7680
tgcaccaccg gcaagctgcc tgtgccatgg cctacactgg tcaccacact gacatacggc 7740
gtgcagtgct tcagcagata ccccgaccat atgaagcagc acgacttctt caagagcgcc 7800
atgcctgagg gctacgtgca agagcggacc atcttcttta aggacgacgg caactacaag 7860
accagggccg aagtgaagtt cgagggcgac accctggtca accggatcga gctgaagggc 7920
atcgacttca aagaggacgg caatatcctg ggccacaagc tcgagtacaa ctacaacagc 7980
cacaacgtgt acatcatggc cgacaagcag aaaaacggca tcaaagtgaa cttcaagatc 8040
cggcacaaca tcgaggacgg ctctgtgcag ctggccgatc actaccagca gaacacaccc 8100
atcggagatg gccctgtgct gctgcccgat aaccactacc tgagcaccca gagcaagctg 8160
agcaaggacc ccaacgagaa gcgggaccac atggtgctgc tggaatttgt gacagccgcc 8220
ggaatcaccc tcggcatgga tgagctgtac aagtga 8256
<210> 17
<211> 11447
<212> DNA
<213> Venezuelan equine encephalitis virus
<220>
<221> misc_feature
<222> (1)..(11447)
<223> VEEV genome L01442.2
<400> 17
atgggcggcg catgagagaa gcccagacca attacctacc caaaatggag aaagttcacg 60
ttgacatcga ggaagacagc ccattcctca gagctttgca gcggagcttc ccgcagtttg 120
aggtagaagc caagcaggtc actgataatg accatgctaa tgccagagcg ttttcgcatc 180
tggcttcaaa actgatcgaa acggaggtgg acccatccga cacgatcctt gacattggaa 240
gtgcgcccgc ccgcagaatg tattctaagc acaagtatca ttgtatctgt ccgatgagat 300
gtgcggaaga tccggacaga ttgtataagt atgcaactaa gctgaagaaa aactgtaagg 360
aaataactga taaggaattg gacaagaaaa tgaaggagct cgccgccgtc atgagcgacc 420
ctgacctgga aactgagact atgtgcctcc acgacgacga gtcgtgtcgc tacgaagggc 480
aagtcgctgt ttaccaggat gtatacgcgg ttgacggacc gacaagtctc tatcaccaag 540
ccaataaggg agttagagtc gcctactgga taggctttga caccacccct tttatgttta 600
agaacttggc tggagcatat ccatcatact ctaccaactg ggccgacgaa accgtgttaa 660
cggctcgtaa cataggccta tgcagctctg acgttatgga gcggtcacgt agagggatgt 720
ccattcttag aaagaagtat ttgaaaccat ccaacaatgt tctattctct gttggctcga 780
ccatctacca cgagaagagg gacttactga ggagctggca cctgccgtct gtatttcact 840
tacgtggcaa gcaaaattac acatgtcggt gtgagactat agttagttgc gacgggtacg 900
tcgttaaaag aatagctatc agtccaggcc tgtatgggaa gccttcaggc tatgctgcta 960
cgatgcaccg cgagggattc ttgtgctgca aagtgacaga cacattgaac ggggagaggg 1020
tctcttttcc cgtgtgcacg tatgtgccag ctacattgtg tgaccaaatg actggcatac 1080
tggcaacaga tgtcagtgcg gacgacgcgc aaaaactgct ggttgggctc aaccagcgta 1140
tagtcgtcaa cggtcgcacc cagagaaaca ccaataccat gaaaaattac cttttgcccg 1200
tagtggccca ggcatttgct aggtgggcaa aggaatataa ggaagatcaa gaagatgaaa 1260
ggccactagg actacgagat agacagttag tcatggggtg ttgttgggct tttagaaggc 1320
acaagataac atctatttat aagcgcccgg atacccaaac catcatcaaa gtgaacagcg 1380
atttccactc attcgtgctg cccaggatag gcagtaacac attggagatc gggctgagaa 1440
caagaatcag gaaaatgtta gaggagcaca aggagccgtc acctctcatt accgccgagg 1500
acgtacaaga agctaagtgc gcagccgatg aggctaagga ggtgcgtgaa gccgaggagt 1560
tgcgcgcagc tctaccacct ttggcagctg atgttgagga gcccactctg gaagccgatg 1620
tcgacttgat gttacaagag gctggggccg gctcagtgga gacacctcgt ggcttgataa 1680
aggttaccag ctacgctggc gaggacaaga tcggctctta cgctgtgctt tctccgcagg 1740
ctgtactcaa gagtgaaaaa ttatcttgca tccaccctct cgctgaacaa gtcatagtga 1800
taacacactc tggccgaaaa gggcgttatg ccgtggaacc ataccatggt aaagtagtgg 1860
tgccagaggg acatgcaata cccgtccagg actttcaagc tctgagtgaa agtgccacca 1920
ttgtgtacaa cgaacgtgag ttcgtaaaca ggtacctgca ccatattgcc acacatggag 1980
gagcgctgaa cactgatgaa gaatattaca aaactgtcaa gcccagcgag cacgacggcg 2040
aatacctgta cgacatcgac aggaaacagt gcgtcaagaa agaactagtc actgggctag 2100
ggctcacagg cgagctggtg gatcctccct tccatgaatt cgcctacgag agtctgagaa 2160
cacgaccagc cgctccttac caagtaccaa ccataggggt gtatggcgtg ccaggatcag 2220
gcaagtctgg catcattaaa agcgcagtca ccaaaaaaga tctagtggtg agcgccaaga 2280
aagaaaactg tgcagaaatt ataagggacg tcaagaaaat gaaagggctg gacgtcaatg 2340
ccagaactgt ggactcagtg ctcttgaatg gatgcaaaca ccccgtagag accctgtata 2400
ttgacgaagc ttttgcttgt catgcaggta ctctcagagc gctcatagcc attataagac 2460
ctaaaaaggc agtgctctgc ggggatccca aacagtgcgg tttttttaac atgatgtgcc 2520
tgaaagtgca ttttaaccac gagatttgca cacaagtctt ccacaaaagc atctctcgcc 2580
gttgcactaa atctgtgact tcggtcgtct caaccttgtt ttacgacaaa aaaatgagaa 2640
cgacgaatcc gaaagagact aagattgtga ttgacactac cggcagtacc aaacctaagc 2700
aggacgatct cattctcact tgtttcagag ggtgggtgaa gcagttgcaa atagattaca 2760
aaggcaacga aataatgacg gcagctgcct ctcaagggct gacccgtaaa ggtgtgtatg 2820
ccgttcggta caaggtgaat gaaaatcctc tgtacgcacc cacctcagaa catgtgaacg 2880
tcctactgac ccgcacggag gaccgcatcg tgtggaaaac actagccggc gacccatgga 2940
taaaaacact gactgccaag taccctggga atttcactgc cacgatagag gagtggcaag 3000
cagagcatga tgccatcatg aggcacatct tggagagacc ggaccctacc gacgtcttcc 3060
agaataaggc aaacgtgtgt tgggccaagg ctttagtgcc ggtgctgaag accgctggca 3120
tagacatgac cactgaacaa tggaacactg tggattattt tgaaacggac aaagctcact 3180
cagcagagat agtattgaac caactatgcg tgaggttctt tggactcgat ctggactccg 3240
gtctattttc tgcacccact gttccgttat ccattaggaa taatcactgg gataactccc 3300
cgtcgcctaa catgtacggg ctgaataaag aagtggtccg tcagctctct cgcaggtacc 3360
cacaactgcc tcgggcagtt gccactggaa gagtctatga catgaacact ggtacactgc 3420
gcaattatga tccgcgcata aacctagtac ctgtaaacag aagactgcct catgctttag 3480
tcctccacca taatgaacac ccacagagtg acttttcttc attcgtcagc aaattgaagg 3540
gcagaactgt cctggtggtc ggggaaaagt tgtccgtccc aggcaaaatg gttgactggt 3600
tgtcagaccg gcctgaggct accttcagag ctcggctgga tttaggcatc ccaggtgatg 3660
tgcccaaata tgacataata tttgttaatg tgaggacccc atataaatac catcactatc 3720
agcagtgtga agaccatgcc attaagctta gcatgttgac caagaaagct tgtctgcatc 3780
tgaatcccgg cggaacctgt gtcagcatag gttatggtta cgctgacagg gccagcgaaa 3840
gcatcattgg tgctatagcg cggcagttca agttttcccg ggtatgcaaa ccgaaatcct 3900
cacttgaaga gacggaagtt ctgtttgtat tcattgggta cgatcgcaag gcccgtacgc 3960
acaatcctta caagctttca tcaaccttga ccaacattta tacaggttcc agactccacg 4020
aagccggatg tgcaccctca tatcatgtgg tgcgagggga tattgccacg gccaccgaag 4080
gagtgattat aaatgctgct aacagcaaag gacaacctgg cggaggggtg tgcggagcgc 4140
tgtataagaa attcccggaa agcttcgatt tacagccgat cgaagtagga aaagcgcgac 4200
tggtcaaagg tgcagctaaa catatcattc atgccgtagg accaaacttc aacaaagttt 4260
cggaggttga aggtgacaaa cagttggcag aggcttatga gtccatcgct aagattgtca 4320
acgataacaa ttacaagtca gtagcgattc cactgttgtc caccggcatc ttttccggga 4380
acaaagatcg actaacccaa tcattgaacc atttgctgac agctttagac accactgatg 4440
cagatgtagc catatactgc agggacaaga aatgggaaat gactctcaag gaagcagtgg 4500
ctaggagaga agcagtggag gagatatgca tatccgacga ctcttcagtg acagaacctg 4560
atgcagagct ggtgagggtg catccgaaga gttctttggc tggaaggaag ggctacagca 4620
caagcgatgg caaaactttc tcatatttgg aagggaccaa gtttcaccag gcggccaagg 4680
atatagcaga aattaatgcc atgtggcccg ttgcaacgga ggccaatgag caggtatgca 4740
tgtatatcct cggagaaagc atgagcagta ttaggtcgaa atgccccgtc gaagagtcgg 4800
aagcctccac accacctagc acgctgcctt gcttgtgcat ccatgccatg actccagaaa 4860
gagtacagcg cctaaaagcc tcacgtccag aacaaattac tgtgtgctca tcctttccat 4920
tgccgaagta tagaatcact ggtgtgcaga agatccaatg ctcccagcct atattgttct 4980
caccgaaagt gcctgcgtat attcatccaa ggaagtatct cgtggaaaca ccaccggtag 5040
acgagactcc ggagccatcg gcagagaacc aatccacaga ggggacacct gaacaaccac 5100
cacttataac cgaggatgag accaggacta gaacgcctga gccgatcatc atcgaagagg 5160
aagaagagga tagcataagt ttgctgtcag atggcccgac ccaccaggtg ctgcaagtcg 5220
aggcagacat tcacgggccg ccctctgtat ctagctcatc ctggtccatt cctcatgcat 5280
ccgactttga tgtggacagt ttatccatac ttgacaccct ggagggagct agcgtgacca 5340
gcggggcaac gtcagccgag actaactctt acttcgcaaa gagtatggag tttctggcgc 5400
gaccggtgcc tgcgcctcga acagtattca ggaaccctcc acatcccgct ccgcgcacaa 5460
gaacaccgtc acttgcaccc agcagggcct gctcgagaac cagcctagtt tccaccccgc 5520
caggcgtgaa tagggtgatc actagagagg agctcgaggc gcttaccccg tcacgcactc 5580
ctagcaggtc ggtctcgaga accagcctgg tctccaaccc gccaggcgta aatagggtga 5640
ttacaagaga ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg 5700
catacatctt ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa 5760
cggtgctatc cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc 5820
tcgaccaaga aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta 5880
acagaagcag ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta 5940
ttctgcaagg cctagggcat tatttgaagg cagaaggaaa agtggagtgc taccgaaccc 6000
tgcatcctgt tcctttgtat tcatctagtg tgaaccgtgc cttttcaagc cccaaggtcg 6060
cagtggaagc ctgtaacgcc atgttgaaag agaactttcc gactgtggct tcttactgta 6120
ttattccaga gtacgatgcc tatttggaca tggttgacgg agcttcatgc tgcttagaca 6180
ctgccagttt ttgccctgca aagctgcgca gctttccaaa gaaacactcc tatttggaac 6240
ccacaatacg atcggcagtg ccttcagcga tccagaacac gctccagaac gtcctggcag 6300
ctgccacaaa aagaaattgc aatgtcacgc aaatgagaga attgcccgta ttggattcgg 6360
cggcctttaa tgtggaatgc ttcaagaaat atgcgtgtaa taatgaatat tgggaaacgt 6420
ttaaagaaaa ccccatcagg cttactgaag aaaacgtggt aaattacatt accaaattaa 6480
aaggaccaaa agctgctgct ctttttgcga agacacataa tttgaatatg ttgcaggaca 6540
taccaatgga caggtttgta atggacttaa agagagacgt gaaagtgact ccaggaacaa 6600
aacatactga agaacggccc aaggtacagg tgatccaggc tgccgatccg ctagcaacag 6660
cgtatctgtg cggaatccac cgagagctgg ttaggagatt aaatgcggtc ctgcttccga 6720
acattcatac actgtttgat atgtcggctg aagactttga cgctattata gccgagcact 6780
tccagcctgg ggattgtgtt ctggaaactg acatcgcgtc gtttgataaa agtgaggacg 6840
acgccatggc tctgaccgcg ttaatgattc tggaagactt aggtgtggac gcagagctgt 6900
tgacgctgat tgaggcggct ttcggcgaaa tttcatcaat acatttgccc actaaaacta 6960
aatttaaatt cggagccatg atgaaatctg gaatgttcct cacactgttt gtgaacacag 7020
tcattaacat tgtaatcgca agcagagtgt tgagagaacg gctaaccgga tcaccatgtg 7080
cagcattcat tggagatgac aatatcgtga aaggagtcaa atcggacaaa ttaatggcag 7140
acaggtgcgc cacctggttg aatatggaag tcaagattat agatgctgtg gtgggcgaga 7200
aagcgcctta tttctgtgga gggtttattt tgtgtgactc cgtgaccggc acagcgtgcc 7260
gtgtggcaga ccccctaaaa aggctgttta agcttggcaa acctctggca gcagacgatg 7320
aacatgatga tgacaggaga agggcattgc atgaagagtc aacacgctgg aaccgagtgg 7380
gtattctttc agagctgtgc aaggcagtag aatcaaggta tgaaaccgta ggaacttcca 7440
tcatagttat ggccatgact actctagcta gcagtgttaa atcattcagc tacctgagag 7500
gggcccctat aactctctac ggctaacctg aatggactac gacatagtct agtccgccaa 7560
gatgttcccg ttccagccaa tgtatccgat gcagccaatg ccctatcgca acccgttcgc 7620
ggccccgcgc aggccctggt tccccagaac cgaccctttt ctggcgatgc aggtgcagga 7680
attaacccgc tcgatggcta acctgacgtt caagcaacgc cgggacgcgc cacctgaggg 7740
gccatccgct aagaaaccga agaaggaggc ctcgcaaaaa cagaaagggg gaggccaagg 7800
gaagaagaag aagaaccaag ggaagaagaa ggctaagaca gggccgccta atccgaaggc 7860
acagaatgga aacaagaaga agaccaacaa gaaaccaggc aagagacagc gcatggtcat 7920
gaaattggaa tctgacaaga cgttcccaat catgttggaa gggaagataa acggctacgc 7980
ttgtgtggtc ggagggaagt tattcaggcc gatgcatgtg gaaggcaaga tcgacaacga 8040
cgttctggcc gcgcttaaga cgaagaaagc atccaaatac gatcttgagt atgcagatgt 8100
gccacagaac atgcgggccg atacattcaa atacacccat gagaaacccc aaggctatta 8160
cagctggcat catggagcag tccaatatga aaatgggcgt ttcacggtgc cgaaaggagt 8220
tggggccaag ggagacagcg gacgacccat tctggataac cagggacggg tggtcgctat 8280
tgtgctggga ggtgtgaatg aaggatctag gacagccctt tcagtcgtca tgtggaacga 8340
gaagggagtt accgtgaagt atactccgga gaactgcgag caatggtcac tagtgaccac 8400
catgtgtctg ctcgccaatg tgacgttccc atgtgctcaa ccaccaattt gctacgacag 8460
aaaaccagca gagactttgg ccatgctcag cgttaacgtt gacaacccgg gctacgatga 8520
gctgctggaa gcagctgtta agtgccccgg aaggaaaagg agatccaccg aggagctgtt 8580
taaggagtat aagctaacgc gcccttacat ggccagatgc atcagatgtg cagttgggag 8640
ctgccatagt ccaatagcaa tcgaggcagt aaagagcgac gggcacgacg gttatgttag 8700
acttcagact tcctcgcagt atggcctgga ttcctccggc aacttaaagg gcaggaccat 8760
gcggtatgac atgcacggga ccattaaaga gataccacta catcaagtgt cactccatac 8820
atctcgcccg tgtcacattg tggatgggca cggttatttc ctgcttgcca ggtgcccggc 8880
aggggactcc atcaccatgg aatttaagaa agattccgtc acacactcct gctcggtgcc 8940
gtatgaagtg aaatttaatc ctgtaggcag agaactctat actcatcccc cagaacacgg 9000
agtagagcaa gcgtgccaag tctacgcaca tgatgcacag aacagaggag cttatgtcga 9060
gatgcacctc ccgggctcag aagtggacag cagtttggtt tccttgagcg gcagttcagt 9120
caccgtgaca cctcctgttg ggactagcgc cctggtggaa tgcgagtgtg gcggcacaaa 9180
gatctccgag accatcaaca agacaaaaca gttcagccag tgcacaaaga aggagcagtg 9240
cagagcatat cggctgcaga acgataagtg ggtgtataat tctgacaaac tgcccaaagc 9300
agcgggagcc accttaaaag gaaaactgca tgtcccattc ttgctggcag acggcaaatg 9360
caccgtgcct ctagcaccag aacctatgat aacctttggt ttcagatcag tgtcactgaa 9420
actgcaccct aagaatccca catatctaac cacccgccaa cttgctgatg agcctcacta 9480
cacgcacgag ctcatatctg aaccagctgt taggaatttt accgtcaccg aaaaagggtg 9540
ggagtttgta tggggaaacc acccgccgaa aaggttttgg gcacaggaaa cagcacccgg 9600
aaatccacat gggctaccgc acgaggtgat aactcattat taccacagat accctatgtc 9660
caccatcctg ggtttgtcaa tttgtgccgc cattgcaacc gtttccgttg cagcgtctac 9720
ctggctgttt tgcagatcta gagttgcgtg cctaactcct taccggctaa cacctaacgc 9780
taggatacca ttttgtctgg ctgtgctttg ctgcgcccgc actgcccggg ccgagaccac 9840
ctgggagtcc ttggatcacc tatggaacaa taaccaacag atgttctgga ttcaattgct 9900
gatccctctg gccgccttga tcgtagtgac tcgcctgctc aggtgcgtgt gctgtgtcgt 9960
gcctttttta gtcatggccg gcgccgcagg cgccggcgcc tacgagcacg cgaccacgat 10020
gccgagccaa gcgggaatct cgtataacac tatagtcaac agagcaggct acgcaccact 10080
ccctatcagc ataacaccaa caaagatcaa gctgatacct acagtgaact tggagtacgt 10140
cacctgccac tacaaaacag gaatggattc accagccatc aaatgctgcg gatctcagga 10200
atgcactcca acttacaggc ctgatgaaca gtgcaaagtc ttcacagggg tttacccgtt 10260
catgtggggt ggtgcatatt gcttttgcga cactgagaac acccaagtca gcaaggccta 10320
cgtaatgaaa tctgacgact gccttgcgga tcatgctgaa gcatataaag cgcacacagc 10380
ctcagtgcag gcgttcctca acatcacagt gggagaacac tctattgtga ctaccgtgta 10440
tgtgaatgga gaaactcctg tgaatttcaa tggggtcaaa ttaactgcag gtccgctttc 10500
cacagcttgg acaccctttg atcgcaaaat cgtgcagtat gccggggaga tctataatta 10560
tgattttcct gagtatgggg caggacaacc aggagcattt ggagatatac aatccagaac 10620
agtctcaagc tcagatctgt atgccaatac caacctagtg ctgcagagac ccaaagcagg 10680
agcgatccac gtgccataca ctcaggcacc ttcgggtttt gagcaatgga agaaagataa 10740
agctccatca ttgaaattta ccgccccttt cggatgcgaa atatatacaa accccattcg 10800
cgccgaaaac tgtgctgtag ggtcaattcc attagccttt gacattcccg acgccttgtt 10860
caccagggtg tcagaaacac cgacactttc agcggccgaa tgcactctta acgagtgcgt 10920
gtattcttcc gactttggtg ggatcgccac ggtcaagtac tcggccagca agtcaggcaa 10980
gtgcgcagtc catgtgccat cagggactgc taccctaaaa gaagcagcag tcgagctaac 11040
cgagcaaggg tcggcgacta tccatttctc gaccgcaaat atccacccgg agttcaggct 11100
ccaaatatgc acatcatatg ttacgtgcaa aggtgattgt caccccccga aagaccatat 11160
tgtgacacac cctcagtatc acgcccaaac atttacagcc gcggtgtcaa aaaccgcgtg 11220
gacgtggtta acatccctgc tgggaggatc agccgtaatt attataattg gcttggtgct 11280
ggctactatt gtggccatgt acgtgctgac caaccagaaa cataattgaa tacagcagca 11340
attggcaagc tgcttacata gaactcgcgg cgattggcat gccgccttaa aatttttatt 11400
ttattttttc ttttcttttc cgaatcggat tttgttttta atatttc 11447
<210> 18
<211> 558
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, VEEV rep RNA nt 6966-7523 with silent
mutations
<400> 18
aaguucggcg ccaugaugaa guccggcaug uuucugaccc uguucgugaa caccgugauc 60
aacaucguga ucgccagccg ggugcugaga gagagacuga caggaucucc uugcgccgcc 120
uucaucggcg acgacaauau cgugaagggc gugaaguccg acaagcugau ggccgauaga 180
ugcgccaccu ggcugaacau ggaagugaag aucaucgacg ccgucguggg cgagaaggcc 240
ccuuauuuuu gcggcggcuu cauccugugc gacagcguga caggcacagc cugcagaguu 300
gccgauccuc ugaagcggcu guucaagcug ggaaaaccuc uggccgccga cgacgagcac 360
gacgacgaua gacguagagc ccugcacgag gaauccacca gauggaacag agugggcauc 420
cugagcgagc ugugcaaggc cguggaaagc agauacgaga cagugggcac cagcaucauu 480
gugauggcaa ugaccacacu ggccagcagc gugaaaagcu ucagcuaccu aaggggcgcc 540
ccuaucacac uguacggc 558
<210> 19
<211> 1821
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, RNA nsP4 engineered RNA +silent mutations
<400> 19
uacaucuuuu ccuccgacac cggucaaggg cauuuacaac aaaaaucagu aaggcaaacg 60
gugcuauccg aagugguguu ggagaggacc gaauuggaga uuucguaugc cccgcgccuc 120
gaccaagaaa aagaagaauu acuacgcaag aaauuacagu uaaaucccac accugcuaac 180
agaagcagau accaguccag gaagguggag aacaugaaag ccauaacagc uagacguauu 240
cugcaaggcc uagggcauua uuugaaggca gaaggaaaag uggagugcua ccgaacccug 300
cauccuguuc cuuuguauuc aucuagugug aaccgugccu uuucaagccc caaggucgca 360
guggaagccu guaacgccau guugaaagag aacuuuccga cuguggcuuc uuacuguauu 420
auuccagagu acgaugccua uuuggacaug guugacggag cuucaugcug cuuagacacu 480
gccaguuuuu gcccugcaaa gcugcgcagc uuuccaaaga aacacuccua uuuggaaccc 540
acaauacgau cggcagugcc uucagcgauc cagaacacgc uccagaacgu ccuggcagcu 600
gccacaaaaa gaaauugcaa ugucacgcaa augagagaau ugcccguauu ggauucggcg 660
gccuuuaaug uggaaugcuu caagaaauau gcguguaaua augaauauug ggaaacguuu 720
aaagaaaacc ccaucaggcu uacugaagaa aacgugguaa auuacauuac caaauuaaaa 780
ggaccaaaag cugcugcucu uuuugcgaag acacauaauu ugaauauguu gcaggacaua 840
ccaauggaca gguuuguaau ggacuuaaag agagacguga aagugacucc aggaacaaaa 900
cauacugaag aacggcccaa gguacaggug auccaggcug ccgauccgcu agcaacagcg 960
uaucugugcg gaauccaccg agagcugguu aggagauuaa augcgguccu gcuuccgaac 1020
auucauacac uguuugauau gucggcugaa gacuuugacg cuauuauagc cgagcacuuc 1080
cagccugggg auuguguucu ggaaacugac aucgcgucgu uugauaaaag ugaggacgac 1140
gccauggcuc ugaccgcguu aaugauucug gaagacuuag guguggacgc agagcuguug 1200
acgcugauug aggcggcuuu cggcgaaauu ucaucaauac auuugcccac uaaaacuaaa 1260
uuuaaguucg gcgccaugau gaaguccggc auguuucuga cccuguucgu gaacaccgug 1320
aucaacaucg ugaucgccag ccgggugcug agagagagac ugacaggauc uccuugcgcc 1380
gccuucaucg gcgacgacaa uaucgugaag ggcgugaagu ccgacaagcu gauggccgau 1440
agaugcgcca ccuggcugaa cauggaagug aagaucaucg acgccgucgu gggcgagaag 1500
gccccuuauu uuugcggcgg cuucauccug ugcgacagcg ugacaggcac agccugcaga 1560
guugccgauc cucugaagcg gcuguucaag cugggaaaac cucuggccgc cgacgacgag 1620
cacgacgacg auagacguag agcccugcac gaggaaucca ccagauggaa cagagugggc 1680
auccugagcg agcugugcaa ggccguggaa agcagauacg agacaguggg caccagcauc 1740
auugugaugg caaugaccac acuggccagc agcgugaaaa gcuucagcua ccuaaggggc 1800
gccccuauca cacuguacgg c 1821
<210> 20
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C01 nsP1-4 ORF and SGP RNA
<400> 20
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaagu acgccugcaa caacgaguac ugggagacau 6420
ucaaagagaa ccccauccgg cugaccgagg aaaacguggu caacuacauc accaagcuga 6480
agggccccaa agccgccgcu cuguuugcca agacacacaa ccugaacaug cugcaggaca 6540
uccccaugga cagauucgug auggaccuga agcgggacgu gaaagugacc ccuggcacca 6600
agcacaccga ggaacggccu aaggugcaag ugauccaggc cgcugauccu cuggccacag 6660
ccuaucugug uggcauccac agagaacucg ugcggagacu gaaugccgug cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg cugaugauuc uggaagaucu cggaguggac gccgagcugc 6900
ugacacugau ugaagccgcc uuuggcgaga ucagcagcau ccaucugccu accaagacca 6960
aguucaaguu cggcgccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaacaggc agcccuugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 21
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C02 nsP1-4 ORF and SGP RNA
<400> 21
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauaugccg uggauggccc uacaagccug uaccaccagg 540
ccaacaaggg cgucagagug gccuacugga ucggcuucga caccacaccu uucauguuca 600
agaaccuggc uggcgcuuac cccagcuaca gcacaaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 22
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C03 nsP1-4 ORF and SGP RNA
<400> 22
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc cuacaaguac caccacuacc 3720
agcagugcga ggaccacgcc aucaagcuga gcaugcugac caagaaggcc ugccugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucaucggcua cgacagaaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 23
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C04 nsP1-4 ORF and SGP RNA
<400> 23
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaaccccua caagcugagc agcacccuga ccaacaucua caccggcagc agacugcacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccaca gccacagaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 24
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C05 nsP1-4 ORF and SGP RNA
<400> 24
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagcgagaag cugagcugca uucacccucu ggccgagcaa gugaucguga 1800
ucacacacag cggccggaag ggcagauaug ccguggaacc uuaucacggc aagguggugg 1860
ugccugaggg acacgcuauu ccagugcagg acuuucaggc ccugagcgag ucugccacca 1920
ucguguacaa cgagcgcgag uucgugaaca gauaccugca ccacauugcc acacacggcg 1980
gagcccugaa caccgacgaa gaguacuaca agaccgugaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggauagcgug cugcugaacg gcugcaagca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccua agcagugcgg cuucuucaac augaugugcc 2520
ugaaggugca cuucaaccac gagaucugca cccagguguu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 25
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C06 nsP1-4 ORF and SGP RNA
<400> 25
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggccagaaa uaucggccug uguagcagcg acgugaugga aagauccaga cggggcauga 720
gcauccugcg gaagaaguac cugaagccua gcaacaacgu gcuguucagc gugggcagca 780
ccaucuacca cgagaagagg gaccugcugc ggagcuggca ucugccuucc guguuucacc 840
ugagaggcaa gcagaacuac accuguagau gcgagacaau cguguccugc gacggcuacg 900
uggucaagcg gaucgccauu ucuccuggcc uguacggcaa gccuucuggc uaugccgcca 960
ccaugcacag agaaggcuuu cuguguugca aagugaccga cacacugaac ggcgagcggg 1020
uguccuuucc uguguguacc uaugugcccg ccacacugug cgaucagaug acaggcauuc 1080
uggccaccga cgugucagcc gacgaugccc agaaacugcu cgugggccug aaccagagaa 1140
ucguggucaa cggcagaacc cagcggaaca ccaacaccau gaagaacuac cugcugccug 1200
ugguggccca ggccuuugcc agaugggcca aagaguacaa agaggaucaa gaggacgagc 1260
ggccccuggg ccugagagau agacaacugg ucaugggcug cugcugggcc uucagaaggc 1320
acaagaucac cagcaucuac aagcggcccg acacacagac caucaucaaa gugaacagcg 1380
acuuccacag cuucgugcug ccucggaucg gcagcaacac acuggaaauc ggccugcgga 1440
cccggaucag aaagaugcug gaagaacaca aagagcccuc uccacugauc accgccgagg 1500
augugcaaga ggccaaaugu gccgccgacg aggcuaaaga agugcgcgaa gccgaggaac 1560
ugagagccgc acuuccuccu cuggccgccg auguugaaga acccacucug gaagccgacg 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 26
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C07 nsP1-4 ORF and SGP RNA
<400> 26
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaagagc aucagcagac 2580
ggugcaccaa gagcgugacc agcguggugu cuacccuguu cuacgacaag aagaugcgga 2640
cgacaaaccc caaagagaca aagaucguca ucgacaccac cggcagcacc aagccuaagc 2700
aggacgaucu gauccugacc ugcuucagag gcugggucaa gcagcugcag aucgacuaca 2760
agggcaacga gaucaugacc gccgcugccu cucagggccu gacaagaaaa ggcguguacg 2820
ccgugcggua caaagugaac gagaacccuc uguacgcccc uaccagcgag caugugaaug 2880
ugcugcugac ccggaccgag gaccggaucg uuuggaaaac acuggccggc gaucccugga 2940
ucaagacccu gacagccaag uaucccggca acuucaccgc caccaucgag gaauggcagg 3000
ccgagcacga ugccaucaug cggcacaucc uggaaagacc cgauccuacc gacguguucc 3060
agaacaaggc caacgugugc ugggccaaag cucuggugcc ugugcugaaa accgccggca 3120
ucgauaugac caccgagcag uggaacaccg uggacuacuu cgagacagac aaggcccaca 3180
gcgccgagau cgugcugaau cagcugugcg ugcgguucuu cggccuggau cuggauagcg 3240
gccuguucuc ugcuccuacc gugccucuga gcauccggaa caaccacugg gacaacagcc 3300
ccucuccuaa uauguacggc cugaacaaag aagucgugcg gcagcugagc agaagauacc 3360
cacagcugcc uagagccgug gccacaggca gaguguacga caugaauacc ggcacacugc 3420
ggaacuacga ccccagaauc aaucuggugc ccgugaacag aaggcugccc cacgcucugg 3480
uucugcacca caaugagcac ccucagagcg acuucagcag cuucgugucc aagcugaagg 3540
gcagaaccgu gcugguugug ggcgagaagc ugucugugcc uggcaagaug guggacuggc 3600
ugagcgauag acccgaggcc accuuuagag ccagacugga ccuuggaauc ccuggcgacg 3660
ugcccaaaua cgacaucauc uucgugaacg ugcggacgcc cuacaaguac caccacuacc 3720
agcagugcga ggaccacgcc aucaagcuga gcaugcugac caagaaggcc ugccugcacc 3780
ugaauccugg cggcaccugu gugucuaucg gcuacggcua ugccgacaga gccagcgagu 3840
cuaucaucgg cgccauugcc agacaguuca aguucagcag agugugcaag cccaagagca 3900
gccuggaaga gacagaggug cuguucgugu ucaucggcua ugaccggaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 27
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C08 nsP1-4 ORF and SGP RNA
<400> 27
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggaa aaggugcacg 60
uggacaucga agaggacagc ccauuccuga gagcccugca gagaagcuuc ccucaguucg 120
agguggaagc caaacaagug accgacaacg aucacgccaa cgcgagggcc uucucucacc 180
ucgcaagcaa gcugaucgag acagaggugg accccagcga caccauccug gauauuggau 240
cugccccugc cagaagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 28
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C09 nsP1-4 ORF and SGP RNA
<400> 28
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggccagaaa uaucggccug uguagcagcg acgugaugga aagauccaga cggggcauga 720
gcauccugcg gaagaaguac cugaagccua gcaacaacgu gcuguucagc gugggcagca 780
ccaucuacca cgagaagagg gaccugcugc ggagcuggca ucugccuucc guguuucacc 840
ugagaggcaa gcagaacuac accuguagau gcgagacaau cguguccugc gacggcuacg 900
uggucaagcg gaucgccauu ucuccuggcc uguacggcaa gccuucuggc uaugccgcca 960
ccaugcacag agaaggcuuu cuguguugca aagugaccga cacacugaac ggcgagcggg 1020
uguccuuucc uguguguacc uaugugcccg ccacacugug cgaucagaug acaggcauuc 1080
uggccaccga cgugucagcc gacgaugccc agaaacugcu cgugggccug aaccagagaa 1140
ucguggucaa cggcagaacc cagcggaaca ccaacaccau gaagaacuac cugcugccug 1200
ugguggccca ggccuuugcc agaugggcca aagaguacaa agaggaucaa gaggacgagc 1260
ggccccuggg ccugagagau agacaacugg ucaugggcug cugcugggcc uucagaaggc 1320
acaagaucac cagcaucuac aagcggcccg acacacagac caucaucaaa gugaacagcg 1380
acuuccacag cuucgugcug ccucggaucg gcagcaacac acuggaaauc ggccugcgga 1440
cccggaucag aaagaugcug gaagaacaca aagagcccuc uccacugauc accgccgagg 1500
augugcaaga ggccaaaugu gccgccgacg aggcuaaaga agugcgcgaa gccgaggaac 1560
ugagagccgc acuuccuccu cuggccgccg auguugaaga acccacucug gaagccgacg 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaagagc aucagcagac 2580
ggugcaccaa gagcgugacc agcguggugu cuacccuguu cuacgacaag aagaugcgga 2640
cgacaaaccc caaagagaca aagaucguca ucgacaccac cggcagcacc aagccuaagc 2700
aggacgaucu gauccugacc ugcuucagag gcugggucaa gcagcugcag aucgacuaca 2760
agggcaacga gaucaugacc gccgcugccu cucagggccu gacaagaaaa ggcguguacg 2820
ccgugcggua caaagugaac gagaacccuc uguacgcccc uaccagcgag caugugaaug 2880
ugcugcugac ccggaccgag gaccggaucg uuuggaaaac acuggccggc gaucccugga 2940
ucaagacccu gacagccaag uaucccggca acuucaccgc caccaucgag gaauggcagg 3000
ccgagcacga ugccaucaug cggcacaucc uggaaagacc cgauccuacc gacguguucc 3060
agaacaaggc caacgugugc ugggccaaag cucuggugcc ugugcugaaa accgccggca 3120
ucgauaugac caccgagcag uggaacaccg uggacuacuu cgagacagac aaggcccaca 3180
gcgccgagau cgugcugaau cagcugugcg ugcgguucuu cggccuggau cuggauagcg 3240
gccuguucuc ugcuccuacc gugccucuga gcauccggaa caaccacugg gacaacagcc 3300
ccucuccuaa uauguacggc cugaacaaag aagucgugcg gcagcugagc agaagauacc 3360
cacagcugcc uagagccgug gccacaggca gaguguacga caugaauacc ggcacacugc 3420
ggaacuacga ccccagaauc aaucuggugc ccgugaacag aaggcugccc cacgcucugg 3480
uucugcacca caaugagcac ccucagagcg acuucagcag cuucgugucc aagcugaagg 3540
gcagaaccgu gcugguugug ggcgagaagc ugucugugcc uggcaagaug guggacuggc 3600
ugagcgauag acccgaggcc accuuuagag ccagacugga ccuuggaauc ccuggcgacg 3660
ugcccaaaua cgacaucauc uucgugaacg ugcggacgcc cuacaaguac caccacuacc 3720
agcagugcga ggaccacgcc aucaagcuga gcaugcugac caagaaggcc ugccugcacc 3780
ugaauccugg cggcaccugu gugucuaucg gcuacggcua ugccgacaga gccagcgagu 3840
cuaucaucgg cgccauugcc agacaguuca aguucagcag agugugcaag cccaagagca 3900
gccuggaaga gacagaggug cuguucgugu ucaucggcua ugaccggaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 29
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C10 nsP1-4 ORF and SGP RNA
<400> 29
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc cuacaaguac caccacuacc 3720
agcagugcga ggaccacgcc aucaagcuga gcaugcugac caagaaggcc ugccugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucaucggcua cgacagaaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaagu acgccugcaa caacgaguac ugggagacau 6420
ucaaagagaa ccccauccgg cugaccgagg aaaacguggu caacuacauc accaagcuga 6480
agggccccaa agccgccgcu cuguuugcca agacacacaa ccugaacaug cugcaggaca 6540
uccccaugga cagauucgug auggaccuga agcgggacgu gaaagugacc ccuggcacca 6600
agcacaccga ggaacggccu aaggugcaag ugauccaggc cgcugauccu cuggccacag 6660
ccuaucugug uggcauccac agagaacucg ugcggagacu gaaugccgug cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg cugaugauuc uggaagaucu cggaguggac gccgagcugc 6900
ugacacugau ugaagccgcc uuuggcgaga ucagcagcau ccaucugccu accaagacca 6960
aguucaaguu cggcgccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaacaggc agcccuugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 30
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C11 nsP1-4 ORF and SGP RNA
<400> 30
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauaugccg uggauggccc uacaagccug uaccaccagg 540
ccaacaaggg cgucagagug gccuacugga ucggcuucga caccacaccu uucauguuca 600
agaaccuggc uggcgcuuac cccagcuaca gcacaaacug ggccgacgaa accguguuaa 660
cggccagaaa uaucggccug uguagcagcg acgugaugga aagauccaga cggggcauga 720
gcauccugcg gaagaaguac cugaagccua gcaacaacgu gcuguucagc gugggcagca 780
ccaucuacca cgagaagagg gaccugcugc ggagcuggca ucugccuucc guguuucacc 840
ugagaggcaa gcagaacuac accuguagau gcgagacaau cguguccugc gacggcuacg 900
uggucaagcg gaucgccauu ucuccuggcc uguacggcaa gccuucuggc uaugccgcca 960
ccaugcacag agaaggcuuu cuguguugca aagugaccga cacacugaac ggcgagcggg 1020
uguccuuucc uguguguacc uaugugcccg ccacacugug cgaucagaug acaggcauuc 1080
uggccaccga cgugucagcc gacgaugccc agaaacugcu cgugggccug aaccagagaa 1140
ucguggucaa cggcagaacc cagcggaaca ccaacaccau gaagaacuac cugcugccug 1200
ugguggccca ggccuuugcc agaugggcca aagaguacaa agaggaucaa gaggacgagc 1260
ggccccuggg ccugagagau agacaacugg ucaugggcug cugcugggcc uucagaaggc 1320
acaagaucac cagcaucuac aagcggcccg acacacagac caucaucaaa gugaacagcg 1380
acuuccacag cuucgugcug ccucggaucg gcagcaacac acuggaaauc ggccugcgga 1440
cccggaucag aaagaugcug gaagaacaca aagagcccuc uccacugauc accgccgagg 1500
augugcaaga ggccaaaugu gccgccgacg aggcuaaaga agugcgcgaa gccgaggaac 1560
ugagagccgc acuuccuccu cuggccgccg auguugaaga acccacucug gaagccgacg 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagcgagaag cugagcugca uucacccucu ggccgagcaa gugaucguga 1800
ucacacacag cggccggaag ggcagauaug ccguggaacc uuaucacggc aagguggugg 1860
ugccugaggg acacgcuauu ccagugcagg acuuucaggc ccugagcgag ucugccacca 1920
ucguguacaa cgagcgcgag uucgugaaca gauaccugca ccacauugcc acacacggcg 1980
gagcccugaa caccgacgaa gaguacuaca agaccgugaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggauagcgug cugcugaacg gcugcaagca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccua agcagugcgg cuucuucaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaagagc aucagcagac 2580
ggugcaccaa gagcgugacc agcguggugu cuacccuguu cuacgacaag aagaugcgga 2640
cgacaaaccc caaagagaca aagaucguca ucgacaccac cggcagcacc aagccuaagc 2700
aggacgaucu gauccugacc ugcuucagag gcugggucaa gcagcugcag aucgacuaca 2760
agggcaacga gaucaugacc gccgcugccu cucagggccu gacaagaaaa ggcguguacg 2820
ccgugcggua caaagugaac gagaacccuc uguacgcccc uaccagcgag caugugaaug 2880
ugcugcugac ccggaccgag gaccggaucg uuuggaaaac acuggccggc gaucccugga 2940
ucaagacccu gacagccaag uaucccggca acuucaccgc caccaucgag gaauggcagg 3000
ccgagcacga ugccaucaug cggcacaucc uggaaagacc cgauccuacc gacguguucc 3060
agaacaaggc caacgugugc ugggccaaag cucuggugcc ugugcugaaa accgccggca 3120
ucgauaugac caccgagcag uggaacaccg uggacuacuu cgagacagac aaggcccaca 3180
gcgccgagau cgugcugaau cagcugugcg ugcgguucuu cggccuggau cuggauagcg 3240
gccuguucuc ugcuccuacc gugccucuga gcauccggaa caaccacugg gacaacagcc 3300
ccucuccuaa uauguacggc cugaacaaag aagucgugcg gcagcugagc agaagauacc 3360
cacagcugcc uagagccgug gccacaggca gaguguacga caugaauacc ggcacacugc 3420
ggaacuacga ccccagaauc aaucuggugc ccgugaacag aaggcugccc cacgcucugg 3480
uucugcacca caaugagcac ccucagagcg acuucagcag cuucgugucc aagcugaagg 3540
gcagaaccgu gcugguugug ggcgagaagc ugucugugcc uggcaagaug guggacuggc 3600
ugagcgauag acccgaggcc accuuuagag ccagacugga ccuuggaauc ccuggcgacg 3660
ugcccaaaua cgacaucauc uucgugaacg ugcggacgcc cuacaaguac caccacuacc 3720
agcagugcga ggaccacgcc aucaagcuga gcaugcugac caagaaggcc ugccugcacc 3780
ugaauccugg cggcaccugu gugucuaucg gcuacggcua ugccgacaga gccagcgagu 3840
cuaucaucgg cgccauugcc agacaguuca aguucagcag agugugcaag cccaagagca 3900
gccuggaaga gacagaggug cuguucgugu ucaucggcua ugaccggaag gcccguacgc 3960
acaaccccua caagcugagc agcacccuga ccaacaucua caccggcagc agacugcacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccaca gccacagaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaagu acgccugcaa caacgaguac ugggagacau 6420
ucaaagagaa ccccauccgg cugaccgagg aaaacguggu caacuacauc accaagcuga 6480
agggccccaa agccgccgcu cuguuugcca agacacacaa ccugaacaug cugcaggaca 6540
uccccaugga cagauucgug auggaccuga agcgggacgu gaaagugacc ccuggcacca 6600
agcacaccga ggaacggccu aaggugcaag ugauccaggc cgcugauccu cuggccacag 6660
ccuaucugug uggcauccac agagaacucg ugcggagacu gaaugccgug cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg cugaugauuc uggaagaucu cggaguggac gccgagcugc 6900
ugacacugau ugaagccgcc uuuggcgaga ucagcagcau ccaucugccu accaagacca 6960
aguucaaguu cggcgccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaacaggc agcccuugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 31
<211> 8428
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C13 nsP1-4_GFP ORF and SGP RNA
<400> 31
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaguu cggcgccaug augaaguccg gcauguuucu gacccuguuc gugaacaccg 7020
ugaucaacau cgugaucgcc agccgggugc ugagagagag acugacagga ucuccuugcg 7080
ccgccuucau cggcgacgac aauaucguga agggcgugaa guccgacaag cugauggccg 7140
auagaugcgc caccuggcug aacauggaag ugaagaucau cgacgccguc gugggcgaga 7200
aggccccuua uuuuugcggc ggcuucaucc ugugcgacag cgugacaggc acagccugca 7260
gaguugccga uccucugaag cggcuguuca agcugggaaa accucuggcc gccgacgacg 7320
agcacgacga cgauagacgu agagcccugc acgaggaauc caccagaugg aacagagugg 7380
gcauccugag cgagcugugc aaggccgugg aaagcagaua cgagacagug ggcaccagca 7440
ucauugugau ggcaaugacc acacuggcca gcagcgugaa aagcuucagc uaccuaaggg 7500
gcgccccuau cacacuguac ggcucuggcg aaggcagagg cagccuucug acauguggcg 7560
acguggaaga gaaccccgga ccugugucua agggcgaaga acuguuuacc ggcguggugc 7620
ccauccuggu ggaacuggau ggggauguga acggccacaa guucagcguu agcggagaag 7680
gcgaaggcga cgccacauac ggaaagcuga cccugaaguu caucugcacc accggcaagc 7740
ugccugugcc auggccuaca cuggucacca cacugacaua cggcgugcag ugcuucagca 7800
gauaccccga ccauaugaag cagcacgacu ucuucaagag cgccaugccu gagggcuacg 7860
ugcaagagcg gaccaucuuc uuuaaggacg acggcaacua caagaccagg gccgaaguga 7920
aguucgaggg cgacacccug gucaaccgga ucgagcugaa gggcaucgac uucaaagagg 7980
acggcaauau ccugggccac aagcucgagu acaacuacaa cagccacaac guguacauca 8040
uggccgacaa gcagaaaaac ggcaucaaag ugaacuucaa gauccggcac aacaucgagg 8100
acggcucugu gcagcuggcc gaucacuacc agcagaacac acccaucgga gauggcccug 8160
ugcugcugcc cgauaaccac uaccugagca cccagagcaa gcugagcaag gaccccaacg 8220
agaagcggga ccacauggug cugcuggaau uugugacagc cgccggaauc acccucggca 8280
uggaugagcu guacaaguga acuuccauca uaguuauggc caugacuacu cuagcuagca 8340
guguuaaauc auucagcuac cugagagggg ccccuauaac ucucuacggc uaaccugaau 8400
ggacuacgac auagucuagu ccgccaag 8428
<210> 32
<211> 8353
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, nsP1-4_3Cpro and SGP_VEEVrep-nsP4[3C]
<400> 32
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaguu cggcgccaug augaaguccg gcauguuucu gacccuguuc gugaacaccg 7020
ugaucaacau cgugaucgcc agccgggugc ugagagagag acugacagga ucuccuugcg 7080
ccgccuucau cggcgacgac aauaucguga agggcgugaa guccgacaag cugauggccg 7140
auagaugcgc caccuggcug aacauggaag ugaagaucau cgacgccguc gugggcgaga 7200
aggccccuua uuuuugcggc ggcuucaucc ugugcgacag cgugacaggc acagccugca 7260
gaguugccga uccucugaag cggcuguuca agcugggaaa accucuggcc gccgacgacg 7320
agcacgacga cgauagacgu agagcccugc acgaggaauc caccagaugg aacagagugg 7380
gcauccugag cgagcugugc aaggccgugg aaagcagaua cgagacagug ggcaccagca 7440
ucauugugau ggcaaugacc acacuggcca gcagcgugaa aagcuucagc uaccuaaggg 7500
gcgccccuau cacacuguac ggcucuggcg aaggcagagg cagccuucug acauguggcg 7560
acguggaaga gaaccccgga ccuaguggug ccccaccgac cgacuugcaa aagaugguca 7620
ugggcaacac aaagccuguu gagcucaucc uugacgggaa gacaguagcc aucuguugug 7680
cuacuggagu guuuggcacu gcuuaccucg ugccucguca ucuuuucgca gagaaguaug 7740
acaagaucau gcuggauggc agagccauga cagacaguga cuacagagug uuugaguuug 7800
agauuaaagu aaaaggacag gacaugcucu cagacgcugc gcucauggug cuccaccgug 7860
ggaaccgcgu gagagauauc acgaaacacu uucgugauac agcaagaaug aagaaaggca 7920
cccccgucgu cggugugguc aacaacgccg acguugggag acugauuuuc ucuggugagg 7980
cccucaccua caaggauauu guagugugca uggacggaga caccaugccu agccucuuug 8040
ccuacaaagc cgccaccaag gcaggcuacu guggaggagc cguucucgcc aaggacgggg 8100
ccgacacuuu caucgucggc acucacuccg caggaggcaa uggaguugga uacugcucau 8160
gcguuuccaa guccaugcuu cucagaauga aggcacacgu ugacccugaa ccacaacacg 8220
aguagacuuc caucauaguu auggccauga cuacucuagc uagcaguguu aaaucauuca 8280
gcuaccugag aggggccccu auaacucucu acggcuaacc ugaauggacu acgacauagu 8340
cuaguccgcc aag 8353
<210> 33
<211> 2322
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, 01 Manisa P1 pr _ RNA coding sequence (SG
ORF)
<400> 33
augggagcug gacagagcuc cccugcuacc ggcagccaga aucaguccgg caacaccggc 60
uccaucauca acaacuacua caugcagcag uaccagaaca gcauggacac ccagcugggc 120
gacaacgcua ccagcggcgg cuccaacgag ggaagcaccg acaccaccuc cacccacacc 180
accaacaccc agaacaacga cugguucucc aagcuggcca gcagcgccuu cagcggccug 240
uucggagccc ugcuggccga caagaagacc gaggagacaa cccugcugga ggaccggauc 300
cugaccacca gaaacggaca cacuaccucc accacccaga gcuccguggg agugaccuac 360
ggauacgcua ccgcugagga cuucgugucc ggaccaaaca ccuccggccu ggagacaagg 420
guggcucagg cugagagauu cuucaagacc caccuguucg acugggugac cagcgaccca 480
uucggaagau gccaccugcu ggagcugccc accgaccaca agggcgugua cggcagccug 540
accgacuccu acgccuacau gagaaacgga ugggacgugg aggugaccgc ugugggaaac 600
caguucaacg gcggaugccu gcugguggcu auggugcccg agcugugcuc cauccagaag 660
agggagcugu accagcugac ccuguucccc caccaguuca ucaaccccag aaccaacaug 720
accgcucaca ucaccgugcc cuucguggga gugaaccggu acgaccagua caaggugcac 780
aagcccugga cccugguggu caugguggug gccccacuga ccgugaacag cgagggagcc 840
ccacagauca agguguacgc caacaucgcu ccaaccaacg ugcacguggc uggagaguuc 900
cccuccaaag agggcaucuu cccuguggcu ugcagcgacg gauacggcgg ccuggugacc 960
accgacccaa agaccgcuga cccagccuac ggcaaggugu ucaacccacc ccggaacaug 1020
cugccuggac gcuucaccaa cuuccuggac guggcugagg ccugcccaac cuuccugcac 1080
uucgagggcg acgugcccua cgugaccacc aagaccgacu ccgacagagu gcuggcccag 1140
uucgaccugu cccuggccgc caagcacaug agcaacaccu uccuggccgg ccuggcccag 1200
uacuacaccc aguacagcgg caccaucaac cugcacuuca uguucaccgg accaaccgac 1260
gcuaaggcua gguacaugau cgcuuacgcc ccacccggaa uggagccccc aaagaccccu 1320
gaggcugcug cucacugcau ccacgccgag ugggacaccg gccugaacag caaguucacc 1380
uucuccaucc ccuaccugag cgcugcugac uacgcuuaca ccgccuccga caccgcugag 1440
acaaccaacg ugcagggcug ggugugccug uuccagauca cccacggcaa ggcugacggc 1500
gacgcucugg uggugcuggc cagcgccggc aaggacuucg agcugaggcu gcccguggac 1560
gcuagaaccc agaccaccag cgcuggagag uccgcugacc cagugaccgc caccguggag 1620
aacuacggcg gcgagacaca ggugcagcgg aggcagcaca ccgacguguc cuucauccug 1680
gacagauucg ugaaggugac ccccaaggac cagaucaacg ugcuggaccu gaugcagacc 1740
ccugcucaca cccugguggg agcccugcug cggaccgcca ccuacuacuu cgccgaccug 1800
gagguggccg ugaagcacga gggcaaccug accugggugc ccaacggagc uccugaggcu 1860
gcccuggaca acaccaccaa ccccaccgcu uaccauaagg ccccacugac ccggcuggcc 1920
cugcccuaca ccgccccaca ccgcgugcug gccaccgugu acaacggcaa cugcaaguac 1980
ggcgacggca ccguggccaa cgugcggggc gaccugcagg ugcuggccca gaaggcugcu 2040
agagcccugc ccaccagcuu caacuacggc gccaucaagg ccaccagagu gaccgagcug 2100
cuguacagga ugaagagagc cgagacauac ugccccaggc cccugcuggc cauccaccca 2160
gaccaggcca gacacaagca gaagaucgug gcccccguga agcagcugcu gaacuucgac 2220
cugcugaagc uggccggcga cguggagucc aacccuggac ccuucuucuu cagcgacgug 2280
cgcagcaacu ucuccaagcu gguggagaca aucaaccagu ga 2322
<210> 34
<211> 8848
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, nsP1-4-STING pr ORF, and
SGP_VEEVrep-nsP4[STING]
<400> 34
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaguu cggcgccaug augaaguccg gcauguuucu gacccuguuc gugaacaccg 7020
ugaucaacau cgugaucgcc agccgggugc ugagagagag acugacagga ucuccuugcg 7080
ccgccuucau cggcgacgac aauaucguga agggcgugaa guccgacaag cugauggccg 7140
auagaugcgc caccuggcug aacauggaag ugaagaucau cgacgccguc gugggcgaga 7200
aggccccuua uuuuugcggc ggcuucaucc ugugcgacag cgugacaggc acagccugca 7260
gaguugccga uccucugaag cggcuguuca agcugggaaa accucuggcc gccgacgacg 7320
agcacgacga cgauagacgu agagcccugc acgaggaauc caccagaugg aacagagugg 7380
gcauccugag cgagcugugc aaggccgugg aaagcagaua cgagacagug ggcaccagca 7440
ucauugugau ggcaaugacc acacuggcca gcagcgugaa aagcuucagc uaccuaaggg 7500
gcgccccuau cacacuguac ggcucuggcg aaggcagagg cagccuucug acauguggcg 7560
acguggaaga gaaccccgga ccuccucaca gcucucugca cccuagcauc ccuuguccua 7620
gaggacacgg cgcccagaaa gcugcucugg uucugcuguc ugccugccug guuacacugu 7680
ggggacuggg agagccuccu gagcacacac ugagauaccu ggugcugcac cuggcuucuc 7740
ugcagcuggg acugcugcug aacggcgugu guucucuggc cgaggaacug agacacaucc 7800
acagcagaua caggggcagc uacuggcgga cagucagagc uugucugggc ugcccucuua 7860
gaagaggcgc ucugcugcug cuguccaucu acuucuacua cagccugccu aacgcugugg 7920
gcccuccuuu cacauggaug cuggcucugc ugggccuguc ucaggcucug aauauccugc 7980
uggggcugaa aggccuggcu ccugcugaaa ucucugccgu gugcgagaag ggcaacuucu 8040
cuguggcuca cggccuggcc ugguccuacu acaucggaua ccugagacug auccugccug 8100
agcugcaggc cagaaucaga accuacaacc agcacuacaa caaccugcug agaggcgcug 8160
ugucccagag acuguacauc cugcugccuc uggacugugg cgugccagac aaccugucua 8220
uggccgaucc uaacauccgg uuucuggaca agcugccuca gcagacaggc gaucacgccg 8280
gcaucaagga cagaguguac agcaacagca ucuacgagcu gcuggaaaac ggccagcgag 8340
ccggaacaug cgugcuggaa uaugccacac cucugcagac ccuguucgcc augagccagu 8400
acucucaggc cggcuucagc cgcgaggaua ugcuggaaca ggccaagcug uucugcagga 8460
cccucgagga cauccuggcu gaugcuccag agagccagaa caacugcaga cugaucgccu 8520
accaagagcc ugccgacgac agcagcuuua gccugagcca agaggugcug agacaccuga 8580
gacaagaaga gaaagaggaa gucaccgucg gcagccugaa aaccagcgcc gugccuagca 8640
ccagcaccau gucucaagaa cccgaacugc ugaucuccgg cauggaaaag ccccugccuc 8700
ugagaaccga cuucagcuga acuuccauca uaguuauggc caugacuacu cuagcuagca 8760
guguuaaauc auucagcuac cugagagggg ccccuauaac ucucuacggc uaaccugaau 8820
ggacuacgac auagucuagu ccgccaag 8848
<210> 35
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C12_nsP1-4 ORF, and SGP
<400> 35
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggccagaaa caucggccug ugcagcagcg acgugaugga acggucuaga cggggcauga 720
gcauccugcg gaagaaguac cugaagccua gcaacaacgu gcuguucagc gugggcagca 780
ccaucuacca cgagaagagg gaccugcugc ggagcuggca ucugccuucc guguuucacc 840
ugagaggcaa gcagaacuac accuguagau gcgagacaau cguguccugc gacggcuacg 900
uggucaagcg gaucgccauu ucuccuggcc uguacggcaa gccuucuggc uaugccgcca 960
ccaugcacag agaaggcuuu cuguguugca aagugaccga cacacugaac ggcgagcggg 1020
uguccuuucc uguguguacc uaugugcccg ccacacugug cgaucagaug acaggcauuc 1080
uggccaccga cgugucagcc gacgaugccc agaaacugcu cgugggccug aaccagagaa 1140
ucguggucaa cggcagaacc cagcggaaca ccaacaccau gaagaacuac cugcugccug 1200
ugguggccca ggccuuugcc agaugggcca aagaguacaa agaggaucaa gaggacgagc 1260
ggccccuggg ccugagagau agacaacugg ucaugggcug cugcugggcc uucagaaggc 1320
acaagaucac cagcaucuac aagcggcccg acacacagac caucaucaaa gugaacagcg 1380
acuuccacag cuucgugcug ccucggaucg gcagcaacac acuggaaauc ggccugcgga 1440
cccggaucag aaagaugcug gaagaacaca aagagcccuc uccacugauc accgccgagg 1500
augugcaaga ggccaaaugu gccgccgacg aggcuaaaga agugcgcgaa gccgaggaac 1560
ugagagccgc acuuccuccu cuggccgccg auguugaaga acccacucug gaagccgacg 1620
uggaccugau gcuucaagaa gccggcgcug gcagcgugga aacaccuaga ggacugauca 1680
aagucaccag cuacgccggc gaggacaaga ucggaucuua ugccgugcug agcccucagg 1740
cugugcugaa gucugagaag cugagcugca uucacccacu ggccgagcaa gugaucguga 1800
ucacacacag cggccggaag ggcagauaug ccguggaacc uuaucacggc aagguggugg 1860
ugccugaggg acacgcuauu ccagugcagg acuuucaggc ccugagcgag ucugccacca 1920
ucguguacaa cgagcgcgag uucgugaaca gauaccugca ccacauugcc acacacggcg 1980
gagcccugaa uaccgacgaa gaguacuaca agaccgugaa gcccagcgag cacgacggcg 2040
aguaccugua cgacaucgac agaaagcagu gcgugaagaa agagcugguc accggcuugg 2100
gacugacagg cgaacuggug gauccuccau uccacgaguu ugccuacgag agccugagaa 2160
ccagaccugc cgcuccuuac caggugccaa caaucggagu guauggcgug ccaggcucug 2220
gcaagagcgg caucauuaag agcgccguga ccaaaaagga ccugguggug uccgccaaga 2280
aagagaacug cgccgagauc auccgggacg ugaagaagau gaagggccuc gacgugaacg 2340
ccagaaccgu ggauagcgug cugcugaacg gaugcaagca ccccguggaa acccuguaca 2400
ucgaugaggc cuucgccugc caugccggaa cacugagagc acugaucgcc aucaucagac 2460
ccaagaaagc cgugcugugc ggcgacccua agcagugugg cuucuucaau augaugugcc 2520
ugaaggugca cuucaaccac gagaucugca cccagguguu ccacaagagc aucagcagac 2580
ggugcaccaa gagcgugacc agcguggugu cuacccuguu cuacgacaag aagaugcgga 2640
cgacaaaccc caaagagacu aagaucguca ucgacaccac cggcuccacc aagccuaagc 2700
aggacgaucu gauccugacc ugcuucagag gcugggucaa gcagcugcag aucgacuaca 2760
agggcaacga gaucaugacc gcugccgcuu cucagggacu gaccagaaaa ggcguguacg 2820
ccgugcggua caaagugaau gagaacccuc uguacgcccc uaccuccgag caugugaaug 2880
uccugcugac caggaccgag gaccggaucg uguggaaaac acuggcuggc gaccccugga 2940
ucaagacccu gacagccaag uaucccggca acuucaccgc cacaaucgag gaauggcagg 3000
ccgagcacga ugccauuaug cggcacaucc uggaacggcc cgauccuacc gauguguucc 3060
agaacaaggc caacgugugc ugggcuaaag cccuggugcc agugcugaaa accgccggca 3120
ucgauaugac caccgagcag uggaauaccg uggacuacuu cgagacagac aaggcccacu 3180
cugccgagau cgugcugaau cagcugugcg ugcgguucuu cggccuggau cuggauagcg 3240
gccuguuuag cgcuccuacc gugccucuga gcauccggaa caaccacugg gacaacagcc 3300
ccucuccuaa uauguacggc cugaacaaag aaguugugcg gcagcugagc agaagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 36
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C13_nsP1-4 ORF, and SGP
<400> 36
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggccagaaa caucggccug ugcagcagcg acgugaugga acggagcaga cggggcauga 720
gcauccugcg gaagaaguac cugaagccca gcaacaacgu gcuguucagc gugggcagca 780
ccaucuacca cgagaagagg gaccugcugc ggagcuggca ccugcccagc guguuccacc 840
ugagaggcaa gcagaacuac accugcagau gcgagacaau cgugagcugc gacggcuacg 900
uggucaagcg gaucgccaua agccccggcc uguacggcaa gcccagcggc uacgccgcca 960
ccaugcacag agaaggcuuc cugugcugca aagugaccga cacacugaac ggcgagcggg 1020
ugagcuuccc cgugugcacc uacgugcccg ccacacugug cgaccagaug acaggcauac 1080
uggccaccga cgugagcgcc gacgacgccc agaaacugcu cgugggccug aaccagagaa 1140
ucguggucaa cggcagaacc cagcggaaca ccaacaccau gaagaacuac cugcugccag 1200
ugguggccca ggccuucgcc agaugggcca aagaguacaa agaggaccaa gaggacgagc 1260
ggccccuggg ccugagagac agacaacugg ucaugggcug cugcugggcc uucagaaggc 1320
acaagaucac cagcaucuac aagcggcccg acacacagac caucaucaaa gugaacagcg 1380
acuuccacag cuucgugcug ccacggaucg gcagcaacac acuggaaauc ggccugcgga 1440
cccggaucag aaagaugcug gaagaacaca aagagcccag cccacugauc accgccgagg 1500
acgugcaaga ggccaaaugc gccgccgacg aggcaaaaga agugcgcgaa gccgaggaac 1560
ugagagccgc acucccccca cuggccgccg acguagaaga acccacccug gaagccgacg 1620
uggaccugau gcuccaagaa gccggcgcag gcagcgugga aacaccgaga ggacugauca 1680
aagucaccag cuacgccggc gaggacaaga ucggaagcua cgccgugcug agcccccagg 1740
ccgugcugaa aagcgagaag cugagcugca uccacccacu ggccgagcaa gugaucguga 1800
ucacacacag cggccggaag ggcagauacg ccguggaacc cuaccacggc aagguggugg 1860
ugccagaggg acacgcaauc ccagugcagg acuuccaggc ccugagcgag agcgccacca 1920
ucguguacaa cgagcgcgag uucgugaaca gauaccugca ccacauagcc acacacggcg 1980
gagcccugaa caccgacgaa gaguacuaca agaccgugaa gcccagcgag cacgacggcg 2040
aguaccugua cgacaucgac agaaagcagu gcgugaagaa agagcugguc accggccuag 2100
gacugacagg cgaacuggug gacccacccu uccacgaguu cgccuacgag agccugagaa 2160
ccagaccagc cgcacccuac caggugccaa caaucggagu guacggcgug ccaggcagcg 2220
gcaagagcgg caucauaaag agcgccguga ccaaaaagga ccugguggug agcgccaaga 2280
aagagaacug cgccgagauc auccgggacg ugaagaagau gaagggccuc gacgugaacg 2340
ccagaaccgu ggacagcgug cugcugaacg gaugcaagca ccccguggaa acccuguaca 2400
ucgacgaggc cuucgccugc cacgccggaa cacugagagc acugaucgcc aucaucagac 2460
ccaagaaagc cgugcugugc ggcgacccca agcagugcgg cuucuucaac augaugugcc 2520
ugaaggugca cuucaaccac gagaucugca cccagguguu ccacaagagc aucagcagac 2580
ggugcaccaa gagcgugacc agcgugguga gcacccuguu cuacgacaag aagaugcgga 2640
cgacaaaccc caaagagacc aagaucguca ucgacaccac cggcagcacc aagccaaagc 2700
aggacgaccu gauccugacc ugcuucagag gcugggucaa gcagcugcag aucgacuaca 2760
agggcaacga gaucaugacc gccgccgcaa gccagggacu gaccagaaaa ggcguguacg 2820
ccgugcggua caaagugaac gagaacccac uguacgcccc caccagcgag cacgugaacg 2880
uccugcugac caggaccgag gaccggaucg uguggaaaac acuggccggc gaccccugga 2940
ucaagacccu gacagccaag uaccccggca acuucaccgc cacaaucgag gaauggcagg 3000
ccgagcacga cgccauaaug cggcacaucc uggaacggcc cgacccaacc gacguguucc 3060
agaacaaggc caacgugugc ugggcaaaag cccuggugcc agugcugaaa accgccggca 3120
ucgacaugac caccgagcag uggaacaccg uggacuacuu cgagacagac aaggcccaca 3180
gcgccgagau cgugcugaac cagcugugcg ugcgguucuu cggccuggac cuggacagcg 3240
gccuguucag cgcacccacc gugccccuga gcauccggaa caaccacugg gacaacagcc 3300
ccagcccaaa cauguacggc cugaacaaag aagucgugcg gcagcugagc agaagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagcgc 4140
uguauaagaa auucccggaa agcuucgauu uacagccgau cgaaguagga aaagcgcgac 4200
uggucaaagg ugcagcuaaa cauaucauuc augccguagg accaaacuuc aacaaaguuu 4260
cggagguuga aggugacaaa caguuggcag aggcuuauga guccaucgcu aagauuguca 4320
acgauaacaa uuacaaguca guagcgauuc cacuguuguc caccggcauc uuuuccggga 4380
acaaagaucg acuaacccaa ucauugaacc auuugcugac agcuuuagac accacugaug 4440
cagauguagc cauauacugc agggacaaga aaugggaaau gacucucaag gaagcagugg 4500
cuaggagaga agcaguggag gagauaugca uauccgacga cucuucagug acagaaccug 4560
augcagagcu ggugagggug cauccgaaga guucuuuggc uggaaggaag ggcuacagca 4620
caagcgaugg caaaacuuuc ucauauuugg aagggaccaa guuucaccag gcggccaagg 4680
auauagcaga aauuaaugcc auguggcccg uugcaacgga ggccaaugag cagguaugca 4740
uguauauccu cggagaaagc augagcagua uuaggucgaa augccccguc gaagagucgg 4800
aagccuccac accaccuagc acgcugccuu gcuugugcau ccaugccaug acuccagaaa 4860
gaguacagcg ccuaaaagcc ucacguccag aacaaauuac ugugugcuca uccuuuccau 4920
ugccgaagua uagaaucacu ggugugcaga agauccaaug cucccagccu auauuguucu 4980
caccgaaagu gccugcguau auucauccaa ggaaguaucu cguggaaaca ccaccgguag 5040
acgagacucc ggagccaucg gcagagaacc aauccacaga ggggacaccu gaacaaccac 5100
cacuuauaac cgaggaugag accaggacua gaacgccuga gccgaucauc aucgaagagg 5160
aagaagagga uagcauaagu uugcugucag auggcccgac ccaccaggug cugcaagucg 5220
aggcagacau ucacgggccg cccucuguau cuagcucauc cugguccauu ccucaugcau 5280
ccgacuuuga uguggacagu uuauccauac uugacacccu ggagggagcu agcgugacca 5340
gcggggcaac gucagccgag acuaacucuu acuucgcaaa gaguauggag uuucuggcgc 5400
gaccggugcc ugcgccucga acaguauuca ggaacccucc acaucccgcu ccgcgcacaa 5460
gaacaccguc acuugcaccc agcagggccu gcucgagaac cagccuaguu uccaccccgc 5520
caggcgugaa uagggugauc acuagagagg agcucgaggc gcuuaccccg ucacgcacuc 5580
cuagcagguc ggucucgaga accagccugg ucuccaaccc gccaggcgua aauaggguga 5640
uuacaagaga ggaguuugag gcguucguag cacaacaaca augacgguuu gaugcgggug 5700
cauacaucuu uuccuccgac accggucaag ggcauuuaca acaaaaauca guaaggcaaa 5760
cggugcuauc cgaaguggug uuggagagga ccgaauugga gauuucguau gccccgcgcc 5820
ucgaccaaga aaaagaagaa uuacuacgca agaaauuaca guuaaauccc acaccugcua 5880
acagaagcag auaccagucc aggaaggugg agaacaugaa agccauaaca gcuagacgua 5940
uucugcaagg ccuagggcau uauuugaagg cagaaggaaa aguggagugc uaccgaaccc 6000
ugcauccugu uccuuuguau ucaucuagug ugaaccgugc cuuuucaagc cccaaggucg 6060
caguggaagc cuguaacgcc auguugaaag agaacuuucc gacuguggcu ucuuacugua 6120
uuauuccaga guacgaugcc uauuuggaca ugguugacgg agcuucaugc ugcuuagaca 6180
cugccaguuu uugcccugca aagcugcgca gcuuuccaaa gaaacacucc uauuuggaac 6240
ccacaauacg aucggcagug ccuucagcga uccagaacac gcuccagaac guccuggcag 6300
cugccacaaa aagaaauugc aaugucacgc aaaugagaga auugcccgua uuggauucgg 6360
cggccuuuaa uguggaaugc uucaagaaau augcguguaa uaaugaauau ugggaaacgu 6420
uuaaagaaaa ccccaucagg cuuacugaag aaaacguggu aaauuacauu accaaauuaa 6480
aaggaccaaa agcugcugcu cuuuuugcga agacacauaa uuugaauaug uugcaggaca 6540
uaccaaugga cagguuugua auggacuuaa agagagacgu gaaagugacu ccaggaacaa 6600
aacauacuga agaacggccc aagguacagg ugauccaggc ugccgauccg cuagcaacag 6660
cguaucugug cggaauccac cgagagcugg uuaggagauu aaaugcgguc cugcuuccga 6720
acauucauac acuguuugau augucggcug aagacuuuga cgcuauuaua gccgagcacu 6780
uccagccugg ggauuguguu cuggaaacug acaucgcguc guuugauaaa agugaggacg 6840
acgccauggc ucugaccgcg uuaaugauuc uggaagacuu agguguggac gcagagcugu 6900
ugacgcugau ugaggcggcu uucggcgaaa uuucaucaau acauuugccc acuaaaacua 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 37
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C14_nsP1-4 ORF, and SGP
<400> 37
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggagggguu ugcggagccc 4140
uguacaagaa guuccccgag agcuucgacc ugcagccuau cgaagugggc aaagccagac 4200
ugguuaaggg cgcugccaag cacaucaucc augccguggg acccaacuuc aacaaggugu 4260
ccgaggugga aggcgacaag cagcuggccg aggccuauga gucuaucgcc aagaucguga 4320
acgacaacaa cuacaagagc guggccauuc cucugcugag caccggcauc uucagcggca 4380
acaaggacag acugacccag agccugaacc aucugcugac agcccuggau accaccgaug 4440
ccgauguggc caucuacugc cgggacaaga aaugggagau gacccugaaa gaagccgugg 4500
ccagacgcga ggccguggaa gagaucugua ucagcgacga cagcagcgug accgagccug 4560
augccgaacu cguuagagug caccccaagu cuagccuggc cggcagaaag ggcuacagca 4620
ccucugaugg caagaccuuc agcuaccugg aaggcaccaa guuccaccag gccgccaagg 4680
auaucgccga gaucaacgcu auguggcccg uggccaccga ggccaaugaa caagugugca 4740
uguacauccu gggcgagagc augagcagca uccgcagcaa guguccugug gaagaguccg 4800
aggccagcac accuccuagc acacugccuu gccugugcau ccacgccaug acaccugaga 4860
gagugcagcg gcugaaggcc ucuagaccug agcagaucac cgugugcagc agcuucccac 4920
ugccuaagua cagaaucacc ggcgugcaga aaauccagug cagccagccu auccuguuca 4980
gcccuaaggu gcccgccuac auucacccca gaaaguaucu gguggaaacc ccaccugugg 5040
acgagacacc ugaaccuagc gccgagaauc agagcaccga gggaacacca gaacagccuc 5100
cacugaucac cgaggacgaa accaggacca gaacacccga gccuaucauc aucgaggaag 5160
aagaagagga cagcaucagc cugcugagcg acggaccuac acaccaggug cugcaagugg 5220
aagccgacau ccauggaccu ccaagcgugu ccagcagcag cuggucuauu ccucacgcca 5280
gcgacuucga cguggacagc cugagcaucc uggacacacu ggaaggcgcc ucugugacau 5340
cuggcgccac auccgccgag acaaacagcu acuucgccaa gagcauggaa uuucuggcca 5400
ggccugugcc ugcuccucgg accguguuua gaaacccucc ucauccugcu ccuagaacca 5460
ggacaccuuc ucuggccccu agcagagccu guagcagaac cagccuggug ucuacaccuc 5520
cuggcgugaa cagagugauc accagagagg aacuggaagc ccugacaccu agcaggaccc 5580
caagcagauc cgugucuaga acaucccugg uguccaaucc uccaggcguc aaccggguca 5640
ucacccggga agaguuugag gccuuugugg cucagcagca gugaagauuc gacgcaggcg 5700
ccuacaucuu cuccagcgau acaggccagg gccaucugca gcagaaaagc gucagacaga 5760
ccgugcuguc cgaaguggug cuggaaagaa ccgagcugga aaucagcuac gccccucggc 5820
uggaccaaga gaaagaagaa cugcugcgga agaaacugca gcugaacccc acaccagcca 5880
acagaagcag auaccagagc cggaaggugg aaaacaugaa ggccaucacc gccagacgga 5940
uccugcaagg ccugggccau uaucugaagg ccgagggcaa aguggaaugc uacagaacac 6000
ugcaccccgu gccucuguac agcagcuccg ugaauagggc cuuuagcagc ccaaaggugg 6060
ccgucgaagc cugcaacgcc augcugaaag aaaacuuccc uaccguggcc uccuacugca 6120
ucauccccga guacgacgcc uaccuggaca ugguggaugg cgcuagcugu ugucuggaua 6180
ccgccagcuu cugccccgcc aagcugagaa gcuuccccaa gaagcacagc uaucuggaac 6240
ccaccaucag auccgccgug ccuuccgcca uucagaauac ccugcagaac gugcuggccg 6300
cugccaccaa gaggaauugc aacgugaccc agaugcgcga gcugcccguu cuggauagcg 6360
ccgccuucaa cguggaaugu uucaagaagu acgccugcaa caacgaguac ugggagacau 6420
ucaaagagaa ccccauccgg cugaccgagg aaaacguggu caacuacauc accaagcuga 6480
agggccccaa agccgccgcu cuguuugcca agacacacaa ccugaacaug cugcaggaca 6540
uccccaugga cagauucgug auggaccuga agcgggacgu gaaagugacc ccuggcacca 6600
agcacaccga ggaacggccu aaagugcaag ugauccaggc cgcugauccu cuggccacag 6660
ccuaucugug uggcauccac agagaacucg ugcggagacu gaaugccgug cugcugccca 6720
acauccacac acuguucgac augagcgccg aggacuucga ugccaucauu gccgagcacu 6780
uccagccugg cgauugcgug cucgagacag auaucgccuc cuucgacaag agcgaggacg 6840
acgccauggc ucugacugcc cugaugaucc uggaagaucu gggcguugac gccgagcugc 6900
ugacacugau ugaagccgcc uuuggcgaga ucagcuccau ccaucugccu accaagacca 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 38
<211> 7561
<212> RNA
<213> Artificial Sequence
<220>
<223> Synthetic construct, C15_nsP1-4 ORF, and SGP
<400> 38
augggcggcg caugagagaa gcccagacca auuaccuacc caaaauggag aaaguucacg 60
uugacaucga ggaagacagc ccauuccuca gagcuuugca gcggagcuuc ccgcaguuug 120
agguagaagc caagcagguc acugauaaug accaugcuaa ugccagagcg uuuucgcauc 180
uggcuucaaa acugaucgaa acggaggugg acccauccga cacgauccuu gacauuggaa 240
gugcgcccgc ccgcagaaug uauucuaagc acaaguauca uuguaucugu ccgaugagau 300
gugcggaaga uccggacaga uuguauaagu augcaacuaa gcugaagaaa aacuguaagg 360
aaauaacuga uaaggaauug gacaagaaaa ugaaggagcu cgccgccguc augagcgacc 420
cugaccugga aacugagacu augugccucc acgacgacga gucgugucgc uacgaagggc 480
aagucgcugu uuaccaggau guauacgcgg uugacggacc gacaagucuc uaucaccaag 540
ccaauaaggg aguuagaguc gccuacugga uaggcuuuga caccaccccu uuuauguuua 600
agaacuuggc uggagcauau ccaucauacu cuaccaacug ggccgacgaa accguguuaa 660
cggcucguaa cauaggccua ugcagcucug acguuaugga gcggucacgu agagggaugu 720
ccauucuuag aaagaaguau uugaaaccau ccaacaaugu ucuauucucu guuggcucga 780
ccaucuacca cgagaagagg gacuuacuga ggagcuggca ccugccgucu guauuucacu 840
uacguggcaa gcaaaauuac acaugucggu gugagacuau aguuaguugc gacggguacg 900
ucguuaaaag aauagcuauc aguccaggcc uguaugggaa gccuucaggc uaugcugcua 960
cgaugcaccg cgagggauuc uugugcugca aagugacaga cacauugaac ggggagaggg 1020
ucucuuuucc cgugugcacg uaugugccag cuacauugug ugaccaaaug acuggcauac 1080
uggcaacaga ugucagugcg gacgacgcgc aaaaacugcu gguugggcuc aaccagcgua 1140
uagucgucaa cggucgcacc cagagaaaca ccaauaccau gaaaaauuac cuuuugcccg 1200
uaguggccca ggcauuugcu aggugggcaa aggaauauaa ggaagaucaa gaagaugaaa 1260
ggccacuagg acuacgagau agacaguuag ucauggggug uuguugggcu uuuagaaggc 1320
acaagauaac aucuauuuau aagcgcccgg auacccaaac caucaucaaa gugaacagcg 1380
auuuccacuc auucgugcug cccaggauag gcaguaacac auuggagauc gggcugagaa 1440
caagaaucag gaaaauguua gaggagcaca aggagccguc accucucauu accgccgagg 1500
acguacaaga agcuaagugc gcagccgaug aggcuaagga ggugcgugaa gccgaggagu 1560
ugcgcgcagc ucuaccaccu uuggcagcug auguugagga gcccacucug gaagccgaug 1620
ucgacuugau guuacaagag gcuggggccg gcucagugga gacaccucgu ggcuugauaa 1680
agguuaccag cuacgcuggc gaggacaaga ucggcucuua cgcugugcuu ucuccgcagg 1740
cuguacucaa gagugaaaaa uuaucuugca uccacccucu cgcugaacaa gucauaguga 1800
uaacacacuc uggccgaaaa gggcguuaug ccguggaacc auaccauggu aaaguagugg 1860
ugccagaggg acaugcaaua cccguccagg acuuucaagc ucugagugaa agugccacca 1920
uuguguacaa cgaacgugag uucguaaaca gguaccugca ccauauugcc acacauggag 1980
gagcgcugaa cacugaugaa gaauauuaca aaacugucaa gcccagcgag cacgacggcg 2040
aauaccugua cgacaucgac aggaaacagu gcgucaagaa agaacuaguc acugggcuag 2100
ggcucacagg cgagcuggug gauccucccu uccaugaauu cgccuacgag agucugagaa 2160
cacgaccagc cgcuccuuac caaguaccaa ccauaggggu guauggcgug ccaggaucag 2220
gcaagucugg caucauuaaa agcgcaguca ccaaaaaaga ucuaguggug agcgccaaga 2280
aagaaaacug ugcagaaauu auaagggacg ucaagaaaau gaaagggcug gacgucaaug 2340
ccagaacugu ggacucagug cucuugaaug gaugcaaaca ccccguagag acccuguaua 2400
uugacgaagc uuuugcuugu caugcaggua cucucagagc gcucauagcc auuauaagac 2460
cuaaaaaggc agugcucugc ggggauccca aacagugcgg uuuuuuuaac augaugugcc 2520
ugaaagugca uuuuaaccac gagauuugca cacaagucuu ccacaaaagc aucucucgcc 2580
guugcacuaa aucugugacu ucggucgucu caaccuuguu uuacgacaaa aaaaugagaa 2640
cgacgaaucc gaaagagacu aagauuguga uugacacuac cggcaguacc aaaccuaagc 2700
aggacgaucu cauucucacu uguuucagag ggugggugaa gcaguugcaa auagauuaca 2760
aaggcaacga aauaaugacg gcagcugccu cucaagggcu gacccguaaa gguguguaug 2820
ccguucggua caaggugaau gaaaauccuc uguacgcacc caccucagaa caugugaacg 2880
uccuacugac ccgcacggag gaccgcaucg uguggaaaac acuagccggc gacccaugga 2940
uaaaaacacu gacugccaag uacccuggga auuucacugc cacgauagag gaguggcaag 3000
cagagcauga ugccaucaug aggcacaucu uggagagacc ggacccuacc gacgucuucc 3060
agaauaaggc aaacgugugu ugggccaagg cuuuagugcc ggugcugaag accgcuggca 3120
uagacaugac cacugaacaa uggaacacug uggauuauuu ugaaacggac aaagcucacu 3180
cagcagagau aguauugaac caacuaugcg ugagguucuu uggacucgau cuggacuccg 3240
gucuauuuuc ugcacccacu guuccguuau ccauuaggaa uaaucacugg gauaacuccc 3300
cgucgccuaa cauguacggg cugaauaaag aagugguccg ucagcucucu cgcagguacc 3360
cacaacugcc ucgggcaguu gccacuggaa gagucuauga caugaacacu gguacacugc 3420
gcaauuauga uccgcgcaua aaccuaguac cuguaaacag aagacugccu caugcuuuag 3480
uccuccacca uaaugaacac ccacagagug acuuuucuuc auucgucagc aaauugaagg 3540
gcagaacugu ccuggugguc ggggaaaagu uguccguccc aggcaaaaug guugacuggu 3600
ugucagaccg gccugaggcu accuucagag cucggcugga uuuaggcauc ccaggugaug 3660
ugcccaaaua ugacauaaua uuuguuaaug ugaggacccc auauaaauac caucacuauc 3720
agcaguguga agaccaugcc auuaagcuua gcauguugac caagaaagcu ugucugcauc 3780
ugaaucccgg cggaaccugu gucagcauag guuaugguua cgcugacagg gccagcgaaa 3840
gcaucauugg ugcuauagcg cggcaguuca aguuuucccg gguaugcaaa ccgaaauccu 3900
cacuugaaga gacggaaguu cuguuuguau ucauugggua cgaucgcaag gcccguacgc 3960
acaauccuua caagcuuuca ucaaccuuga ccaacauuua uacagguucc agacuccacg 4020
aagccggaug ugcacccuca uaucaugugg ugcgagggga uauugccacg gccaccgaag 4080
gagugauuau aaaugcugcu aacagcaaag gacaaccugg cggaggggug ugcggagccc 4140
uguacaagaa guuccccgag agcuucgacc ugcagccaau cgaagugggc aaagccagac 4200
uggucaaggg cgcagccaag cacaucaucc acgccguggg acccaacuuc aacaagguga 4260
gcgaggugga aggcgacaag cagcuggccg aggccuacga gagcaucgcc aagaucguga 4320
acgacaacaa cuacaagagc guggccauac cccugcugag caccggcauc uucagcggca 4380
acaaggacag acugacccag agccugaacc accugcugac agcccuggac accaccgacg 4440
ccgacguggc caucuacugc cgggacaaga aaugggagau gacccugaaa gaagccgugg 4500
ccagacgcga ggccguggaa gagaucugca ucagcgacga cagcagcgug accgagccag 4560
acgccgaacu cgucagagug caccccaaga gcagccuggc cggcagaaag ggcuacagca 4620
ccagcgacgg caagaccuuc agcuaccugg aaggcaccaa guuccaccag gccgccaagg 4680
acaucgccga gaucaacgca auguggcccg uggccaccga ggccaacgaa caagugugca 4740
uguacauccu gggcgagagc augagcagca uccgcagcaa gugccccgug gaagagagcg 4800
aggccagcac accacccagc acacugcccu gccugugcau ccacgccaug acaccagaga 4860
gagugcagcg gcugaaggcc agcagacccg agcagaucac cgugugcagc agcuucccac 4920
ugcccaagua cagaaucacc ggcgugcaga aaauccagug cagccagcca auccuguuca 4980
gccccaaggu gcccgccuac auacacccca gaaaguaccu gguggaaacc ccacccgugg 5040
acgagacacc cgaaccaagc gccgagaacc agagcaccga gggaacacca gaacagcccc 5100
cacugaucac cgaggacgaa accaggacca gaacacccga gccaaucauc aucgaggaag 5160
aagaagagga cagcaucagc cugcugagcg acggacccac acaccaggug cugcaagugg 5220
aagccgacau ccacggaccc ccaagcguga gcagcagcag cuggagcauc ccccacgcca 5280
gcgacuucga cguggacagc cugagcaucc uggacacacu ggaaggcgcc agcgugacaa 5340
gcggcgccac aagcgccgag acaaacagcu acuucgccaa gagcauggaa uuccuggcca 5400
ggcccgugcc agcaccccgg accguguuca gaaacccccc ucacccagcc cccagaacca 5460
ggacacccag ccuggcccca agcagagccu gcagcagaac cagccuggug agcacacccc 5520
ccggcgugaa cagagugauc accagagagg aacuggaagc ccugacaccc agcaggaccc 5580
caagcagaag cgugagcaga acaagccugg ugagcaaccc accaggcguc aaccggguca 5640
ucacccggga agaguucgag gccuucgugg cacagcagca gugaagauuc gacgcaggcg 5700
ccuacaucuu cagcagcgac acaggccagg gccaccugca gcagaaaagc gucagacaga 5760
ccgugcugag cgaaguggug cuggaaagaa ccgagcugga aaucagcuac gccccccggc 5820
uggaccaaga gaaagaagaa cugcugcgga agaaacugca gcugaacccc acaccagcca 5880
acagaagcag auaccagagc cggaaggugg aaaacaugaa ggccaucacc gccagacgga 5940
uccugcaagg ccugggccac uaccugaagg ccgagggcaa aguggaaugc uacagaacac 6000
ugcaccccgu gccacuguac agcagcagcg ugaacagggc cuucagcagc ccaaaggugg 6060
ccgucgaagc cugcaacgcc augcugaaag aaaacuuccc caccguggcc agcuacugca 6120
ucauccccga guacgacgcc uaccuggaca ugguggacgg cgcaagcugc ugccuggaca 6180
ccgccagcuu cugccccgcc aagcugagaa gcuuccccaa gaagcacagc uaccuggaac 6240
ccaccaucag aagcgccgug ccaagcgcca uucagaacac ccugcagaac gugcuggccg 6300
cagccaccaa gaggaacugc aacgugaccc agaugcgcga gcugcccguc cuggacagcg 6360
ccgccuucaa cguggaaugc uucaagaagu acgccugcaa caacgaguac ugggagacau 6420
ucaaagagaa ccccauccgg cugaccgagg aaaacguggu caacuacauc accaagcuga 6480
agggccccaa agccgcagcc cuguucgcca agacacacaa ccugaacaug cugcaggaca 6540
uccccaugga cagauucgug auggaccuga agcgggacgu gaaagugacc ccaggcacca 6600
agcacaccga ggaacggccc aaagugcaag ugauccaggc cgcagacccc cuggccacag 6660
ccuaccugug cggcauccac agagaacucg ugcggagacu gaacgccgug cugcugccca 6720
acauccacac acuguucgac augagcgccg aggacuucga cgccaucaua gccgagcacu 6780
uccagccagg cgacugcgug cucgagacag acaucgccag cuucgacaag agcgaggacg 6840
acgccauggc acugaccgcc cugaugaucc uggaagaccu gggcguggac gccgagcugc 6900
ugacacugau agaagccgcc uucggcgaga ucagcagcau ccaccugccc accaagacca 6960
aauuuaaauu cggagccaug augaaaucug gaauguuccu cacacuguuu gugaacacag 7020
ucauuaacau uguaaucgca agcagagugu ugagagaacg gcuaaccgga ucaccaugug 7080
cagcauucau uggagaugac aauaucguga aaggagucaa aucggacaaa uuaauggcag 7140
acaggugcgc caccugguug aauauggaag ucaagauuau agaugcugug gugggcgaga 7200
aagcgccuua uuucugugga ggguuuauuu ugugugacuc cgugaccggc acagcgugcc 7260
guguggcaga cccccuaaaa aggcuguuua agcuuggcaa accucuggca gcagacgaug 7320
aacaugauga ugacaggaga agggcauugc augaagaguc aacacgcugg aaccgagugg 7380
guauucuuuc agagcugugc aaggcaguag aaucaaggua ugaaaccgua ggaacuucca 7440
ucauaguuau ggccaugacu acucuagcua gcaguguuaa aucauucagc uaccugagag 7500
gggccccuau aacucucuac ggcuaaccug aauggacuac gacauagucu aguccgccaa 7560
g 7561
<210> 39
<211> 627
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic construct, VEEV nsP4-2A polyprotein
<400> 39
Tyr Ile Phe Ser Ser Asp Thr Gly Gln Gly His Leu Gln Gln Lys Ser
1 5 10 15
Val Arg Gln Thr Val Leu Ser Glu Val Val Leu Glu Arg Thr Glu Leu
20 25 30
Glu Ile Ser Tyr Ala Pro Arg Leu Asp Gln Glu Lys Glu Glu Leu Leu
35 40 45
Arg Lys Lys Leu Gln Leu Asn Pro Thr Pro Ala Asn Arg Ser Arg Tyr
50 55 60
Gln Ser Arg Lys Val Glu Asn Met Lys Ala Ile Thr Ala Arg Arg Ile
65 70 75 80
Leu Gln Gly Leu Gly His Tyr Leu Lys Ala Glu Gly Lys Val Glu Cys
85 90 95
Tyr Arg Thr Leu His Pro Val Pro Leu Tyr Ser Ser Ser Val Asn Arg
100 105 110
Ala Phe Ser Ser Pro Lys Val Ala Val Glu Ala Cys Asn Ala Met Leu
115 120 125
Lys Glu Asn Phe Pro Thr Val Ala Ser Tyr Cys Ile Ile Pro Glu Tyr
130 135 140
Asp Ala Tyr Leu Asp Met Val Asp Gly Ala Ser Cys Cys Leu Asp Thr
145 150 155 160
Ala Ser Phe Cys Pro Ala Lys Leu Arg Ser Phe Pro Lys Lys His Ser
165 170 175
Tyr Leu Glu Pro Thr Ile Arg Ser Ala Val Pro Ser Ala Ile Gln Asn
180 185 190
Thr Leu Gln Asn Val Leu Ala Ala Ala Thr Lys Arg Asn Cys Asn Val
195 200 205
Thr Gln Met Arg Glu Leu Pro Val Leu Asp Ser Ala Ala Phe Asn Val
210 215 220
Glu Cys Phe Lys Lys Tyr Ala Cys Asn Asn Glu Tyr Trp Glu Thr Phe
225 230 235 240
Lys Glu Asn Pro Ile Arg Leu Thr Glu Glu Asn Val Val Asn Tyr Ile
245 250 255
Thr Lys Leu Lys Gly Pro Lys Ala Ala Ala Leu Phe Ala Lys Thr His
260 265 270
Asn Leu Asn Met Leu Gln Asp Ile Pro Met Asp Arg Phe Val Met Asp
275 280 285
Leu Lys Arg Asp Val Lys Val Thr Pro Gly Thr Lys His Thr Glu Glu
290 295 300
Arg Pro Lys Val Gln Val Ile Gln Ala Ala Asp Pro Leu Ala Thr Ala
305 310 315 320
Tyr Leu Cys Gly Ile His Arg Glu Leu Val Arg Arg Leu Asn Ala Val
325 330 335
Leu Leu Pro Asn Ile His Thr Leu Phe Asp Met Ser Ala Glu Asp Phe
340 345 350
Asp Ala Ile Ile Ala Glu His Phe Gln Pro Gly Asp Cys Val Leu Glu
355 360 365
Thr Asp Ile Ala Ser Phe Asp Lys Ser Glu Asp Asp Ala Met Ala Leu
370 375 380
Thr Ala Leu Met Ile Leu Glu Asp Leu Gly Val Asp Ala Glu Leu Leu
385 390 395 400
Thr Leu Ile Glu Ala Ala Phe Gly Glu Ile Ser Ser Ile His Leu Pro
405 410 415
Thr Lys Thr Lys Phe Lys Phe Gly Ala Met Met Lys Ser Gly Met Phe
420 425 430
Leu Thr Leu Phe Val Asn Thr Val Ile Asn Ile Val Ile Ala Ser Arg
435 440 445
Val Leu Arg Glu Arg Leu Thr Gly Ser Pro Cys Ala Ala Phe Ile Gly
450 455 460
Asp Asp Asn Ile Val Lys Gly Val Lys Ser Asp Lys Leu Met Ala Asp
465 470 475 480
Arg Cys Ala Thr Trp Leu Asn Met Glu Val Lys Ile Ile Asp Ala Val
485 490 495
Val Gly Glu Lys Ala Pro Tyr Phe Cys Gly Gly Phe Ile Leu Cys Asp
500 505 510
Ser Val Thr Gly Thr Ala Cys Arg Val Ala Asp Pro Leu Lys Arg Leu
515 520 525
Phe Lys Leu Gly Lys Pro Leu Ala Ala Asp Asp Glu His Asp Asp Asp
530 535 540
Arg Arg Arg Ala Leu His Glu Glu Ser Thr Arg Trp Asn Arg Val Gly
545 550 555 560
Ile Leu Ser Glu Leu Cys Lys Ala Val Glu Ser Arg Tyr Glu Thr Val
565 570 575
Gly Thr Ser Ile Ile Val Met Ala Met Thr Thr Leu Ala Ser Ser Val
580 585 590
Lys Ser Phe Ser Tyr Leu Arg Gly Ala Pro Ile Thr Leu Tyr Gly Ser
595 600 605
Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn
610 615 620
Pro Gly Pro
625
<210> 40
<211> 840
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic construct, VEEV nsP4-2A-3Cpro
<400> 40
Tyr Ile Phe Ser Ser Asp Thr Gly Gln Gly His Leu Gln Gln Lys Ser
1 5 10 15
Val Arg Gln Thr Val Leu Ser Glu Val Val Leu Glu Arg Thr Glu Leu
20 25 30
Glu Ile Ser Tyr Ala Pro Arg Leu Asp Gln Glu Lys Glu Glu Leu Leu
35 40 45
Arg Lys Lys Leu Gln Leu Asn Pro Thr Pro Ala Asn Arg Ser Arg Tyr
50 55 60
Gln Ser Arg Lys Val Glu Asn Met Lys Ala Ile Thr Ala Arg Arg Ile
65 70 75 80
Leu Gln Gly Leu Gly His Tyr Leu Lys Ala Glu Gly Lys Val Glu Cys
85 90 95
Tyr Arg Thr Leu His Pro Val Pro Leu Tyr Ser Ser Ser Val Asn Arg
100 105 110
Ala Phe Ser Ser Pro Lys Val Ala Val Glu Ala Cys Asn Ala Met Leu
115 120 125
Lys Glu Asn Phe Pro Thr Val Ala Ser Tyr Cys Ile Ile Pro Glu Tyr
130 135 140
Asp Ala Tyr Leu Asp Met Val Asp Gly Ala Ser Cys Cys Leu Asp Thr
145 150 155 160
Ala Ser Phe Cys Pro Ala Lys Leu Arg Ser Phe Pro Lys Lys His Ser
165 170 175
Tyr Leu Glu Pro Thr Ile Arg Ser Ala Val Pro Ser Ala Ile Gln Asn
180 185 190
Thr Leu Gln Asn Val Leu Ala Ala Ala Thr Lys Arg Asn Cys Asn Val
195 200 205
Thr Gln Met Arg Glu Leu Pro Val Leu Asp Ser Ala Ala Phe Asn Val
210 215 220
Glu Cys Phe Lys Lys Tyr Ala Cys Asn Asn Glu Tyr Trp Glu Thr Phe
225 230 235 240
Lys Glu Asn Pro Ile Arg Leu Thr Glu Glu Asn Val Val Asn Tyr Ile
245 250 255
Thr Lys Leu Lys Gly Pro Lys Ala Ala Ala Leu Phe Ala Lys Thr His
260 265 270
Asn Leu Asn Met Leu Gln Asp Ile Pro Met Asp Arg Phe Val Met Asp
275 280 285
Leu Lys Arg Asp Val Lys Val Thr Pro Gly Thr Lys His Thr Glu Glu
290 295 300
Arg Pro Lys Val Gln Val Ile Gln Ala Ala Asp Pro Leu Ala Thr Ala
305 310 315 320
Tyr Leu Cys Gly Ile His Arg Glu Leu Val Arg Arg Leu Asn Ala Val
325 330 335
Leu Leu Pro Asn Ile His Thr Leu Phe Asp Met Ser Ala Glu Asp Phe
340 345 350
Asp Ala Ile Ile Ala Glu His Phe Gln Pro Gly Asp Cys Val Leu Glu
355 360 365
Thr Asp Ile Ala Ser Phe Asp Lys Ser Glu Asp Asp Ala Met Ala Leu
370 375 380
Thr Ala Leu Met Ile Leu Glu Asp Leu Gly Val Asp Ala Glu Leu Leu
385 390 395 400
Thr Leu Ile Glu Ala Ala Phe Gly Glu Ile Ser Ser Ile His Leu Pro
405 410 415
Thr Lys Thr Lys Phe Lys Phe Gly Ala Met Met Lys Ser Gly Met Phe
420 425 430
Leu Thr Leu Phe Val Asn Thr Val Ile Asn Ile Val Ile Ala Ser Arg
435 440 445
Val Leu Arg Glu Arg Leu Thr Gly Ser Pro Cys Ala Ala Phe Ile Gly
450 455 460
Asp Asp Asn Ile Val Lys Gly Val Lys Ser Asp Lys Leu Met Ala Asp
465 470 475 480
Arg Cys Ala Thr Trp Leu Asn Met Glu Val Lys Ile Ile Asp Ala Val
485 490 495
Val Gly Glu Lys Ala Pro Tyr Phe Cys Gly Gly Phe Ile Leu Cys Asp
500 505 510
Ser Val Thr Gly Thr Ala Cys Arg Val Ala Asp Pro Leu Lys Arg Leu
515 520 525
Phe Lys Leu Gly Lys Pro Leu Ala Ala Asp Asp Glu His Asp Asp Asp
530 535 540
Arg Arg Arg Ala Leu His Glu Glu Ser Thr Arg Trp Asn Arg Val Gly
545 550 555 560
Ile Leu Ser Glu Leu Cys Lys Ala Val Glu Ser Arg Tyr Glu Thr Val
565 570 575
Gly Thr Ser Ile Ile Val Met Ala Met Thr Thr Leu Ala Ser Ser Val
580 585 590
Lys Ser Phe Ser Tyr Leu Arg Gly Ala Pro Ile Thr Leu Tyr Gly Ser
595 600 605
Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn
610 615 620
Pro Gly Pro Ser Gly Ala Pro Pro Thr Asp Leu Gln Lys Met Val Met
625 630 635 640
Gly Asn Thr Lys Pro Val Glu Leu Ile Leu Asp Gly Lys Thr Val Ala
645 650 655
Ile Cys Cys Ala Thr Gly Val Phe Gly Thr Ala Tyr Leu Val Pro Arg
660 665 670
His Leu Phe Ala Glu Lys Tyr Asp Lys Ile Met Leu Asp Gly Arg Ala
675 680 685
Met Thr Asp Ser Asp Tyr Arg Val Phe Glu Phe Glu Ile Lys Val Lys
690 695 700
Gly Gln Asp Met Leu Ser Asp Ala Ala Leu Met Val Leu His Arg Gly
705 710 715 720
Asn Arg Val Arg Asp Ile Thr Lys His Phe Arg Asp Thr Ala Arg Met
725 730 735
Lys Lys Gly Thr Pro Val Val Gly Val Val Asn Asn Ala Asp Val Gly
740 745 750
Arg Leu Ile Phe Ser Gly Glu Ala Leu Thr Tyr Lys Asp Ile Val Val
755 760 765
Cys Met Asp Gly Asp Thr Met Pro Ser Leu Phe Ala Tyr Lys Ala Ala
770 775 780
Thr Lys Ala Gly Tyr Cys Gly Gly Ala Val Leu Ala Lys Asp Gly Ala
785 790 795 800
Asp Thr Phe Ile Val Gly Thr His Ser Ala Gly Gly Asn Gly Val Gly
805 810 815
Tyr Cys Ser Cys Val Ser Lys Ser Met Leu Leu Arg Met Lys Ala His
820 825 830
Val Asp Pro Glu Pro Gln His Glu
835 840
<210> 41
<211> 865
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic construct, VEEV nsP4-2A-GFP
<400> 41
Tyr Ile Phe Ser Ser Asp Thr Gly Gln Gly His Leu Gln Gln Lys Ser
1 5 10 15
Val Arg Gln Thr Val Leu Ser Glu Val Val Leu Glu Arg Thr Glu Leu
20 25 30
Glu Ile Ser Tyr Ala Pro Arg Leu Asp Gln Glu Lys Glu Glu Leu Leu
35 40 45
Arg Lys Lys Leu Gln Leu Asn Pro Thr Pro Ala Asn Arg Ser Arg Tyr
50 55 60
Gln Ser Arg Lys Val Glu Asn Met Lys Ala Ile Thr Ala Arg Arg Ile
65 70 75 80
Leu Gln Gly Leu Gly His Tyr Leu Lys Ala Glu Gly Lys Val Glu Cys
85 90 95
Tyr Arg Thr Leu His Pro Val Pro Leu Tyr Ser Ser Ser Val Asn Arg
100 105 110
Ala Phe Ser Ser Pro Lys Val Ala Val Glu Ala Cys Asn Ala Met Leu
115 120 125
Lys Glu Asn Phe Pro Thr Val Ala Ser Tyr Cys Ile Ile Pro Glu Tyr
130 135 140
Asp Ala Tyr Leu Asp Met Val Asp Gly Ala Ser Cys Cys Leu Asp Thr
145 150 155 160
Ala Ser Phe Cys Pro Ala Lys Leu Arg Ser Phe Pro Lys Lys His Ser
165 170 175
Tyr Leu Glu Pro Thr Ile Arg Ser Ala Val Pro Ser Ala Ile Gln Asn
180 185 190
Thr Leu Gln Asn Val Leu Ala Ala Ala Thr Lys Arg Asn Cys Asn Val
195 200 205
Thr Gln Met Arg Glu Leu Pro Val Leu Asp Ser Ala Ala Phe Asn Val
210 215 220
Glu Cys Phe Lys Lys Tyr Ala Cys Asn Asn Glu Tyr Trp Glu Thr Phe
225 230 235 240
Lys Glu Asn Pro Ile Arg Leu Thr Glu Glu Asn Val Val Asn Tyr Ile
245 250 255
Thr Lys Leu Lys Gly Pro Lys Ala Ala Ala Leu Phe Ala Lys Thr His
260 265 270
Asn Leu Asn Met Leu Gln Asp Ile Pro Met Asp Arg Phe Val Met Asp
275 280 285
Leu Lys Arg Asp Val Lys Val Thr Pro Gly Thr Lys His Thr Glu Glu
290 295 300
Arg Pro Lys Val Gln Val Ile Gln Ala Ala Asp Pro Leu Ala Thr Ala
305 310 315 320
Tyr Leu Cys Gly Ile His Arg Glu Leu Val Arg Arg Leu Asn Ala Val
325 330 335
Leu Leu Pro Asn Ile His Thr Leu Phe Asp Met Ser Ala Glu Asp Phe
340 345 350
Asp Ala Ile Ile Ala Glu His Phe Gln Pro Gly Asp Cys Val Leu Glu
355 360 365
Thr Asp Ile Ala Ser Phe Asp Lys Ser Glu Asp Asp Ala Met Ala Leu
370 375 380
Thr Ala Leu Met Ile Leu Glu Asp Leu Gly Val Asp Ala Glu Leu Leu
385 390 395 400
Thr Leu Ile Glu Ala Ala Phe Gly Glu Ile Ser Ser Ile His Leu Pro
405 410 415
Thr Lys Thr Lys Phe Lys Phe Gly Ala Met Met Lys Ser Gly Met Phe
420 425 430
Leu Thr Leu Phe Val Asn Thr Val Ile Asn Ile Val Ile Ala Ser Arg
435 440 445
Val Leu Arg Glu Arg Leu Thr Gly Ser Pro Cys Ala Ala Phe Ile Gly
450 455 460
Asp Asp Asn Ile Val Lys Gly Val Lys Ser Asp Lys Leu Met Ala Asp
465 470 475 480
Arg Cys Ala Thr Trp Leu Asn Met Glu Val Lys Ile Ile Asp Ala Val
485 490 495
Val Gly Glu Lys Ala Pro Tyr Phe Cys Gly Gly Phe Ile Leu Cys Asp
500 505 510
Ser Val Thr Gly Thr Ala Cys Arg Val Ala Asp Pro Leu Lys Arg Leu
515 520 525
Phe Lys Leu Gly Lys Pro Leu Ala Ala Asp Asp Glu His Asp Asp Asp
530 535 540
Arg Arg Arg Ala Leu His Glu Glu Ser Thr Arg Trp Asn Arg Val Gly
545 550 555 560
Ile Leu Ser Glu Leu Cys Lys Ala Val Glu Ser Arg Tyr Glu Thr Val
565 570 575
Gly Thr Ser Ile Ile Val Met Ala Met Thr Thr Leu Ala Ser Ser Val
580 585 590
Lys Ser Phe Ser Tyr Leu Arg Gly Ala Pro Ile Thr Leu Tyr Gly Ser
595 600 605
Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn
610 615 620
Pro Gly Pro Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
625 630 635 640
Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val
645 650 655
Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys
660 665 670
Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
675 680 685
Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His
690 695 700
Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val
705 710 715 720
Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg
725 730 735
Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu
740 745 750
Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu
755 760 765
Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln
770 775 780
Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp
785 790 795 800
Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
805 810 815
Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser
820 825 830
Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
835 840 845
Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
850 855 860
Lys
865
<210> 42
<211> 1005
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic construct, VEEV nsP4-2A-STING
<400> 42
Tyr Ile Phe Ser Ser Asp Thr Gly Gln Gly His Leu Gln Gln Lys Ser
1 5 10 15
Val Arg Gln Thr Val Leu Ser Glu Val Val Leu Glu Arg Thr Glu Leu
20 25 30
Glu Ile Ser Tyr Ala Pro Arg Leu Asp Gln Glu Lys Glu Glu Leu Leu
35 40 45
Arg Lys Lys Leu Gln Leu Asn Pro Thr Pro Ala Asn Arg Ser Arg Tyr
50 55 60
Gln Ser Arg Lys Val Glu Asn Met Lys Ala Ile Thr Ala Arg Arg Ile
65 70 75 80
Leu Gln Gly Leu Gly His Tyr Leu Lys Ala Glu Gly Lys Val Glu Cys
85 90 95
Tyr Arg Thr Leu His Pro Val Pro Leu Tyr Ser Ser Ser Val Asn Arg
100 105 110
Ala Phe Ser Ser Pro Lys Val Ala Val Glu Ala Cys Asn Ala Met Leu
115 120 125
Lys Glu Asn Phe Pro Thr Val Ala Ser Tyr Cys Ile Ile Pro Glu Tyr
130 135 140
Asp Ala Tyr Leu Asp Met Val Asp Gly Ala Ser Cys Cys Leu Asp Thr
145 150 155 160
Ala Ser Phe Cys Pro Ala Lys Leu Arg Ser Phe Pro Lys Lys His Ser
165 170 175
Tyr Leu Glu Pro Thr Ile Arg Ser Ala Val Pro Ser Ala Ile Gln Asn
180 185 190
Thr Leu Gln Asn Val Leu Ala Ala Ala Thr Lys Arg Asn Cys Asn Val
195 200 205
Thr Gln Met Arg Glu Leu Pro Val Leu Asp Ser Ala Ala Phe Asn Val
210 215 220
Glu Cys Phe Lys Lys Tyr Ala Cys Asn Asn Glu Tyr Trp Glu Thr Phe
225 230 235 240
Lys Glu Asn Pro Ile Arg Leu Thr Glu Glu Asn Val Val Asn Tyr Ile
245 250 255
Thr Lys Leu Lys Gly Pro Lys Ala Ala Ala Leu Phe Ala Lys Thr His
260 265 270
Asn Leu Asn Met Leu Gln Asp Ile Pro Met Asp Arg Phe Val Met Asp
275 280 285
Leu Lys Arg Asp Val Lys Val Thr Pro Gly Thr Lys His Thr Glu Glu
290 295 300
Arg Pro Lys Val Gln Val Ile Gln Ala Ala Asp Pro Leu Ala Thr Ala
305 310 315 320
Tyr Leu Cys Gly Ile His Arg Glu Leu Val Arg Arg Leu Asn Ala Val
325 330 335
Leu Leu Pro Asn Ile His Thr Leu Phe Asp Met Ser Ala Glu Asp Phe
340 345 350
Asp Ala Ile Ile Ala Glu His Phe Gln Pro Gly Asp Cys Val Leu Glu
355 360 365
Thr Asp Ile Ala Ser Phe Asp Lys Ser Glu Asp Asp Ala Met Ala Leu
370 375 380
Thr Ala Leu Met Ile Leu Glu Asp Leu Gly Val Asp Ala Glu Leu Leu
385 390 395 400
Thr Leu Ile Glu Ala Ala Phe Gly Glu Ile Ser Ser Ile His Leu Pro
405 410 415
Thr Lys Thr Lys Phe Lys Phe Gly Ala Met Met Lys Ser Gly Met Phe
420 425 430
Leu Thr Leu Phe Val Asn Thr Val Ile Asn Ile Val Ile Ala Ser Arg
435 440 445
Val Leu Arg Glu Arg Leu Thr Gly Ser Pro Cys Ala Ala Phe Ile Gly
450 455 460
Asp Asp Asn Ile Val Lys Gly Val Lys Ser Asp Lys Leu Met Ala Asp
465 470 475 480
Arg Cys Ala Thr Trp Leu Asn Met Glu Val Lys Ile Ile Asp Ala Val
485 490 495
Val Gly Glu Lys Ala Pro Tyr Phe Cys Gly Gly Phe Ile Leu Cys Asp
500 505 510
Ser Val Thr Gly Thr Ala Cys Arg Val Ala Asp Pro Leu Lys Arg Leu
515 520 525
Phe Lys Leu Gly Lys Pro Leu Ala Ala Asp Asp Glu His Asp Asp Asp
530 535 540
Arg Arg Arg Ala Leu His Glu Glu Ser Thr Arg Trp Asn Arg Val Gly
545 550 555 560
Ile Leu Ser Glu Leu Cys Lys Ala Val Glu Ser Arg Tyr Glu Thr Val
565 570 575
Gly Thr Ser Ile Ile Val Met Ala Met Thr Thr Leu Ala Ser Ser Val
580 585 590
Lys Ser Phe Ser Tyr Leu Arg Gly Ala Pro Ile Thr Leu Tyr Gly Ser
595 600 605
Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn
610 615 620
Pro Gly Pro Pro His Ser Ser Leu His Pro Ser Ile Pro Cys Pro Arg
625 630 635 640
Gly His Gly Ala Gln Lys Ala Ala Leu Val Leu Leu Ser Ala Cys Leu
645 650 655
Val Thr Leu Trp Gly Leu Gly Glu Pro Pro Glu His Thr Leu Arg Tyr
660 665 670
Leu Val Leu His Leu Ala Ser Leu Gln Leu Gly Leu Leu Leu Asn Gly
675 680 685
Val Cys Ser Leu Ala Glu Glu Leu Arg His Ile His Ser Arg Tyr Arg
690 695 700
Gly Ser Tyr Trp Arg Thr Val Arg Ala Cys Leu Gly Cys Pro Leu Arg
705 710 715 720
Arg Gly Ala Leu Leu Leu Leu Ser Ile Tyr Phe Tyr Tyr Ser Leu Pro
725 730 735
Asn Ala Val Gly Pro Pro Phe Thr Trp Met Leu Ala Leu Leu Gly Leu
740 745 750
Ser Gln Ala Leu Asn Ile Leu Leu Gly Leu Lys Gly Leu Ala Pro Ala
755 760 765
Glu Ile Ser Ala Val Cys Glu Lys Gly Asn Phe Ser Val Ala His Gly
770 775 780
Leu Ala Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu
785 790 795 800
Leu Gln Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu
805 810 815
Arg Gly Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys
820 825 830
Gly Val Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu
835 840 845
Asp Lys Leu Pro Gln Gln Thr Gly Asp His Ala Gly Ile Lys Asp Arg
850 855 860
Val Tyr Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala
865 870 875 880
Gly Thr Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala
885 890 895
Met Ser Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Met Leu Glu
900 905 910
Gln Ala Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala
915 920 925
Pro Glu Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala
930 935 940
Asp Asp Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg
945 950 955 960
Gln Glu Glu Lys Glu Glu Val Thr Val Gly Ser Leu Lys Thr Ser Ala
965 970 975
Val Pro Ser Thr Ser Thr Met Ser Gln Glu Pro Glu Leu Leu Ile Ser
980 985 990
Gly Met Glu Lys Pro Leu Pro Leu Arg Thr Asp Phe Ser
995 1000 1005
<210> 43
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic construct, thosea asigna virus 2A protein
<400> 43
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
<210> 44
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic construct, porcine teschovirus -1_2A protein
<400> 44
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 45
<211> 22
<212> PRT
<213> Foot-and-mouth disease virus
<220>
<221> MISC_FEATURE
<222> (1)..(22)
<223> FMDV 2A protein
<400> 45
Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val
1 5 10 15
Glu Ser Asn Pro Gly Pro
20
<210> 46
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> Syntehtic construct, equine rhinitis A virus 2A protein
<400> 46
Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser
1 5 10 15
Asn Pro Gly Pro
20

Claims (64)

1.一种合成的甲病毒衍生复制子核酸分子，其包含：(i)第一核酸，其编码甲病毒非结构蛋白nsP1、nsP2、nsP3和nsP4，并且包含在如SEQ D NO：17所示的甲病毒基因组序列中从nt 503至nt 658、nt 658至nt 1620、nt 1620至nt 2560、nt 2560至nt 3954、nt 3954至nt4120、nt 6381至nt 7083和nt 6966至nt 7526的区域内的任意位置处引入的至少一个沉默突变；以及(ii)第二核酸，其包含经过修饰的亚基因组开放阅读框(ORF)。
2.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，经过修饰的亚基因组ORF包含编码第一异源蛋白的序列。
3.根据权利要求2所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码经过修改的nsP4的多核苷酸，所述经过修改的nsP4包含第二异源蛋白。
4.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 503至nt 658的区域内的任意位置处引入的至少一个沉默突变。
5.根据权利要求4所述的合成的甲病毒属衍生复制子核酸分子，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：21所示的参考序列具有至少90％同一性的序列的多核苷酸。
6.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 658至nt 1620的区域内的任意位置处引入的至少一个沉默突变。
7.根据权利要求6所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：25所示的参考序列具有至少90％同一性的序列的多核苷酸。
8.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 1620至nt 2560的区域内的任意位置处引入的至少一个沉默突变。
9.根据权利要求8所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：24所示的参考序列具有至少90％同一性的序列的多核苷酸。
10.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 3694至nt 3954的区域内的任意位置处引入的至少一个沉默突变。
11.根据权利要求10所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：22所示的参考序列具有至少90％同一性的序列的多核苷酸。
12.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 2560至nt 3954的区域内的任意位置处引入的至少一个沉默突变。
13.根据权利要求12所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：26所示的参考序列具有至少90％同一性的序列的多核苷酸。
14.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 3954至nt 4120的区域内的任意位置处引入的至少一个沉默突变。
15.根据权利要求14所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP2和nsP3的接合物并且含有与如SEQ ID NO：23所示的参考序列具有至少90％同一性的序列的多核苷酸。
16.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 6381至nt 7083的区域内的任意位置处引入的至少一个沉默突变。
17.根据权利要求16所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP4并且含有与如SEQ ID NO：20所示的参考序列具有至少90％同一性的序列的多核苷酸。
18.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 658至nt 1620和nt 2560至nt 3954的区域内的任意位置处引入的至少一个沉默突变。
19.根据权利要求18所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：25所示的参考序列具有至少90％同一性的序列的多核苷酸，以及编码nsP2并且含有与如SEQ ID NO：26所示的参考序列具有至少90％同一性的序列的多核苷酸。
20.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含在甲病毒基因组中从nt 3694至nt 3954和nt 6381至nt 7083的区域内的任意位置处引入的至少一个沉默突变。
21.根据权利要求20所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP2并且含有与如SEQ ID NO：22所示的参考序列具有至少90％同一性的序列的多核苷酸，以及编码nsP4并且含有与如SEQ ID NO：20所示的参考序列具有至少90％同一性的序列的多核苷酸。
22.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码nsP1并且含有与如SEQ ID NO：21所示的参考序列具有至少90％同一性的序列的多核苷酸；编码nsP1并且含有与如SEQ ID NO：25所示的参考序列具有至少90％同一性的序列的多核苷酸，编码nsP1和nsP2的接合物并且含有与如SEQ ID NO：24所示的参考序列具有至少90％同一性的序列的多核苷酸；编码nsP2并且含有与如SEQ ID NO：26所示的参考序列具有至少90％同一性的序列的多核苷酸；编码nsP2和nsP3的接合物并且含有与如SEQ D NO：23所示的参考序列具有至少90％同一性的序列的多核苷酸；以及编码nsP4并且含有与如SEQID NO：20所示的参考序列具有至少90％同一性的序列的多核苷酸。
23.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含编码所述非结构蛋白并且含有与如SEQ ID NO：30所示的参考序列具有至少90％同一性的多核苷酸序列的多核苷酸。
24.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，第一核酸分子包含在甲病毒基因组中从nt 6966到nt 7523的区域内的任意位置处引入的至少一个沉默突变。
25.根据权利要求24所述的合成的甲病毒衍生复制子核酸分子，第一核酸分子包含与如SEQ D NO：18所示的参考序列具有至少90％同一性的多核苷酸序列。
26.根据权利要求1所述的合成的甲病毒衍生复制子核酸分子，其中，第一核酸包含引入编码nsP4蛋白的多核苷酸的至少一个沉默突变。
27.根据权利要求26所述的合成的甲病毒衍生复制子核酸分子，第一核酸分子包含与如SEQ D NO：19所示的参考序列具有至少90％同一性的多核苷酸序列。
28.根据权利要求3所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白或第二异源蛋白包含细胞代谢、免疫或信号传导功能的病毒衍生的或哺乳动物细胞衍生的调节剂。
29.根据权利要求3所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白或第二异源蛋白影响细胞抗病毒先天免疫应答并增强甲病毒复制子RNA分子的复制。
30.根据权利要求3所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白或第二异源蛋白包含自病毒病原体分离或衍生的抗原蛋白。
31.根据权利要求30所述的合成的甲病毒衍生复制子核酸分子，其中，病毒病原体选自由：小核糖核酸病毒、腺病毒、鼻病毒、轮状病毒、西尼罗病毒、寨卡病毒、疱疹病毒或冠状病毒(CoV)组成的组中。
32.根据权利要求31所述的合成的甲病毒衍生复制子核酸分子，其中，病毒病原体为小核糖核酸病毒。
33.根据权利要求3所述的合成的甲病毒衍生复制子核酸分子，其中，第二异源蛋白包含3C蛋白酶蛋白，并且经过修改的nsP4包含与如SEQ ID NO：40所示的参考序列具有至少90％同一性的氨基酸序列。
34.根据权利要求33所述的合成的甲病毒衍生复制子核酸分子，其中，经过修改的nsP4由与如SEQ ID NO：32所示的参考序列具有至少90％同一性的多核苷酸序列编码。
35.根据权利要求3所述的合成的甲病毒衍生复制子核酸分子，其中，第二核酸包含编码O1 ManisaP1蛋白的多核苷酸。
36.根据权利要求35所述的合成的甲病毒衍生复制子核酸分子，其中，所述多核苷酸包含与如SEQ nt NO：33所示的参考序列具有至少90％同一性的序列。
37.根据权利要求3所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白包含O1 ManisaP1蛋白并且第二异源蛋白包含3C蛋白酶。
38.根据权利要求3所述的合成的甲病毒衍生复制子核酸分子，其中，第二核酸包含编码抗原蛋白的多核苷酸。
39.根据权利要求3所述的合成的甲病毒衍生复制子核酸分子，其中，第二异源蛋白包含STING蛋白，所述经过修改的nsP4包含与如SEQ ID NO：42所示的参考序列具有至少90％同一性的氨基酸序列。
40.根据权利要求3所述的合成的甲病毒衍生复制子核酸分子，其中，第一异源蛋白包含抗原蛋白并且第二异源蛋白包含STING蛋白。
41.一种甲病毒衍生RNA复制子表达系统，其包含权利要求1-40中任一项所述的合成的甲病毒衍生复制子核酸分子和宿主细胞。
42.一种药学上可接受的组合物，其包含权利要求1-40中任一项所述的甲病毒衍生复制子和药学上可接受的载体或赋形剂。
43.一种在细胞中产生至少一种异源蛋白的方法，其包括在宿主细胞中表达权利要求1-40中任一项所述的合成的甲病毒衍生复制子核酸分子。
44.一种预防、抑制或治疗受试者的疾病或病症的症状的方法，其包括：提供合成的甲病毒衍生复制子核酸分子，其包含：
(i)第一核酸，其编码甲病毒非结构蛋白nsP1、nsP2、nsP3和nsP4，并且包含在如SEQ IDNO：17所示的甲病毒基因组序列中从nt 503至nt 658、nt 658至nt 1620、nt 1620至nt2560、nt 2560至nt 3954、nt 3954至nt 4120、nt 6381至nt 7083和nt 6966至nt 7526的区域内的任意位置处引入的至少一个沉默突变；以及
(ii)第二核酸，其包含经过修饰的亚基因组开放阅读框(ORF)；以及
将治疗有效量的合成的甲病毒衍生复制子核酸分子施用给受试者。
45.根据权利要求44所述的方法，其中，第一核酸包含与选自由SEQ ID NOS：20-30组成的组中的参考序列具有至少90％同一性的至少一个序列。
46.根据权利要求44所述的方法，其中，经过修饰的亚基因组ORF包含编码第一异源蛋白的序列。
47.根据权利要求44所述的方法，其中，第一核酸包含编码经过修改的nsP4的多核苷酸，所述经过修改的nsP4包含第二异源蛋白。
48.根据权利要求47所述的方法，其中，第一异源蛋白或第二异源蛋白包含细胞代谢、免疫或信号传导功能的病毒衍生的或哺乳动物细胞衍生的调节剂。
49.根据权利要求47所述的方法，其中，第一异源蛋白或第二异源蛋白影响细胞抗病毒先天免疫应答并增强甲病毒复制子RNA分子的复制。
50.根据权利要求47所述的方法，其中，第一异源蛋白或第二异源蛋白包含自病毒病原体分离或衍生的抗原蛋白。
51.根据权利要求50所述的方法，其中，病毒病原体选自由：小核糖核酸病毒、腺病毒、鼻病毒、轮状病毒、西尼罗病毒、寨卡病毒、疱疹病毒或冠状病毒(CoV)组成的组中。
52.根据权利要求51所述的方法，其中，病毒病原体为小核糖核酸病毒。
53.根据权利要求47所述的方法，其中，第一异源蛋白包含O1Manisa P1蛋白并且第二异源蛋白包含3C蛋白酶。
54.根据权利要求47所述的方法，其中，第一异源蛋白或第二异源蛋白包含STING蛋白。
55.根据权利要求47所述的方法，其中，第二异源蛋白包含STING蛋白并且第一异源蛋白包含抗原蛋白。
56.根据权利要求44所述的方法，其中，施用步骤使得受试者的疾病得到预防、治疗、严重性减轻或病程发展减缓。
57.根据权利要求44所述的方法，其中，疾病为癌症。
58.根据权利要求57所述的方法，其中，癌症为黑素瘤或上皮性肿瘤。
59.根据权利要求58所述的方法，其中，该方法还包括向受试者共同施用一种或多种另外的抗癌疗法。
60.根据权利要求59所述的方法，其中，另外的抗癌疗法选自由外科手术、化学疗法、放射疗法、温热疗法、免疫疗法、激素疗法、激光疗法、抗血管生成疗法及其任何组合组成的组中。
61.根据权利要求44所述的方法，其中，疾病为FMD。
62.根据权利要求44所述的方法，其中，受试者为哺乳动物。
63.根据权利要求62所述的方法，其中，哺乳动物为偶蹄动物或人。
64.根据权利要求63所述的方法，其中，偶蹄动物选自：牛、水牛、绵羊、山羊、猪、羚羊、鹿、野牛、刺猬、大象、美洲驼和羊驼组成的组中。
CN202280022669.3A 2021-03-19 2022-03-18 人工甲病毒衍生的rna复制子表达系统 Pending CN117083071A (zh)

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