重组核酸的线粒体递送的制作方法

文档序号:17485577发布日期:2019-04-20 06:47阅读:1259来源:国知局
重组核酸的线粒体递送的制作方法

本申请要求于2016年5月6日提交的新加坡临时申请10201603628q的优先权,为所有目的将其全部内容通过引用并入此处。

本公开内容涉及分子生物学领域。特别地,本发明涉及线粒体靶向序列用于转运核酸序列的用途。



背景技术:

线粒体是几乎所有活的生物体中参与呼吸循环末端部分的细胞器。线粒体的基因组组织是独特的且编码37种基因,其中22种是trna基因,2种是线粒体核糖体rna(12s和16s)基因,13种是呼吸酶亚基的基因。这些呼吸基因的缺陷与许多神经退行性疾病有关,例如共济失调、视神经病变、帕金森病,也与衰老有关。但是,被线粒体摄取的核酸用于线粒体保护和调节的研究很少,并且尚未鉴定出有效的线粒体递送载体。因此,需要一种能够将有效负载(例如,由递送系统携带的核酸序列)递送到细胞线粒体中的递送系统。



技术实现要素:

在一个方面,本发明涉及核酸递送构建体,该核酸递送构建体包括人巨细胞病毒β2.7rna的至少一个正义或反义rna亚结构域,其中每个亚结构域能够定位在线粒体内。

在另一方面,本发明涉及包括本文公开的核酸序列的载体、重组细胞或重组生物体。

在又一方面,本发明涉及核酸序列,该核酸序列包括选自由结构域1(d1;seqidno:3或7)、结构域2(d2;seqidno:4或8)、结构域3(d3;seqidno:5或9)和结构域4(d4;seqidno:6或10)组成的组的人巨细胞病毒β2.7rna的至少一种或多种正义或反义rna序列。

在另一方面,本发明涉及增强线粒体基因功能或抑制有缺陷的线粒体基因功能或两者(条件是在这种情况下线粒体基因彼此不同)的方法,该方法包括向受试者(subject)施用如本文所公开的核酸递送构建体,其中线粒体基因功能彼此不同。

在另一方面,本发明涉及治疗线粒体病症的方法,该方法包括向受试者施用本文公开的核酸递送构建体。

附图说明

当结合非限制性示例和附图考虑时,参考详细描述将更好地理解本发明,其中:

图1显示了结构和功能分析,其鉴定了控制全长β2.7rna的完整线粒体定位活性的单个结构性亚结构域。a)用体外转录的β2.7rna转染hepg2细胞。纯化细胞质和线粒体部分(fraction),并使用实时rt-pcr测定每部分中β2.7rna的相对分布。数据代表3次独立实验的平均值±sd(*p<0.05,学生氏t检验(student’st-test))。b)使用foldsplit(husar)算法和200nt的窗口大小和int的相移增量对cmvtowne株的β2.7rna序列进行热力学分析。将每个窗口的二级结构形成的吉布斯自由能(ag)值相对于序列窗口的位置作图。δg最小值表示四个热力学上保守的和潜在功能相关的结构性亚结构域d1至d4。c)如mfold所预测的,明确定义的二级结构亚结构域d1至d4可以被分配给towneβ2.7rna的t7聚合酶转录物的最小自由能结构(mfe)内的每个能量最小值。d)显示了β2-7rna结构域结构以及单结构域缺失构建体(sddc)和单结构域构建体(sdc)的示意图。e)显示了用体外转录的全长β2.7rna或sddc转染的hepg2细胞的结果。使用rtrt-pcr监测线粒体摄取。数据代表3次独立实验的平均值±sd(***p<0.001,****p<0.0001单因素方差分析(one-wayanova),dunnett多重比较检验)。f)显示了用在体外转录的全长β2.7rna或sdc转染的hepg2细胞的结果,并使用rtrt-pcr和结构域特异性引物测量线粒体摄取。数据代表3次独立实验的±sd(*p<0.01,***p<0.001,学生氏t检验)。

图2显示了结构和功能分析,表明β2.7rna与其反义rna之间具有显著的同源性。a)使用foldsplit(husar)的towneβ2.7反义rna的热力学分析鉴定了互补链中对应于结构域d1至d4的热力学保守的结构性亚结构域d1as至d4as。b)用体外转录正义(t7)或反义(sp6)全长β2.7rna转染hepg2细胞,并使用rtrt-pcr和结构域特异性引物(对于d1至d4)从左到右定量线粒体摄取。数据代表三次独立实验的平均值±sd(***p<0.001,学生氏t检验)。c)使用rtrt-pcr检测的全长β2.7反义rna和反义sddc的线粒体定位。数据代表三次独立实验的平均值±sd(*p<0.05,**p<0.01,单因素方差分析,dunnett多重比较检验)。d)使用rtrt-pcr和结构域特异性引物测量全长β2.7rna、全长β2.7反义rna和反义sdc的线粒体定位。数据代表3次独立实验的平均值±sd(*p<0.05,**p<0.01,***p<0.001,***p<0.0001,单因素方差分析,tukey多重比较检验)。

图3显示了β2.7rna介导的gfpmrna线粒体靶向的结果,其触发线粒体gfp表达。a)β2.7-gfp融合构建体和对照的设计。将β2.7rna序列与具有基因组(ggfp)或线粒体(mtgfp)起始密码子和终止密码子的gfp序列基因融合。为了在与gfp序列融合时保留β2.7rna的亚结构域d1至d4的结构,设计并插入了间隔序列。为了将任何细胞质mtgfp靶向细胞核(nucleus),设计了构建体,其中mtgfp蛋白与核定位序列(nls)融合。将相应的核或线粒体翻译终止密码子(由三角形表示)置于相应的gfp或nls序列的下游,使得只翻译gfp蛋白或gfp-nls融合体。b)使用由mfold预测的rna二级结构的实施例证明间隔序列的功能性。在gfpmrna和β2.7rna之间插入间隔子(spacer)恢复β2.7rna亚结构域d2和d3。c)相对于β2.7阴性对照或亲本β2.7rna,β2.7融合rna的线粒体靶向活性。d)用体外转录的rna转染hepg2细胞,并使用rtrt-pcr监测线粒体定位。数据代表3次独立实验的平均值(****p<0.0001,单因素方差分析,dunnett多重比较检验)。e)至f)包括散点图和直方图移位的流式细胞术分析。用表达gfp_β2.7融合rna的质粒dna载体转染的hepg2细胞并在第3天监测gfp表达。h)流式细胞术数据的总结。显示了几何平均值、gfp阳性细胞的百分比和中位数。数据代表3次独立实验(*p<0.05,**p<0.01,***p<0.001,****p<0.0001,单因素方差分析,tukey多重比较检验和学生t检验)。i-k,用nls(+)或nls(-)gfp-β2.7rna融合构建体转染hepg2细胞,并在共聚焦显微镜图像中分析gfp表达和线粒体染色(mitotrackerred)的共定位,并使用imagej算法jacop:i,pearson系数;j,mander系数;和k,重叠系数,以三种不同的方式呈现。分析每种五个代表性图像(*p<0.05,**p<0.01,***p<0.001,****p<0.0001,双因素方差分析,tukey多重比较检验)。

图4显示β2.7rna介导的反义rna的递送可以触发mtatp6和mtatp8的敲低。a)由mfold预测的靶向线粒体基因mtatp6和mtatp8的计算选择的非结构化反义rna的最小自由能(mfe)二级结构。b)β2.7_反义融合rna和对照的设计。间隔子s6和s8确保反义rna结构域在与β2.7rna融合时保持开放。c)由mfold预测的asatp6_β2.7和β2.7_asatp8融合rna的mfe结构。d)在用β2.7_反义融合rna(asatp6或asatp8)或对照rna转染的hek293t细胞中使用rtrt-pcr测量的mtatp6或mtatp8mrna水平的敲低。数据代表3次独立实验的平均值±sd(*p<0.05,***p<0.001,单因素方差分析,dunnett多重比较检验)。e)使用celltitre-glo试验检测rna转染的hek293t细胞中atp水平的降低。数据代表3次独立实验的平均值+sd(*p<0.05,**p<0.05,***p<0.001,单因素方差分析,dunnett多重比较检验)。f,rna转染24小时后通过alamarblue细胞活力测定确定hek293t细胞的细胞活力。数据代表3次独立实验的平均值±sd(*p<0.05,**p<0.05,***p<0.001,单因素方差分析,dunnett多重比较检验)。

图5显示的结果显示β2.7rna亚结构域2和3的不同串联重复(tandemrepeats)显示增强的线粒体摄取并且可以保护线粒体复合物i。a)显示mfold预测的结构域2(d2x4)和b)结构域3(d3x4)的串联重复的mfe结构的示意图。间隔子s1至s4用于稳定串联重复构建体d2x4和d3x4中的亚结构域d2和d3的结构。d)显示了包含结构域2(d2x4)或3(d3x4)的四聚体重复、其组合(d3x4_d2x4或d2x4_d3x4)或结构域3/2重复((d3_d2)x4)的构建体的示意性、示例性表示。c)显示描绘中值ct比率的线图。将体外转录的rna连续稀释、逆转录并进行rtpcr以确定每种稀释度的ct比率。每条标准曲线有效检测到低至104个分子。e)显示柱状图,其描绘了通过rtrt-pcr测定的相对于转染的hepg2细胞中的亲本β2.7rna的结构域2或3单体或四聚体的线粒体摄取的相对摄取水平。f)显示柱状图,其描绘了通过rtrt-pcr测定的、转染的hepg2细胞中相对于亲本β2.7rna的不同串联重复rna的线粒体摄取的相对摄取水平。g)和h)显示了使用alamarblue细胞活力测定的细胞活力染色的结果,从而监测用体外转录的rna或质粒表达载体转染hek293t细胞后24小时(g))或48小时(h))用200μm鱼藤酮处理时对线粒体复合物i的保护。相对于未处理的对照将数据归一化(normalize)。e)至h)中显示的数据表示为3次独立实验的平均值加标准偏差(*p<0.05,**p<0.01,***p<0.001,****p<0.0001,单因素方差分析,tukey多重比较检验)。

图6显示了使用cmvβ2.7rna衍生的序列/结构(mβ2.7)用于重组核酸的线粒体递送的示意性实例。mβ2.7可以是全长β2.7rna、其结构性亚结构域、β2.7rna的重复。a)显示将mβ2.7融合至重组非编码或编码rna的5’端。b)显示将mβ2.7融合至重组非编码或编码rna的3’端。c)显示将mβ2.7通过互补碱基配对(即结合结构域)与双链dna(基因)结合。d)显示通过互补结合结构域(dna或rna)将mβ2.7连接至环状单链dna或重组线粒体基因组。e)显示通过互补结合结构域的mβ2.7的连接,如d)中所示,但ssdna被酶促转化为dsdna并被连接。f)显示了通过互补结合结构域的mβ2.7的连接,如d)中所示,但将两个或更多个mβ2.7rna连接至环状ssdna。

图7显示了用于本发明的pvax1载体的示意图。ndei和bcll位点用于将构建体克隆到pvax1载体中。

图8显示了用于产生单结构域缺失构建体的重叠延伸聚合酶链反应(oepcr)的示意图,其分两步进行。在第一步中,使用携带nhei位点的通用(common)正向引物和oe反向引物pcr扩增缺失结构域(δd)上游的区域,oe反向引物引入用于δd下游区域的引发位点。随后,使用oe正向引物和携带bamhi位点的通用反向引物pcr扩增δd下游的结构域,oe正向引物引入用于δd上游区域的引发位点。然后将两种pcr产物凝胶纯化并以等摩尔量混合在一起,然后用pcr重新扩增,其中oe引入的引发位点充当其各自对应部分的引物,从而得到具有所需缺失的全长构建体。

图9显示了概述单结构域构建体的合成和克隆策略示意图。设计正向和反向引物以分别引入bspei和hindiii位点。随后用bspei和hindiii消化pcr产物,纯化并使用bspei和hindiii位点将其克隆到pvax1载体中。

图10显示了用于测试线粒体gfp递送的不同构建体的示意图。为了促进线粒体特异性gfp表达,将基因组起始和终止密码子(分别)pcr修饰为线粒体特异性起始(ata)和终止密码子。

图11显示了pcr引入核定位序列(nls)的示意图。通过pcr,使用正向和反向引物从pebfp-nuc质粒pcr扩增nls序列,并插入终止密码子(aga)的上游。

图12显示了用于合成结构域2和结构域3串联重复序列的克隆策略示意图。首先使用pcr引物分4组来合成各种拷贝。由于特定串联重复的所有引物共享相同的结合区域,因此对于特定串联重复的所有组,tm是相同的。每组都有自己的一组间隔子,如图中的s1-s4所示。选择限制性位点使得一组3’端的位点与串联重复内的下一组的5’端的位点匹配。使用的限制性位点是nhei、ecori、kpni、agel和hindiii。pcr后,将产物进行pcr纯化,并用各自的限制性位点消化。然后将消化的pcr产物在等摩尔量的消化的载体骨架中混合,并用t4dna连接酶连接。

图13显示合成d3x4_d2x4以及d2x4_d3x4的策略示意图。从携带d2x4的pvax1载体pcr扩增结构域2串联重复(d2x4)。为了防止较短片段的扩增,设计引物使得引物的大部分结合区域位于载体骨架自身上。每个引物在两端引入hindiii位点。将pcr产物消化并在携带d3x4序列的pvax1载体的hindiii位点内重新连接。为了防止载体重新连接,用碱性磷酸酶将消化的载体去磷酸化。图13的第二部分显示了用于d2x4_d3x4的策略示意图:类似地,从携带d3x4的pvax1载体pcr扩增结构域3串联重复(d3x4)。使用的引物设计策略与上文概述的结构域2串联重复(d2x4)使用的相同。将pcr产物消化并在携带d2x4序列的去磷酸化pvax1载体的hindiii位点内重新连接。

图14显示了用于合成(d3_d2)x4的策略示意图。在第一步中,使用相同的限制性位点将d3x4的第一个d3克隆到d2x4的第一个重复位置。在第二步中,使用相同的限制性位点将d3x4的第三个d3克隆到d2x4的第三个位置。由此获得的步骤2产物接着使用在两端引入hindiii位点的引物pcr扩增。然后将pcr产物连接到步骤2产物的下游。

图15显示了用于分离反义rna靶向的序列的策略示意图。用基因特异性反向引物序列(atp6rv和atp8rv)逆转录线粒体rna以获得第一链atp6和atp8cdna池(pool)。随后,将这些池用作pcr的模板以获得代表靶元件的双链dna序列。

图16显示了用于合成atp6_β2.7的策略示意图。通过pcr重新扩增atp6靶序列以引入与β2.7_pvax1序列相反方向的限制性位点。将所得产物用nhei和bspel消化,然后连接到β2.7_pvax1载体骨架中以获得atp6_无间隔子_β2.7。通过用ndei和nhei消化筛选产物。随后通过2步巢式pcr将间隔序列引入该构建体的下游。在第一步中,用通用正向引物和引入一部分间隔序列(sf1)的反向引物pcr扩增β2.7(β2.7f)的片段。在下一步中,步骤1产物用与步骤i中相同的正向引物和反向引物进行pcr扩增,所述反向引物结合sf1并同时引入剩余的间隔序列(sf2)和hindiii位点用于克隆。这重构了间隔序列。将步骤2的pcr产物克隆到atp6_无间隔子_β2.7中以获得atp6_β2.7。

图17显示了用于产生β2.7_atp8构建体的策略示意图。用反向引物pcr扩增纯化的atp8靶序列,从而引入spei位点。同时,通过pcr从β2.7扩增片段,使得所得产物分别在5’端和3’端携带bamhi和hindiii位点。将β2.7pcr产物和atp8pcr产物分别用hindiii和spei单独消化,以等摩尔量混合并在22℃下连接4小时。spei位点可以通过2个碱基填充连接到hindiii位点,这有效地破坏了两个位点。连接后,从凝胶中纯化出正确尺寸的产物,其具有与反义atp8序列融合的β2.7片段。然后,pcr扩增该连接产物以引入hindiii位点并克隆回β2.7_pvax1载体骨架以产生完整序列。

图18显示mt-atp6反义串联重复融合rnaatp6_(d3)4_(d2)4在线粒体中定位并触发高效功能性mt-atp6敲低的数据和示意图。a)显示atp6_(d3)4_(d2)4融合rna(上图)和atp6_(d3)4_(d2)4mfe结构(下图)的示意图。b)显示体外转录rnaatp6_pvax、atp6_β2.7和atp6_(d3)4_(d2)4的琼脂糖凝胶电泳的结果。c)显示hek293t细胞的蛋白质印迹结果,所述hek293t细胞用反义融合rna转染。转染后24小时通过蛋白质印迹分析监测atp6蛋白的水平。mt-coxii(细胞色素c氧化酶多肽ii)用作线粒体负载对照。d)显示了描绘hek293t细胞中atp水平降低的柱状图,所述细胞用反义融合rna和对照rna转染。使用celltitre-glo试验在转染后24小时测定atp水平的降低。使用仅有细胞的对照来归一化数据集(dunnett多重比较检验)。e)显示等摩尔量的体外转录的荧光素-12尿嘧啶标记的rna的无溴化乙锭琼脂糖凝胶电泳的图像。f)提供柱形图,显示荧光素标记的rna的荧光强度对尿嘧啶比率。g)描绘了hek293t或hepg2(h))细胞的mander重叠系数,细胞用荧光素-12尿嘧啶标记的rna转染。使用imagej算法jacop从共焦显微镜图像定量荧光素-12尿嘧啶标记的rna与mitotracker-orange染色的线粒体的共定位。显示了由mander重叠系数表示的共定位。显示了hek293t(i))和hepg2(j))中相对于mander重叠系数调节的rna标记强度。五个图像用于分析(tukey多重比较检验)。数据代表每五个代表性图像的平均值±sd。使用单因素方差分析和dunnett多重比较检验测试g)和h)中数据的显著性。使用tukey多重比较检验测试i)和j)中数据的显著性。

图19显示用荧光素-12尿嘧啶标记的体外转录的rna(绿色)转染hek293t(a)或hepg2(b)细胞,用hoechst33342(蓝色)染色细胞核,用mitotracker(红色)染色线粒体。通过覆盖图像(overlayimage)中的黄色信号指示转染的rna与线粒体的共定位。

图20显示了串联重复rna的最小自由能(mfe)结构,即(d2)4、(d3)4、(d3)4_(d2)4、(d2)4_(d3)4和(d3_d2)4。这些结构正如mfold预测的。

术语的定义

术语“裸核酸”是指核酸(dna或rna),与非病毒或病毒载体相反,其不与任何其它化合物复合,不与组蛋白、蛋白质、脂质、糖、纳米颗粒结构、病毒衣壳或包膜复合,例如,在细胞间转移或用核酸序列转化细胞时出现的核酸。

术语“编码/非编码核酸序列”是指编码和非编码核酸序列两者。非编码核酸(例如rna或dna)是不翻译成蛋白质的核酸分子。相反,编码核酸分子是翻译成蛋白质的核酸分子。例如,当用于rna时,这些非编码rna也称为非蛋白质编码rna(npcrna)、非信使rna(nmrna)和功能性rna(frna)。例如,转录功能性非编码rna的dna序列通常称为rna基因。非编码rna基因的实例包括但不限于高丰度且功能重要的rna,例如转运rna(trna)、核糖体rna(rrna),以及rna诸如小核仁rna(snorna)、microrna、小干扰rna(sirnas)、反义rna(asrna)、小核rna(snrna或u-rna)、外来体/细胞外rna(exrna)、piwi-相互作用rna(pirna)、小cajal体rna基因(scarna)和长的非编码rna(ncrna或incrna),其包括例如但不限于x-无活性特异性转录物(xist)和hox转录物反义rna(hotair)的实例。在人类基因组内编码的非编码rna的数量是未知的;但是,转录组学和生物信息学研究表明存在数千种非编码rna。由于许多新鉴定的非编码rna尚未验证其功能,因此许多非编码rna可能是非功能性的。许多非编码rna也可能是非功能性的(通常称为“垃圾rna”),并且是假转录的结果。

术语“正义”和“反义”是指用于比较核酸分子(例如dna或rna)与其它核酸分子的极性的概念。取决于上下文,与核酸序列的常见5’-3’命名惯例相比,这些正义和反义分子可以指不同的分子。例如,在双链dna(dsdna)中,如果相同序列的rna形式翻译或可翻译成蛋白质,则dna的单链可称为正义链(或正(+)链)。该正dna链的互补链称为反义(或负(-)链)。不要将其与如上定义的编码和非编码核酸序列的概念混淆。例如,双链dna(dsdna)的两条互补链通常被划分为“正义”链和“反义”链。dna正义链看起来像信使rna(mrna),可用于读取预期的蛋白质代码;例如,正义dna中的atg可以对应于mrna中的aug密码子,编码氨基酸蛋氨酸。但是,dna正义链本身不用于通过细胞制造蛋白质。作为蛋白质编码来源的是dna反义链,因为,其具有与dna正义链互补的碱基,它被用作mrna的模板。由于转录产生与dna模板链互补的rna产物,因此mrna与dna反义链互补。mrna是用于翻译(蛋白质合成)的物质。在rna的实例中,反义rna是与内源mrna互补的rna序列(或转录物)。换言之,它是与rna的编码序列互补的非编码链。引入编码反义rna的转基因是例如用于阻断目标基因表达的技术。

具体实施方式

线粒体是在所有真核生物中发现的双膜结合细胞器,通常与所有活的真核细胞中的atp产生相关。但是,形成呼吸循环一部分的酶的缺陷导致例如线粒体相关疾病,由于线粒体基因组不易接近而难以治疗。因此,为了治疗与例如线粒体基因组中的缺陷相关的此类疾病,本公开内容鉴定了例如人巨细胞病毒(cmv)β2.7rna内的rna序列的亚结构域和组合,用于将rna靶向递送进入线粒体,利用人类巨细胞病毒β2.7rna的倾向性靶向和共定位到线粒体中。因此,本文公开了将重组编码rna线粒体递送到线粒体中,其导致例如重组线粒体基因表达。本文还公开了线粒体递送,例如,非编码反义rna递送进入线粒体,其触发线粒体基因表达的功能性敲低。鉴定的序列用于基因治疗,例如,抑制线粒体功能障碍,或恢复神经退行性疾病或其它线粒体相关疾病中的线粒体基因功能,或用于抗衰老目的。

结果显示人巨细胞病毒β2.7rna的5’末端部分,即所谓的p137rna,与线粒体复合物i共定位保护复合物i的活性,但是,功能性编码或非编码rna尚未使用β2.7rna序列递送。允许将重组核酸递送到线粒体中的载体系统的产生允许例如线粒体相关但不可治愈的人类疾病的遗传疗法。发现源自人巨细胞病毒的独特的非编码rna,即所谓的β2.7rna,定位于哺乳动物细胞的线粒体并与线粒体复合物i结合。因此,在一个实例中,如本文所公开的核酸递送系统包括rna,或dna,或其组合。在另一个实例中,核酸递送系统包括rna。在另一个实例中,核酸递送系统包括dna。

因此,在一个实例中,本发明涉及核酸递送构建体,该构建体包括人巨细胞病毒β2.7rna的至少一个正义或反义rna亚结构域,其中每个亚结构域能够定位在线粒体内。在另一个实例中,核酸递送构建体包括人巨细胞病毒β2.7rna的至少一个正义rna亚结构域。在另一个实例中,核酸递送构建体包括人巨细胞病毒β2.7rna的至少一个反义rna亚结构域。在又一个实例中,核酸递送构建体包括人巨细胞病毒β2.7rna的一个或多个正义或反义rna亚结构域。

如本文所公开的核酸递送构建体可以包括多个正义或反义rna亚结构域。在一个实例中,核酸递送构建体中的亚结构域的数目是,但不限于,1至10个亚结构域、5至15个亚结构域、12至22个亚结构域,或1、2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个亚结构域。换言之,根据本公开内容的核酸递送构建体包括1至10个rna序列、5至15个rna序列、12至22个rna序列,或1、2、3、4、5、6、7、8、9、10、11、12、13、14、15或16个rna序列。

本公开内容涉及靶向递送至线粒体的cmvβ2.7rna内的rna序列的鉴定。也就是说,当引入细胞时,人巨细胞病毒β2.7rna找到并进入线粒体,结果,在所述细胞的细胞质中未发现。对于人巨细胞病毒β2.7rna的每个rna亚结构域也可以这样说。鉴定的rna序列由四个热力学保守的结构性亚结构域(d1至d4)组成。从这些亚结构域构建串联重复和rna序列的组合。串联重复由功能相关结构域构建,例如结构域2(d2x4)和结构域3(d3x4),其中例如结构域3(d3x4)表现出增强的线粒体定位潜力。串联重复的组合构建为例如(d3x4_d2x4或d2x4_d3x4),其中(d3x4_d2x4)表现出最高的线粒体靶向潜力。结构域1和4在反义转录物上显示相似的结构,并且反义结构域as1和as4表现出巨大的线粒体定位潜力。将带有编码rna的cmvβ2.7rna衍生序列递送到线粒体中导致重组线粒体基因表达。例如,结构域d3和d2的双重四聚体(d3x4_d2x4)以比野生型β2.7rna更高的效率保护线粒体复合物i。

因此,在一个实例中,核酸序列如本文所公开,其中每个亚结构域能够定位在线粒体内但不定位于细胞质中。

本文公开了人巨细胞病毒p137rna的分离的rna序列,其是人巨细胞病毒β2.7rna的5’末端。人巨细胞病毒β2.7rna序列的这个5’末端分别包含四个热力学保守的结构性亚结构域,分别命名为d1至d4,每个亚结构域都能够靶向细胞的线粒体。因此,在一个实例中,如本文所公开的核酸递送构建体包括来自人巨细胞病毒β2.7rna的rna序列,其包括但不限于β2.7rna(seqidno:1或seqidno:2)、β2.7rna的结构域1(d1;seqidno:3或seqidno:7)、β2.7rna的结构域2(d2;seqidno:4或seqidno:8)、β2.7rna的结构域3(d3;seqidno:5或seqidno:9)、β2.7rna的结构域4(d4;seqidno:6或seqidno:10)及其组合。在一个实例中,核酸递送构建体包括如本文所公开的一种rna序列类型。rna序列的类型的实例包括但不限于正义rna、反义rna、信使rna(mrna)、转运rna(trna)、microrna(mirna)、小干扰rna(sirna)、小核仁rna(snrna)、piwi-相互作用rna(pirna)、trna衍生rna(tsrna)、小rdna衍生rna(srrna)、核糖体rna(rrna)、长非编码rna(incrna)、短发夹rna(shrna)和转运-信使rna(tmrna)。在一个实例中,核酸递送构建体包括正义rna。在另一个实例中,核酸递送构建体包括反义rna。在另一个实例中,核酸递送构建体包括正义和反义rna的组合。在另一个实例中,核酸递送构建体包括本文公开的rna序列的组合。在又一个实例中,核酸递送构建体包括如本文公开的rna序列的组合,其中核酸递送构建体可包括单个rna序列的多个重复。在另一个实例中,核酸递送构建体包括β2.7rna的全长序列(seqidno:1(正义))。在另一个实例中,核酸递送构建体包括β2.7rna的全长序列(seqidno:2(反义))。在又一个实例中,核酸递送构建体包括β2.7rna的结构域1(seqidno:3(正义))。在又一个实例中,核酸递送构建体包括β2.7rna的结构域1(seqidno:7(反义))。在另一个实例中,核酸递送构建体包括β2.7rna的结构域2(seqidno:4(正义))。在一个实例中,核酸递送构建体包括β2.7rna的结构域2(seqidno:8(反义))。在另一个实例中,核酸递送构建体包括β2.7rna的结构域3(seqidno:5(正义))。在又一个实例中,核酸递送构建体包括β2.7rna的结构域3(seqidno:9(反义))。在另一个实例中,核酸递送构建体包括β2.7rna的结构域4(seqidno:6(正义))。在一个实例中,核酸递送构建体包括β2.7rna的结构域4(seqidno:10(反义))。在另一个实例中,核酸递送构建体包括β2.7rna的结构域2、结构域3和结构域4(seqidno:11(正义)或seqidno:39(反义))。在另一个实例中,核酸递送构建体包括β2.7rna的结构域1、结构域3和结构域4(seqidno:12(正义)或seqidno:40(反义))。在另一个实例中,核酸递送构建体包括β2.7rna的结构域1、结构域2和结构域4(seqidno:13(正义)或seqidno:41(反义))。在另一个实例中,核酸递送构建体包括β2.7rna的结构域1、结构域2和结构域3(seqidno:14(正义)或seqidno:42(反义))。

如本文所用的,术语“序列同一性”是指两条多核苷酸或氨基酸序列相同或比较窗口上具有许多相同残基(即,在核苷酸-核苷酸或残基-残基的基础上)的情况。通过比较比较窗口上的两个最佳对齐序列,确定两个序列中相同核酸碱基(例如,a、t、c、g、u或i)或残基出现的位置数,以产生匹配位置的数量,将匹配位置的数量除以比较窗口中的位置总数(即窗口大小),并将结果乘以100得到序列同一性的百分比,来计算术语“序列同一性的百分比”。如本文所用的,术语“序列同一性”表示多核苷酸或氨基酸序列的特征,其中多核苷酸或氨基酸可包括具有至少85%序列同一性,优选至少90%至95%序列同一性,更通常的是,至少99%的序列同一性的序列,与至少18个核苷酸(6个氨基酸)位置的比较窗口上的参考序列相比,通常在至少24至48个核苷酸(8至16个氨基酸)位置的窗口上进行比较,其中序列同一性的百分比通过将参考序列与该序列比较来计算,可包括缺失或添加,其在比较窗口上总共为参考序列的20%或更少。参考序列可以是较大序列的子集。因此,在另一个实例中,如本文所公开的核酸递送系统包括来自人巨细胞病毒β2.7rna的rna序列,其中该rna序列具有本文公开的一种或多种rna序列的70%至99%、75%至85%、78%至88%、80%至89%、约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%或约99%的序列同一性。在一个实例中,核酸递送构建体如本文所公开,其中来自人巨细胞病毒β2.7rna的rna序列具有一种或多种rna序列的约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%或约99%的序列同一性,所述一种或多种rna序列包括但不限于β2.7rna(seqidno:1或seqidno:2)、β2.7rna的结构域1(d1;seqidno:3或seqidno:7)、β2.7rna的结构域2(d2;seqidno:4或seqidno:8)、β2.7rna的结构域3(d3;seqidno:5或seqidno:9)、β2.7rna的结构域(d4;seqidno:6或seqidno:10)及其组合。

如本文所公开的核酸递送构建体还可以进一步包括核酸序列中的一个或多个改变。在一个实例中,这种改变是rna序列中的突变。在另一个实例中,核酸递送构建体包括一个或多个非结构突变。在又一个实例中,核酸递送构建体包括一个或多个结构中性突变。如本文所用的,术语“非结构突变”或“结构中性突变”是指核酸序列中的突变,其改变核酸序列的序列,但与未突变的序列相比,突变序列的功能性结构得到保留。

从这些亚结构域构建rna序列的串联重复和组合。如本文所用的,术语“串联重复”是指核酸序列内的部分,其中重复一个或多个核苷酸的模式并且这些重复直接彼此相邻。例如,连续重复3次atggc序列,因此产生包含atggcatggcatggc的序列,应理解为串联重复。基于本文公开的发明,序列由功能性rna结构域构建,也就是来自任何亚结构域d1、d2、d3或d4。这种构建的结构域的实例是,但不限于,结构域2(结构域2的四次重复,换言之d2x4)和结构域3(结构域3的四次重复,换言之d3x4)。因此,在一个实例中,本文公开的核酸递送系统包括本文公开的rna序列的组合和/或倍数,包括但不限于两次重复(2)、三次重复(3)、四次重复(4)、五次重复(5)、六次重复(6)、七次重复(7)、八次重复(8)或更长的单个结构域的重复。换言之,本文公开的rna序列的组合和/或倍数包括但不限于二聚体、三聚体、四聚体或单结构域的聚合物。

如本文所用的,术语“间隔子”是指在构建体内插入核酸序列的5’或3’端或核酸序列的两端的核酸序列。将间隔子插入核酸序列内的限定位置,以确保保留核酸序列(例如rna序列)的结构完整性。这确保了rna序列的功能或特征的保留,例如核酸构建体的线粒体靶向能力。间隔子还起到防止任何空间效应发生的作用,并使核酸序列能够达到其天然的三级结构,从而也有助于保持其功能。因此,在一个实例中,如本文中公开的核酸递送构建体包括1至10个、5至15个、8至24个、至少一个、至少两个、至少三个、至少四个、至少5个、约6个、约7个、约8个或约9个间隔序列。在一个实例中,核酸递送构建体包括约6个间隔序列。在另一个实例中,核酸递送构建体包括约7个间隔序列。在又一个实例中,核酸递送构建体包括约8个间隔序列。在另一个实例中,核酸递送构建体包括约9个间隔序列。

如上所述,间隔序列可以置于核酸序列内的任何位置。例如,可以在rna序列的开头(即5’端)找到间隔子。也可以在rna序列的结尾(即3’端)找到间隔子。当使用多于一个间隔子时,也可以在rna序列的5’和3’两端发现这些间隔子。因此,在一个实例中,当核酸递送构建体包括至少两个或更多个间隔子时,至少一个间隔序列位于人巨细胞病毒β2.7rna的rna序列的5’端,并且至少一个其它间隔序列位于人巨细胞病毒β2.7rna的rna序列的3’端。

间隔子的长度例如通过间隔子旨在实现的特定功能来定义。例如,如果间隔子的功能是为了防止两个或更多个rna序列之间的空间位阻,则该间隔子可以在数十至数百个核苷酸长之间,这取决于所得rna结构的大小。换言之,本发明中公开的间隔序列足够长以防止在相邻rna亚结构域之间产生任何空间位阻和/或其中间隔序列的长度足够长以允许相邻rna亚结构域折叠成其热力学优选的结构。尽管如此,本领域技术人员将理解,间隔子长度取决于如本文公开的核酸递送系统中公开的至少一个正义或反义rna亚结构域的长度、结构和组合。在一个实例中,间隔序列长度为5至40个核苷酸、5至30个核苷酸、6至10个核苷酸、8至14个核苷酸、15至20个核苷酸、22至28个核苷酸、25至37个、28至39个核苷酸、约7个核苷酸、约9个核苷酸、约11个核苷酸、约12个核苷酸、约13个核苷酸、约15个核苷酸、约17个核苷酸、约21个核苷酸、约27个核苷酸、约29个核苷酸、约30个核苷酸、约34个核苷酸、约36个核苷酸。在一个实例中,间隔序列长5个核苷酸。在另一个实例中,间隔序列长6个核苷酸。在又一个实例中,间隔序列长13个核苷酸。在另一个实例中,间隔序列长17个核苷酸。在一个实例中,间隔序列长24个核苷酸,而另一个实例,间隔序列长32个核苷酸。

在一个实例中,间隔序列是但不限于sla(seqidno:26)、s1b(seqidno:27),s2a(seqidno:28)、s2b(seqidno:29)、s3a(seqidno:30)、s3b(seqidno:31)、s4a(seqidno:32)、s4b(seqidno:33)、s6a(seqidno:34)、s6b(seqidno:35)、s8a(seqidno:36)、s8b(seqidno:37)和间隔子f3a(seqidno:38),及其组合。

间隔序列还可以含有功能性核酸序列,或其它结构或功能基序(motif)。例如,间隔序列可以进一步任选地包括终止密码子。

如本文所用的,术语“核定位信号”或“nls”是指编码一种或多种另外的分泌信号或信号肽的核酸序列。可以将这些核定位序列添加到核酸序列的5’或3’端,从而导致在肽的c-末端或n-末端或c-和n-两末端的这种核定位序列的表达。本领域已知的一个实例是添加核定位序列(nls),其指导新生蛋白质从细胞质进入细胞核。存在许多不同版本的核定位序列,它们的长度和组成取决于它们被分离的细胞类型。例如,核质素(nucleoplasmin)的核定位序列是“avkrpaatkkagqakkkkld”,而c-myc的核定位序列是“paakrvkld”。因此,在一个实例中,核酸递送构建体进一步任选地包括核定位信号(nls)。

在一个实例中,本文公开的核酸递送系统是根据式i的rna序列:

其中每个x独立地是但不限于d1(seqidno:3或seqidno:7)、d2(seqidno:4或seqidno:8)、d3(seqidno:5或seqidno:9)、d4(seqidno:6或seqidno:10)或其组合,包括两次重复、三次重复、四次重复、五次重复、六次重复、七次重复、八次重复或更长的单个结构域的重复;并且其中每个x任选在其之前或之后或侧接至少一个或多个如本文所定义的间隔序列。换言之,在一个实例中,核酸递送系统是二聚体,其中每个x独立地是但不限于d1(seqidno:3或seqidno:7)、d2(seqidno:4或seqidno:8)、d3(seqidno:5或seqidno:9)、d4(seqidno:6或seqidno:10)或其组合;并且其中每个x任选在其之前或之后或侧接至少一个或多个如本文所定义的间隔序列。

在一个实例中,本文公开的核酸递送系统是根据式ii的rna序列:

其中每个x独立地是但不限于d1(seqidno:3或seqidno:7)、d2(seqidno:4或seqidno:8)、d3(seqidno:5或seqidno:9)、d4(seqidno:6或seqidno:10),或其组合,包括两次重复、三次重复、四次重复、五次重复、六次重复、七次重复、八次重复或更长的单个结构域的重复;其中间隔序列s1a、s1b、s2a、s2b、s3a、s3b、s4a和s4b如本文所公开的。在一个实例中,x是d2(seqidno:4或seqidno:8)。在另一个实例中,x是d3(seqidno:5或seqidno:9)。在又一个实例中,根据式ii的核酸递送构建体包括根据seqidno:15或seqidno:51的核酸序列。在另一个实例中,根据式ii的核酸递送构建体包括根据seqidno:16或seqidno:52的核酸序列。换言之,在一个实例中,核酸递送系统是四聚体,其中每个x独立地是但不限于d1(seqidno:3或seqidno:7)、d2(seqidno:4或seqidno:8)、d3(seqidno:5或seqidno:9)、d4(seqidno:6或seqidno:10)或其组合;并且其中每个x任选在其之前或之后或侧接至少一个或多个如本文所定义的间隔序列。

在一个实例中,本文公开的核酸递送系统是根据式iii的rna序列:

其中x和y彼此不同,其中每个x和每个y独立地是但不限于d1(seqidno:3或seqidno:7)、d2(seqidno:4或seqidno:8)、d3(seqidno:5或seqidno:9)、d4(seqidno:6或seqidno:10)或其组合,包括两次重复、三次重复、四次重复、五次重复、六次重复、七次重复、八次重复或更长的单个结构域的重复;其中间隔序列s1a、s1b、s2a、s2b、s3a、s3b、s4a和s4b如本文所定义的。在一个实例中,x是d3(seqidno:5或seqidno:9),y是d2(seqidno:4或seqidno:8)。在另一个实例中,x是d2(seqidno:4或seqidno:8),y是d3(seqidno:5或seqidno:9)。在另一个实例中,根据式iii的核酸递送构建体包括根据seqidno:17或seqidno:53的核酸序列。在另一个实例中,根据式iii的核酸递送构建体包括根据seqidno:18的核酸序列。换言之,在一个实例中,核酸递送系统是四聚体,其中每个x和y独立地是但不限于d1(seqidno:3或seqidno:7)、d2(seqidno:4或seqidno:8)、d3(seqidno:5或seqidno:9)、d4(seqidno:6或seqidno:10)或其组合;并且其中每个x和y任选地在其之前或之后或侧接至少一个或多个如本文所定义的间隔序列。

在一个实例中,本文公开的核酸递送系统是根据式iv的rna序列的八聚体:

其中x和y彼此不同,其中每个x和每个y独立地是但不限于d1(seqidno:3或seqidno:7)、d2(seqidno:4或seqidno:8)、d3(seqidno:5或seqidno:9)、d4(seqidno:6或seqidno:10)或其组合,包括两次重复、三次重复、四次重复、五次重复、六次重复、七次重复、八次重复或更长的单个结构域的重复;其中间隔序列s1a、s1b、s2a、s2b、s3a、s3b、s4a和s4b如本文所定义的。在一个实例中,x是d3(seqidno:5或seqidno:9),y是d2(seqidno:4或seqidno:8)。在另一个实例中,根据式iii的核酸递送构建体包括根据seqidno:19的核酸序列。

为了要求保护的核酸递送系统起递送系统的作用,核酸序列需要进一步包括有效负载。如本文所用的,术语“有效负载”是指可以插入核酸递送系统的序列中的一个或多个核酸序列,其由于其插入而作用于细胞的线粒体上或内部。有效负载可以是但不限于重组核酸序列、rna、dna、修饰的核酸、核酸类似物和核酸模拟物,包括吡喃糖基(pyranosyl)核酸(p-rna)、苏糖核酸(tna)、乙二醇核酸(gna)、肽核酸(pna)、丙氨酸核酸(ana)、锁核酸(lna)、吗啉代氨基磷酸酯(morpholinophosphoramidate)(mf)、非核酸基分子,包括肽、蛋白质、脂质、碳水化合物、合成聚合物、小分子量化合物等。在一个实例中,重组核酸序列是但不限于非编码核酸序列、编码核酸序列、单链核酸序列、线性双链核酸序列、反义核酸序列、正义核酸序列环状单链核酸序列和环状双链核酸序列。在另一个实例中,重组核酸序列是完整的、天然的或重组的线粒体基因组。在又一个实例中,核酸递送构建体包括根据seqidno:20至seqidno:82中任一个的序列。

此外,所述有效负载需要附着到核酸递送系统以便能够被输送。在一个实例中,有效负载与核酸递送系统共价连接。在另一个实例中,有效负载与核酸递送系统非共价连接。非共价连接可以通过以下实现:静电相互作用,包括离子相互作用,氢键,或卤素键合,通过范德华力,包括偶极-偶极相互作用,诱导偶极相互作用,或伦敦色散力,通过π效应,包括π-π相互作用,阳离子-或阴离子-π相互作用,或极性-π相互作用,或通过疏水作用。另一方面,共价连接是涉及原子间电子对共享的连接。共价键或连接的实例包括多种相互作用,包括但不限于σ-键合、π-键合、金属-金属键合、元结效应(agosticinteraction)、弯键和三中心双电子键。

类似于管理例如有效负载运输的航空概念的物理概念,本领域技术人员将理解,有效负载的大小决定了携带这种有效负载到达其预定目的地所需的载体(即核酸递送系统)的大小。因此,在一个实例中,核酸递送构建体的总大小与有效负载的大小成比例。这意味着用于例如长度为200个核酸的有效负载的核酸递送系统将是仅有50个核酸长的有效负载的核酸递送系统的四倍大。相反,仅10个核酸长的有效负载可以利用核酸递送系统,该核酸递送系统为用于20个核酸长的有效负载的核酸递送系统的一半大小。因此,在一个实例中,核酸递送系统是可缩放的。在另一个实例中,核酸递送系统可根据有效负载的大小或与其成比例地缩放。

研究了最活跃的rna((d3)4_(d2)4)将针对mt-atp6的反义rna共递送到线粒体中的潜力。为此,通过间隔子将mt-atp6反义rna与(d3)4_(d2)4的5’端融合,从而产生构建体atp6_(d3)4_(d2)4。选择间隔子以根据mfold的预测保留反义rna的开放结构以及(d3)4_(d2)4内的结构域结构(图18a)。用体外转录的rna转染hek293t细胞,并在转染后24小时使用蛋白质印迹监测蛋白质水平的靶基因敲低(图18b、c)。与atp6_β2.7rna相比,atp6_(d3)4_(d2)4rna触发了mt-atp6蛋白的显著更强的敲低,导致细胞atp水平降低2.1倍(图18d)。该数据表明线粒体靶向是可缩放的并且不受靶向载体长度增加的损害的限制。为了直接证明线粒体rna靶向,在体外转录期间用荧光素-12-尿嘧啶标记rna,并且使用琼脂糖凝胶电泳评估rna的完整性和标记效率(图18e)。使用imagejv1.48软件量化条带强度(图19),并计算强度对尿嘧啶计数或长度比(图18f)。所有rna的这些比率相当,表明相似的标记效率。随后,用标记的rna转染hek293t或hepg2细胞,并用mitotrackerorange染色线粒体(图19)。mander重叠系数(moc)被确定为标记的rna和线粒体的共定位度量(图18g、h)。与β2.7-阴性rna相比,所有β2.7-rna/-结构域嵌合体显示出显著水平的共定位。当比较hek293t(图18i)或hepg2细胞(图18j)中rna标记、强度调整的mander重叠系数时,与含有rna的β2.7-rna相比,具有(d3)4_(d2)4串联重复结构的rna显示出显著更强的共定位效应。

本公开内容还包括载体、重组细胞、重组生物体和核酸序列,其包括或表达如本文公开的核酸递送系统。在一个实例中,载体包括本文公开的核酸递送系统。在另一个实例中,载体包括裸核酸,或非病毒载体,或病毒载体,或其组合。

在一个实例中,重组细胞包含本文公开的核酸序列。在另一个实例中,重组细胞以连续(consecutive)方式(即,连续地)表达核酸序列。在又一个实例中,重组细胞以非连续方式(即,非连续地)表达核酸序列。在一个实例中,重组生物体包含本文公开的核酸序列。在另一个实例中,重组生物体以连续方式(即,连续地)表达核酸序列。在又一个实例中,重组生物体以非连续方式(即,非连续地)表达核酸序列。

在一个实例中,核酸序列包括人巨细胞病毒β2.7rna的至少一个或多个正义或反义rna序列。在另一个实例中,人巨细胞病毒β2.7rna的rna序列是但不限于结构域1(d1;seqidno:3或7)、结构域2(d2;seqidno:4或8)、结构域3(d3;seqidno:5或9)和结构域4(d4;seqidno:6或10)。

在本发明范围内还公开了治疗疾病的方法。线粒体病症通常由缺陷线粒体dna(mdna)的不均等分离(segregation)导致的异质性引起。因此,一种治疗方法是减少缺陷信使rna(mrna)的丰度,从而允许野生型信使rna重新填充线粒体。因此,鉴定的序列可用于基因治疗以抑制线粒体功能障碍,或恢复神经退行性疾病或其它线粒体相关疾病中的线粒体基因功能,或用于抗衰老目的。

亲本人巨细胞病毒β2.7rna可以保护线粒体复合物i免受某些抑制剂的影响,从而保护线粒体免受氧化应激和dna损伤,从而增加细胞活力。还发现亲本人巨细胞病毒β2.7rna可防止脑中多巴胺能神经元的死亡。大脑中多巴胺能神经元的死亡被认为是帕金森病的标志。已经表明,例如,短的100个核苷酸长的亚结构域(β2.7rna的结构域2)成功地保护线粒体复合物i至与亲本β2.7rna序列相似的程度。因此,在本文公开的其它结构域中,结构域2可以在帕金森病的治疗中实施。术语亲本序列是指源自人巨细胞病毒毒株towne(genbank:fj616285.1)的原始β2.7rna序列。

细胞穿透肽可用于成功地将β2.7衍生的序列递送到例如人和动物模型的肺组织中。例如,该递送用于治疗受损的氧化磷酸化(oxphos),并且在另一个实例中,增加与慢性阻塞性肺病(copd)相关的活性氧物质(ros)水平。另外,反义rna可用于靶向肺中的遗传性线粒体缺陷。

β2.7rna或其亚结构域也可用于传递完整的线粒体基因组,用于治疗线粒体dna(mdna)缺失的疾病,如卡恩斯-塞尔综合征(keams-sayresyndrome)(kss)、皮尔逊综合征(pearsonsyndrome)和进行性眼肌麻痹(peo),所有这些病共有重叠表型,并且与线粒体dna内共同的4977碱基对缺失相关。

因此,在一个实例中,公开了一种治疗线粒体病症的方法。在另一个实例中,治疗线粒体病症的方法包括向受试者施用本文公开的核酸递送构建体。在又一个实例中,该方法包括使用核酸递送构建体递送反义rna。线粒体病症可以是但不限于母系遗传性糖尿病、leber遗传性视神经病变(lhon)、神经病变、共济失调、色素性视网膜炎、肌阵挛性癫痫伴破碎红纤维(myoclonicepilepsywithraggedredfibers)(merrf)、线粒体肌病脑病伴乳酸性酸中毒和中风样症状(mitochondrialmyopathyencephalopathylacticacidosisandstrokelikesymptoms)(melas)、帕金森病、慢性阻塞性肺病(copd)、卡恩斯-塞尔综合征(kss)、皮尔逊综合征和进行性眼肌麻痹(peo)。

所述线粒体病症可以以各种方式治疗,例如,通过靶向一个或多个线粒体基因,其是(但不限于)mt-tl1(trna亮氨酸)、mt-nd1、mt-nd4、mt-nd6、mt-atp6、mt-tk(trna赖氨酸)、mt-nd1、mt-nd5、mt-th(组氨酸)、mt-tl1(亮氨酸)、mt-tv(缬氨酸)及其组合。

治疗线粒体病症的一个实例是反义介导的对缺陷线粒体基因/基因功能的抑制。这涉及,例如,使用如本文公开的核酸递送系统将反义rna递送到线粒体中。例如,在本申请中已经证明了人巨细胞病毒β2.7介导的反义rna递送成功地敲低线粒体内的mt-atp6mrna的能力。mt-atp6的缺陷与例如但不限于神经病变、共济失调和色素性视网膜炎(narp)有关。在narp的实例中,β2.7介导的反义rna递送系统己被用于成功敲低与narp相关的缺陷mrna,从而允许野生型atp6mrna重新填充线粒体。因此,本文公开的核酸系统可用于治疗narp。

要求保护的发明如何用于治疗线粒体病症的另一个实例是线粒体基因的递送。作为使用反义rna抑制缺陷mrna的替代,本文公开的核酸递送系统可用于将完整(线粒体)基因递送到线粒体中以增加完整mrna与缺陷rna的比率。下面提供了靶基因及其相关疾病的非穷尽列表。

表1:靶基因和相关疾病的列表。

mt-tl1:线粒体编码的trna亮氨酸;mt-tv:线粒体编码的trna缬氨酸;mt-tk:线粒体编码的trna赖氨酸;mt-th:线粒体编码的trna组氨酸;mt-nd1:线粒体编码的nadh脱氢酶1;mt-nd4:线粒体编码的nadh脱氢酶1;mt-nd5:线粒体编码的nadh脱氢酶5;mt-nd6:线粒体编码的nadh脱氢酶6;mt-atp6:线粒体编码的atp合成酶6。

治疗线粒体病症的另一个实例是抑制缺陷线粒体基因功能和将完整基因递送到线粒体中以增加完整和缺陷基因之间的比例的组合。

如本文所公开的,本发明的治疗应用可以是递送反义序列以抑制缺陷基因表达,或者,可以递送完整基因以补充正确的基因功能,或两者的组合。

因此,在一个实例中,公开了增强线粒体基因功能或抑制有缺陷的线粒体基因功能或两者(假设在这种情况下,线粒体基因彼此不同)的方法。在增强线粒体基因功能并抑制缺陷线粒体基因功能的情况下,基因功能的增强和抑制可以同时或按顺序发生。在一个实例中,基因功能的增强和抑制同时发生。在另一个实例中,线粒体基因功能彼此不同。在又一个实例中,所述方法包括向受试者施用本文公开的核酸递送序列。

例如,结构域3(d3x4)表现出增强的线粒体定位潜力。串联重复的组合构建为例如d3x4_d2x4或d2x4_d3x4,由此显示d3x4_d2x4表现出最高的线粒体靶向潜力。进一步显示,结构域1和4的反义转录物显示相似的结构,并且反义结构域as1和as4表现出巨大的线粒体定位潜力。

将具有编码rna的cmvβ2.7rna衍生序列递送到线粒体中导致重组线粒体基因表达。例如,结构域d3和d2的双重四聚体(表示为d3x4_d2x4)以比例如野生型β2.7rna更高的效率保护线粒体复合物i。

使用计算方法,鉴定了β2.7rna内的四个热力学保守的结构性亚结构域。所有四个结构域都显示出显著的线粒体定位,并且显示全长β2.7rna的完整线粒体定位活性也可以通过例如使用称为结构域3的单个亚结构域来实现。此外,四个结构域中的两个(例如,结构域1和4)在反义转录物上显示高度相似的结构,并且反义结构域as1和as4表现出显著的线粒体定位潜力。发现例如正义结构域3的四聚体具有高两倍的线粒体定位活性,并且在另一个实例中,结构域3的四聚体后接着是结构域2的四聚体,其表现出例如比β2.7rna或结构域3高三倍的活性。β2.7rna衍生的序列用于将重组核酸递送到线粒体中以触发线粒体特异性表型:首先,在一个实例中,编码rna具有线粒体特异性起始和终止密码子,导致线粒体特异性重组基因表达;其次,靶向线粒体特异性基因的反义rna用于触发线粒体特异性基因表达的功能性敲低。因此,该技术可用于线粒体基因治疗,或例如用于非核酸化合物的线粒体递送。

作为使用所要求保护的发明的实例,一个示例性方法涉及将带有编码rna的cmvβ2.7rna衍生序列递送到线粒体中,其进而导致重组线粒体基因表达。将cmvβ2.7rna衍生序列(例如,带有反义rna)递送到线粒体中触发线粒体基因表达的功能性敲低。这种递送构建体的一个实例是β2.7rna亚结构域3的四聚体重复,其已经显示出表现了增强的线粒体定位潜力。例如β2.7rna亚结构域3和2的两个四聚体重复(d3x4_d2x4)的示例性排列,其表现出高线粒体靶向潜力。示例性的结构域d3和d2的双四聚体(d3x4_d2x4)表现出比野生型β2.7rna更高的保护线粒体复合物i的效率。概述方法的示例性应用是在遗传治疗中,例如,抑制线粒体功能障碍,或者在另一个实例中,恢复线粒体基因功能。应用实例还包括用于神经退行性疾病或其它线粒体相关疾病或用于抗衰老。

使用所要求保护的发明的另一个实例包括将要求保护的核酸递送系统与crispr/cas技术一起使用,即使用要求保护的核酸递送系统递送编码cas9核酸内切酶的mrna和单一导向(sg)rna,或用于递送编码这些组分的各个基因。然后,cas9酶与sgrna一起形成可以特异性裂解和功能性灭活缺陷线粒体基因或基因组的核糖核蛋白复合物。

另一个应用是提供基于质粒的线粒体靶向载体。可以将用于编码或非编码rna转录的目的序列插入线粒体靶向序列的上游或下游。然后可以在体外从dna模板转录嵌合rna,然后将其递送到靶细胞中,或在用dna载体转染靶细胞后内源性转录嵌合rna。还设想了用于产生病毒递送颗粒的这些载体的变体,例如但不限于慢病毒、腺病毒、腺相关病毒。

本文说明性描述的发明可以在缺少本文未具体公开的任何元素或多个元素、限制或多个限制的情况下适当地实施。因此,例如,术语“包括”、“包含”、“含有”等应当被广泛地阅读而不受限制。另外,本文使用的术语和表达被用作描述而非限制的术语,并且没有意图使用这样的术语和表达来排除所显示和描述的特征的任何等同物或其部分,但是认识到在本发明所要求保护的范围内可以进行各种修改。因此,应该理解,尽管已经通过优选实施例和可选特征具体公开了本发明,但是本领域技术人员可以对本文公开的发明进行修改和变化,并且这些修改和变化是被认为是在本发明的范围内。

在此广泛和一般地描述了本发明。落入一般公开内容中的每个较窄种类和亚属组群也构成本发明的一部分。这包括本发明的附带条件或从属中去除任何主题的否定限制的一般描述,无论是否在本文中具体叙述了被切除的材料。

其它实施方式在以下权利要求和非限制性实施例内。另外,在根据马库什群组描述本发明的特征或方面的情况下,本领域技术人员将认识到,本发明也因此以马库什群组的任何单个成员或成员子群描述。

贯穿本公开内容,某些实施方式可以以范围格式公开。应当理解,范围格式的描述仅仅是为了方便和简洁,并且不应该被解释为对所公开范围的范围的不能转变的限制。因此,应该认为范围的描述具体公开了所有可能的子范围以及范围内的各个数值。例如,应当认为例如1至6的范围的描述具体公开了子范围,例如1至3、1至4、1至5、2至4、2至6、3至6等,以及范围内的各个数值,例如,1、2、3、4、5和6。无论范围的宽度如何,这都适用。

实验部分

用cmvβ2.7rna鉴定热力学保守的结构性亚结构域

使用软件foldsplit鉴定了cmv的非编码β2.7rna内的四个热力学保守的结构性亚结构域(分别称为d1至d4)(图1b)。热力学保守性经常与rna功能相关。可以将这四个结构域中的每一个指定为对完整β2.7rna预测的rna二级结构内明确定义的结构性亚结构域(图1c)。

cmvβ2.7rna及其不同的功能性亚结构域定位于人细胞的线粒体

例如,调查了(i)全长β2.7rna的线粒体定位、(ii)四个单结构域d1至d4中的每一个的线粒体定位,和(iii)单结构域缺失构建体的线粒体定位(图1d-f)。虽然所有缺失突变体和四个单结构域中的三个显示减少的线粒体靶向,但结构域3显示出全长β2.7rna的完整线粒体靶向潜力。

不同的cmvβ2.7rna反义结构域定位于人细胞的线粒体

本文公开的构建体的反义序列被考虑用作负对照,因此也在其结构和热力学保守性方面进行了研究。出乎意料的是,与正义β2.7rna的结构相比,反义β2.7rna的结构高度对称。观察到高度相似的热力学保守性,并且可以在β2.7rna反义序列内的相应位置鉴定四个保守的结构性亚结构域(d1_as至d4_as)(图2a)。详细的结构分析特别地揭示,反义结构域d1_as和d4_as在结构上几乎与相应的正义结构域d1和d4相同。调查了(i)全长反义β2.7rna的线粒体定位,(ii)四个单反义结构域d1_as至d4_as中的每一个的线粒体定位,和(iii)反义单结构域缺失构建体的线粒体定位(图2b-d)。与正义rna相比,全长反义β2.7rna显示出显著较弱的线粒体摄取(图2b),并且四个反义结构域d1_as至d4_as中的每一个的缺失进一步降低了线粒体靶向(图2c)。当测试单个反义结构域时,显示反义结构域d1_as和d4_as表现出比全长反义rna高约5倍的活性,达到正义全长β2.7rna的线粒体靶向活性的约70%。(图2d)。

cmvβ2.7rna将重组编码rna共递送到线粒体中,导致重组蛋白的线粒体表达

通过间隔序列将β2.7rna全长rna与egfpmrna的3’端融合,确保β2.7rna的活性结构在与egfp序列融合时不会发生变化(图3a、b)。考虑了两种不同版本的egfpmrna:1.常规mrna,其配备有通常的(regular)起始和终止密码子用于细胞质表达;和2.配备有线粒体起始和终止密码子的修饰版本,其只能在细胞的线粒体中翻译而不能在细胞质中翻译。此外,产生了一种线粒体egfp序列的版本,其中egfp蛋白与核定位肽融合,使得任何到达细胞质的egfp蛋白都将靶向细胞核,以抑制任何来自细胞质和线粒体的重叠egfp信号。然后使用实时rt-pcr测试所有序列和对照的线粒体靶向(图3c)或使用流式细胞术(facs)和共聚焦显微镜(图3d-j)测试所有序列和对照的egfp表达。β2.7rna的线粒体靶向没有因将其与egfpmrna融合而受到阻碍(图3c)。facs分析表明单独的线粒体egfp序列(mtgfp_s)不会触发任何egfp表达。显著egfp表达(必须源自线粒体)能够在将其融合至β2.7rna(mtgfp_s_β2.7)后被检测到(图3g)。共聚焦成像的灵敏度不足以显示egfp和线粒体特异性染色(mitotrackerred)的共定位。但是,三种不同的共定位系数表明在mtgfp_s_β2.7的情况下egfp和mitotrackerred的显著共定位,但对于mtgfp_s对照没有(图3h-j)。

cmvβ2.7rna将反义rna共递送到线粒体中,导致抑制重组蛋白的线粒体表达

接下来,将β2.7rna与计算选择的靶向线粒体基因的非结构化反义rna融合,所述基因例如mt-atp6和mt-atp8,它们都参与线粒体atp合成(图4)。通过间隔子将反义rna与β2.7rna融合,以确保在融合过程中维持两种序列(反义和β2.7rna)的活性结构。然后使用rtrt-pcr来测量线粒体rna靶向(图4e),测量线粒体atp合成的敲低(图4f)和由于atp水平降低导致的细胞活力降低(图4g)。融合反义rna的β2.7rna靶向线粒体,触发线粒体mt-atp6或mt-atp8mrna水平的敲低,并显著降低细胞活力。

β2.7rna亚结构域3的四聚体重复表现出增强的线粒体定位潜力

目的在于改善β2.7rna的线粒体靶向潜力,例如通过使用多个重复的最有活性的β2.7rna亚结构域(在一个实例中是d3和d2)。构建结构域d3或d2的四聚体重复,并使用rtrt-pcr研究线粒体靶向(图5a-f)。虽然d2四聚体的线粒体靶向与全长β2.7rna的线粒体靶向相当,但d3四聚体显示出两倍高的线粒体靶向活性。

β2.7rna亚结构域3和2的两个四聚体重复序列的排列(d3x4_d2x4)表现出最高的线粒体靶向潜力

接下来测试了构建体,所述构建体包括(i)d3的四个重复,后接d2的四个重复,(d3x4_d2x4),或(ii)d2的四个重复,后接d3的四个重复,(d2x4_d3x4),或(iii)交替的结构域d3和d2[(d3_d2)x4]。虽然d2x4_d3x4和(d3_d2)x4表现出与全长β2.7rna可比的降低的线粒体靶向活性,但发现构建体d3x4_d2x4的活性约为3倍(图5g)。

结构域d3和d2的双重四聚体(d3x4_d2x4)保护线粒体复合物i的效率高于野生型β2.7rna

早先报道了β2.7rna的5’末端部分保护线粒体复合物i免受诸如鱼藤酮的抑制剂的影响。研究了单独的结构域d3或d2或它们的多聚体排列是否能够在递送例如体外合成的rna或者内源性表达rna的质粒时保护复合物i免受鱼藤酮的影响(图5h、i)。虽然d2、d3、d3x4和d3x4_d2x4rna在直接递送rna时的保护程度与全长β2.7rna一样,但发现d3x4_d2x4在递送rna表达质粒dna时更具活性。

使用线粒体靶向序列将重组核酸递送到线粒体中的策略

重组rna或dna可以与线粒体靶向序列共价连接,或者通过互补碱基配对非共价连接。重组核酸可以是单链、线性双链、环状单链或环状双链。一个或多个线粒体靶向序列可用于形成一种线粒体靶向复合物。(图6)

材料和方法

设计亲本构建体

设计构建体的目的是将其克隆到pvax1(invitrogen)和pegfp-cl(addgene)载体中以分别研究结构域特征和转基因递送。使用在线算法nebcutterv2.0选择限制性位点。构建体由geneart(lifetechnologies)合成。随后将构建体克隆到pvax1载体中。cmv启动子序列获自pvax1质粒载体。cmv启动子促进rna在动物细胞中的表达。t7启动子促进亲本构建体的体外转录,而sp6启动子被倒置以促进反义rna序列的体外转录。对于t7转录,hindiii用于线性化质粒,对于sp6转录,bspe1用于线性化质粒。nhei、xbai和bcli位点用于克隆。添加sv40polya信号以促进mrna的多聚腺苷酸化和核输出。间隔子代表卡那霉素启动子和polya信号之间的载体序列,kanap是启动子的一部分,其在使用bcli克隆期间首先从载体中除去。纳入draiii以促进克隆到pegfp-c1载体中(参见图7)。

合成单结构域缺失构建体(sddc)

为了研究单个结构域对β2.7rna功能的影响,产生了缺失单个结构域的构建体。如图8所示,用于缺失结构域的策略是重叠延伸pcr。

合成sddc的重叠延伸(oe)pcr策略

重叠延伸聚合酶链反应(oepcr)分两步进行。在第一步中,使用携带nhei位点的通用正向引物和oe反向引物pcr扩增缺失结构域(dd)的上游区域,oe反向引物引入dd下游区域的引发位点。随后,使用oe正向引物(其引入dd上游区域的引发位点)和通用的反向引物(其携带bamh1位点)pcr扩增dd下游的结构域。然后将两种pcr产物凝胶纯化并以等摩尔量混合在一起,然后使用pcr重新扩增,其中oe引入的引发位点充当其各自对应部分的引物,从而得到具有所需缺失的全长构建体。

合成单结构域构建体(sdc)

从亲本β2.7_pvax1pcr扩增单结构域。每个单结构域的pcr条件如下:单结构域1(sdl_pvax1):使用β2.7_pvax1作为模板使用d1f和d1r进行pcr,然后使用bspel和hindiii位点克隆pcr产物。pcr条件反应混合物包括10ng模板、5μl的10xtaq缓冲液、4mm的mgc12、每种dntp各0.2mm、每种引物各300nm和0.25μl的taq聚合酶(thermoscientific)。用超纯无核酸酶的水(invitrogen)将最终体积补足至50μl。所用的反应条件是94℃5分钟,25个循环的94℃30秒,56℃30秒,72℃30秒,72℃5分钟。单结构域2(sd2_pvax1)、单结构域3(sd3_pvax1)和单结构域4(sd4_pvax1)构建体以相同的方式产生,但是使用特定的pcr引物d2f和d2r、d3f和d3r或d4f和d4r代替(参见图9)。

单结构域扩增策略

设计正向和反向引物以分别引入bspei和hindiii位点。随后用bspei和hindiii消化pcr产物并纯化,并使用bspei和hindiii位点克隆到pvaxl载体中。

合成gfp融合构建体

设计gfp融合构建体:使用pegfp-c1(addgene)质粒载体设计所有gfp融合构建体。使用限制性位点bspei和draiii将β2.7rna序列克隆到质粒中egfp序列的下游。接下来,从质粒中pcr扩增egfp序列,以在任一端引入限制性位点以及间隔序列。为了实现线粒体特异性基因表达,用线粒体特异性起始和终止密码子修饰egfp信息的起始和终止密码子。由于gfp表达的分析将使用质粒载体进行,因此主要使用mfold分析cmv转录物的结构的结构性保存。pcr条件描述如下:建立50μlpcr反应,其具有10ng的pegfp-c1模板、5mm的10xtaq缓冲液、4mm的mgcl2、每种引物300nm、每种dntp0.2mm和0.25μl的taq聚合酶(thermoscientific)。将循环仪设定在94℃5分钟,25个循环的94℃30秒,55℃30秒,72℃1分钟,72℃10分钟。从β2.7_pvaxl载体切下具有sv40polya的亲本β2.7序列,凝胶纯化并使用如图10中所示的bspei和draiii位点连接到egfp序列的下游。

上述克隆过程产生基因组gfp+β2.7。第二个对照即基因组gfp+间隔子+β2.7是通过pcr使用pcr引物扩增来自原始pegfp-c1载体的egfp序列而产生的,其中反向引物引入间隔序列和nhei位点,之后将其克隆回基因组gfp+β2.7载体中。为了实现线粒体特异性表达,使用pcr引物修饰egfp序列的起始和终止密码子,正向引物引入mt起始密码子,反向引物引入间隔序列和线粒体特异性终止密码子,之后使用nhei位点将其克隆回携带β2.7的pegfp-cl载体中以产生mtgfp+间隔子+β2.7。在这两种情况下,使用单个nhei限制性位点将gfp序列克隆到载体中,因此为了防止载体骨架重新连接,在用nhei消化期间同时将其用碱性磷酸酶去磷酸化。作为阴性对照,使用分别携带ndei和bamhi位点的引物,从mtgfp+间隔子+β2.7质粒pcr扩增具有cmv启动子的一部分的mtgfp+间隔序列。同时,在ndei位点和bamhi位点之间消化pegfp-c1质粒载体以除去原始gfp序列和cmv启动子的一部分。然后用ndei和bamhi消化pcr产物,纯化并连接回凝胶纯化的载体骨架(其重构cmv启动子并用mtgfp+间隔序列取代egfp序列)。

将sv40核定位信号添加到gfp融合构建体中

为了通过共聚焦显微镜促进成像,从pebfp_nuc(addgene)pcr扩增sv40核定位信号(nls)并克隆到所有gfp表达构建体(包括线粒体构建体)中gfp序列的下游。nls在框架中携带线粒体特异性终止密码子,因此,只有细胞质gfp靶向细胞核,而线粒体内gfp仍然位于线粒体中。这是因为线粒体中的翻译产生了不完整的nls。pebfp-nuc质粒携带nls序列的3个串联重复序列,因此设计pcr引物以侧接串联重复序列。pcr扩增和设计策略在图中解释。克隆后,使用单一xhoi消化筛选构建体。pcr条件如下。使用pebfp_nuc作为模板,使用nls_cloningfw和nls_cloningrv进行pcr,然后将样品进行pcr纯化并克隆到bsp1407i和bspei位点之间的gfp序列的下游。使用的pcr条件如下:10ng的pebfp-nuc模板、5mm的taq缓冲液、4mm的mgc12、每种dntp2mm、分别为300nm的nls_cloningfw和nls_cloningrv、0.25μl的taq聚合酶(thermoscientific),用超纯无核酸酶的水(invitrogen)将体积补成50μl。使用的循环条件是94℃5分钟,25个循环的94℃30秒,52℃30秒,72℃5分钟。

使用分别引入bsrgi和bspei位点的正向和反向引物,通过pcr从pebfp-nuc质粒pcr扩增nls序列(参见图11)。此外,正向引物还通过单个t-g取代破坏了nls上游的额外bspei位点。然后分别用bsrgi和bspei消化pcr产物,并连接到所有构建体gfp序列的下游。pcr产物携带xhoi位点,其用于通过消化筛选克隆。

合成串联重复构建体

为了研究两个功能结构域的结构域串联重复的相互作用,在该实施例中,即产生了结构域2和3。下面产生了四种不同的排列。

构建体分别具有结构域3(d3x4)和结构域2(d2x4)的四个拷贝

产生构建体的策略如图12所示。使用nebcutterv2.0选择限制性位点。使用间隔序列稳定这些串联重复序列的t7和cmv转录物的结构,并使用rnafold和mfold进行验证。使用pcr引物(4组)合成每种重复序列及其相关的间隔子,该pcr引物插入间隔子和相应的限制性位点,然后克隆到pvax1载体中。由于相同串联重复内的每组引物具有相同的结合区域,因此对于特定的串联重复,所有pcr组的退火温度保持相同。pcr中使用的循环条件在下面提供。

结构域2串联重复

使用多重连接过程构建串联重复。

首先使用pcr引物合成4组各个拷贝(参见图12)。由于特定串联重复的所有引物共享相同的结合区域,因此对于特定串联重复,所有组的tm是相同的。每组具有其自己的一组间隔子,如图中的s1-s4所示。选择限制性位点使得串联重复内的一组的3’端位点与下一组的5’端位点匹配。pcr后,将产物进行pcr纯化,并用其各自的限制性位点消化。同时用nhei和hindiii消化pvax1载体,凝胶纯化载体骨架。然后将消化的pcr产物与等摩尔量的消化的载体骨架混合,并用t4dna连接酶连接。

d3x4_d2x4的构建策略

类似地,从携带d2x4的pvax1载体pcr扩增结构域2串联重复(d2x4)(参见图13)。为了防止较短片段的扩增,设计引物使得引物的大部分结合区域位于载体骨架本身上。每个引物在任一端引入hindiii位点。将pcr产物消化并在携带d3x4序列的pvax1载体的hindiii位点内重新连接。为了防止载体重新连接,用碱性磷酸酶将载体去磷酸化。

d2x4_d3x4的构建策略

类似地,从携带d3x4的pvax1载体pcr扩增结构域3串联重复(d3x4)。采用的引物设计策略与d3x4_d2x4使用的相同。将pcr产物消化并在携带d2x4序列的去磷酸化pvax1载体的hindiii位点内重新连接。

每个(d3_d2)4具有结构域3和结构域2的交替重复的构建体

交替重复构建体分四步产生。d3x4和d2x4共享同一组限制性位点。在克隆之前,使用mfold和rnafold验证cmv和t7转录物。详细的克隆方案描述于图14中。

在第一步中,使用nhei和ecori位点克隆结构域3的单个拷贝。在第二步中,在kpni和agei位点之间克隆另一个结构域3的拷贝。如此获得的产物现在用作使用结构域3串联fw和结构域2串联rv(两者都带有hindiii位点)的pcr模板。将正确尺寸的pcr产物凝胶纯化,并连接回携带步骤2产物的去磷酸化pvax1质粒中。然后通过消化筛选克隆并通过测序验证(aitbiotech)。

用于线粒体靶向的反义rna的设计

选择靶标和选择候选物

使用的线粒体靶向的候选物是mt-atp6和mt-atp8,它们基本上是复合物v(atp酶)的亚基。靶向这两个基因的反义rna使用husar折叠分析设计,窗口大小为100、200和300,并且移位为1个核苷酸,随后选择在5’或3’端具有最大未配对碱基数的候选物。随后基于开放端的位置将这些序列与β2.7rna的5’端或3’端融合,并使用mfold分析结构保存。

纯化靶序列

使用下图中描述的程序从分离的线粒体rna获得靶序列。逆转录的反应条件如下:将500ng的mtrna与1μl的10mm的dntp混合物、1μ1的2um基因特异性反向引物混合,用无rnase的水补足至终体积14μl。将混合物在65℃加热5分钟,然后在冰上快速冷却2分钟。然后将该反应混合物与4μl的5x第一链缓冲液、1μl的100mm的dtt、20u的rnaseout(invitrogen)、0.5μl的superscriptiv混合,反应的最终体积为20μl,并在55℃孵育1小时,然后在70℃下热灭活15分钟。

分离用于产生反义序列的靶序列

用基因特异性反向引物序列(atp6rv和atp8rv)逆转录线粒体rna以获得第一链atp6和atp8cdna池。随后将这些池用作pcr的模板以获得代表靶元件的双链dna序列(参见图15)。

克隆靶序列以产生反义序列

克隆atp6反义序列

由于atp6反义序列在5’端具有最大数目的未配对碱基,因此必须与β2.7序列的5’端融合。为了获得反义序列,将从线粒体rna池获得的靶序列逆转并克隆到β2.7_pvax1载体中,处于t7启动子下游和β2.7序列的上游。使用atp6_cloning_fw和atp6_cloning_rv对纯化的atp6靶进行pcr扩增。克隆策略如图16所示。

atp6反义序列的合成和克隆

通过pcr重新扩增atp6靶序列以引入与β2.7_pvax1序列相反方向的限制性位点。用nhei和bspei消化所得产物,然后连接到β2.7_pvax1载体骨架中。通过用ndei和nhei消化筛选产物。随后通过巢式pcr将间隔序列引入该构建体的下游,然后通过测序验证(aitbiotech)。

克隆atp8反义序列

将atp8靶序列克隆到β2.7序列的下游,因为未配对的碱基主要在3’端。通过巢式pcr扩增atp8靶标以引入所需的限制性位点和稳定化间隔序列,并克隆到β2.7序列的下游(参见图17)。

产生β2.7_atp8融合构建体的克隆策略

用反向引物pcr扩增纯化的atp8靶序列引入spei位点。同时,通过pcr从β2.7扩增片段,使得所得产物分别在5’端和3’端携带bamhi和hindiii位点。将β2.7pcr产物和atp8pcr产物分别用hindii和spei单一消化,以等摩尔量混合,并使用t4dna连接酶(thermoscientific)在22℃连接3小时。spei位点可以通过2碱基填充连接到hindiii位点,这有效地破坏了两个限制性位点。连接后,从凝胶中纯化出正确尺寸的产物,该产物具有与反义atp8序列融合的β2.7片段。将该连接产物进行pcr扩增,分别引入bamhi和hindiii位点,并克隆回β2.7_pvax1载体骨架中以产生完整序列。通过bamhi和hindiii双重消化验证克隆,然后通过测序验证(aitbiotech)。

通过体外转录合成rna

使用t7(thennoscientific)和sp6rna聚合酶(thermoscientific)进行体外转录。sp6聚合酶用于合成反义rna和β2.7_gfprna。使用t7rna聚合酶合成剩余的rna。如前所述分别选择t7和sp6启动子的最后6个碱基,并最终分别成为t7和sp6转录物的一部分。

制备体外转录的模板

将用于体外转录的所有质粒载体用苯酚∶氯仿∶异戊醇(25∶24∶1)提取过夜,并在第二天用乙醇沉淀。通过pcr或通过质粒线性化制备用于体外转录的模板。用于质粒线性化的所有限制酶产生5’突出端以防止形成失控转录物。在pcr合成的模板的情况下,正向引物引入t7/sp6启动子的序列用于体外转录。通过pcr纯化试剂盒(qiagen)纯化pcr模板和线性化质粒。

体外转录(ivt)

使用t7/sp6rna聚合酶(thermoscientific)进行ivt。将1ug的线性化的质粒/纯化的pcr模板与1x转录缓冲液、10mm的ntp混合物、20u的rnaseout(invitrogen)和30u的sp6/t7rna聚合酶一起在37℃孵育2小时,最终体积为50μl。孵育2小时后,将3u的dnasei加入到反应混合物中并在37℃下孵育至少30分钟。然后通过苯酚氯仿提取纯化rna。

转染

所有转染均使用lipofectamine2000(invitrogen)在opti-mem(gibco)中进行。

转染hepg2细胞用于分析rna摄取

hepg2细胞在t75烧瓶含有抗生素的dmem中生长,并在90%汇合度(confluency)时转染。转染在opti-mem(gibco)中进行。根据制造商的方案转染lug当量的β2.7rna及其衍生物。转染后6小时更换培养基。转染后24小时,分离线粒体rna用于通过实时pcr分析。

转染hek293t细胞用于分析线粒体rna敲低

将105的293thek293t细胞在具有抗生素的dmem的24孔板中培养,并在第二天转染。转染在opti-mem(gibco)中进行。按照制造商的方案转染800ng当量的反义_β2.7融合rna。转染后6小时更换培养基。转染后24小时,分离总rna并通过实时pcr分析。

转染hepg2细胞用于facs分析和共聚焦显微镜

对于facs分析,将30,000个hepg2细胞在24孔板中培养,并在30%汇合度时转染。转染在opti-mem(gibco)中进行。按照制造商的方案转染800ng对照pegfp-c1和β2.7_gfp融合质粒(+nls)。转染后6小时更换培养基。转染后24小时,用胰蛋白酶处理细胞并使用流式细胞术分析gfp表达。对于共聚焦显微镜分析,将20,000个hepg2细胞接种在1.5um腔室载玻片(chamberslides)(ibio)中。载玻片中腔室的面积与48孔板中孔的面积相同,相应进行转染。按照制造商的方案转染400ng对照pegfp_c1和β2.7_gfp融合质粒(+nls)。转染后24小时,用相应的染料染色细胞并使用共聚焦显微镜分析。

转染hek293t用于细胞活力分析

将50000个hek293t细胞接种在24孔板中。向每个孔中加入800ng当量的β2.7rna,并使用先前从未处理的hek293t细胞分离的rna将总rna/孔调节至800ng。6小时后更换培养基。将105个hek293t细胞接种在24孔板中,并用1.5μg的β2.7rna_反义融合rna转染。由于先前分离的rna可含有靶序列,因此使用饲养(feeder)rna(酵母trna)代替分离的rna将总rna/孔调节至1.5μg。随后在转染后6小时更换培养基。

转染hek293t用于celltiter-glo测定

将25000个hek293t细胞接种在96孔板中,并用300ng当量的β2.7rna_反义rna转染。使用饲养rna调节每孔的rna量。转染后6小时更换培养基。

rna分离和实时pcr

线粒体rna的分离

根据制造商的指南,使用线粒体分离试剂盒(biochain)从hepg2细胞中分离线粒体。将分离的线粒体重悬于1x线粒体分离缓冲液中。为了去除污染的细胞质rna,如前所述用rnasea(thermoscientific)处理线粒体悬浮液。孵育后,通过加入2x体积的trizol试剂使rnasea失活,然后根据制造商的指南使用trizol试剂(invitrogen)提取rna。

总rna的分离

接种在24孔板中的hek293t细胞用1xpbs洗涤。随后,加入200μltrizol试剂。将24孔板中的混合物重新悬浮直至溶液失去粘度。然后将组分转移到eppendorf管中,并按照制造商的指南分离rna。

dnase处理和cdna合成

将500ng分离的rna(线粒体/总)与ludnasei(thermoscientific)和20u的rnaseout在37℃孵育30分钟。孵育后,加入乙二胺四乙酸(edta)至终浓度为3.75mm,并在75c孵育12分钟以使dnasei失活。然后用1xrt缓冲液、5.5mm的mgcl2、20u的rnaseout、每种dntp500μm、200ng的随机引物(invitrogen)和25u的multiscribe逆转录酶(abi)在终体积20μl,37℃下保持2小时逆转录该反应混合物,然后在85℃下加热灭活15分钟。

实时pcr

将1μ1的cdna与5μ1的2xsybrcfx主混合物(mastermix)和400nm各(正向和反向)引物混合。在abi7900ht实时pcr仪中使用以下热循环条件进行实时pcr:50℃2分钟,95℃10分钟,40个循环的95℃15秒和60℃60秒。每个样品都进行重复操作。

相对量化

12srrna用作归一化的内部对照。使用δδct方法测定相对rna水平。通过与未处理的线粒体样品比较来确定细胞质污染物β-肌动蛋白的水平。

绝对量化

将体外转录的rna(待转染的rna)从1010个分子至1001个分子作连续稀释,逆转录并如上所述进行实时pcr。通过将中值ct值对每个反应的分子数作图来制备rna标准曲线。通过使用sds2.4软件与各标准曲线比较,确定每种rna的拷贝数(分离的rna每微克(μg)的分子数)。

流式细胞术分析gfp表达

样品制备

将接种在24孔板中的hepg2细胞用胰蛋白酶消化,然后通过加入完全dmem使胰蛋白酶失活。将dmem中的细胞重悬浮8至10次(times)直至无细胞团块,随后在离心机中以6000g沉淀5分钟。然后用1xpbs洗涤细胞并重悬于完全dmem中用于流式细胞术分析。

流式细胞术分析

使用beckmanncoultercyanadp流式细胞仪进行流式细胞术分析。用488nm激光照射细胞,并使用正向(fsc-a)和侧向散射(ssc-a)进行设门(gate),并使用fsc脉冲宽度分析进行双重排除。使用510nm至540nm带通滤波器测量gfp表达。在转染后第1、3和5天测量了多达20,000个细胞。使用flowjo7.6.1分析数据。

共聚焦显微镜

将细胞在腔室载玻片中转染并在转染后第3天进行分析。根据制造商的指南,用hoechst33342(molecularprobes)和/或mitotrackerorangecmh2tmros(molecularprobes)对细胞染色。然后用hcscellmaskdeepredstain(molecularprobes)对细胞进行复染色。使用olympusfluoviewfv1000(olympus,japan)激光扫描共聚焦显微镜拍摄图像,使用60x/1.00水物镜,405nm固态激光二极管(hoechst),488nm氩离子激光(gfp),543nmhenegreen激光(mitoctracker)和633nmhenered(cellmask)。随后使用imagejv1.48分析图像。

鱼藤酮诱导细胞死亡

在无水dmso(sigma)中制备200mm鱼藤酮储备溶液。在完全dmem中将鱼藤酮储备溶液稀释至终浓度200um,并使用0.22um过滤器过滤。转染后24小时,将鱼藤酮_dmem施用于hek293t细胞,并使用alamarblue试验在转染后24、48和72小时测定细胞死亡。细胞死亡评估后,用pbs洗涤细胞,并施用新鲜药物用于在随后的时间点进行分析。

alamarblue细胞活力测定

根据制造商的指南,将接种在24孔板中的hek293t细胞进行alamarblue(invitrogen)细胞活力测定。使用bioteksynergyhi读板器在发射/激发(530/590)下测量荧光,灵敏度设定为60。通过与仅细胞的对照比较来确定细胞活力降低的百分比(%)。

测量atp水平

将hek293t细胞在96孔板中转染,并使用发光细胞活力测定法(promega)在转染后24小时测定atp水平。使用tecaninfinitem200pro读板器测量发光。根据制造商的指南制备的atp标准曲线测定绝对atp水平。通过与仅细胞的对照比较来确定atp水平的相对变化。

统计分析

使用graphpadprismsoftware6.0进行统计分析。所有数值均表示为三次独立实验的平均值±sd(平均值加标准偏差)。使用学生氏t检验和anova确定统计学显著性。

产生构建体atp6_(d3)4_(d2)4

将稳定化间隔序列插入最终d2重复序列下游的(d2)4中,得到(d2)4_s构建体。用在两端引入hindiii位点的引物pcr扩增(d2)4_s。随后将pcr产物用hindiii消化并克隆到hindiii位点内以产生atp6_(d3)4_(d2)4构建体。

荧光素-12尿嘧啶-标记的rna的体外转录(ivt)

通过t7rna聚合酶(thermoscientifictm)使用荧光素-12-utp(enzo)通过体外转录合成荧光rna。将1μg线性化质粒/纯化的pcr模板与5μl的5x转录缓冲液、5μl的10mm的ntp混合物(10mmgtp、10mmctp、10mmatp、7.5mmutp、2.5mm荧光素-12utp)、i0urnaseouttm(invitrogen)和20u的t7rna聚合酶,终体积为25μl,在37℃一起孵育3小时。将3u的dnasei加入到反应混合物中并在37℃孵育至少30分钟以除去dna模板。然后使用pcr纯化试剂盒(qiagen)纯化rna。在不含溴化乙锭的1.5%琼脂糖凝胶上分析rna的质量。在uv透射照明器上照射凝胶并使用samsunggalaxys7智能手机拍摄。使用imagejv1.48测定条带的强度。

共聚焦显微镜

将细胞在腔室载玻片中转染并在转染后24小时进行分析。根据制造商的指南,用hoechst33342(molecularprobes)、mitotrackerorangecmh2tmros(molecularprobes)对细胞染色。使用olympusfluoviewfv1000(olympus,japan)激光扫描共聚焦显微镜拍摄图像,使用60x/1.00水物镜,405nm固态激光二极管(hoechst),488nm氩离子激光(gfp),543nmhenegreen激光(mitotracker)。随后使用imagejv1.48分析图像。使用插件jacop确定gfp(绿色)在线粒体(红色)部分内的共定位程度,并报告了mander重叠系数(moc)。

表格

表2:呈现在所附序列表中的序列概览。“as”代表反义;“endo”代表内源转录的rna。这些序列作为质粒进行测试。“_core”代表核心序列,其是可操作的最小多核苷酸序列(没有间隔子)。

表3:序列。测试以下序列1至26和44至47作为体外转录的rna,使用线性化的dna(以切割位点结束的rna)或pct产物(以pcr模板的末端结束的rna)作为dna模板。作为启动子,使用t7rna(rna以ggcgcu开始)或sp6(rna以ggaguc开始)启动子。在6个转录的启动子核苷酸之后,存在或不存在限制性位点。

使用cmv启动子和sv40polya位点将以下序列作为质粒dna转染。因此,内源性转录的rna全都以ucagaucc开始(该ucagaucc是cmv启动子的转录核苷酸,后面接着是切割位点),它们全都以uuuuuuucacugc(a)n结束。粗体c表示两个polya切割位点,后接polya延伸(a)n。所以序列的正确末端可能是..uuuuuuucacugc(a)n或...u(a)n。

序列表

<110>新加坡国立大学

<120>重组核酸的线粒体递送

<130>10104sg1156

<160>87

<170>patentinversion3.5

<210>1

<211>2512

<212>rna

<213>人工序列

<220>

<223>全长β2.7rna(正义)

<400>1

ggcgcuuccggaagagcuagcuccccagaucgcugcugccccggcguucuccagaagccc60

cggcgggcgaaucggccggcuggucggucggcgcucggacggauggggagaacggcggug120

acuuagccgcccguggccgggagaagauggaggagccgagaugacaacggcagucgugga180

agggucgccaagccccgguccuucucuucugucuggucgaaucucguuuucuuuuuucaa240

ccgcucuuuuuaucaccuuuuuaugugaguuucucuuccgcgucucccggccguaccauc300

cacccaugcagcaugcacgcguguauguaugcaucgucucuccuccgucccgacuaccau360

cagcagcaccacuaccgccacccccagcgccaccaccgcugccgucgccaccgcguuauc420

cguuccucguaggcugguccuggggaacgggucggcggccggucggcuucuguuuuauua480

uuuuuuuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccua540

ccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaaagcuacagacauuug600

gguaccucagcuuuccgauaacucgaagaauucaaagucgacgauucccaacaagagaaa660

acagaacaaaaacaaggucauuuuuauuuauccucaucgucaacaacaacuaccgacaac720

aacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccug780

ucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguc840

cgguccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaauc900

auaggaaaugaaccagaagaacaaaaagaucaucucucucgguguauagcaacaccaaca960

acaaccgcaucgcaacaucuucauccgcaagacggaaagaaaacaacaauaaugagaaug1020

aaaucaccacaaccaagccagauuucacguccaugaguuuuuauuauauuauuaucaaaa1080

cgaaaaacagaaaaacugucauagauaaauauaaaaaaaaauagaaaccacaaacgacua1140

cuaguacuccaaucuuagauguauaugcuccuagauaagauuuaguauuaccauaaucau1200

cgaagaaugaaagacgacgaugauuccuuaccgcuccugccacccggucuguauguagag1260

agagaagagagaaaacggugaauccaagauccccgggucggcgucggcaugccgcugauc1320

gcaguggccccaccucggcaugccggcgccgggcgaggaauugcucaugaaaaaaguauc1380

uuucuguaaaaaaagaaaacaauacaugauuaaccgaaaagaaaccaacaaaaagaaccc1440

gagaucagucgauuucgaucacuacgauaaacacauggaagauuucuugaaaaaagaaaa1500

gagaaagagaccaccuucccggcggcggacacgcuccucuccgucgccguucugcaccau1560

gauucgaucaauaacaacaucaucaucggagaccaucuuuuaaucaaucagcguugcagu1620

agucgacucccuggacacgaaggagucauccauuuuuauccucgcacuucuucgcucuca1680

aagccgccuuuaaaguugaaaugaaaggauggaaacauggaauacaguuuuaauugcacg1740

uaucaccauuuuacuacaaaaagaaaaaaaaacaacuuacacauaguauuaccuuagguu1800

uacggauaaguagaguguaggcguuuuugaaacaguucagccaaugcaaucuugucucgg1860

cauaaucacucuuucugcauauaauaguaguaguagauuuauucacaucaacacagcgaa1920

aaacuccagcaucaaaguacaccuagagacagcccuuaaaauauaguuugcagcuuuuag1980

auguacuuacaccaaagaagauuaccguccuuacgagaaaacagauacucggauauagga2040

aucaagacagcucugcacugaaaacacacucuccugucacgacaccgcgccacaccagag2100

gcguacgcgugacuucaucgcaacgauccaucgugaugucccucgcagaaccuaaaaaga2160

ccaaaaaaaaaucuuggaccacaguugucgauucuugaagacaauauucucgugagaacu2220

uugagauucgcacuugaaaccucuuaggauccacaaaaacaacaaccucuguauggaaaa2280

ugcgcuauuuuaucucagcuuuucucccaaaccucgguuucuuccuauucuuaaguuuuc2340

ccuaguauauuugccuccuuauaagaaaagaagcacaagcucggucgcacggauuauucc2400

uucugcuaaucuauuauuuuguuccuuuuuuuuuuguuugccuucacccccuucacuccc2460

uguagcaacacagaguaguagacacaauaaaugagaaguuugcaugcaagcu2512

<210>2

<211>2511

<212>rna

<213>人工序列

<220>

<223>全长β2.7rna(反义)

<400>2

ggagucaagcuugcaugcaaacuucucauuuauugugucuacuacucuguguugcuacag60

ggagugaagggggugaaggcaaacaaaaaaaaaaggaacaaaauaauagauuagcagaag120

gaauaauccgugcgaccgagcuugugcuucuuuucuuauaaggaggcaaauauacuaggg180

aaaacuuaagaauaggaagaaaccgagguuugggagaaaagcugagauaaaauagcgcau240

uuuccauacagagguuguuguuuuuguggauccuaagagguuucaagugcgaaucucaaa300

guucucacgagaauauugucuucaagaaucgacaacugugguccaagauuuuuuuuuggu360

cuuuuuagguucugcgagggacaucacgauggaucguugcgaugaagucacgcguacgcc420

ucugguguggcgcggugucgugacaggagaguguguuuucagugcagagcugucuugauu480

ccuauauccgaguaucuguuuucucguaaggacgguaaucuucuuugguguaaguacauc540

uaaaagcugcaaacuauauuuuaagggcugucucuagguguacuuugaugcuggaguuuu600

ucgcuguguugaugugaauaaaucuacuacuacuauuauaugcagaaagagugauuaugc660

cgagacaagauugcauuggcugaacuguuucaaaaacgccuacacucuacuuauccguaa720

accuaagguaauacuauguguaaguuguuuuuuuuucuuuuuguaguaaaauggugauac780

gugcaauuaaaacuguauuccauguuuccauccuuucauuucaacuuuaaaggcggcuuu840

gagagcgaagaagugcgaggauaaaaauggaugacuccuucguguccagggagucgacua900

cugcaacgcugauugauuaaaagauggucuccgaugaugauguuguuauugaucgaauca960

uggugcagaacggcgacggagaggagcguguccgccgccgggaagguggucucuuucucu1020

uuucuuuuuucaagaaaucuuccauguguuuaucguagugaucgaaaucgacugaucucg1080

gguucuuuuuguugguuucuuuucgguuaaucauguauuguuuucuuuuuuuacagaaag1140

auacuuuuuucaugagcaauuccucgcccggcgccggcaugccgagguggggccacugcg1200

aucagcggcaugccgacgccgacccggggaucuuggauucaccguuuucucucuucucuc1260

ucuacauacagaccggguggcaggagcgguaaggaaucaucgucgucuuucauucuucga1320

ugauuaugguaauacuaaaucuuaucuaggagcauauacaucuaagauuggaguacuagu1380

agucguuugugguuucuauuuuuuuuuauauuuaucuaugacaguuuuucuguuuuucgu1440

uuugauaauaauauaauaaaaacucauggacgugaaaucuggcuugguuguggugauuuc1500

auucucauuauuguuguuuucuuuccgucuugcggaugaagauguugcgaugcgguuguu1560

guugguguugcuauacaccgagagagaugaucuuuuuguucuucugguucauuuccuaug1620

auuguuuggcugcugaccgacgcgucaggaugugcagggcaugcggggaaucaggaccgg1680

acacgggauaauuucaucuaccuauacggagaucgcgguccucgccaugaggaucgcgac1740

aggcgcgucgagggggcaggaacacccuugcggauugacauucuuggugguguuucguug1800

uugucgguaguuguuguugacgaugaggauaaauaaaaaugaccuuguuuuuguucuguu1860

uucucuuguugggaaucgucgacuuugaauucuucgaguuaucggaaagcugagguaccc1920

aaaugucuguagcuuuuuucuuuuuacccucuuguuuaucaucugcgauucgugguaggu1980

aggagagggaaaugauaauccgagauuaaggaaaggagaagauaaaaaauaaaaaaaaau2040

aauaaaacagaagccgaccggccgccgacccguuccccaggaccagccuacgaggaacgg2100

auaacgcgguggcgacggcagcggugguggcgcuggggguggcgguaguggugcugcuga2160

ugguagucgggacggaggagagacgaugcauacauacacgcgugcaugcugcaugggugg2220

augguacggccgggagacgcggaagagaaacucacauaaaaaggugauaaaaagagcggu2280

ugaaaaaagaaaacgagauucgaccagacagaagagaaggaccggggcuuggcgacccuu2340

ccacgacugccguugucaucucggcuccuccaucuucucccggccacgggcggcuaaguc2400

accgccguucuccccauccguccgagcgccgaccgaccagccggccgauucgcccgccgg2460

ggcuucuggagaacgccggggcagcagcgaucuggggaugugcuagcuccg2511

<210>3

<211>438

<212>rna

<213>人工序列

<220>

<223>正义结构域1:d1

<400>3

ggcgcuuccggacagaagccccggcgggcgaaucggccggcuggucggucggcgcucgga60

cggauggggagaacggcggugacuuagccgcccguggccgggagaagauggaggagccga120

gaugacaacggcagucguggaagggucgccaagccccgguccuucucuucugucuggucg180

aaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuuuaugugaguuucucuucc240

gcgucucccggccguaccauccacccaugcagcaugcacgcguguauguaugcaucgucu300

cuccuccgucccgacuaccaucagcagcaccacuaccgccacccccagcgccaccaccgc360

ugccgucgccaccgcguuauccguuccucguaggcugguccuggggaacgggucggcggc420

cggucggcuucugaagcu438

<210>4

<211>117

<212>rna

<213>人工序列

<220>

<223>正义结构域2:d2

<400>4

ggcgcuuccggauuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuuccc60

ucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaaaagcu117

<210>5

<211>255

<212>rna

<213>人工序列

<220>

<223>正义结构域3:d3

<400>5

ggcgcuuccggaggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaac60

gaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucg120

cgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgg180

uccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucaua240

ggaaaugaacaagcu255

<210>6

<211>384

<212>rna

<213>人工序列

<220>

<223>正义结构域4:d4

<400>6

ggcgcuuccggacggcgucggcaugccgcugaucgcaguggccccaccucggcaugccgg60

cgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaacaauaca120

ugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucgaucacuacg180

auaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccuucccggcggc240

ggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaacaacaucaucau300

cggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggacacgaaggagu360

cauccauuuuuauccucgcaagcu384

<210>7

<211>438

<212>rna

<213>人工序列

<220>

<223>反义结构域1:d1as

<400>7

ggagucaagcuucagaagccgaccggccgccgacccguuccccaggaccagccuacgagg60

aacggauaacgcgguggcgacggcagcggugguggcgcuggggguggcgguaguggugcu120

gcugaugguagucgggacggaggagagacgaugcauacauacacgcgugcaugcugcaug180

gguggaugguacggccgggagacgcggaagagaaacucacauaaaaaggugauaaaaaga240

gcgguugaaaaaagaaaacgagauucgaccagacagaagagaaggaccggggcuuggcga300

cccuuccacgacugccguugucaucucggcuccuccaucuucucccggccacgggcggcu360

aagucaccgccguucuccccauccguccgagcgccgaccgaccagccggccgauucgccc420

gccggggcuucuguccgg438

<210>8

<211>117

<212>rna

<213>人工序列

<220>

<223>反义结构域2:d2as

<400>8

ggagucaagcuuuuuuucuuuuuacccucuuguuuaucaucugcgauucgugguagguag60

gagagggaaaugauaauccgagauuaaggaaaggagaagauaaaaaauaaaauccgg117

<210>9

<211>255

<212>rna

<213>人工序列

<220>

<223>反义结构域3:d3as

<400>9

ggagucaagcuuguucauuuccuaugauuguuuggcugcugaccgacgcgucaggaugug60

cagggcaugcggggaaucaggaccggacacgggauaauuucaucuaccuauacggagauc120

gcgguccucgccaugaggaucgcgacaggcgcgucgagggggcaggaacacccuugcgga180

uugacauucuuggugguguuucguuguugucgguaguuguuguugacgaugaggauaaau240

aaaaaugaccuccgg255

<210>10

<211>384

<212>rna

<213>人工序列

<220>

<223>反义结构域4:d4as

<400>10

ggagucaagcuugcgaggauaaaaauggaugacuccuucguguccagggagucgacuacu60

gcaacgcugauugauuaaaagauggucuccgaugaugauguuguuauugaucgaaucaug120

gugcagaacggcgacggagaggagcguguccgccgccgggaagguggucucuuucucuuu180

ucuuuuuucaagaaaucuuccauguguuuaucguagugaucgaaaucgacugaucucggg240

uucuuuuuguugguuucuuuucgguuaaucauguauuguuuucuuuuuuuacagaaagau300

acuuuuuucaugagcaauuccucgcccggcgccggcaugccgagguggggccacugcgau360

cagcggcaugccgacgccguccgg384

<210>11

<211>2091

<212>rna

<213>人工序列

<220>

<223>正义结构域1缺失突变体:δd1

<400>11

ggcgcuuccggaagagcuagcuccccagaucgcugcugccccggcguucucuuuuauuau60

uuuuuuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuac120

cuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaaagcuacagacauuugg180

guaccucagcuuuccgauaacucgaagaauucaaagucgacgauucccaacaagagaaaa240

cagaacaaaaacaaggucauuuuuauuuauccucaucgucaacaacaacuaccgacaaca300

acgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugu360

cgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccgugucc420

gguccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaauca480

uaggaaaugaaccagaagaacaaaaagaucaucucucucgguguauagcaacaccaacaa540

caaccgcaucgcaacaucuucauccgcaagacggaaagaaaacaacaauaaugagaauga600

aaucaccacaaccaagccagauuucacguccaugaguuuuuauuauauuauuaucaaaac660

gaaaaacagaaaaacugucauagauaaauauaaaaaaaaauagaaaccacaaacgacuac720

uaguacuccaaucuuagauguauaugcuccuagauaagauuuaguauuaccauaaucauc780

gaagaaugaaagacgacgaugauuccuuaccgcuccugccacccggucuguauguagaga840

gagaagagagaaaacggugaauccaagauccccgggucggcgucggcaugccgcugaucg900

caguggccccaccucggcaugccggcgccgggcgaggaauugcucaugaaaaaaguaucu960

uucuguaaaaaaagaaaacaauacaugauuaaccgaaaagaaaccaacaaaaagaacccg1020

agaucagucgauuucgaucacuacgauaaacacauggaagauuucuugaaaaaagaaaag1080

agaaagagaccaccuucccggcggcggacacgcuccucuccgucgccguucugcaccaug1140

auucgaucaauaacaacaucaucaucggagaccaucuuuuaaucaaucagcguugcagua1200

gucgacucccuggacacgaaggagucauccauuuuuauccucgcacuucuucgcucucaa1260

agccgccuuuaaaguugaaaugaaaggauggaaacauggaauacaguuuuaauugcacgu1320

aucaccauuuuacuacaaaaagaaaaaaaaacaacuuacacauaguauuaccuuagguuu1380

acggauaaguagaguguaggcguuuuugaaacaguucagccaaugcaaucuugucucggc1440

auaaucacucuuucugcauauaauaguaguaguagauuuauucacaucaacacagcgaaa1500

aacuccagcaucaaaguacaccuagagacagcccuuaaaauauaguuugcagcuuuuaga1560

uguacuuacaccaaagaagauuaccguccuuacgagaaaacagauacucggauauaggaa1620

ucaagacagcucugcacugaaaacacacucuccugucacgacaccgcgccacaccagagg1680

cguacgcgugacuucaucgcaacgauccaucgugaugucccucgcagaaccuaaaaagac1740

caaaaaaaaaucuuggaccacaguugucgauucuugaagacaauauucucgugagaacuu1800

ugagauucgcacuugaaaccucuuaggauccacaaaaacaacaaccucuguauggaaaau1860

gcgcuauuuuaucucagcuuuucucccaaaccucgguuucuuccuauucuuaaguuuucc1920

cuaguauauuugccuccuuauaagaaaagaagcacaagcucggucgcacggauuauuccu1980

ucugcuaaucuauuauuuuguuccuuuuuuuuuuguuugccuucacccccuucacucccu2040

guagcaacacagaguaguagacacaauaaaugagaaguuugcaugcaagcu2091

<210>12

<211>2412

<212>rna

<213>人工序列

<220>

<223>正义结构域2缺失突变体:δd2

<400>12

ggcgcuuccggaagagcuagcuccccagaucgcugcugccccggcguucuccagaagccc60

cggcgggcgaaucggccggcuggucggucggcgcucggacggauggggagaacggcggug120

acuuagccgcccguggccgggagaagauggaggagccgagaugacaacggcagucgugga180

agggucgccaagccccgguccuucucuucugucuggucgaaucucguuuucuuuuuucaa240

ccgcucuuuuuaucaccuuuuuaugugaguuucucuuccgcgucucccggccguaccauc300

cacccaugcagcaugcacgcguguauguaugcaucgucucuccuccgucccgacuaccau360

cagcagcaccacuaccgccacccccagcgccaccaccgcugccgucgccaccgcguuauc420

cguuccucguaggcugguccuggggaacgggucggcggccggucggcuucuguuuuauua480

uuuuuagcuacagacauuuggguaccucagcuuuccgauaacucgaagaauucaaagucg540

acgauucccaacaagagaaaacagaacaaaaacaaggucauuuuuauuuauccucaucgu600

caacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuc660

cugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauaggu720

agaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugacgcgu780

cggucagcagccaaacaaucauaggaaaugaaccagaagaacaaaaagaucaucucucuc840

gguguauagcaacaccaacaacaaccgcaucgcaacaucuucauccgcaagacggaaaga900

aaacaacaauaaugagaaugaaaucaccacaaccaagccagauuucacguccaugaguuu960

uuauuauauuauuaucaaaacgaaaaacagaaaaacugucauagauaaauauaaaaaaaa1020

auagaaaccacaaacgacuacuaguacuccaaucuuagauguauaugcuccuagauaaga1080

uuuaguauuaccauaaucaucgaagaaugaaagacgacgaugauuccuuaccgcuccugc1140

cacccggucuguauguagagagagaagagagaaaacggugaauccaagauccccgggucg1200

gcgucggcaugccgcugaucgcaguggccccaccucggcaugccggcgccgggcgaggaa1260

uugcucaugaaaaaaguaucuuucuguaaaaaaagaaaacaauacaugauuaaccgaaaa1320

gaaaccaacaaaaagaacccgagaucagucgauuucgaucacuacgauaaacacauggaa1380

gauuucuugaaaaaagaaaagagaaagagaccaccuucccggcggcggacacgcuccucu1440

ccgucgccguucugcaccaugauucgaucaauaacaacaucaucaucggagaccaucuuu1500

uaaucaaucagcguugcaguagucgacucccuggacacgaaggagucauccauuuuuauc1560

cucgcacuucuucgcucucaaagccgccuuuaaaguugaaaugaaaggauggaaacaugg1620

aauacaguuuuaauugcacguaucaccauuuuacuacaaaaagaaaaaaaaacaacuuac1680

acauaguauuaccuuagguuuacggauaaguagaguguaggcguuuuugaaacaguucag1740

ccaaugcaaucuugucucggcauaaucacucuuucugcauauaauaguaguaguagauuu1800

auucacaucaacacagcgaaaaacuccagcaucaaaguacaccuagagacagcccuuaaa1860

auauaguuugcagcuuuuagauguacuuacaccaaagaagauuaccguccuuacgagaaa1920

acagauacucggauauaggaaucaagacagcucugcacugaaaacacacucuccugucac1980

gacaccgcgccacaccagaggcguacgcgugacuucaucgcaacgauccaucgugauguc2040

ccucgcagaaccuaaaaagaccaaaaaaaaaucuuggaccacaguugucgauucuugaag2100

acaauauucucgugagaacuuugagauucgcacuugaaaccucuuaggauccacaaaaac2160

aacaaccucuguauggaaaaugcgcuauuuuaucucagcuuuucucccaaaccucgguuu2220

cuuccuauucuuaaguuuucccuaguauauuugccuccuuauaagaaaagaagcacaagc2280

ucggucgcacggauuauuccuucugcuaaucuauuauuuuguuccuuuuuuuuuuguuug2340

ccuucacccccuucacucccuguagcaacacagaguaguagacacaauaaaugagaaguu2400

ugcaugcaagcu2412

<210>13

<211>2273

<212>rna

<213>人工序列

<220>

<223>正义结构域3缺失突变体:δd3

<400>13

ggcgcuuccggaagagcuagcuccccagaucgcugcugccccggcguucuccagaagccc60

cggcgggcgaaucggccggcuggucggucggcgcucggacggauggggagaacggcggug120

acuuagccgcccguggccgggagaagauggaggagccgagaugacaacggcagucgugga180

agggucgccaagccccgguccuucucuucugucuggucgaaucucguuuucuuuuuucaa240

ccgcucuuuuuaucaccuuuuuaugugaguuucucuuccgcgucucccggccguaccauc300

cacccaugcagcaugcacgcguguauguaugcaucgucucuccuccgucccgacuaccau360

cagcagcaccacuaccgccacccccagcgccaccaccgcugccgucgccaccgcguuauc420

cguuccucguaggcugguccuggggaacgggucggcggccggucggcuucuguuuuauua480

uuuuuuuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccua540

ccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaaagcuacagacauuug600

gguaccucagcuuuccgauaacucgaagaauucaaagucgacgauucccaacaagagaaa660

acagaacaaaaacaaagaagaacaaaaagaucaucucucucgguguauagcaacaccaac720

aacaaccgcaucgcaacaucuucauccgcaagacggaaagaaaacaacaauaaugagaau780

gaaaucaccacaaccaagccagauuucacguccaugaguuuuuauuauauuauuaucaaa840

acgaaaaacagaaaaacugucauagauaaauauaaaaaaaaauagaaaccacaaacgacu900

acuaguacuccaaucuuagauguauaugcuccuagauaagauuuaguauuaccauaauca960

ucgaagaaugaaagacgacgaugauuccuuaccgcuccugccacccggucuguauguaga1020

gagagaagagagaaaacggugaauccaagauccccgggucggcgucggcaugccgcugau1080

cgcaguggccccaccucggcaugccggcgccgggcgaggaauugcucaugaaaaaaguau1140

cuuucuguaaaaaaagaaaacaauacaugauuaaccgaaaagaaaccaacaaaaagaacc1200

cgagaucagucgauuucgaucacuacgauaaacacauggaagauuucuugaaaaaagaaa1260

agagaaagagaccaccuucccggcggcggacacgcuccucuccgucgccguucugcacca1320

ugauucgaucaauaacaacaucaucaucggagaccaucuuuuaaucaaucagcguugcag1380

uagucgacucccuggacacgaaggagucauccauuuuuauccucgcacuucuucgcucuc1440

aaagccgccuuuaaaguugaaaugaaaggauggaaacauggaauacaguuuuaauugcac1500

guaucaccauuuuacuacaaaaagaaaaaaaaacaacuuacacauaguauuaccuuaggu1560

uuacggauaaguagaguguaggcguuuuugaaacaguucagccaaugcaaucuugucucg1620

gcauaaucacucuuucugcauauaauaguaguaguagauuuauucacaucaacacagcga1680

aaaacuccagcaucaaaguacaccuagagacagcccuuaaaauauaguuugcagcuuuua1740

gauguacuuacaccaaagaagauuaccguccuuacgagaaaacagauacucggauauagg1800

aaucaagacagcucugcacugaaaacacacucuccugucacgacaccgcgccacaccaga1860

ggcguacgcgugacuucaucgcaacgauccaucgugaugucccucgcagaaccuaaaaag1920

accaaaaaaaaaucuuggaccacaguugucgauucuugaagacaauauucucgugagaac1980

uuugagauucgcacuugaaaccucuuaggauccacaaaaacaacaaccucuguauggaaa2040

augcgcuauuuuaucucagcuuuucucccaaaccucgguuucuuccuauucuuaaguuuu2100

cccuaguauauuugccuccuuauaagaaaagaagcacaagcucggucgcacggauuauuc2160

cuucugcuaaucuauuauuuuguuccuuuuuuuuuuguuugccuucacccccuucacucc2220

cuguagcaacacagaguaguagacacaauaaaugagaaguuugcaugcaagcu2273

<210>14

<211>2145

<212>rna

<213>人工序列

<220>

<223>正义结构域4缺失突变体:δd4

<400>14

ggcgcuuccggaagagcuagcuccccagaucgcugcugccccggcguucuccagaagccc60

cggcgggcgaaucggccggcuggucggucggcgcucggacggauggggagaacggcggug120

acuuagccgcccguggccgggagaagauggaggagccgagaugacaacggcagucgugga180

agggucgccaagccccgguccuucucuucugucuggucgaaucucguuuucuuuuuucaa240

ccgcucuuuuuaucaccuuuuuaugugaguuucucuuccgcgucucccggccguaccauc300

cacccaugcagcaugcacgcguguauguaugcaucgucucuccuccgucccgacuaccau360

cagcagcaccacuaccgccacccccagcgccaccaccgcugccgucgccaccgcguuauc420

cguuccucguaggcugguccuggggaacgggucggcggccggucggcuucuguuuuauua480

uuuuuuuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccua540

ccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaaagcuacagacauuug600

gguaccucagcuuuccgauaacucgaagaauucaaagucgacgauucccaacaagagaaa660

acagaacaaaaacaaggucauuuuuauuuauccucaucgucaacaacaacuaccgacaac720

aacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccug780

ucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguc840

cgguccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaauc900

auaggaaaugaaccagaagaacaaaaagaucaucucucucgguguauagcaacaccaaca960

acaaccgcaucgcaacaucuucauccgcaagacggaaagaaaacaacaauaaugagaaug1020

aaaucaccacaaccaagccagauuucacguccaugaguuuuuauuauauuauuaucaaaa1080

cgaaaaacagaaaaacugucauagauaaauauaaaaaaaaauagaaaccacaaacgacua1140

cuaguacuccaaucuuagauguauaugcuccuagauaagauuuaguauuaccauaaucau1200

cgaagaaugaaagacgacgaugauuccuuaccgcuccugccacccggucuguauguagag1260

agagaagagagaaaacggugaauccaagauccccggguacuucuucgcucucaaagccgc1320

cuuuaaaguugaaaugaaaggauggaaacauggaauacaguuuuaauugcacguaucacc1380

auuuuacuacaaaaagaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggau1440

aaguagaguguaggcguuuuugaaacaguucagccaaugcaaucuugucucggcauaauc1500

acucuuucugcauauaauaguaguaguagauuuauucacaucaacacagcgaaaaacucc1560

agcaucaaaguacaccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacu1620

uacaccaaagaagauuaccguccuuacgagaaaacagauacucggauauaggaaucaaga1680

cagcucugcacugaaaacacacucuccugucacgacaccgcgccacaccagaggcguacg1740

cgugacuucaucgcaacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaa1800

aaaaucuuggaccacaguugucgauucuugaagacaauauucucgugagaacuuugagau1860

ucgcacuugaaaccucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcua1920

uuuuaucucagcuuuucucccaaaccucgguuucuuccuauucuuaaguuuucccuagua1980

uauuugccuccuuauaagaaaagaagcacaagcucggucgcacggauuauuccuucugcu2040

aaucuauuauuuuguuccuuuuuuuuuuguuugccuucacccccuucacucccuguagca2100

acacagaguaguagacacaauaaaugagaaguuugcaugcaagcu2145

<210>15

<211>491

<212>rna

<213>人工序列

<220>

<223>多聚体序列d2x4

<400>15

ggcgcuuccggaagagcuagcuuuuauuuuuuaucuucuccuuuccuuaaucucggauua60

ucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaa120

aagcgcgaauuccgaucuuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucau180

uucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaagau240

cguauccgguaccugugguuuuauuuuuuaucuucuccuuuccuuaaucucggauuauca300

uuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaacc360

acaccuccaccggugggccguuuuauuuuuuaucuucuccuuuccuuaaucucggauuau420

cauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaa480

cggcccaagcu491

<210>16

<211>1068

<212>rna

<213>人工序列

<220>

<223>多聚体序列d3x4

<400>16

ggcgcuuccggaagagcuagcccggcggucauuuuuauuuauccucaucgucaacaacaa60

cuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccu120

cgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaau180

uaucccguguccgguccugauuccccgcaugcccugcacauccugacgcgucggucagca240

gccaaacaaucauaggaaaugaaccgccgggaagcgccgaauucauaucgaucggucauu300

uuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugu360

caauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggac420

cgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcc480

cugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccgaucgaau540

aaagguaccuguggggucauuuuuauuuauccucaucgucaacaacaacuaccgacaaca600

acgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugu660

cgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccgugucc720

gguccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaauca780

uaggaaaugaaccccacauuuuaccgguaauaccggggggucauuuuuauuuauccucau840

cgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaagggug900

uuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguaua960

gguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugacg1020

cgucggucagcagccaaacaaucauaggaaaugaaccccccggaagcu1068

<210>17

<211>1584

<212>rna

<213>人工序列

<220>

<223>多聚体序列d3x4_d2x4

<400>17

ggcgcuuccggaagagcuagcccggcggucauuuuuauuuauccucaucgucaacaacaa60

cuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccu120

cgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaau180

uaucccguguccgguccugauuccccgcaugcccugcacauccugacgcgucggucagca240

gccaaacaaucauaggaaaugaaccgccgggaagcgccgaauucauaucgaucggucauu300

uuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugu360

caauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggac420

cgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcc480

cugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccgaucgaau540

aaagguaccuguggggucauuuuuauuuauccucaucgucaacaacaacuaccgacaaca600

acgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugu660

cgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccgugucc720

gguccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaauca780

uaggaaaugaaccccacauuuuaccgguaauaccggggggucauuuuuauuuauccucau840

cgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaagggug900

uuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguaua960

gguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugacg1020

cgucggucagcagccaaacaaucauaggaaaugaaccccccggaagcuuuccggaagagc1080

uagcuuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuac1140

cuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaaagcgcgaauuccgauc1200

uuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuac1260

cacgaaucgcagaugauaaacaagaggguaaaaagaaaaagaucguauccgguaccugug1320

guuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccua1380

ccacgaaucgcagaugauaaacaagaggguaaaaagaaaaaccacaccuccaccgguggg1440

ccguuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuacc1500

uaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaacggcccaagcuugacuc1560

cuauagugucaccuaaaugucuag1584

<210>18

<211>1569

<212>rna

<213>人工序列

<220>

<223>多聚体序列d2x4_d3x4

<400>18

ggcgcuuccggaagagcuagcuuuuauuuuuuaucuucuccuuuccuuaaucucggauua60

ucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaa120

aagcgcgaauuccgaucuuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucau180

uucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaagau240

cguauccgguaccugugguuuuauuuuuuaucuucuccuuuccuuaaucucggauuauca300

uuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaacc360

acaccuccaccggugggccguuuuauuuuuuaucuucuccuuuccuuaaucucggauuau420

cauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaa480

cggcccaagcuuccggcggucauuuuuauuuauccucaucgucaacaacaacuaccgaca540

acaacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgcc600

ugucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccgug660

uccgguccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaa720

ucauaggaaaugaaccgccgggaagcgccgaauucauaucgaucggucauuuuuauuuau780

ccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgca840

aggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuc900

cguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauc960

cugacgcgucggucagcagccaaacaaucauaggaaaugaaccgaucgaauaaagguacc1020

uguggggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacac1080

caccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccu1140

cauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugau1200

uccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaug1260

aaccccacauuuuaccgguaauaccggggggucauuuuuauuuauccucaucgucaacaa1320

caacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuccugccc1380

ccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauagguagauga1440

aauuaucccguguccgguccugauuccccgcaugcccugcacauccugacgcgucgguca1500

gcagccaaacaaucauaggaaaugaaccccccggaagcuugacuccuauagugucaccua1560

aaugucuag1569

<210>19

<211>1587

<212>rna

<213>人工序列

<220>

<223>多聚体序列(d3_d2)x4

<400>19

ggcgcuuccggaagagcuagcccggcggucauuuuuauuuauccucaucgucaacaacaa60

cuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccu120

cgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaau180

uaucccguguccgguccugauuccccgcaugcccugcacauccugacgcgucggucagca240

gccaaacaaucauaggaaaugaaccgccgggaagcgccgaauuccgaucuuuuauuuuuu300

aucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgca360

gaugauaaacaagaggguaaaaagaaaaagaucguauccgguaccuguggggucauuuuu420

auuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaa480

uccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgc540

gaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccug600

cacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccccacauuuuac660

cggugggccguuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccuc720

uccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaacggcccaagc780

uuuccggaagagcuagcccggcggucauuuuuauuuauccucaucgucaacaacaacuac840

cgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgac900

gcgccugucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuauc960

ccguguccgguccugauuccccgcaugcccugcacauccugacgcgucggucagcagcca1020

aacaaucauaggaaaugaaccgccgggaagcgccgaauuccgaucuuuuauuuuuuaucu1080

ucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaug1140

auaaacaagaggguaaaaagaaaaagaucguauccgguaccuguggggucauuuuuauuu1200

auccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccg1260

caaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgauc1320

uccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcaca1380

uccugacgcgucggucagcagccaaacaaucauaggaaaugaaccccacauuuuaccggu1440

gggccguuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccu1500

accuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaacggcccaagcuuga1560

cuccuauagugucaccuaaaugucuag1587

<210>20

<211>3251

<212>rna

<213>人工序列

<220>

<223>ggfp_β2.7

<400>20

ggagucucagauccgcuagcgcuaccggucgccaccauggugagcaagggcgaggagcug60

uucaccgggguggugcccauccuggucgagcuggacggcgacguaaacggccacaaguuc120

agcguguccggcgagggcgagggcgaugccaccuacggcaagcugacccugaaguucauc180

ugcaccaccggcaagcugcccgugcccuggcccacccucgugaccacccugaccuacggc240

gugcagugcuucagccgcuaccccgaccacaugaagcagcacgacuucuucaaguccgcc300

augcccgaaggcuacguccaggagcgcaccaucuucuucaaggacgacggcaacuacaag360

acccgcgccgaggugaaguucgagggcgacacccuggugaaccgcaucgagcugaagggc420

aucgacuucaaggaggacggcaacauccuggggcacaagcuggaguacaacuacaacagc480

cacaacgucuauaucauggccgacaagcagaagaacggcaucaaggugaacuucaagauc540

cgccacaacaucgaggacggcagcgugcagcucgccgaccacuaccagcagaacaccccc600

aucggcgacggccccgugcugcugcccgacaaccacuaccugagcacccaguccgcccug660

agcaaagaccccaacgagaagcgcgaucacaugguccugcuggaguucgugaccgccgcc720

gggaucacucucggcauggacgagcuguacaaguccggaugagcuagcuccccagaucgc780

ugcugccccggcguucuccagaagccccggcgggcgaaucggccggcuggucggucggcg840

cucggacggauggggagaacggcggugacuuagccgcccguggccgggagaagauggagg900

agccgagaugacaacggcagucguggaagggucgccaagccccgguccuucucuucuguc960

uggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuuuaugugaguuuc1020

ucuuccgcgucucccggccguaccauccacccaugcagcaugcacgcguguauguaugca1080

ucgucucuccuccgucccgacuaccaucagcagcaccacuaccgccacccccagcgccac1140

caccgcugccgucgccaccgcguuauccguuccucguaggcugguccuggggaacggguc1200

ggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuucuccuuuccuua1260

aucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggg1320

uaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaacucgaagaauuc1380

aaagucgacgauucccaacaagagaaaacagaacaaaaacaaggucauuuuuauuuaucc1440

ucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaag1500

gguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccg1560

uauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccu1620

gacgcgucggucagcagccaaacaaucauaggaaaugaaccagaagaacaaaaagaucau1680

cucucucgguguauagcaacaccaacaacaaccgcaucgcaacaucuucauccgcaagac1740

ggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagccagauuucacgucca1800

ugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacugucauagauaaauaua1860

aaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuagauguauaugcuccua1920

gauaagauuuaguauuaccauaaucaucgaagaaugaaagacgacgaugauuccuuaccg1980

cuccugccacccggucuguauguagagagagaagagagaaaacggugaauccaagauccc2040

cgggucggcgucggcaugccgcugaucgcaguggccccaccucggcaugccggcgccggg2100

cgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaacaauacaugauuaa2160

ccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucgaucacuacgauaaaca2220

cauggaagauuucuugaaaaaagaaaagagaaagagaccaccuucccggcggcggacacg2280

cuccucuccgucgccguucugcaccaugauucgaucaauaacaacaucaucaucggagac2340

caucuuuuaaucaaucagcguugcaguagucgacucccuggacacgaaggagucauccau2400

uuuuauccucgcacuucuucgcucucaaagccgccuuuaaaguugaaaugaaaggaugga2460

aacauggaauacaguuuuaauugcacguaucaccauuuuacuacaaaaagaaaaaaaaac2520

aacuuacacauaguauuaccuuagguuuacggauaaguagaguguaggcguuuuugaaac2580

aguucagccaaugcaaucuugucucggcauaaucacucuuucugcauauaauaguaguag2640

uagauuuauucacaucaacacagcgaaaaacuccagcaucaaaguacaccuagagacagc2700

ccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaagaagauuaccguccuua2760

cgagaaaacagauacucggauauaggaaucaagacagcucugcacugaaaacacacucuc2820

cugucacgacaccgcgccacaccagaggcguacgcgugacuucaucgcaacgauccaucg2880

ugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggaccacaguugucgauu2940

cuugaagacaauauucucgugagaacuuugagauucgcacuugaaaccucuuaggaucca3000

caaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcuuuucucccaaacc3060

ucgguuucuuccuauucuuaaguuuucccuaguauauuugccuccuuauaagaaaagaag3120

cacaagcucggucgcacggauuauuccuucugcuaaucuauuauuuuguuccuuuuuuuu3180

uuguuugccuucacccccuucacucccuguagcaacacagaguaguagacacaauaaaug3240

agaaguaagcu3251

<210>21

<211>3275

<212>rna

<213>人工序列

<220>

<223>ggfp_s_β2.7

<400>21

ggagucucagauccgcuagcgcuaccggucgccaccauggugagcaagggcgaggagcug60

uucaccgggguggugcccauccuggucgagcuggacggcgacguaaacggccacaaguuc120

agcguguccggcgagggcgagggcgaugccaccuacggcaagcugacccugaaguucauc180

ugcaccaccggcaagcugcccgugcccuggcccacccucgugaccacccugaccuacggc240

gugcagugcuucagccgcuaccccgaccacaugaagcagcacgacuucuucaaguccgcc300

augcccgaaggcuacguccaggagcgcaccaucuucuucaaggacgacggcaacuacaag360

acccgcgccgaggugaaguucgagggcgacacccuggugaaccgcaucgagcugaagggc420

aucgacuucaaggaggacggcaacauccuggggcacaagcuggaguacaacuacaacagc480

cacaacgucuauaucauggccgacaagcagaagaacggcaucaaggugaacuucaagauc540

cgccacaacaucgaggacggcagcgugcagcucgccgaccacuaccagcagaacaccccc600

aucggcgacggccccgugcugcugcccgacaaccacuaccugagcacccaguccgcccug660

agcaaagaccccaacgagaagcgcgaucacaugguccugcuggaguucgugaccgccgcc720

gggaucacucucggcauggacgagcuguacaaguccggaugacaccaccccggagaacag780

cuccucgcuagcuccccagaucgcugcugccccggcguucuccagaagccccggcgggcg840

aaucggccggcuggucggucggcgcucggacggauggggagaacggcggugacuuagccg900

cccguggccgggagaagauggaggagccgagaugacaacggcagucguggaagggucgcc960

aagccccgguccuucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuu1020

uuaucaccuuuuuaugugaguuucucuuccgcgucucccggccguaccauccacccaugc1080

agcaugcacgcguguauguaugcaucgucucuccuccgucccgacuaccaucagcagcac1140

cacuaccgccacccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucg1200

uaggcugguccuggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuu1260

auuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacg1320

aaucgcagaugauaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccuca1380

gcuuuccgauaacucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaa1440

aaacaaggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaaca1500

ccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgaucc1560

ucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccuga1620

uuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaau1680

gaaccagaagaacaaaaagaucaucucucucgguguauagcaacaccaacaacaaccgca1740

ucgcaacaucuucauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucacca1800

caaccaagccagauuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaaca1860

gaaaaacugucauagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuc1920

caaucuuagauguauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaug1980

aaagacgacgaugauuccuuaccgcuccugccacccggucuguauguagagagagaagag2040

agaaaacggugaauccaagauccccgggucggcgucggcaugccgcugaucgcaguggcc2100

ccaccucggcaugccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaa2160

aaaaagaaaacaauacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagu2220

cgauuucgaucacuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagag2280

accaccuucccggcggcggacacgcuccucuccgucgccguucugcaccaugauucgauc2340

aauaacaacaucaucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacuc2400

ccuggacacgaaggagucauccauuuuuauccucgcacuucuucgcucucaaagccgccu2460

uuaaaguugaaaugaaaggauggaaacauggaauacaguuuuaauugcacguaucaccau2520

uuuacuacaaaaagaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaa2580

guagaguguaggcguuuuugaaacaguucagccaaugcaaucuugucucggcauaaucac2640

ucuuucugcauauaauaguaguaguagauuuauucacaucaacacagcgaaaaacuccag2700

caucaaaguacaccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuua2760

caccaaagaagauuaccguccuuacgagaaaacagauacucggauauaggaaucaagaca2820

gcucugcacugaaaacacacucuccugucacgacaccgcgccacaccagaggcguacgcg2880

ugacuucaucgcaacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaa2940

aaucuuggaccacaguugucgauucuugaagacaauauucucgugagaacuuugagauuc3000

gcacuugaaaccucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauu3060

uuaucucagcuuuucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguaua3120

uuugccuccuuauaagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaa3180

ucuauuauuuuguuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaac3240

acagaguaguagacacaauaaaugagaaguaagcu3275

<210>22

<211>3276

<212>rna

<213>人工序列

<220>

<223>mtgfp_s_β2.7

<400>22

ggagucucagauccgcuagcgcuaccggucgccaccauagugagcaagggcgaggagcug60

uucaccgggguggugcccauccuggucgagcuggacggcgacguaaacggccacaaguuc120

agcguguccggcgagggcgagggcgaugccaccuacggcaagcugacccugaaguucauc180

ugcaccaccggcaagcugcccgugcccuggcccacccucgugaccacccugaccuacggc240

gugcagugcuucagccgcuaccccgaccacaugaagcagcacgacuucuucaaguccgcc300

augcccgaaggcuacguccaggagcgcaccaucuucuucaaggacgacggcaacuacaag360

acccgcgccgaggugaaguucgagggcgacacccuggugaaccgcaucgagcugaagggc420

aucgacuucaaggaggacggcaacauccuggggcacaagcuggaguacaacuacaacagc480

cacaacgucuauaucauggccgacaagcagaagaacggcaucaaggugaacuucaagauc540

cgccacaacaucgaggacggcagcgugcagcucgccgaccacuaccagcagaacaccccc600

aucggcgacggccccgugcugcugcccgacaaccacuaccugagcacccaguccgcccug660

agcaaagaccccaacgagaagcgcgaucacaugguccugcuggaguucgugaccgccgcc720

gggaucacucucggcauggacgagcuguacaaguccggaaagacaccaccccggagaaca780

gcuccucgcuagcuccccagaucgcugcugccccggcguucuccagaagccccggcgggc840

gaaucggccggcuggucggucggcgcucggacggauggggagaacggcggugacuuagcc900

gcccguggccgggagaagauggaggagccgagaugacaacggcagucguggaagggucgc960

caagccccgguccuucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuu1020

uuuaucaccuuuuuaugugaguuucucuuccgcgucucccggccguaccauccacccaug1080

cagcaugcacgcguguauguaugcaucgucucuccuccgucccgacuaccaucagcagca1140

ccacuaccgccacccccagcgccaccaccgcugccgucgccaccgcguuauccguuccuc1200

guaggcugguccuggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuu1260

uauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccac1320

gaaucgcagaugauaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccuc1380

agcuuuccgauaacucgaagaauucaaagucgacgauucccaacaagagaaaacagaaca1440

aaaacaaggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaac1500

accaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauc1560

cucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccug1620

auuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaa1680

ugaaccagaagaacaaaaagaucaucucucucgguguauagcaacaccaacaacaaccgc1740

aucgcaacaucuucauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucacc1800

acaaccaagccagauuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaac1860

agaaaaacugucauagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacu1920

ccaaucuuagauguauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaau1980

gaaagacgacgaugauuccuuaccgcuccugccacccggucuguauguagagagagaaga2040

gagaaaacggugaauccaagauccccgggucggcgucggcaugccgcugaucgcaguggc2100

cccaccucggcaugccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucugua2160

aaaaaagaaaacaauacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucag2220

ucgauuucgaucacuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaaga2280

gaccaccuucccggcggcggacacgcuccucuccgucgccguucugcaccaugauucgau2340

caauaacaacaucaucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacu2400

cccuggacacgaaggagucauccauuuuuauccucgcacuucuucgcucucaaagccgcc2460

uuuaaaguugaaaugaaaggauggaaacauggaauacaguuuuaauugcacguaucacca2520

uuuuacuacaaaaagaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggaua2580

aguagaguguaggcguuuuugaaacaguucagccaaugcaaucuugucucggcauaauca2640

cucuuucugcauauaauaguaguaguagauuuauucacaucaacacagcgaaaaacucca2700

gcaucaaaguacaccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuu2760

acaccaaagaagauuaccguccuuacgagaaaacagauacucggauauaggaaucaagac2820

agcucugcacugaaaacacacucuccugucacgacaccgcgccacaccagaggcguacgc2880

gugacuucaucgcaacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaa2940

aaaucuuggaccacaguugucgauucuugaagacaauauucucgugagaacuuugagauu3000

cgcacuugaaaccucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuau3060

uuuaucucagcuuuucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguau3120

auuugccuccuuauaagaaaagaagcacaagcucggucgcacggauuauuccuucugcua3180

aucuauuauuuuguuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaa3240

cacagaguaguagacacaauaaaugagaaguaagcu3276

<210>23

<211>786

<212>rna

<213>人工序列

<220>

<223>mtgfp_s

<400>23

ggagucucagauccgcuagcgcuaccggucgccaccauagugagcaagggcgaggagcug60

uucaccgggguggugcccauccuggucgagcuggacggcgacguaaacggccacaaguuc120

agcguguccggcgagggcgagggcgaugccaccuacggcaagcugacccugaaguucauc180

ugcaccaccggcaagcugcccgugcccuggcccacccucgugaccacccugaccuacggc240

gugcagugcuucagccgcuaccccgaccacaugaagcagcacgacuucuucaaguccgcc300

augcccgaaggcuacguccaggagcgcaccaucuucuucaaggacgacggcaacuacaag360

acccgcgccgaggugaaguucgagggcgacacccuggugaaccgcaucgagcugaagggc420

aucgacuucaaggaggacggcaacauccuggggcacaagcuggaguacaacuacaacagc480

cacaacgucuauaucauggccgacaagcagaagaacggcaucaaggugaacuucaagauc540

cgccacaacaucgaggacggcagcgugcagcucgccgaccacuaccagcagaacaccccc600

aucggcgacggccccgugcugcugcccgacaaccacuaccugagcacccaguccgcccug660

agcaaagaccccaacgagaagcgcgaucacaugguccugcuggaguucgugaccgccgcc720

gggaucacucucggcauggacgagcuguacaaguccggaagacaccaccccggagaacag780

cuccuc786

<210>24

<211>2661

<212>rna

<213>人工序列

<220>

<223>asatp6_s6_β2.7

<400>24

ggagucuccggaauaagcgccaguagaauuagaauugugaagaugauaaguguagaggga60

agguuaaugguugauauugcuaggguggcgcuuccaauuaggugcgugaguagguggccu120

gcaguaauguuaaagcaaggguuauuccgaauugggcuagcuccccagaucgcugcugcc180

ccggcguucuccagaagccccggcgggcgaaucggccggcuggucggucggcgcucggac240

ggauggggagaacggcggugacuuagccgcccguggccgggagaagauggaggagccgag300

augacaacggcagucguggaagggucgccaagccccgguccuucucuucugucuggucga360

aucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuuuaugugaguuucucuuccg420

cgucucccggccguaccauccacccaugcagcaugcacgcguguauguaugcaucgucuc480

uccuccgucccgacuaccaucagcagcaccacuaccgccacccccagcgccaccaccgcu540

gccgucgccaccgcguuauccguuccucguaggcugguccuggggaacgggucggcggcc600

ggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuucuccuuuccuuaaucucgg660

auuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaag720

aaaaaagcuacagacauuuggguaccucagcuuuccgauaacucgaagaauucaaagucg780

acgauucccaacaagagaaaacagaacaaaaacaaggucauuuuuauuuauccucaucgu840

caacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuc900

cugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauaggu960

agaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugacgcgu1020

cggucagcagccaaacaaucauaggaaaugaaccagaagaacaaaaagaucaucucucuc1080

gguguauagcaacaccaacaacaaccgcaucgcaacaucuucauccgcaagacggaaaga1140

aaacaacaauaaugagaaugaaaucaccacaaccaagccagauuucacguccaugaguuu1200

uuauuauauuauuaucaaaacgaaaaacagaaaaacugucauagauaaauauaaaaaaaa1260

auagaaaccacaaacgacuacuaguacuccaaucuuagauguauaugcuccuagauaaga1320

uuuaguauuaccauaaucaucgaagaaugaaagacgacgaugauuccuuaccgcuccugc1380

cacccggucuguauguagagagagaagagagaaaacggugaauccaagauccccgggucg1440

gcgucggcaugccgcugaucgcaguggccccaccucggcaugccggcgccgggcgaggaa1500

uugcucaugaaaaaaguaucuuucuguaaaaaaagaaaacaauacaugauuaaccgaaaa1560

gaaaccaacaaaaagaacccgagaucagucgauuucgaucacuacgauaaacacauggaa1620

gauuucuugaaaaaagaaaagagaaagagaccaccuucccggcggcggacacgcuccucu1680

ccgucgccguucugcaccaugauucgaucaauaacaacaucaucaucggagaccaucuuu1740

uaaucaaucagcguugcaguagucgacucccuggacacgaaggagucauccauuuuuauc1800

cucgcacuucuucgcucucaaagccgccuuuaaaguugaaaugaaaggauggaaacaugg1860

aauacaguuuuaauugcacguaucaccauuuuacuacaaaaagaaaaaaaaacaacuuac1920

acauaguauuaccuuagguuuacggauaaguagaguguaggcguuuuugaaacaguucag1980

ccaaugcaaucuugucucggcauaaucacucuuucugcauauaauaguaguaguagauuu2040

auucacaucaacacagcgaaaaacuccagcaucaaaguacaccuagagacagcccuuaaa2100

auauaguuugcagcuuuuagauguacuuacaccaaagaagauuaccguccuuacgagaaa2160

acagauacucggauauaggaaucaagacagcucugcacugaaaacacacucuccugucac2220

gacaccgcgccacaccagaggcguacgcgugacuucaucgcaacgauccaucgugauguc2280

ccucgcagaaccuaaaaagaccaaaaaaaaaucuuggaccacaguugucgauucuugaag2340

acaauauucucgugagaacuuugagauucgcacuugaaaccucuuaggauccacaaaaac2400

aacaaccucuguauggaaaaugcgcuauuuuaucucagcuuuucucccaaaccucgguuu2460

cuuccuauucuuaaguuuucccuaguauauuugccuccuuauaagaaaagaagcacaagc2520

ucggucgcacggauuauuccuucugcuaaucuauuauuuuguuccuuuuuuuuuuguuug2580

ccuucacccccuucacucccuguagcaacacagaguaguagacacaauaaaugagaaguc2640

caauucggaauaacccaagcu2661

<210>25

<211>2657

<212>rna

<213>人工序列

<220>

<223>β2.7_s8_asatp8

<400>25

ggcgcuuccggaauagguuggauugggggcuagcuccccagaucgcugcugccccggcgu60

ucuccagaagccccggcgggcgaaucggccggcuggucggucggcgcucggacggauggg120

gagaacggcggugacuuagccgcccguggccgggagaagauggaggagccgagaugacaa180

cggcagucguggaagggucgccaagccccgguccuucucuucugucuggucgaaucucgu240

uuucuuuuuucaaccgcucuuuuuaucaccuuuuuaugugaguuucucuuccgcgucucc300

cggccguaccauccacccaugcagcaugcacgcguguauguaugcaucgucucuccuccg360

ucccgacuaccaucagcagcaccacuaccgccacccccagcgccaccaccgcugccgucg420

ccaccgcguuauccguuccucguaggcugguccuggggaacgggucggcggccggucggc480

uucuguuuuauuauuuuuuuuuauuuuuuaucuucuccuuuccuuaaucucggauuauca540

uuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaaag600

cuacagacauuuggguaccucagcuuuccgauaacucgaagaauucaaagucgacgauuc660

ccaacaagagaaaacagaacaaaaacaaggucauuuuuauuuauccucaucgucaacaac720

aacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccc780

cucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaa840

auuaucccguguccgguccugauuccccgcaugcccugcacauccugacgcgucggucag900

cagccaaacaaucauaggaaaugaaccagaagaacaaaaagaucaucucucucgguguau960

agcaacaccaacaacaaccgcaucgcaacaucuucauccgcaagacggaaagaaaacaac1020

aauaaugagaaugaaaucaccacaaccaagccagauuucacguccaugaguuuuuauuau1080

auuauuaucaaaacgaaaaacagaaaaacugucauagauaaauauaaaaaaaaauagaaa1140

ccacaaacgacuacuaguacuccaaucuuagauguauaugcuccuagauaagauuuagua1200

uuaccauaaucaucgaagaaugaaagacgacgaugauuccuuaccgcuccugccacccgg1260

ucuguauguagagagagaagagagaaaacggugaauccaagauccccgggucggcgucgg1320

caugccgcugaucgcaguggccccaccucggcaugccggcgccgggcgaggaauugcuca1380

ugaaaaaaguaucuuucuguaaaaaaagaaaacaauacaugauuaaccgaaaagaaacca1440

acaaaaagaacccgagaucagucgauuucgaucacuacgauaaacacauggaagauuucu1500

ugaaaaaagaaaagagaaagagaccaccuucccggcggcggacacgcuccucuccgucgc1560

cguucugcaccaugauucgaucaauaacaacaucaucaucggagaccaucuuuuaaucaa1620

ucagcguugcaguagucgacucccuggacacgaaggagucauccauuuuuauccucgcac1680

uucuucgcucucaaagccgccuuuaaaguugaaaugaaaggauggaaacauggaauacag1740

uuuuaauugcacguaucaccauuuuacuacaaaaagaaaaaaaaacaacuuacacauagu1800

auuaccuuagguuuacggauaaguagaguguaggcguuuuugaaacaguucagccaaugc1860

aaucuugucucggcauaaucacucuuucugcauauaauaguaguaguagauuuauucaca1920

ucaacacagcgaaaaacuccagcaucaaaguacaccuagagacagcccuuaaaauauagu1980

uugcagcuuuuagauguacuuacaccaaagaagauuaccguccuuacgagaaaacagaua2040

cucggauauaggaaucaagacagcucugcacugaaaacacacucuccugucacgacaccg2100

cgccacaccagaggcguacgcgugacuucaucgcaacgauccaucgugaugucccucgca2160

gaaccuaaaaagaccaaaaaaaaaucuuggaccacaguugucgauucuugaagacaauau2220

ucucgugagaacuuugagauucgcacuugaaaccucuuaggauccacaaaaacaacaacc2280

ucuguauggaaaaugcgcuauuuuaucucagcuuuucucccaaaccucgguuucuuccua2340

uucuuaaguuuucccuaguauauuugccuccuuauaagaaaagaagcacaagcucggucg2400

cacggauuauuccuucugcuaaucuauuauuuuguuccuuuuuuuuuuguuugccuucac2460

ccccuucacucccuguagcaacacagaguaguagacacaauaaaugagaaguaagcuucc2520

ccaaaccaaccucuaccggaagcgccuuuuuuucguucauuuugguucucaggguuuguu2580

auaauuuuuuauuuuuaugggcuuuggugagggagguaggugguaguuuguguuuaauau2640

uuuuaguugggugauga2657

<210>26

<211>1757

<212>rna

<213>人工序列

<220>

<223>atp6_d34_d24

<400>26

ggagucuccggaauaagcgccaguagaauuagaauugugaagaugauaaguguagaggga60

agguuaaugguugauauugcuaggguggcgcuuccaauuaggugcgugaguagguggccu120

gcaguaauguuaaagcaaggguuauuccgaauugggcuagcccggcggucauuuuuauuu180

auccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccg240

caaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgauc300

uccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcaca360

uccugacgcgucggucagcagccaaacaaucauaggaaaugaaccgccgggaagcgccga420

auucauaucgaucggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaa480

cgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccuguc540

gcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccg600

guccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucau660

aggaaaugaaccgaucgaauaaagguaccuguggggucauuuuuauuuauccucaucguc720

aacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguucc780

ugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauaggua840

gaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugacgcguc900

ggucagcagccaaacaaucauaggaaaugaaccccacauuuuaccgguaauaccgggggg960

ucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaag1020

aaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcg1080

aggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgc1140

augcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccccc1200

cggaagcuuuccggaagagcuagcuuuuauuuuuuaucuucuccuuuccuuaaucucgga1260

uuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaaga1320

aaaaagcgcgaauuccgaucuuuuauuuuuuaucuucuccuuuccuuaaucucggauuau1380

cauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaa1440

gaucguauccgguaccugugguuuuauuuuuuaucuucuccuuuccuuaaucucggauua1500

ucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaa1560

accacaccuccaccggugggccguuuuauuuuuuaucuucuccuuuccuuaaucucggau1620

uaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaa1680

aaacggcccccaugaauaaggcccaagcuugacuccuauagugucaccuaaaugucuaga1740

uacuaagggagucuugc1757

<210>27

<211>5

<212>rna

<213>人工序列

<220>

<223>s1a

<400>27

ccggc5

<210>28

<211>5

<212>rna

<213>人工序列

<220>

<223>s1b

<400>28

gccgg5

<210>29

<211>5

<212>rna

<213>人工序列

<220>

<223>s2a

<400>29

cgauc5

<210>30

<211>5

<212>rna

<213>人工序列

<220>

<223>s2b

<400>30

gaucg5

<210>31

<211>5

<212>rna

<213>人工序列

<220>

<223>s3a

<400>31

ugugg5

<210>32

<211>5

<212>rna

<213>人工序列

<220>

<223>s3b

<400>32

ccaca5

<210>33

<211>6

<212>rna

<213>人工序列

<220>

<223>s4a

<400>33

ccgggg6

<210>34

<211>6

<212>rna

<213>人工序列

<220>

<223>s4b

<400>34

ccccgg6

<210>35

<211>23

<212>rna

<213>人工序列

<220>

<223>s6a

<400>35

aagcaaggguuauuccgaauugg23

<210>36

<211>17

<212>rna

<213>人工序列

<220>

<223>s6b

<400>36

ccaauucggaauaaccc17

<210>37

<211>13

<212>rna

<213>人工序列

<220>

<223>s8a

<400>37

gguuggauugggg13

<210>38

<211>32

<212>rna

<213>人工序列

<220>

<223>s8b

<400>38

ccccaaaccaaccucuaccggaagcgccuuuu32

<210>39

<211>24

<212>dna

<213>人工序列

<220>

<223>间隔子图3a

<400>39

caccaccccggagaacagctcctc24

<210>40

<211>2091

<212>rna

<213>人工序列

<220>

<223>反义结构域1缺失突变体:δd1as

<400>40

ggagucaagcuugcaugcaaacuucucauuuauugugucuacuacucuguguugcuacag60

ggagugaagggggugaaggcaaacaaaaaaaaaaggaacaaaauaauagauuagcagaag120

gaauaauccgugcgaccgagcuugugcuucuuuucuuauaaggaggcaaauauacuaggg180

aaaacuuaagaauaggaagaaaccgagguuugggagaaaagcugagauaaaauagcgcau240

uuuccauacagagguuguuguuuuuguggauccuaagagguuucaagugcgaaucucaaa300

guucucacgagaauauugucuucaagaaucgacaacugugguccaagauuuuuuuuuggu360

cuuuuuagguucugcgagggacaucacgauggaucguugcgaugaagucacgcguacgcc420

ucugguguggcgcggugucgugacaggagaguguguuuucagugcagagcugucuugauu480

ccuauauccgaguaucuguuuucucguaaggacgguaaucuucuuugguguaaguacauc540

uaaaagcugcaaacuauauuuuaagggcugucucuagguguacuuugaugcuggaguuuu600

ucgcuguguugaugugaauaaaucuacuacuacuauuauaugcagaaagagugauuaugc660

cgagacaagauugcauuggcugaacuguuucaaaaacgccuacacucuacuuauccguaa720

accuaagguaauacuauguguaaguuguuuuuuuuucuuuuuguaguaaaauggugauac780

gugcaauuaaaacuguauuccauguuuccauccuuucauuucaacuuuaaaggcggcuuu840

gagagcgaagaagugcgaggauaaaaauggaugacuccuucguguccagggagucgacua900

cugcaacgcugauugauuaaaagauggucuccgaugaugauguuguuauugaucgaauca960

uggugcagaacggcgacggagaggagcguguccgccgccgggaagguggucucuuucucu1020

uuucuuuuuucaagaaaucuuccauguguuuaucguagugaucgaaaucgacugaucucg1080

gguucuuuuuguugguuucuuuucgguuaaucauguauuguuuucuuuuuuuacagaaag1140

auacuuuuuucaugagcaauuccucgcccggcgccggcaugccgagguggggccacugcg1200

aucagcggcaugccgacgccgacccggggaucuuggauucaccguuuucucucuucucuc1260

ucuacauacagaccggguggcaggagcgguaaggaaucaucgucgucuuucauucuucga1320

ugauuaugguaauacuaaaucuuaucuaggagcauauacaucuaagauuggaguacuagu1380

agucguuugugguuucuauuuuuuuuuauauuuaucuaugacaguuuuucuguuuuucgu1440

uuugauaauaauauaauaaaaacucauggacgugaaaucuggcuugguuguggugauuuc1500

auucucauuauuguuguuuucuuuccgucuugcggaugaagauguugcgaugcgguuguu1560

guugguguugcuauacaccgagagagaugaucuuuuuguucuucugguucauuuccuaug1620

auuguuuggcugcugaccgacgcgucaggaugugcagggcaugcggggaaucaggaccgg1680

acacgggauaauuucaucuaccuauacggagaucgcgguccucgccaugaggaucgcgac1740

aggcgcgucgagggggcaggaacacccuugcggauugacauucuuggugguguuucguug1800

uugucgguaguuguuguugacgaugaggauaaauaaaaaugaccuuguuuuuguucuguu1860

uucucuuguugggaaucgucgacuuugaauucuucgaguuaucggaaagcugagguaccc1920

aaaugucuguagcuuuuuucuuuuuacccucuuguuuaucaucugcgauucgugguaggu1980

aggagagggaaaugauaauccgagauuaaggaaaggagaagauaaaaaauaaaaaaaaau2040

aauaaaagagaacgccggggcagcagcgaucuggggaugugcuagcuccgg2091

<210>41

<211>2412

<212>rna

<213>人工序列

<220>

<223>反义结构域2缺失突变体:δd2as

<400>41

ggagucaagcuugcaugcaaacuucucauuuauugugucuacuacucuguguugcuacag60

ggagugaagggggugaaggcaaacaaaaaaaaaaggaacaaaauaauagauuagcagaag120

gaauaauccgugcgaccgagcuugugcuucuuuucuuauaaggaggcaaauauacuaggg180

aaaacuuaagaauaggaagaaaccgagguuugggagaaaagcugagauaaaauagcgcau240

uuuccauacagagguuguuguuuuuguggauccuaagagguuucaagugcgaaucucaaa300

guucucacgagaauauugucuucaagaaucgacaacugugguccaagauuuuuuuuuggu360

cuuuuuagguucugcgagggacaucacgauggaucguugcgaugaagucacgcguacgcc420

ucugguguggcgcggugucgugacaggagaguguguuuucagugcagagcugucuugauu480

ccuauauccgaguaucuguuuucucguaaggacgguaaucuucuuugguguaaguacauc540

uaaaagcugcaaacuauauuuuaagggcugucucuagguguacuuugaugcuggaguuuu600

ucgcuguguugaugugaauaaaucuacuacuacuauuauaugcagaaagagugauuaugc660

cgagacaagauugcauuggcugaacuguuucaaaaacgccuacacucuacuuauccguaa720

accuaagguaauacuauguguaaguuguuuuuuuuucuuuuuguaguaaaauggugauac780

gugcaauuaaaacuguauuccauguuuccauccuuucauuucaacuuuaaaggcggcuuu840

gagagcgaagaagugcgaggauaaaaauggaugacuccuucguguccagggagucgacua900

cugcaacgcugauugauuaaaagauggucuccgaugaugauguuguuauugaucgaauca960

uggugcagaacggcgacggagaggagcguguccgccgccgggaagguggucucuuucucu1020

uuucuuuuuucaagaaaucuuccauguguuuaucguagugaucgaaaucgacugaucucg1080

gguucuuuuuguugguuucuuuucgguuaaucauguauuguuuucuuuuuuuacagaaag1140

auacuuuuuucaugagcaauuccucgcccggcgccggcaugccgagguggggccacugcg1200

aucagcggcaugccgacgccgacccggggaucuuggauucaccguuuucucucuucucuc1260

ucuacauacagaccggguggcaggagcgguaaggaaucaucgucgucuuucauucuucga1320

ugauuaugguaauacuaaaucuuaucuaggagcauauacaucuaagauuggaguacuagu1380

agucguuugugguuucuauuuuuuuuuauauuuaucuaugacaguuuuucuguuuuucgu1440

uuugauaauaauauaauaaaaacucauggacgugaaaucuggcuugguuguggugauuuc1500

auucucauuauuguuguuuucuuuccgucuugcggaugaagauguugcgaugcgguuguu1560

guugguguugcuauacaccgagagagaugaucuuuuuguucuucugguucauuuccuaug1620

auuguuuggcugcugaccgacgcgucaggaugugcagggcaugcggggaaucaggaccgg1680

acacgggauaauuucaucuaccuauacggagaucgcgguccucgccaugaggaucgcgac1740

aggcgcgucgagggggcaggaacacccuugcggauugacauucuuggugguguuucguug1800

uugucgguaguuguuguugacgaugaggauaaauaaaaaugaccuuguuuuuguucuguu1860

uucucuuguugggaaucgucgacuuugaauucuucgaguuaucggaaagcugagguaccc1920

aaaugucuguagcuaaaaauaauaaaacagaagccgaccggccgccgacccguuccccag1980

gaccagccuacgaggaacggauaacgcgguggcgacggcagcggugguggcgcugggggu2040

ggcgguaguggugcugcugaugguagucgggacggaggagagacgaugcauacauacacg2100

cgugcaugcugcauggguggaugguacggccgggagacgcggaagagaaacucacauaaa2160

aaggugauaaaaagagcgguugaaaaaagaaaacgagauucgaccagacagaagagaagg2220

accggggcuuggcgacccuuccacgacugccguugucaucucggcuccuccaucuucucc2280

cggccacgggcggcuaagucaccgccguucuccccauccguccgagcgccgaccgaccag2340

ccggccgauucgcccgccggggcuucuggagaacgccggggcagcagcgaucuggggaug2400

ugcuagcuccgg2412

<210>42

<211>2273

<212>rna

<213>人工序列

<220>

<223>反义结构域3缺失突变体:δd3as

<400>42

ggagucaagcuugcaugcaaacuucucauuuauugugucuacuacucuguguugcuacag60

ggagugaagggggugaaggcaaacaaaaaaaaaaggaacaaaauaauagauuagcagaag120

gaauaauccgugcgaccgagcuugugcuucuuuucuuauaaggaggcaaauauacuaggg180

aaaacuuaagaauaggaagaaaccgagguuugggagaaaagcugagauaaaauagcgcau240

uuuccauacagagguuguuguuuuuguggauccuaagagguuucaagugcgaaucucaaa300

guucucacgagaauauugucuucaagaaucgacaacugugguccaagauuuuuuuuuggu360

cuuuuuagguucugcgagggacaucacgauggaucguugcgaugaagucacgcguacgcc420

ucugguguggcgcggugucgugacaggagaguguguuuucagugcagagcugucuugauu480

ccuauauccgaguaucuguuuucucguaaggacgguaaucuucuuugguguaaguacauc540

uaaaagcugcaaacuauauuuuaagggcugucucuagguguacuuugaugcuggaguuuu600

ucgcuguguugaugugaauaaaucuacuacuacuauuauaugcagaaagagugauuaugc660

cgagacaagauugcauuggcugaacuguuucaaaaacgccuacacucuacuuauccguaa720

accuaagguaauacuauguguaaguuguuuuuuuuucuuuuuguaguaaaauggugauac780

gugcaauuaaaacuguauuccauguuuccauccuuucauuucaacuuuaaaggcggcuuu840

gagagcgaagaagugcgaggauaaaaauggaugacuccuucguguccagggagucgacua900

cugcaacgcugauugauuaaaagauggucuccgaugaugauguuguuauugaucgaauca960

uggugcagaacggcgacggagaggagcguguccgccgccgggaagguggucucuuucucu1020

uuucuuuuuucaagaaaucuuccauguguuuaucguagugaucgaaaucgacugaucucg1080

gguucuuuuuguugguuucuuuucgguuaaucauguauuguuuucuuuuuuuacagaaag1140

auacuuuuuucaugagcaauuccucgcccggcgccggcaugccgagguggggccacugcg1200

aucagcggcaugccgacgccgacccggggaucuuggauucaccguuuucucucuucucuc1260

ucuacauacagaccggguggcaggagcgguaaggaaucaucgucgucuuucauucuucga1320

ugauuaugguaauacuaaaucuuaucuaggagcauauacaucuaagauuggaguacuagu1380

agucguuugugguuucuauuuuuuuuuauauuuaucuaugacaguuuuucuguuuuucgu1440

uuugauaauaauauaauaaaaacucauggacgugaaaucuggcuugguuguggugauuuc1500

auucucauuauuguuguuuucuuuccgucuugcggaugaagauguugcgaugcgguuguu1560

guugguguugcuauacaccgagagagaugaucuuuuuguucuucuuuguuuuuguucugu1620

uuucucuuguugggaaucgucgacuuugaauucuucgaguuaucggaaagcugagguacc1680

caaaugucuguagcuuuuuucuuuuuacccucuuguuuaucaucugcgauucgugguagg1740

uaggagagggaaaugauaauccgagauuaaggaaaggagaagauaaaaaauaaaaaaaaa1800

uaauaaaacagaagccgaccggccgccgacccguuccccaggaccagccuacgaggaacg1860

gauaacgcgguggcgacggcagcggugguggcgcuggggguggcgguaguggugcugcug1920

augguagucgggacggaggagagacgaugcauacauacacgcgugcaugcugcaugggug1980

gaugguacggccgggagacgcggaagagaaacucacauaaaaaggugauaaaaagagcgg2040

uugaaaaaagaaaacgagauucgaccagacagaagagaaggaccggggcuuggcgacccu2100

uccacgacugccguugucaucucggcuccuccaucuucucccggccacgggcggcuaagu2160

caccgccguucuccccauccguccgagcgccgaccgaccagccggccgauucgcccgccg2220

gggcuucuggagaacgccggggcagcagcgaucuggggaugugcuagcuccgg2273

<210>43

<211>2145

<212>rna

<213>人工序列

<220>

<223>反义结构域4缺失突变体:δd4as

<400>43

ggagucaagcuugcaugcaaacuucucauuuauugugucuacuacucuguguugcuacag60

ggagugaagggggugaaggcaaacaaaaaaaaaaggaacaaaauaauagauuagcagaag120

gaauaauccgugcgaccgagcuugugcuucuuuucuuauaaggaggcaaauauacuaggg180

aaaacuuaagaauaggaagaaaccgagguuugggagaaaagcugagauaaaauagcgcau240

uuuccauacagagguuguuguuuuuguggauccuaagagguuucaagugcgaaucucaaa300

guucucacgagaauauugucuucaagaaucgacaacugugguccaagauuuuuuuuuggu360

cuuuuuagguucugcgagggacaucacgauggaucguugcgaugaagucacgcguacgcc420

ucugguguggcgcggugucgugacaggagaguguguuuucagugcagagcugucuugauu480

ccuauauccgaguaucuguuuucucguaaggacgguaaucuucuuugguguaaguacauc540

uaaaagcugcaaacuauauuuuaagggcugucucuagguguacuuugaugcuggaguuuu600

ucgcuguguugaugugaauaaaucuacuacuacuauuauaugcagaaagagugauuaugc660

cgagacaagauugcauuggcugaacuguuucaaaaacgccuacacucuacuuauccguaa720

accuaagguaauacuauguguaaguuguuuuuuuuucuuuuuguaguaaaauggugauac780

gugcaauuaaaacuguauuccauguuuccauccuuucauuucaacuuuaaaggcggcuuu840

gagagcgaagaaguacccggggaucuuggauucaccguuuucucucuucucucucuacau900

acagaccggguggcaggagcgguaaggaaucaucgucgucuuucauucuucgaugauuau960

gguaauacuaaaucuuaucuaggagcauauacaucuaagauuggaguacuaguagucguu1020

ugugguuucuauuuuuuuuuauauuuaucuaugacaguuuuucuguuuuucguuuugaua1080

auaauauaauaaaaacucauggacgugaaaucuggcuugguuguggugauuucauucuca1140

uuauuguuguuuucuuuccgucuugcggaugaagauguugcgaugcgguuguuguuggug1200

uugcuauacaccgagagagaugaucuuuuuguucuucugguucauuuccuaugauuguuu1260

ggcugcugaccgacgcgucaggaugugcagggcaugcggggaaucaggaccggacacggg1320

auaauuucaucuaccuauacggagaucgcgguccucgccaugaggaucgcgacaggcgcg1380

ucgagggggcaggaacacccuugcggauugacauucuuggugguguuucguuguugucgg1440

uaguuguuguugacgaugaggauaaauaaaaaugaccuuguuuuuguucuguuuucucuu1500

guugggaaucgucgacuuugaauucuucgaguuaucggaaagcugagguacccaaauguc1560

uguagcuuuuuucuuuuuacccucuuguuuaucaucugcgauucgugguagguaggagag1620

ggaaaugauaauccgagauuaaggaaaggagaagauaaaaaauaaaaaaaaauaauaaaa1680

cagaagccgaccggccgccgacccguuccccaggaccagccuacgaggaacggauaacgc1740

gguggcgacggcagcggugguggcgcuggggguggcgguaguggugcugcugaugguagu1800

cgggacggaggagagacgaugcauacauacacgcgugcaugcugcauggguggaugguac1860

ggccgggagacgcggaagagaaacucacauaaaaaggugauaaaaagagcgguugaaaaa1920

agaaaacgagauucgaccagacagaagagaaggaccggggcuuggcgacccuuccacgac1980

ugccguugucaucucggcuccuccaucuucucccggccacgggcggcuaagucaccgccg2040

uucuccccauccguccgagcgccgaccgaccagccggccgauucgcccgccggggcuucu2100

ggagaacgccggggcagcagcgaucuggggaugugcuagcuccgg2145

<210>44

<211>3358

<212>rna

<213>人工序列

<220>

<223>ggfp_β2.7_endo

<400>44

ucagaucgcuagcgcuaccggucgccaccauggugagcaagggcgaggagcuguucaccg60

ggguggugcccauccuggucgagcuggacggcgacguaaacggccacaaguucagcgugu120

ccggcgagggcgagggcgaugccaccuacggcaagcugacccugaaguucaucugcacca180

ccggcaagcugcccgugcccuggcccacccucgugaccacccugaccuacggcgugcagu240

gcuucagccgcuaccccgaccacaugaagcagcacgacuucuucaaguccgccaugcccg300

aaggcuacguccaggagcgcaccaucuucuucaaggacgacggcaacuacaagacccgcg360

ccgaggugaaguucgagggcgacacccuggugaaccgcaucgagcugaagggcaucgacu420

ucaaggaggacggcaacauccuggggcacaagcuggaguacaacuacaacagccacaacg480

ucuauaucauggccgacaagcagaagaacggcaucaaggugaacuucaagauccgccaca540

acaucgaggacggcagcgugcagcucgccgaccacuaccagcagaacacccccaucggcg600

acggccccgugcugcugcccgacaaccacuaccugagcacccaguccgcccugagcaaag660

accccaacgagaagcgcgaucacaugguccugcuggaguucgugaccgccgccgggauca720

cucucggcauggacgagcuguacaaguccggaagagcuagcuccccagaucgcugcugcc780

ccggcguucuccagaagccccggcgggcgaaucggccggcuggucggucggcgcucggac840

ggauggggagaacggcggugacuuagccgcccguggccgggagaagauggaggagccgag900

augacaacggcagucguggaagggucgccaagccccgguccuucucuucugucuggucga960

aucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuuuaugugaguuucucuuccg1020

cgucucccggccguaccauccacccaugcagcaugcacgcguguauguaugcaucgucuc1080

uccuccgucccgacuaccaucagcagcaccacuaccgccacccccagcgccaccaccgcu1140

gccgucgccaccgcguuauccguuccucguaggcugguccuggggaacgggucggcggcc1200

ggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuucuccuuuccuuaaucucgg1260

auuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaag1320

aaaaaagcuacagacauuuggguaccucagcuuuccgauaacucgaagaauucaaagucg1380

acgauucccaacaagagaaaacagaacaaaaacaaggucauuuuuauuuauccucaucgu1440

caacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuc1500

cugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauaggu1560

agaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugacgcgu1620

cggucagcagccaaacaaucauaggaaaugaaccagaagaacaaaaagaucaucucucuc1680

gguguauagcaacaccaacaacaaccgcaucgcaacaucuucauccgcaagacggaaaga1740

aaacaacaauaaugagaaugaaaucaccacaaccaagccagauuucacguccaugaguuu1800

uuauuauauuauuaucaaaacgaaaaacagaaaaacugucauagauaaauauaaaaaaaa1860

auagaaaccacaaacgacuacuaguacuccaaucuuagauguauaugcuccuagauaaga1920

uuuaguauuaccauaaucaucgaagaaugaaagacgacgaugauuccuuaccgcuccugc1980

cacccggucuguauguagagagagaagagagaaaacggugaauccaagauccccgggucg2040

gcgucggcaugccgcugaucgcaguggccccaccucggcaugccggcgccgggcgaggaa2100

uugcucaugaaaaaaguaucuuucuguaaaaaaagaaaacaauacaugauuaaccgaaaa2160

gaaaccaacaaaaagaacccgagaucagucgauuucgaucacuacgauaaacacauggaa2220

gauuucuugaaaaaagaaaagagaaagagaccaccuucccggcggcggacacgcuccucu2280

ccgucgccguucugcaccaugauucgaucaauaacaacaucaucaucggagaccaucuuu2340

uaaucaaucagcguugcaguagucgacucccuggacacgaaggagucauccauuuuuauc2400

cucgcacuucuucgcucucaaagccgccuuuaaaguugaaaugaaaggauggaaacaugg2460

aauacaguuuuaauugcacguaucaccauuuuacuacaaaaagaaaaaaaaacaacuuac2520

acauaguauuaccuuagguuuacggauaaguagaguguaggcguuuuugaaacaguucag2580

ccaaugcaaucuugucucggcauaaucacucuuucugcauauaauaguaguaguagauuu2640

auucacaucaacacagcgaaaaacuccagcaucaaaguacaccuagagacagcccuuaaa2700

auauaguuugcagcuuuuagauguacuuacaccaaagaagauuaccguccuuacgagaaa2760

acagauacucggauauaggaaucaagacagcucugcacugaaaacacacucuccugucac2820

gacaccgcgccacaccagaggcguacgcgugacuucaucgcaacgauccaucgugauguc2880

ccucgcagaaccuaaaaagaccaaaaaaaaaucuuggaccacaguugucgauucuugaag2940

acaauauucucgugagaacuuugagauucgcacuugaaaccucuuaggauccacaaaaac3000

aacaaccucuguauggaaaaugcgcuauuuuaucucagcuuuucucccaaaccucgguuu3060

cuuccuauucuuaaguuuucccuaguauauuugccuccuuauaagaaaagaagcacaagc3120

ucggucgcacggauuauuccuucugcuaaucuauuauuuuguuccuuuuuuuuuuguuug3180

ccuucacccccuucacucccuguagcaacacagaguaguagacacaauaaaugagaagua3240

agcuugacuccuauagugucaccuaaaugucuagaaacuuguuuauugcagcuuauaaug3300

guuacaaauaaagcaauagcaucacaaauuucacaaauaaagcauuuuuuucacugca3358

<210>45

<211>3382

<212>rna

<213>人工序列

<220>

<223>ggfp_s_β2.7_endo

<400>45

ucagaucgcuagcgcuaccggucgccaccauggugagcaagggcgaggagcuguucaccg60

ggguggugcccauccuggucgagcuggacggcgacguaaacggccacaaguucagcgugu120

ccggcgagggcgagggcgaugccaccuacggcaagcugacccugaaguucaucugcacca180

ccggcaagcugcccgugcccuggcccacccucgugaccacccugaccuacggcgugcagu240

gcuucagccgcuaccccgaccacaugaagcagcacgacuucuucaaguccgccaugcccg300

aaggcuacguccaggagcgcaccaucuucuucaaggacgacggcaacuacaagacccgcg360

ccgaggugaaguucgagggcgacacccuggugaaccgcaucgagcugaagggcaucgacu420

ucaaggaggacggcaacauccuggggcacaagcuggaguacaacuacaacagccacaacg480

ucuauaucauggccgacaagcagaagaacggcaucaaggugaacuucaagauccgccaca540

acaucgaggacggcagcgugcagcucgccgaccacuaccagcagaacacccccaucggcg600

acggccccgugcugcugcccgacaaccacuaccugagcacccaguccgcccugagcaaag660

accccaacgagaagcgcgaucacaugguccugcuggaguucgugaccgccgccgggauca720

cucucggcauggacgagcuguacaaguccggaugacaccaccccggagaacagcuccucg780

cuagcuccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggc840

cggcuggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccgugg900

ccgggagaagauggaggagccgagaugacaacggcagucguggaagggucgccaagcccc960

gguccuucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucac1020

cuuuuuaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugc1080

acgcguguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuacc1140

gccacccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcug1200

guccuggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuu1260

aucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgca1320

gaugauaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuucc1380

gauaacucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaag1440

gucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaa1500

gaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggc1560

gaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccg1620

caugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccag1680

aagaacaaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaac1740

aucuucauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaa1800

gccagauuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaac1860

ugucauagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuu1920

agauguauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacg1980

acgaugauuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaac2040

ggugaauccaagauccccgggucggcgucggcaugccgcugaucgcaguggccccaccuc2100

ggcaugccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaaga2160

aaacaauacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuuc2220

gaucacuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccu2280

ucccggcggcggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaaca2340

acaucaucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggac2400

acgaaggagucauccauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaagu2460

ugaaaugaaaggauggaaacauggaauacaguuuuaauugcacguaucaccauuuuacua2520

caaaaagaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaaguagagu2580

guaggcguuuuugaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucu2640

gcauauaauaguaguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaa2700

guacaccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaa2760

gaagauuaccguccuuacgagaaaacagauacucggauauaggaaucaagacagcucugc2820

acugaaaacacacucuccugucacgacaccgcgccacaccagaggcguacgcgugacuuc2880

aucgcaacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuug2940

gaccacaguugucgauucuugaagacaauauucucgugagaacuuugagauucgcacuug3000

aaaccucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucuc3060

agcuuuucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccu3120

ccuuauaagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaaucuauua3180

uuuuguuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaacacagagu3240

aguagacacaauaaaugagaaguaagcuugacuccuauagugucaccuaaaugucuagaa3300

acuuguuuauugcagcuuauaaugguuacaaauaaagcaauagcaucacaaauuucacaa3360

auaaagcauuuuuuucacugca3382

<210>46

<211>3382

<212>rna

<213>人工序列

<220>

<223>mtgfp_s_β2.7_endo

<400>46

ucagaucgcuagcgcuaccggucgccaccauagugagcaagggcgaggagcuguucaccg60

ggguggugcccauccuggucgagcuggacggcgacguaaacggccacaaguucagcgugu120

ccggcgagggcgagggcgaugccaccuacggcaagcugacccugaaguucaucugcacca180

ccggcaagcugcccgugcccuggcccacccucgugaccacccugaccuacggcgugcagu240

gcuucagccgcuaccccgaccacaugaagcagcacgacuucuucaaguccgccaugcccg300

aaggcuacguccaggagcgcaccaucuucuucaaggacgacggcaacuacaagacccgcg360

ccgaggugaaguucgagggcgacacccuggugaaccgcaucgagcugaagggcaucgacu420

ucaaggaggacggcaacauccuggggcacaagcuggaguacaacuacaacagccacaacg480

ucuauaucauggccgacaagcagaagaacggcaucaaggugaacuucaagauccgccaca540

acaucgaggacggcagcgugcagcucgccgaccacuaccagcagaacacccccaucggcg600

acggccccgugcugcugcccgacaaccacuaccugagcacccaguccgcccugagcaaag660

accccaacgagaagcgcgaucacaugguccugcuggaguucgugaccgccgccgggauca720

cucucggcauggacgagcuguacaaguccggaagacaccaccccggagaacagcuccucg780

cuagcuccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggc840

cggcuggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccgugg900

ccgggagaagauggaggagccgagaugacaacggcagucguggaagggucgccaagcccc960

gguccuucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucac1020

cuuuuuaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugc1080

acgcguguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuacc1140

gccacccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcug1200

guccuggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuu1260

aucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgca1320

gaugauaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuucc1380

gauaacucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaag1440

gucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaa1500

gaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggc1560

gaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccg1620

caugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccag1680

aagaacaaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaac1740

aucuucauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaa1800

gccagauuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaac1860

ugucauagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuu1920

agauguauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacg1980

acgaugauuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaac2040

ggugaauccaagauccccgggucggcgucggcaugccgcugaucgcaguggccccaccuc2100

ggcaugccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaaga2160

aaacaauacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuuc2220

gaucacuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccu2280

ucccggcggcggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaaca2340

acaucaucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggac2400

acgaaggagucauccauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaagu2460

ugaaaugaaaggauggaaacauggaauacaguuuuaauugcacguaucaccauuuuacua2520

caaaaagaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaaguagagu2580

guaggcguuuuugaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucu2640

gcauauaauaguaguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaa2700

guacaccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaa2760

gaagauuaccguccuuacgagaaaacagauacucggauauaggaaucaagacagcucugc2820

acugaaaacacacucuccugucacgacaccgcgccacaccagaggcguacgcgugacuuc2880

aucgcaacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuug2940

gaccacaguugucgauucuugaagacaauauucucgugagaacuuugagauucgcacuug3000

aaaccucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucuc3060

agcuuuucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccu3120

ccuuauaagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaaucuauua3180

uuuuguuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaacacagagu3240

aguagacacaauaaaugagaaguaagcuugacuccuauagugucaccuaaaugucuagaa3300

acuuguuuauugcagcuuauaaugguuacaaauaaagcaauagcaucacaaauuucacaa3360

auaaagcauuuuuuucacugca3382

<210>47

<211>3500

<212>rna

<213>人工序列

<220>

<223>mtgfp_nls_s_β2.7_endo

<400>47

ucagaucgcuagcgcuaccggucgccaccauagugagcaagggcgaggagcuguucaccg60

ggguggugcccauccuggucgagcuggacggcgacguaaacggccacaaguucagcgugu120

ccggcgagggcgagggcgaugccaccuacggcaagcugacccugaaguucaucugcacca180

ccggcaagcugcccgugcccuggcccacccucgugaccacccugaccuacggcgugcagu240

gcuucagccgcuaccccgaccacaugaagcagcacgacuucuucaaguccgccaugcccg300

aaggcuacguccaggagcgcaccaucuucuucaaggacgacggcaacuacaagacccgcg360

ccgaggugaaguucgagggcgacacccuggugaaccgcaucgagcugaagggcaucgacu420

ucaaggaggacggcaacauccuggggcacaagcuggaguacaacuacaacagccacaacg480

ucuauaucauggccgacaagcagaagaacggcaucaaggugaacuucaagauccgccaca540

acaucgaggacggcagcgugcagcucgccgaccacuaccagcagaacacccccaucggcg600

acggccccgugcugcugcccgacaaccacuaccugagcacccaguccgcccugagcaaag660

accccaacgagaagcgcgaucacaugguccugcuggaguucgugaccgccgccgggauca720

cucucggcauggacgagcuguacaaggccggacucagaucucgagcugauccaaaaaaga780

agagaaagguagauccaaaaaagaagagaaagguagauccaaaaaagaagagaaagguaa840

ggauccaccggaucuagauaacuguccggaagacaccaccccggagaacagcuccucgcu900

agcuccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggccg960

gcuggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccguggcc1020

gggagaagauggaggagccgagaugacaacggcagucguggaagggucgccaagccccgg1080

uccuucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccu1140

uuuuaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugcac1200

gcguguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuaccgc1260

cacccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcuggu1320

ccuggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuau1380

cuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcaga1440

ugauaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccga1500

uaacucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaaggu1560

cauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaaga1620

augucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcga1680

ggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgca1740

ugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccagaa1800

gaacaaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaacau1860

cuucauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagc1920

cagauuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacug1980

ucauagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuag2040

auguauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacgac2100

gaugauuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaacgg2160

ugaauccaagauccccgggucggcgucggcaugccgcugaucgcaguggccccaccucgg2220

caugccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaa2280

acaauacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucga2340

ucacuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccuuc2400

ccggcggcggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaacaac2460

aucaucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggacac2520

gaaggagucauccauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaaguug2580

aaaugaaaggauggaaacauggaauacaguuuuaauugcacguaucaccauuuuacuaca2640

aaaagaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaaguagagugu2700

aggcguuuuugaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucugc2760

auauaauaguaguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaagu2820

acaccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaaga2880

agauuaccguccuuacgagaaaacagauacucggauauaggaaucaagacagcucugcac2940

ugaaaacacacucuccugucacgacaccgcgccacaccagaggcguacgcgugacuucau3000

cgcaacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuugga3060

ccacaguugucgauucuugaagacaauauucucgugagaacuuugagauucgcacuugaa3120

accucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucag3180

cuuuucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccucc3240

uuauaagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaaucuauuauu3300

uuguuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaacacagaguag3360

uagacacaauaaaugagaaguaagcuugacuccuauagugucaccuaaaugucuagaaac3420

uuguuuauugcagcuuauaaugguuacaaauaaagcaauagcaucacaaauuucacaaau3480

aaagcauuuuuuucacugca3500

<210>48

<211>991

<212>rna

<213>人工序列

<220>

<223>mtgfp_s_endo

<400>48

ucagaucgcuagcgcuaccggucgccaccauagugagcaagggcgaggagcuguucaccg60

ggguggugcccauccuggucgagcuggacggcgacguaaacggccacaaguucagcgugu120

ccggcgagggcgagggcgaugccaccuacggcaagcugacccugaaguucaucugcacca180

ccggcaagcugcccgugcccuggcccacccucgugaccacccugaccuacggcgugcagu240

gcuucagccgcuaccccgaccacaugaagcagcacgacuucuucaaguccgccaugcccg300

aaggcuacguccaggagcgcaccaucuucuucaaggacgacggcaacuacaagacccgcg360

ccgaggugaaguucgagggcgacacccuggugaaccgcaucgagcugaagggcaucgacu420

ucaaggaggacggcaacauccuggggcacaagcuggaguacaacuacaacagccacaacg480

ucuauaucauggccgacaagcagaagaacggcaucaaggugaacuucaagauccgccaca540

acaucgaggacggcagcgugcagcucgccgaccacuaccagcagaacacccccaucggcg600

acggccccgugcugcugcccgacaaccacuaccugagcacccaguccgcccugagcaaag660

accccaacgagaagcgcgaucacaugguccugcuggaguucgugaccgccgccgggauca720

cucucggcauggacgagcuguacaaguccggaagacaccaccccggagaacagcuccucg780

gauccaccggaucuagauaacugaucauaaucagccauaccacauuuguagagguuuuac840

uugcuuuaaaaaaccucccacaccucccccugaaccugaaacauaaaaugaaugcaauug900

uuguuguuaacuuguuuauugcagcuuauaaugguuacaaauaaagcaauagcaucacaa960

auuucacaaauaaagcauuuuuuucacugca991

<210>49

<211>2649

<212>rna

<213>人工序列

<220>

<223>全长β2.7rna_endo(正义)

<400>49

ucagaucuaauacgacucacuauaggcgcuuccggaagagcuagcuccccagaucgcugc60

ugccccggcguucuccagaagccccggcgggcgaaucggccggcuggucggucggcgcuc120

ggacggauggggagaacggcggugacuuagccgcccguggccgggagaagauggaggagc180

cgagaugacaacggcagucguggaagggucgccaagccccgguccuucucuucugucugg240

ucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuuuaugugaguuucucu300

uccgcgucucccggccguaccauccacccaugcagcaugcacgcguguauguaugcaucg360

ucucuccuccgucccgacuaccaucagcagcaccacuaccgccacccccagcgccaccac420

cgcugccgucgccaccgcguuauccguuccucguaggcugguccuggggaacgggucggc480

ggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuucuccuuuccuuaauc540

ucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaa600

aaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaacucgaagaauucaaa660

gucgacgauucccaacaagagaaaacagaacaaaaacaaggucauuuuuauuuauccuca720

ucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaagggu780

guuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguau840

agguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugac900

gcgucggucagcagccaaacaaucauaggaaaugaaccagaagaacaaaaagaucaucuc960

ucucgguguauagcaacaccaacaacaaccgcaucgcaacaucuucauccgcaagacgga1020

aagaaaacaacaauaaugagaaugaaaucaccacaaccaagccagauuucacguccauga1080

guuuuuauuauauuauuaucaaaacgaaaaacagaaaaacugucauagauaaauauaaaa1140

aaaaauagaaaccacaaacgacuacuaguacuccaaucuuagauguauaugcuccuagau1200

aagauuuaguauuaccauaaucaucgaagaaugaaagacgacgaugauuccuuaccgcuc1260

cugccacccggucuguauguagagagagaagagagaaaacggugaauccaagauccccgg1320

gucggcgucggcaugccgcugaucgcaguggccccaccucggcaugccggcgccgggcga1380

ggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaacaauacaugauuaaccg1440

aaaagaaaccaacaaaaagaacccgagaucagucgauuucgaucacuacgauaaacacau1500

ggaagauuucuugaaaaaagaaaagagaaagagaccaccuucccggcggcggacacgcuc1560

cucuccgucgccguucugcaccaugauucgaucaauaacaacaucaucaucggagaccau1620

cuuuuaaucaaucagcguugcaguagucgacucccuggacacgaaggagucauccauuuu1680

uauccucgcacuucuucgcucucaaagccgccuuuaaaguugaaaugaaaggauggaaac1740

auggaauacaguuuuaauugcacguaucaccauuuuacuacaaaaagaaaaaaaaacaac1800

uuacacauaguauuaccuuagguuuacggauaaguagaguguaggcguuuuugaaacagu1860

ucagccaaugcaaucuugucucggcauaaucacucuuucugcauauaauaguaguaguag1920

auuuauucacaucaacacagcgaaaaacuccagcaucaaaguacaccuagagacagcccu1980

uaaaauauaguuugcagcuuuuagauguacuuacaccaaagaagauuaccguccuuacga2040

gaaaacagauacucggauauaggaaucaagacagcucugcacugaaaacacacucuccug2100

ucacgacaccgcgccacaccagaggcguacgcgugacuucaucgcaacgauccaucguga2160

ugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggaccacaguugucgauucuu2220

gaagacaauauucucgugagaacuuugagauucgcacuugaaaccucuuaggauccacaa2280

aaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcuuuucucccaaaccucg2340

guuucuuccuauucuuaaguuuucccuaguauauuugccuccuuauaagaaaagaagcac2400

aagcucggucgcacggauuauuccuucugcuaaucuauuauuuuguuccuuuuuuuuuug2460

uuugccuucacccccuucacucccuguagcaacacagaguaguagacacaauaaaugaga2520

aguuugcaugcagcuugacuccuauagugucaccuaaaugucuagaaacuuguuuauugc2580

agcuuauaaugguuacaaauaaagcaauagcaucacaaauuucacaaauaaagcauuuuu2640

uucacugca2649

<210>50

<211>264

<212>rna

<213>人工序列

<220>

<223>β2.7rna结构域2_endo(正义)

<400>50

ucagaucuaauacgacucacuauaggcgcuuccggaagagcuagcuuuuauuuuuuaucu60

ucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaug120

auaaacaagaggguaaaaagaaaaaaagcuugacuccuauagugucaccuaaaugucuag180

aaacuuguuuauugcagcuuauaaugguuacaaauaaagcaauagcaucacaaauuucac240

aaauaaagcauuuuuuucacugca264

<210>51

<211>403

<212>rna

<213>人工序列

<220>

<223>β2.7rna结构域3_endo(正义)

<400>51

ucagaucuaauacgacucacuauaggcgcuuccggaagagcuagcggucauuuuuauuua60

uccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgc120

aaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucu180

ccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacau240

ccugacgcgucggucagcagccaaacaaucauaggaaaugaaccaagcuugacuccuaua300

gugucaccuaaaugucuagaaacuuguuuauugcagcuuauaaugguuacaaauaaagca360

auagcaucacaaauuucacaaauaaagcauuuuuuucacugca403

<210>52

<211>629

<212>rna

<213>人工序列

<220>

<223>多聚体序列d2x4_endo

<400>52

ucagaucuaauacgacucacuauaggcgcuuccggaagagcuagcuuuuauuuuuuaucu60

ucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaug120

auaaacaagaggguaaaaagaaaaaagcgcgaauuccgaucuuuuauuuuuuaucuucuc180

cuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaa240

acaagaggguaaaaagaaaaagaucguauccgguaccugugguuuuauuuuuuaucuucu300

ccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugaua360

aacaagaggguaaaaagaaaaaccacaccuccaccggugggccguuuuauuuuuuaucuu420

cuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagauga480

uaaacaagaggguaaaaagaaaaacggcccaagcuugacuccuauagugucaccuaaaug540

ucuagaaacuuguuuauugcagcuuauaaugguuacaaauaaagcaauagcaucacaaau600

uucacaaauaaagcauuuuuuucacugca629

<210>53

<211>1206

<212>rna

<213>人工序列

<220>

<223>多聚体序列d3x4_endo

<400>53

ucagaucuaauacgacucacuauaggcgcuuccggaagagcuagcccggcggucauuuuu60

auuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaa120

uccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgc180

gaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccug240

cacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccgccgggaagcg300

ccgaauucauaucgaucggucauuuuuauuuauccucaucgucaacaacaacuaccgaca360

acaacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgcc420

ugucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccgug480

uccgguccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaa540

ucauaggaaaugaaccgaucgaauaaagguaccuguggggucauuuuuauuuauccucau600

cgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaagggug660

uuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguaua720

gguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugacg780

cgucggucagcagccaaacaaucauaggaaaugaaccccacauuuuaccgguaauaccgg840

ggggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccac900

caagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucau960

ggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauucc1020

ccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaac1080

cccccggaagcuugacuccuauagugucaccuaaaugucuagaaacuuguuuauugcagc1140

uuauaaugguuacaaauaaagcaauagcaucacaaauuucacaaauaaagcauuuuuuuc1200

acugca1206

<210>54

<211>1692

<212>rna

<213>人工序列

<220>

<223>多聚体序列d3x4_d2x4_endo

<400>54

ucagaucuaauacgacucacuauaggcgcuuccggaagagcuagcccggcggucauuuuu60

auuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaa120

uccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgc180

gaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccug240

cacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccgccgggaagcg300

ccgaauucauaucgaucggucauuuuuauuuauccucaucgucaacaacaacuaccgaca360

acaacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgcc420

ugucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccgug480

uccgguccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaa540

ucauaggaaaugaaccgaucgaauaaagguaccuguggggucauuuuuauuuauccucau600

cgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaagggug660

uuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguaua720

gguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugacg780

cgucggucagcagccaaacaaucauaggaaaugaaccccacauuuuaccgguaauaccgg840

ggggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccac900

caagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucau960

ggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauucc1020

ccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaac1080

cccccggaagcuuuccggaagagcuagcuuuuauuuuuuaucuucuccuuuccuuaaucu1140

cggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaa1200

aagaaaaaagcgcgaauuccgaucuuuuauuuuuuaucuucuccuuuccuuaaucucgga1260

uuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaaga1320

aaaagaucguauccgguaccugugguuuuauuuuuuaucuucuccuuuccuuaaucucgg1380

auuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaag1440

aaaaaccacaccuccaccggugggccguuuuauuuuuuaucuucuccuuuccuuaaucuc1500

ggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaa1560

agaaaaacggcccaagcuugacuccuauagugucaccuaaaugucuagaaacuuguuuau1620

ugcagcuuauaaugguuacaaauaaagcaauagcaucacaaauuucacaaauaaagcauu1680

uuuuucacugca1692

<210>55

<211>2486

<212>rna

<213>人工序列

<220>

<223>β2.7_core

<400>55

uccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggccggcu60

ggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccguggccggg120

agaagauggaggagccgagaugacaacggcagucguggaagggucgccaagccccggucc180

uucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuu240

uaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugcacgcg300

uguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuaccgccac360

ccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcugguccu420

ggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuu480

cuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagauga540

uaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaa600

cucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaaggucau660

uuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaug720

ucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgagga780

ccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugc840

ccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccagaagaa900

caaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaacaucuu960

cauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagccag1020

auuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacuguca1080

uagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuagaug1140

uauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacgacgau1200

gauuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaacgguga1260

auccaagauccccgggucggcgucggcaugccgcugaucgcaguggccccaccucggcau1320

gccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaaca1380

auacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucgauca1440

cuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccuucccg1500

gcggcggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaacaacauc1560

aucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggacacgaa1620

ggagucauccauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaaguugaaa1680

ugaaaggauggaaacauggaauacaguuuuaauugcacguaucaccauuuuacuacaaaa1740

agaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaaguagaguguagg1800

cguuuuugaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucugcaua1860

uaauaguaguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaaguaca1920

ccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaagaaga1980

uuaccguccuuacgagaaaacagauacucggauauaggaaucaagacagcucugcacuga2040

aaacacacucuccugucacgacaccgcgccacaccagaggcguacgcgugacuucaucgc2100

aacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggacca2160

caguugucgauucuugaagacaauauucucgugagaacuuugagauucgcacuugaaacc2220

ucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcuu2280

uucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccuccuua2340

uaagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaaucuauuauuuug2400

uuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaacacagaguaguag2460

acacaauaaaugagaaguuugcaugc2486

<210>56

<211>421

<212>rna

<213>人工序列

<220>

<223>d1_core

<400>56

cagaagccccggcgggcgaaucggccggcuggucggucggcgcucggacggauggggaga60

acggcggugacuuagccgcccguggccgggagaagauggaggagccgagaugacaacggc120

agucguggaagggucgccaagccccgguccuucucuucugucuggucgaaucucguuuuc180

uuuuuucaaccgcucuuuuuaucaccuuuuuaugugaguuucucuuccgcgucucccggc240

cguaccauccacccaugcagcaugcacgcguguauguaugcaucgucucuccuccguccc300

gacuaccaucagcagcaccacuaccgccacccccagcgccaccaccgcugccgucgccac360

cgcguuauccguuccucguaggcugguccuggggaacgggucggcggccggucggcuucu420

g421

<210>57

<211>100

<212>rna

<213>人工序列

<220>

<223>d2_core

<400>57

uuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuac60

cacgaaucgcagaugauaaacaagaggguaaaaagaaaaa100

<210>58

<211>238

<212>rna

<213>人工序列

<220>

<223>d3_core

<400>58

ggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccacca60

agaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucaugg120

cgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauucccc180

gcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaac238

<210>59

<211>367

<212>rna

<213>人工序列

<220>

<223>d4_core

<400>59

cggcgucggcaugccgcugaucgcaguggccccaccucggcaugccggcgccgggcgagg60

aauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaacaauacaugauuaaccgaa120

aagaaaccaacaaaaagaacccgagaucagucgauuucgaucacuacgauaaacacaugg180

aagauuucuugaaaaaagaaaagagaaagagaccaccuucccggcggcggacacgcuccu240

cuccgucgccguucugcaccaugauucgaucaauaacaacaucaucaucggagaccaucu300

uuuaaucaaucagcguugcaguagucgacucccuggacacgaaggagucauccauuuuua360

uccucgc367

<210>60

<211>2065

<212>rna

<213>人工序列

<220>

<223>δd1_core

<400>60

uccccagaucgcugcugccccggcguucucuuuuauuauuuuuuuuuauuuuuuaucuuc60

uccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugau120

aaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaac180

ucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaaggucauu240

uuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugu300

caauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggac360

cgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcc420

cugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccagaagaac480

aaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaacaucuuc540

auccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagccaga600

uuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacugucau660

agauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuagaugu720

auaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacgacgaug780

auuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaacggugaa840

uccaagauccccgggucggcgucggcaugccgcugaucgcaguggccccaccucggcaug900

ccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaacaa960

uacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucgaucac1020

uacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccuucccgg1080

cggcggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaacaacauca1140

ucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggacacgaag1200

gagucauccauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaaguugaaau1260

gaaaggauggaaacauggaauacaguuuuaauugcacguaucaccauuuuacuacaaaaa1320

gaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaaguagaguguaggc1380

guuuuugaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucugcauau1440

aauaguaguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaaguacac1500

cuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaagaagau1560

uaccguccuuacgagaaaacagauacucggauauaggaaucaagacagcucugcacugaa1620

aacacacucuccugucacgacaccgcgccacaccagaggcguacgcgugacuucaucgca1680

acgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggaccac1740

aguugucgauucuugaagacaauauucucgugagaacuuugagauucgcacuugaaaccu1800

cuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcuuu1860

ucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccuccuuau1920

aagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaaucuauuauuuugu1980

uccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaacacagaguaguaga2040

cacaauaaaugagaaguuugcaugc2065

<210>61

<211>2386

<212>rna

<213>人工序列

<220>

<223>δd2_core

<400>61

uccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggccggcu60

ggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccguggccggg120

agaagauggaggagccgagaugacaacggcagucguggaagggucgccaagccccggucc180

uucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuu240

uaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugcacgcg300

uguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuaccgccac360

ccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcugguccu420

ggggaacgggucggcggccggucggcuucuguuuuauuauuuuuagcuacagacauuugg480

guaccucagcuuuccgauaacucgaagaauucaaagucgacgauucccaacaagagaaaa540

cagaacaaaaacaaggucauuuuuauuuauccucaucgucaacaacaacuaccgacaaca600

acgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugu660

cgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccgugucc720

gguccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaauca780

uaggaaaugaaccagaagaacaaaaagaucaucucucucgguguauagcaacaccaacaa840

caaccgcaucgcaacaucuucauccgcaagacggaaagaaaacaacaauaaugagaauga900

aaucaccacaaccaagccagauuucacguccaugaguuuuuauuauauuauuaucaaaac960

gaaaaacagaaaaacugucauagauaaauauaaaaaaaaauagaaaccacaaacgacuac1020

uaguacuccaaucuuagauguauaugcuccuagauaagauuuaguauuaccauaaucauc1080

gaagaaugaaagacgacgaugauuccuuaccgcuccugccacccggucuguauguagaga1140

gagaagagagaaaacggugaauccaagauccccgggucggcgucggcaugccgcugaucg1200

caguggccccaccucggcaugccggcgccgggcgaggaauugcucaugaaaaaaguaucu1260

uucuguaaaaaaagaaaacaauacaugauuaaccgaaaagaaaccaacaaaaagaacccg1320

agaucagucgauuucgaucacuacgauaaacacauggaagauuucuugaaaaaagaaaag1380

agaaagagaccaccuucccggcggcggacacgcuccucuccgucgccguucugcaccaug1440

auucgaucaauaacaacaucaucaucggagaccaucuuuuaaucaaucagcguugcagua1500

gucgacucccuggacacgaaggagucauccauuuuuauccucgcacuucuucgcucucaa1560

agccgccuuuaaaguugaaaugaaaggauggaaacauggaauacaguuuuaauugcacgu1620

aucaccauuuuacuacaaaaagaaaaaaaaacaacuuacacauaguauuaccuuagguuu1680

acggauaaguagaguguaggcguuuuugaaacaguucagccaaugcaaucuugucucggc1740

auaaucacucuuucugcauauaauaguaguaguagauuuauucacaucaacacagcgaaa1800

aacuccagcaucaaaguacaccuagagacagcccuuaaaauauaguuugcagcuuuuaga1860

uguacuuacaccaaagaagauuaccguccuuacgagaaaacagauacucggauauaggaa1920

ucaagacagcucugcacugaaaacacacucuccugucacgacaccgcgccacaccagagg1980

cguacgcgugacuucaucgcaacgauccaucgugaugucccucgcagaaccuaaaaagac2040

caaaaaaaaaucuuggaccacaguugucgauucuugaagacaauauucucgugagaacuu2100

ugagauucgcacuugaaaccucuuaggauccacaaaaacaacaaccucuguauggaaaau2160

gcgcuauuuuaucucagcuuuucucccaaaccucgguuucuuccuauucuuaaguuuucc2220

cuaguauauuugccuccuuauaagaaaagaagcacaagcucggucgcacggauuauuccu2280

ucugcuaaucuauuauuuuguuccuuuuuuuuuuguuugccuucacccccuucacucccu2340

guagcaacacagaguaguagacacaauaaaugagaaguuugcaugc2386

<210>62

<211>2247

<212>rna

<213>人工序列

<220>

<223>δd3_core

<400>62

uccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggccggcu60

ggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccguggccggg120

agaagauggaggagccgagaugacaacggcagucguggaagggucgccaagccccggucc180

uucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuu240

uaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugcacgcg300

uguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuaccgccac360

ccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcugguccu420

ggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuu480

cuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagauga540

uaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaa600

cucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaaagaaga660

acaaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaacaucu720

ucauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagcca780

gauuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacuguc840

auagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuagau900

guauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacgacga960

ugauuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaacggug1020

aauccaagauccccgggucggcgucggcaugccgcugaucgcaguggccccaccucggca1080

ugccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaac1140

aauacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucgauc1200

acuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccuuccc1260

ggcggcggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaacaacau1320

caucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggacacga1380

aggagucauccauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaaguugaa1440

augaaaggauggaaacauggaauacaguuuuaauugcacguaucaccauuuuacuacaaa1500

aagaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaaguagaguguag1560

gcguuuuugaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucugcau1620

auaauaguaguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaaguac1680

accuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaagaag1740

auuaccguccuuacgagaaaacagauacucggauauaggaaucaagacagcucugcacug1800

aaaacacacucuccugucacgacaccgcgccacaccagaggcguacgcgugacuucaucg1860

caacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggacc1920

acaguugucgauucuugaagacaauauucucgugagaacuuugagauucgcacuugaaac1980

cucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcu2040

uuucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccuccuu2100

auaagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaaucuauuauuuu2160

guuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaacacagaguagua2220

gacacaauaaaugagaaguuugcaugc2247

<210>63

<211>2119

<212>rna

<213>人工序列

<220>

<223>δd4_core

<400>63

uccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggccggcu60

ggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccguggccggg120

agaagauggaggagccgagaugacaacggcagucguggaagggucgccaagccccggucc180

uucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuu240

uaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugcacgcg300

uguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuaccgccac360

ccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcugguccu420

ggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuu480

cuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagauga540

uaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaa600

cucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaaggucau660

uuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaug720

ucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgagga780

ccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugc840

ccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccagaagaa900

caaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaacaucuu960

cauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagccag1020

auuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacuguca1080

uagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuagaug1140

uauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacgacgau1200

gauuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaacgguga1260

auccaagauccccggguacuucuucgcucucaaagccgccuuuaaaguugaaaugaaagg1320

auggaaacauggaauacaguuuuaauugcacguaucaccauuuuacuacaaaaagaaaaa1380

aaaacaacuuacacauaguauuaccuuagguuuacggauaaguagaguguaggcguuuuu1440

gaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucugcauauaauagu1500

aguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaaguacaccuagag1560

acagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaagaagauuaccgu1620

ccuuacgagaaaacagauacucggauauaggaaucaagacagcucugcacugaaaacaca1680

cucuccugucacgacaccgcgccacaccagaggcguacgcgugacuucaucgcaacgauc1740

caucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggaccacaguugu1800

cgauucuugaagacaauauucucgugagaacuuugagauucgcacuugaaaccucuuagg1860

auccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcuuuucuccc1920

aaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccuccuuauaagaaa1980

agaagcacaagcucggucgcacggauuauuccuucugcuaaucuauuauuuuguuccuuu2040

uuuuuuuguuugccuucacccccuucacucccuguagcaacacagaguaguagacacaau2100

aaaugagaaguuugcaugc2119

<210>64

<211>2486

<212>rna

<213>人工序列

<220>

<223>β2.7rna_as_core

<400>64

gcaugcaaacuucucauuuauugugucuacuacucuguguugcuacagggagugaagggg60

gugaaggcaaacaaaaaaaaaaggaacaaaauaauagauuagcagaaggaauaauccgug120

cgaccgagcuugugcuucuuuucuuauaaggaggcaaauauacuagggaaaacuuaagaa180

uaggaagaaaccgagguuugggagaaaagcugagauaaaauagcgcauuuuccauacaga240

gguuguuguuuuuguggauccuaagagguuucaagugcgaaucucaaaguucucacgaga300

auauugucuucaagaaucgacaacugugguccaagauuuuuuuuuggucuuuuuagguuc360

ugcgagggacaucacgauggaucguugcgaugaagucacgcguacgccucugguguggcg420

cggugucgugacaggagaguguguuuucagugcagagcugucuugauuccuauauccgag480

uaucuguuuucucguaaggacgguaaucuucuuugguguaaguacaucuaaaagcugcaa540

acuauauuuuaagggcugucucuagguguacuuugaugcuggaguuuuucgcuguguuga600

ugugaauaaaucuacuacuacuauuauaugcagaaagagugauuaugccgagacaagauu660

gcauuggcugaacuguuucaaaaacgccuacacucuacuuauccguaaaccuaagguaau720

acuauguguaaguuguuuuuuuuucuuuuuguaguaaaauggugauacgugcaauuaaaa780

cuguauuccauguuuccauccuuucauuucaacuuuaaaggcggcuuugagagcgaagaa840

gugcgaggauaaaaauggaugacuccuucguguccagggagucgacuacugcaacgcuga900

uugauuaaaagauggucuccgaugaugauguuguuauugaucgaaucauggugcagaacg960

gcgacggagaggagcguguccgccgccgggaagguggucucuuucucuuuucuuuuuuca1020

agaaaucuuccauguguuuaucguagugaucgaaaucgacugaucucggguucuuuuugu1080

ugguuucuuuucgguuaaucauguauuguuuucuuuuuuuacagaaagauacuuuuuuca1140

ugagcaauuccucgcccggcgccggcaugccgagguggggccacugcgaucagcggcaug1200

ccgacgccgacccggggaucuuggauucaccguuuucucucuucucucucuacauacaga1260

ccggguggcaggagcgguaaggaaucaucgucgucuuucauucuucgaugauuaugguaa1320

uacuaaaucuuaucuaggagcauauacaucuaagauuggaguacuaguagucguuugugg1380

uuucuauuuuuuuuuauauuuaucuaugacaguuuuucuguuuuucguuuugauaauaau1440

auaauaaaaacucauggacgugaaaucuggcuugguuguggugauuucauucucauuauu1500

guuguuuucuuuccgucuugcggaugaagauguugcgaugcgguuguuguugguguugcu1560

auacaccgagagagaugaucuuuuuguucuucugguucauuuccuaugauuguuuggcug1620

cugaccgacgcgucaggaugugcagggcaugcggggaaucaggaccggacacgggauaau1680

uucaucuaccuauacggagaucgcgguccucgccaugaggaucgcgacaggcgcgucgag1740

ggggcaggaacacccuugcggauugacauucuuggugguguuucguuguugucgguaguu1800

guuguugacgaugaggauaaauaaaaaugaccuuguuuuuguucuguuuucucuuguugg1860

gaaucgucgacuuugaauucuucgaguuaucggaaagcugagguacccaaaugucuguag1920

cuuuuuucuuuuuacccucuuguuuaucaucugcgauucgugguagguaggagagggaaa1980

ugauaauccgagauuaaggaaaggagaagauaaaaaauaaaaaaaaauaauaaaacagaa2040

gccgaccggccgccgacccguuccccaggaccagccuacgaggaacggauaacgcggugg2100

cgacggcagcggugguggcgcuggggguggcgguaguggugcugcugaugguagucggga2160

cggaggagagacgaugcauacauacacgcgugcaugcugcauggguggaugguacggccg2220

ggagacgcggaagagaaacucacauaaaaaggugauaaaaagagcgguugaaaaaagaaa2280

acgagauucgaccagacagaagagaaggaccggggcuuggcgacccuuccacgacugccg2340

uugucaucucggcuccuccaucuucucccggccacgggcggcuaagucaccgccguucuc2400

cccauccguccgagcgccgaccgaccagccggccgauucgcccgccggggcuucuggaga2460

acgccggggcagcagcgaucugggga2486

<210>65

<211>421

<212>rna

<213>人工序列

<220>

<223>d1as_core

<400>65

cagaagccgaccggccgccgacccguuccccaggaccagccuacgaggaacggauaacgc60

gguggcgacggcagcggugguggcgcuggggguggcgguaguggugcugcugaugguagu120

cgggacggaggagagacgaugcauacauacacgcgugcaugcugcauggguggaugguac180

ggccgggagacgcggaagagaaacucacauaaaaaggugauaaaaagagcgguugaaaaa240

agaaaacgagauucgaccagacagaagagaaggaccggggcuuggcgacccuuccacgac300

ugccguugucaucucggcuccuccaucuucucccggccacgggcggcuaagucaccgccg360

uucuccccauccguccgagcgccgaccgaccagccggccgauucgcccgccggggcuucu420

g421

<210>66

<211>100

<212>rna

<213>人工序列

<220>

<223>d2as_core

<400>66

uuuuucuuuuuacccucuuguuuaucaucugcgauucgugguagguaggagagggaaaug60

auaauccgagauuaaggaaaggagaagauaaaaaauaaaa100

<210>67

<211>238

<212>rna

<213>人工序列

<220>

<223>d3as_core

<400>67

guucauuuccuaugauuguuuggcugcugaccgacgcgucaggaugugcagggcaugcgg60

ggaaucaggaccggacacgggauaauuucaucuaccuauacggagaucgcgguccucgcc120

augaggaucgcgacaggcgcgucgagggggcaggaacacccuugcggauugacauucuug180

gugguguuucguuguugucgguaguuguuguugacgaugaggauaaauaaaaaugacc238

<210>68

<211>367

<212>rna

<213>人工序列

<220>

<223>d4as_core

<400>68

gcgaggauaaaaauggaugacuccuucguguccagggagucgacuacugcaacgcugauu60

gauuaaaagauggucuccgaugaugauguuguuauugaucgaaucauggugcagaacggc120

gacggagaggagcguguccgccgccgggaagguggucucuuucucuuuucuuuuuucaag180

aaaucuuccauguguuuaucguagugaucgaaaucgacugaucucggguucuuuuuguug240

guuucuuuucgguuaaucauguauuguuuucuuuuuuuacagaaagauacuuuuuucaug300

agcaauuccucgcccggcgccggcaugccgagguggggccacugcgaucagcggcaugcc360

gacgccg367

<210>69

<211>2065

<212>rna

<213>人工序列

<220>

<223>δd1as_core

<400>69

gcaugcaaacuucucauuuauugugucuacuacucuguguugcuacagggagugaagggg60

gugaaggcaaacaaaaaaaaaaggaacaaaauaauagauuagcagaaggaauaauccgug120

cgaccgagcuugugcuucuuuucuuauaaggaggcaaauauacuagggaaaacuuaagaa180

uaggaagaaaccgagguuugggagaaaagcugagauaaaauagcgcauuuuccauacaga240

gguuguuguuuuuguggauccuaagagguuucaagugcgaaucucaaaguucucacgaga300

auauugucuucaagaaucgacaacugugguccaagauuuuuuuuuggucuuuuuagguuc360

ugcgagggacaucacgauggaucguugcgaugaagucacgcguacgccucugguguggcg420

cggugucgugacaggagaguguguuuucagugcagagcugucuugauuccuauauccgag480

uaucuguuuucucguaaggacgguaaucuucuuugguguaaguacaucuaaaagcugcaa540

acuauauuuuaagggcugucucuagguguacuuugaugcuggaguuuuucgcuguguuga600

ugugaauaaaucuacuacuacuauuauaugcagaaagagugauuaugccgagacaagauu660

gcauuggcugaacuguuucaaaaacgccuacacucuacuuauccguaaaccuaagguaau720

acuauguguaaguuguuuuuuuuucuuuuuguaguaaaauggugauacgugcaauuaaaa780

cuguauuccauguuuccauccuuucauuucaacuuuaaaggcggcuuugagagcgaagaa840

gugcgaggauaaaaauggaugacuccuucguguccagggagucgacuacugcaacgcuga900

uugauuaaaagauggucuccgaugaugauguuguuauugaucgaaucauggugcagaacg960

gcgacggagaggagcguguccgccgccgggaagguggucucuuucucuuuucuuuuuuca1020

agaaaucuuccauguguuuaucguagugaucgaaaucgacugaucucggguucuuuuugu1080

ugguuucuuuucgguuaaucauguauuguuuucuuuuuuuacagaaagauacuuuuuuca1140

ugagcaauuccucgcccggcgccggcaugccgagguggggccacugcgaucagcggcaug1200

ccgacgccgacccggggaucuuggauucaccguuuucucucuucucucucuacauacaga1260

ccggguggcaggagcgguaaggaaucaucgucgucuuucauucuucgaugauuaugguaa1320

uacuaaaucuuaucuaggagcauauacaucuaagauuggaguacuaguagucguuugugg1380

uuucuauuuuuuuuuauauuuaucuaugacaguuuuucuguuuuucguuuugauaauaau1440

auaauaaaaacucauggacgugaaaucuggcuugguuguggugauuucauucucauuauu1500

guuguuuucuuuccgucuugcggaugaagauguugcgaugcgguuguuguugguguugcu1560

auacaccgagagagaugaucuuuuuguucuucugguucauuuccuaugauuguuuggcug1620

cugaccgacgcgucaggaugugcagggcaugcggggaaucaggaccggacacgggauaau1680

uucaucuaccuauacggagaucgcgguccucgccaugaggaucgcgacaggcgcgucgag1740

ggggcaggaacacccuugcggauugacauucuuggugguguuucguuguugucgguaguu1800

guuguugacgaugaggauaaauaaaaaugaccuuguuuuuguucuguuuucucuuguugg1860

gaaucgucgacuuugaauucuucgaguuaucggaaagcugagguacccaaaugucuguag1920

cuuuuuucuuuuuacccucuuguuuaucaucugcgauucgugguagguaggagagggaaa1980

ugauaauccgagauuaaggaaaggagaagauaaaaaauaaaaaaaaauaauaaaagagaa2040

cgccggggcagcagcgaucugggga2065

<210>70

<211>2386

<212>rna

<213>人工序列

<220>

<223>δd2as_core

<400>70

gcaugcaaacuucucauuuauugugucuacuacucuguguugcuacagggagugaagggg60

gugaaggcaaacaaaaaaaaaaggaacaaaauaauagauuagcagaaggaauaauccgug120

cgaccgagcuugugcuucuuuucuuauaaggaggcaaauauacuagggaaaacuuaagaa180

uaggaagaaaccgagguuugggagaaaagcugagauaaaauagcgcauuuuccauacaga240

gguuguuguuuuuguggauccuaagagguuucaagugcgaaucucaaaguucucacgaga300

auauugucuucaagaaucgacaacugugguccaagauuuuuuuuuggucuuuuuagguuc360

ugcgagggacaucacgauggaucguugcgaugaagucacgcguacgccucugguguggcg420

cggugucgugacaggagaguguguuuucagugcagagcugucuugauuccuauauccgag480

uaucuguuuucucguaaggacgguaaucuucuuugguguaaguacaucuaaaagcugcaa540

acuauauuuuaagggcugucucuagguguacuuugaugcuggaguuuuucgcuguguuga600

ugugaauaaaucuacuacuacuauuauaugcagaaagagugauuaugccgagacaagauu660

gcauuggcugaacuguuucaaaaacgccuacacucuacuuauccguaaaccuaagguaau720

acuauguguaaguuguuuuuuuuucuuuuuguaguaaaauggugauacgugcaauuaaaa780

cuguauuccauguuuccauccuuucauuucaacuuuaaaggcggcuuugagagcgaagaa840

gugcgaggauaaaaauggaugacuccuucguguccagggagucgacuacugcaacgcuga900

uugauuaaaagauggucuccgaugaugauguuguuauugaucgaaucauggugcagaacg960

gcgacggagaggagcguguccgccgccgggaagguggucucuuucucuuuucuuuuuuca1020

agaaaucuuccauguguuuaucguagugaucgaaaucgacugaucucggguucuuuuugu1080

ugguuucuuuucgguuaaucauguauuguuuucuuuuuuuacagaaagauacuuuuuuca1140

ugagcaauuccucgcccggcgccggcaugccgagguggggccacugcgaucagcggcaug1200

ccgacgccgacccggggaucuuggauucaccguuuucucucuucucucucuacauacaga1260

ccggguggcaggagcgguaaggaaucaucgucgucuuucauucuucgaugauuaugguaa1320

uacuaaaucuuaucuaggagcauauacaucuaagauuggaguacuaguagucguuugugg1380

uuucuauuuuuuuuuauauuuaucuaugacaguuuuucuguuuuucguuuugauaauaau1440

auaauaaaaacucauggacgugaaaucuggcuugguuguggugauuucauucucauuauu1500

guuguuuucuuuccgucuugcggaugaagauguugcgaugcgguuguuguugguguugcu1560

auacaccgagagagaugaucuuuuuguucuucugguucauuuccuaugauuguuuggcug1620

cugaccgacgcgucaggaugugcagggcaugcggggaaucaggaccggacacgggauaau1680

uucaucuaccuauacggagaucgcgguccucgccaugaggaucgcgacaggcgcgucgag1740

ggggcaggaacacccuugcggauugacauucuuggugguguuucguuguugucgguaguu1800

guuguugacgaugaggauaaauaaaaaugaccuuguuuuuguucuguuuucucuuguugg1860

gaaucgucgacuuugaauucuucgaguuaucggaaagcugagguacccaaaugucuguag1920

cuaaaaauaauaaaacagaagccgaccggccgccgacccguuccccaggaccagccuacg1980

aggaacggauaacgcgguggcgacggcagcggugguggcgcuggggguggcgguaguggu2040

gcugcugaugguagucgggacggaggagagacgaugcauacauacacgcgugcaugcugc2100

auggguggaugguacggccgggagacgcggaagagaaacucacauaaaaaggugauaaaa2160

agagcgguugaaaaaagaaaacgagauucgaccagacagaagagaaggaccggggcuugg2220

cgacccuuccacgacugccguugucaucucggcuccuccaucuucucccggccacgggcg2280

gcuaagucaccgccguucuccccauccguccgagcgccgaccgaccagccggccgauucg2340

cccgccggggcuucuggagaacgccggggcagcagcgaucugggga2386

<210>71

<211>2247

<212>rna

<213>人工序列

<220>

<223>δd3as_core

<400>71

gcaugcaaacuucucauuuauugugucuacuacucuguguugcuacagggagugaagggg60

gugaaggcaaacaaaaaaaaaaggaacaaaauaauagauuagcagaaggaauaauccgug120

cgaccgagcuugugcuucuuuucuuauaaggaggcaaauauacuagggaaaacuuaagaa180

uaggaagaaaccgagguuugggagaaaagcugagauaaaauagcgcauuuuccauacaga240

gguuguuguuuuuguggauccuaagagguuucaagugcgaaucucaaaguucucacgaga300

auauugucuucaagaaucgacaacugugguccaagauuuuuuuuuggucuuuuuagguuc360

ugcgagggacaucacgauggaucguugcgaugaagucacgcguacgccucugguguggcg420

cggugucgugacaggagaguguguuuucagugcagagcugucuugauuccuauauccgag480

uaucuguuuucucguaaggacgguaaucuucuuugguguaaguacaucuaaaagcugcaa540

acuauauuuuaagggcugucucuagguguacuuugaugcuggaguuuuucgcuguguuga600

ugugaauaaaucuacuacuacuauuauaugcagaaagagugauuaugccgagacaagauu660

gcauuggcugaacuguuucaaaaacgccuacacucuacuuauccguaaaccuaagguaau720

acuauguguaaguuguuuuuuuuucuuuuuguaguaaaauggugauacgugcaauuaaaa780

cuguauuccauguuuccauccuuucauuucaacuuuaaaggcggcuuugagagcgaagaa840

gugcgaggauaaaaauggaugacuccuucguguccagggagucgacuacugcaacgcuga900

uugauuaaaagauggucuccgaugaugauguuguuauugaucgaaucauggugcagaacg960

gcgacggagaggagcguguccgccgccgggaagguggucucuuucucuuuucuuuuuuca1020

agaaaucuuccauguguuuaucguagugaucgaaaucgacugaucucggguucuuuuugu1080

ugguuucuuuucgguuaaucauguauuguuuucuuuuuuuacagaaagauacuuuuuuca1140

ugagcaauuccucgcccggcgccggcaugccgagguggggccacugcgaucagcggcaug1200

ccgacgccgacccggggaucuuggauucaccguuuucucucuucucucucuacauacaga1260

ccggguggcaggagcgguaaggaaucaucgucgucuuucauucuucgaugauuaugguaa1320

uacuaaaucuuaucuaggagcauauacaucuaagauuggaguacuaguagucguuugugg1380

uuucuauuuuuuuuuauauuuaucuaugacaguuuuucuguuuuucguuuugauaauaau1440

auaauaaaaacucauggacgugaaaucuggcuugguuguggugauuucauucucauuauu1500

guuguuuucuuuccgucuugcggaugaagauguugcgaugcgguuguuguugguguugcu1560

auacaccgagagagaugaucuuuuuguucuucuuuguuuuuguucuguuuucucuuguug1620

ggaaucgucgacuuugaauucuucgaguuaucggaaagcugagguacccaaaugucugua1680

gcuuuuuucuuuuuacccucuuguuuaucaucugcgauucgugguagguaggagagggaa1740

augauaauccgagauuaaggaaaggagaagauaaaaaauaaaaaaaaauaauaaaacaga1800

agccgaccggccgccgacccguuccccaggaccagccuacgaggaacggauaacgcggug1860

gcgacggcagcggugguggcgcuggggguggcgguaguggugcugcugaugguagucggg1920

acggaggagagacgaugcauacauacacgcgugcaugcugcauggguggaugguacggcc1980

gggagacgcggaagagaaacucacauaaaaaggugauaaaaagagcgguugaaaaaagaa2040

aacgagauucgaccagacagaagagaaggaccggggcuuggcgacccuuccacgacugcc2100

guugucaucucggcuccuccaucuucucccggccacgggcggcuaagucaccgccguucu2160

ccccauccguccgagcgccgaccgaccagccggccgauucgcccgccggggcuucuggag2220

aacgccggggcagcagcgaucugggga2247

<210>72

<211>2119

<212>rna

<213>人工序列

<220>

<223>δd4as_core

<400>72

gcaugcaaacuucucauuuauugugucuacuacucuguguugcuacagggagugaagggg60

gugaaggcaaacaaaaaaaaaaggaacaaaauaauagauuagcagaaggaauaauccgug120

cgaccgagcuugugcuucuuuucuuauaaggaggcaaauauacuagggaaaacuuaagaa180

uaggaagaaaccgagguuugggagaaaagcugagauaaaauagcgcauuuuccauacaga240

gguuguuguuuuuguggauccuaagagguuucaagugcgaaucucaaaguucucacgaga300

auauugucuucaagaaucgacaacugugguccaagauuuuuuuuuggucuuuuuagguuc360

ugcgagggacaucacgauggaucguugcgaugaagucacgcguacgccucugguguggcg420

cggugucgugacaggagaguguguuuucagugcagagcugucuugauuccuauauccgag480

uaucuguuuucucguaaggacgguaaucuucuuugguguaaguacaucuaaaagcugcaa540

acuauauuuuaagggcugucucuagguguacuuugaugcuggaguuuuucgcuguguuga600

ugugaauaaaucuacuacuacuauuauaugcagaaagagugauuaugccgagacaagauu660

gcauuggcugaacuguuucaaaaacgccuacacucuacuuauccguaaaccuaagguaau720

acuauguguaaguuguuuuuuuuucuuuuuguaguaaaauggugauacgugcaauuaaaa780

cuguauuccauguuuccauccuuucauuucaacuuuaaaggcggcuuugagagcgaagaa840

guacccggggaucuuggauucaccguuuucucucuucucucucuacauacagaccgggug900

gcaggagcgguaaggaaucaucgucgucuuucauucuucgaugauuaugguaauacuaaa960

ucuuaucuaggagcauauacaucuaagauuggaguacuaguagucguuugugguuucuau1020

uuuuuuuuauauuuaucuaugacaguuuuucuguuuuucguuuugauaauaauauaauaa1080

aaacucauggacgugaaaucuggcuugguuguggugauuucauucucauuauuguuguuu1140

ucuuuccgucuugcggaugaagauguugcgaugcgguuguuguugguguugcuauacacc1200

gagagagaugaucuuuuuguucuucugguucauuuccuaugauuguuuggcugcugaccg1260

acgcgucaggaugugcagggcaugcggggaaucaggaccggacacgggauaauuucaucu1320

accuauacggagaucgcgguccucgccaugaggaucgcgacaggcgcgucgagggggcag1380

gaacacccuugcggauugacauucuuggugguguuucguuguugucgguaguuguuguug1440

acgaugaggauaaauaaaaaugaccuuguuuuuguucuguuuucucuuguugggaaucgu1500

cgacuuugaauucuucgaguuaucggaaagcugagguacccaaaugucuguagcuuuuuu1560

cuuuuuacccucuuguuuaucaucugcgauucgugguagguaggagagggaaaugauaau1620

ccgagauuaaggaaaggagaagauaaaaaauaaaaaaaaauaauaaaacagaagccgacc1680

ggccgccgacccguuccccaggaccagccuacgaggaacggauaacgcgguggcgacggc1740

agcggugguggcgcuggggguggcgguaguggugcugcugaugguagucgggacggagga1800

gagacgaugcauacauacacgcgugcaugcugcauggguggaugguacggccgggagacg1860

cggaagagaaacucacauaaaaaggugauaaaaagagcgguugaaaaaagaaaacgagau1920

ucgaccagacagaagagaaggaccggggcuuggcgacccuuccacgacugccguugucau1980

cucggcuccuccaucuucucccggccacgggcggcuaagucaccgccguucuccccaucc2040

guccgagcgccgaccgaccagccggccgauucgcccgccggggcuucuggagaacgccgg2100

ggcagcagcgaucugggga2119

<210>73

<211>400

<212>rna

<213>人工序列

<220>

<223>多聚体序列d2x4_core

<400>73

uuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuac60

cacgaaucgcagaugauaaacaagaggguaaaaagaaaaauuuuauuuuuuaucuucucc120

uuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaa180

caagaggguaaaaagaaaaauuuuauuuuuuaucuucuccuuuccuuaaucucggauuau240

cauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaa300

uuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuac360

cacgaaucgcagaugauaaacaagaggguaaaaagaaaaa400

<210>74

<211>954

<212>rna

<213>人工序列

<220>

<223>多聚体序列d3x4_core

<400>74

ggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccacca60

agaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucaugg120

cgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauucccc180

gcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccg240

gucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaa300

gaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggc360

gaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccg420

caugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaagguc480

auuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaa540

ugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgag600

gaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcau660

gcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccgguca720

uuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaau780

gucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgagg840

accgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaug900

cccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaacc954

<210>75

<211>1360

<212>rna

<213>人工序列

<220>

<223>多聚体序列d3x4_d2x4_core

<400>75

ggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccacca60

agaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucaugg120

cgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauucccc180

gcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccg240

gucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaa300

gaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggc360

gaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccg420

caugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaagguc480

auuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaa540

ugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgag600

gaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcau660

gcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccgguca720

uuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaau780

gucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgagg840

accgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaug900

cccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccuuuuau960

uuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaa1020

ucgcagaugauaaacaagaggguaaaaagaaaaauuuuauuuuuuaucuucuccuuuccu1080

uaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagag1140

gguaaaaagaaaaauuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuuc1200

ccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaauuuuau1260

uuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaa1320

ucgcagaugauaaacaagaggguaaaaagaaaaacggccc1360

<210>76

<211>1326

<212>rna

<213>人工序列

<220>

<223>多聚体序列d2x4_d3x4_core

<400>76

uuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuac60

cacgaaucgcagaugauaaacaagaggguaaaaagaaaaauuuuauuuuuuaucuucucc120

uuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcuuuuauuuuu180

uaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgc240

agaugauaaacaagaggguaaaaagaaaaauuuuauuuuuuaucuucuccuuuccuuaau300

cucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagagggua360

aaaagaaaaaggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacga420

aacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcg480

auccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguc540

cugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucauagg600

aaaugaaccggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaa660

acaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcga720

uccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccggucc780

ugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucauagga840

aaugaaccggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaa900

caccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgau960

ccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccu1020

gauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaa1080

augaaccggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaac1140

accaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauc1200

cucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccug1260

auuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaa1320

ugaacc1326

<210>77

<211>1356

<212>rna

<213>人工序列

<220>

<223>多聚体序列(d3_d2)x4_core

<400>77

ggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccacca60

agaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucaugg120

cgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauucccc180

gcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccu240

uuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuacc300

acgaaucgcagaugauaaacaagaggguaaaaagaaaaaggucauuuuuauuuauccuca360

ucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaagggu420

guuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguau480

agguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugac540

gcgucggucagcagccaaacaaucauaggaaaugaaccuuuuauuuuuuaucuucuccuu600

uccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaaca660

agaggguaaaaagaaaaaggucauuuuuauuuauccucaucgucaacaacaacuaccgac720

aacaacgaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgc780

cugucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccgu840

guccgguccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaaca900

aucauaggaaaugaaccuuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucau960

uucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaaggu1020

cauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaaga1080

augucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcga1140

ggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgca1200

ugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccuuuu1260

auuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacg1320

aaucgcagaugauaaacaagaggguaaaaagaaaaa1356

<210>78

<211>3198

<212>rna

<213>人工序列

<220>

<223>ggfp_β2.7_core

<400>78

auggugagcaagggcgaggagcuguucaccgggguggugcccauccuggucgagcuggac60

ggcgacguaaacggccacaaguucagcguguccggcgagggcgagggcgaugccaccuac120

ggcaagcugacccugaaguucaucugcaccaccggcaagcugcccgugcccuggcccacc180

cucgugaccacccugaccuacggcgugcagugcuucagccgcuaccccgaccacaugaag240

cagcacgacuucuucaaguccgccaugcccgaaggcuacguccaggagcgcaccaucuuc300

uucaaggacgacggcaacuacaagacccgcgccgaggugaaguucgagggcgacacccug360

gugaaccgcaucgagcugaagggcaucgacuucaaggaggacggcaacauccuggggcac420

aagcuggaguacaacuacaacagccacaacgucuauaucauggccgacaagcagaagaac480

ggcaucaaggugaacuucaagauccgccacaacaucgaggacggcagcgugcagcucgcc540

gaccacuaccagcagaacacccccaucggcgacggccccgugcugcugcccgacaaccac600

uaccugagcacccaguccgcccugagcaaagaccccaacgagaagcgcgaucacaugguc660

cugcuggaguucgugaccgccgccgggaucacucucggcauggacgagcuguacaaguga720

uccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggccggcu780

ggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccguggccggg840

agaagauggaggagccgagaugacaacggcagucguggaagggucgccaagccccggucc900

uucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuu960

uaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugcacgcg1020

uguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuaccgccac1080

ccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcugguccu1140

ggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuu1200

cuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagauga1260

uaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaa1320

cucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaaggucau1380

uuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaug1440

ucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgagga1500

ccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugc1560

ccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccagaagaa1620

caaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaacaucuu1680

cauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagccag1740

auuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacuguca1800

uagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuagaug1860

uauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacgacgau1920

gauuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaacgguga1980

auccaagauccccgggucggcgucggcaugccgcugaucgcaguggccccaccucggcau2040

gccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaaca2100

auacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucgauca2160

cuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccuucccg2220

gcggcggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaacaacauc2280

aucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggacacgaa2340

ggagucauccauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaaguugaaa2400

ugaaaggauggaaacauggaauacaguuuuaauugcacguaucaccauuuuacuacaaaa2460

agaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaaguagaguguagg2520

cguuuuugaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucugcaua2580

uaauaguaguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaaguaca2640

ccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaagaaga2700

uuaccguccuuacgagaaaacagauacucggauauaggaaucaagacagcucugcacuga2760

aaacacacucuccugucacgacaccgcgccacaccagaggcguacgcgugacuucaucgc2820

aacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggacca2880

caguugucgauucuugaagacaauauucucgugagaacuuugagauucgcacuugaaacc2940

ucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcuu3000

uucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccuccuua3060

uaagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaaucuauuauuuug3120

uuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaacacagaguaguag3180

acacaauaaaugagaagu3198

<210>79

<211>3198

<212>rna

<213>人工序列

<220>

<223>ggfp_s_β2.7_core

<400>79

auggugagcaagggcgaggagcuguucaccgggguggugcccauccuggucgagcuggac60

ggcgacguaaacggccacaaguucagcguguccggcgagggcgagggcgaugccaccuac120

ggcaagcugacccugaaguucaucugcaccaccggcaagcugcccgugcccuggcccacc180

cucgugaccacccugaccuacggcgugcagugcuucagccgcuaccccgaccacaugaag240

cagcacgacuucuucaaguccgccaugcccgaaggcuacguccaggagcgcaccaucuuc300

uucaaggacgacggcaacuacaagacccgcgccgaggugaaguucgagggcgacacccug360

gugaaccgcaucgagcugaagggcaucgacuucaaggaggacggcaacauccuggggcac420

aagcuggaguacaacuacaacagccacaacgucuauaucauggccgacaagcagaagaac480

ggcaucaaggugaacuucaagauccgccacaacaucgaggacggcagcgugcagcucgcc540

gaccacuaccagcagaacacccccaucggcgacggccccgugcugcugcccgacaaccac600

uaccugagcacccaguccgcccugagcaaagaccccaacgagaagcgcgaucacaugguc660

cugcuggaguucgugaccgccgccgggaucacucucggcauggacgagcuguacaaguga720

uccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggccggcu780

ggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccguggccggg840

agaagauggaggagccgagaugacaacggcagucguggaagggucgccaagccccggucc900

uucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuu960

uaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugcacgcg1020

uguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuaccgccac1080

ccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcugguccu1140

ggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuu1200

cuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagauga1260

uaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaa1320

cucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaaggucau1380

uuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaug1440

ucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgagga1500

ccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugc1560

ccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccagaagaa1620

caaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaacaucuu1680

cauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagccag1740

auuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacuguca1800

uagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuagaug1860

uauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacgacgau1920

gauuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaacgguga1980

auccaagauccccgggucggcgucggcaugccgcugaucgcaguggccccaccucggcau2040

gccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaaca2100

auacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucgauca2160

cuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccuucccg2220

gcggcggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaacaacauc2280

aucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggacacgaa2340

ggagucauccauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaaguugaaa2400

ugaaaggauggaaacauggaauacaguuuuaauugcacguaucaccauuuuacuacaaaa2460

agaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaaguagaguguagg2520

cguuuuugaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucugcaua2580

uaauaguaguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaaguaca2640

ccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaagaaga2700

uuaccguccuuacgagaaaacagauacucggauauaggaaucaagacagcucugcacuga2760

aaacacacucuccugucacgacaccgcgccacaccagaggcguacgcgugacuucaucgc2820

aacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggacca2880

caguugucgauucuugaagacaauauucucgugagaacuuugagauucgcacuugaaacc2940

ucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcuu3000

uucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccuccuua3060

uaagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaaucuauuauuuug3120

uuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaacacagaguaguag3180

acacaauaaaugagaagu3198

<210>80

<211>3198

<212>rna

<213>人工序列

<220>

<223>mtgfp_s_β2.7_core

<400>80

auagugagcaagggcgaggagcuguucaccgggguggugcccauccuggucgagcuggac60

ggcgacguaaacggccacaaguucagcguguccggcgagggcgagggcgaugccaccuac120

ggcaagcugacccugaaguucaucugcaccaccggcaagcugcccgugcccuggcccacc180

cucgugaccacccugaccuacggcgugcagugcuucagccgcuaccccgaccacaugaag240

cagcacgacuucuucaaguccgccaugcccgaaggcuacguccaggagcgcaccaucuuc300

uucaaggacgacggcaacuacaagacccgcgccgaggugaaguucgagggcgacacccug360

gugaaccgcaucgagcugaagggcaucgacuucaaggaggacggcaacauccuggggcac420

aagcuggaguacaacuacaacagccacaacgucuauaucauggccgacaagcagaagaac480

ggcaucaaggugaacuucaagauccgccacaacaucgaggacggcagcgugcagcucgcc540

gaccacuaccagcagaacacccccaucggcgacggccccgugcugcugcccgacaaccac600

uaccugagcacccaguccgcccugagcaaagaccccaacgagaagcgcgaucacaugguc660

cugcuggaguucgugaccgccgccgggaucacucucggcauggacgagcuguacaagaga720

uccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggccggcu780

ggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccguggccggg840

agaagauggaggagccgagaugacaacggcagucguggaagggucgccaagccccggucc900

uucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuu960

uaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugcacgcg1020

uguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuaccgccac1080

ccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcugguccu1140

ggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuu1200

cuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagauga1260

uaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaa1320

cucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaaggucau1380

uuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaug1440

ucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgagga1500

ccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugc1560

ccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccagaagaa1620

caaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaacaucuu1680

cauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagccag1740

auuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacuguca1800

uagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuagaug1860

uauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacgacgau1920

gauuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaacgguga1980

auccaagauccccgggucggcgucggcaugccgcugaucgcaguggccccaccucggcau2040

gccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaaca2100

auacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucgauca2160

cuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccuucccg2220

gcggcggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaacaacauc2280

aucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggacacgaa2340

ggagucauccauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaaguugaaa2400

ugaaaggauggaaacauggaauacaguuuuaauugcacguaucaccauuuuacuacaaaa2460

agaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaaguagaguguagg2520

cguuuuugaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucugcaua2580

uaauaguaguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaaguaca2640

ccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaagaaga2700

uuaccguccuuacgagaaaacagauacucggauauaggaaucaagacagcucugcacuga2760

aaacacacucuccugucacgacaccgcgccacaccagaggcguacgcgugacuucaucgc2820

aacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggacca2880

caguugucgauucuugaagacaauauucucgugagaacuuugagauucgcacuugaaacc2940

ucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcuu3000

uucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccuccuua3060

uaagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaaucuauuauuuug3120

uuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaacacagaguaguag3180

acacaauaaaugagaagu3198

<210>81

<211>720

<212>rna

<213>人工序列

<220>

<223>mtgfp_s_core

<400>81

auagugagcaagggcgaggagcuguucaccgggguggugcccauccuggucgagcuggac60

ggcgacguaaacggccacaaguucagcguguccggcgagggcgagggcgaugccaccuac120

ggcaagcugacccugaaguucaucugcaccaccggcaagcugcccgugcccuggcccacc180

cucgugaccacccugaccuacggcgugcagugcuucagccgcuaccccgaccacaugaag240

cagcacgacuucuucaaguccgccaugcccgaaggcuacguccaggagcgcaccaucuuc300

uucaaggacgacggcaacuacaagacccgcgccgaggugaaguucgagggcgacacccug360

gugaaccgcaucgagcugaagggcaucgacuucaaggaggacggcaacauccuggggcac420

aagcuggaguacaacuacaacagccacaacgucuauaucauggccgacaagcagaagaac480

ggcaucaaggugaacuucaagauccgccacaacaucgaggacggcagcgugcagcucgcc540

gaccacuaccagcagaacacccccaucggcgacggccccgugcugcugcccgacaaccac600

uaccugagcacccaguccgcccugagcaaagaccccaacgagaagcgcgaucacaugguc660

cugcuggaguucgugaccgccgccgggaucacucucggcauggacgagcuguacaagaga720

<210>82

<211>2589

<212>rna

<213>人工序列

<220>

<223>asatp6_s6_β2.7_core

<400>82

aguagaauuagaauugugaagaugauaaguguagagggaagguuaaugguugauauugcu60

aggguggcgcuuccaauuaggugcgugaguagguggccugcaguaauguuauccccagau120

cgcugcugccccggcguucuccagaagccccggcgggcgaaucggccggcuggucggucg180

gcgcucggacggauggggagaacggcggugacuuagccgcccguggccgggagaagaugg240

aggagccgagaugacaacggcagucguggaagggucgccaagccccgguccuucucuucu300

gucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuuuaugugagu360

uucucuuccgcgucucccggccguaccauccacccaugcagcaugcacgcguguauguau420

gcaucgucucuccuccgucccgacuaccaucagcagcaccacuaccgccacccccagcgc480

caccaccgcugccgucgccaccgcguuauccguuccucguaggcugguccuggggaacgg540

gucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuucuccuuucc600

uuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaaga660

ggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaacucgaagaa720

uucaaagucgacgauucccaacaagagaaaacagaacaaaaacaaggucauuuuuauuua780

uccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgc840

aaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucu900

ccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacau960

ccugacgcgucggucagcagccaaacaaucauaggaaaugaaccagaagaacaaaaagau1020

caucucucucgguguauagcaacaccaacaacaaccgcaucgcaacaucuucauccgcaa1080

gacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagccagauuucacgu1140

ccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacugucauagauaaau1200

auaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuagauguauaugcuc1260

cuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacgacgaugauuccuua1320

ccgcuccugccacccggucuguauguagagagagaagagagaaaacggugaauccaagau1380

ccccgggucggcgucggcaugccgcugaucgcaguggccccaccucggcaugccggcgcc1440

gggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaacaauacaugau1500

uaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucgaucacuacgauaa1560

acacauggaagauuucuugaaaaaagaaaagagaaagagaccaccuucccggcggcggac1620

acgcuccucuccgucgccguucugcaccaugauucgaucaauaacaacaucaucaucgga1680

gaccaucuuuuaaucaaucagcguugcaguagucgacucccuggacacgaaggagucauc1740

cauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaaguugaaaugaaaggau1800

ggaaacauggaauacaguuuuaauugcacguaucaccauuuuacuacaaaaagaaaaaaa1860

aacaacuuacacauaguauuaccuuagguuuacggauaaguagaguguaggcguuuuuga1920

aacaguucagccaaugcaaucuugucucggcauaaucacucuuucugcauauaauaguag1980

uaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaaguacaccuagagac2040

agcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaagaagauuaccgucc2100

uuacgagaaaacagauacucggauauaggaaucaagacagcucugcacugaaaacacacu2160

cuccugucacgacaccgcgccacaccagaggcguacgcgugacuucaucgcaacgaucca2220

ucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggaccacaguugucg2280

auucuugaagacaauauucucgugagaacuuugagauucgcacuugaaaccucuuaggau2340

ccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcuuuucucccaa2400

accucgguuucuuccuauucuuaaguuuucccuaguauauuugccuccuuauaagaaaag2460

aagcacaagcucggucgcacggauuauuccuucugcuaaucuauuauuuuguuccuuuuu2520

uuuuuguuugccuucacccccuucacucccuguagcaacacagaguaguagacacaauaa2580

augagaagu2589

<210>83

<211>2585

<212>rna

<213>人工序列

<220>

<223>β2.7_s8_asatp8_core

<400>83

uccccagaucgcugcugccccggcguucuccagaagccccggcgggcgaaucggccggcu60

ggucggucggcgcucggacggauggggagaacggcggugacuuagccgcccguggccggg120

agaagauggaggagccgagaugacaacggcagucguggaagggucgccaagccccggucc180

uucucuucugucuggucgaaucucguuuucuuuuuucaaccgcucuuuuuaucaccuuuu240

uaugugaguuucucuuccgcgucucccggccguaccauccacccaugcagcaugcacgcg300

uguauguaugcaucgucucuccuccgucccgacuaccaucagcagcaccacuaccgccac360

ccccagcgccaccaccgcugccgucgccaccgcguuauccguuccucguaggcugguccu420

ggggaacgggucggcggccggucggcuucuguuuuauuauuuuuuuuuauuuuuuaucuu480

cuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagauga540

uaaacaagaggguaaaaagaaaaaagcuacagacauuuggguaccucagcuuuccgauaa600

cucgaagaauucaaagucgacgauucccaacaagagaaaacagaacaaaaacaaggucau660

uuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaug720

ucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgagga780

ccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugc840

ccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccagaagaa900

caaaaagaucaucucucucgguguauagcaacaccaacaacaaccgcaucgcaacaucuu960

cauccgcaagacggaaagaaaacaacaauaaugagaaugaaaucaccacaaccaagccag1020

auuucacguccaugaguuuuuauuauauuauuaucaaaacgaaaaacagaaaaacuguca1080

uagauaaauauaaaaaaaaauagaaaccacaaacgacuacuaguacuccaaucuuagaug1140

uauaugcuccuagauaagauuuaguauuaccauaaucaucgaagaaugaaagacgacgau1200

gauuccuuaccgcuccugccacccggucuguauguagagagagaagagagaaaacgguga1260

auccaagauccccgggucggcgucggcaugccgcugaucgcaguggccccaccucggcau1320

gccggcgccgggcgaggaauugcucaugaaaaaaguaucuuucuguaaaaaaagaaaaca1380

auacaugauuaaccgaaaagaaaccaacaaaaagaacccgagaucagucgauuucgauca1440

cuacgauaaacacauggaagauuucuugaaaaaagaaaagagaaagagaccaccuucccg1500

gcggcggacacgcuccucuccgucgccguucugcaccaugauucgaucaauaacaacauc1560

aucaucggagaccaucuuuuaaucaaucagcguugcaguagucgacucccuggacacgaa1620

ggagucauccauuuuuauccucgcacuucuucgcucucaaagccgccuuuaaaguugaaa1680

ugaaaggauggaaacauggaauacaguuuuaauugcacguaucaccauuuuacuacaaaa1740

agaaaaaaaaacaacuuacacauaguauuaccuuagguuuacggauaaguagaguguagg1800

cguuuuugaaacaguucagccaaugcaaucuugucucggcauaaucacucuuucugcaua1860

uaauaguaguaguagauuuauucacaucaacacagcgaaaaacuccagcaucaaaguaca1920

ccuagagacagcccuuaaaauauaguuugcagcuuuuagauguacuuacaccaaagaaga1980

uuaccguccuuacgagaaaacagauacucggauauaggaaucaagacagcucugcacuga2040

aaacacacucuccugucacgacaccgcgccacaccagaggcguacgcgugacuucaucgc2100

aacgauccaucgugaugucccucgcagaaccuaaaaagaccaaaaaaaaaucuuggacca2160

caguugucgauucuugaagacaauauucucgugagaacuuugagauucgcacuugaaacc2220

ucuuaggauccacaaaaacaacaaccucuguauggaaaaugcgcuauuuuaucucagcuu2280

uucucccaaaccucgguuucuuccuauucuuaaguuuucccuaguauauuugccuccuua2340

uaagaaaagaagcacaagcucggucgcacggauuauuccuucugcuaaucuauuauuuug2400

uuccuuuuuuuuuuguuugccuucacccccuucacucccuguagcaacacagaguaguag2460

acacaauaaaugagaaguuuucguucauuuugguucucaggguuuguuauaauuuuuuau2520

uuuuaugggcuuuggugagggagguaggugguaguuuguguuuaauauuuuuaguugggu2580

gauga2585

<210>84

<211>1466

<212>rna

<213>人工序列

<220>

<223>atp6_d34_d24(core)

<400>84

aguagaauuagaauugugaagaugauaaguguagagggaagguuaaugguugauauugcu60

aggguggcgcuuccaauuaggugcgugaguagguggccugcaguaauguuggucauuuuu120

auuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaa180

uccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgc240

gaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccug300

cacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccggucauuuuua360

uuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaau420

ccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcg480

aucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugc540

acauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccggucauuuuuau600

uuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauc660

cgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcga720

ucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugca780

cauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccggucauuuuuauu840

uauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaaucc900

gcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgau960

cuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcac1020

auccugacgcgucggucagcagccaaacaaucauaggaaaugaaccuuuuauuuuuuauc1080

uucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagau1140

gauaaacaagaggguaaaaagaaaaauuuuauuuuuuaucuucuccuuuccuuaaucucg1200

gauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaa1260

gaaaaauuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccu1320

accuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaauuuuauuuuuuauc1380

uucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagau1440

gauaaacaagaggguaaaaagaaaaa1466

<210>85

<211>1741

<212>rna

<213>人工序列

<220>

<223>体外转录的atp6_(d3)4_(d2)4rna的序列

<400>85

ggcgcuuccggaagaaagcgccaguagaauuagaauugugaagaugauaaguguagaggg60

aagguuaaugguugauauugcuaggggggcgcuuccaauuaggugcaugaguagguggcc120

ugcaguaauguuaaagcaagggccuuauucaugggcuagcccggcggucauuuuuauuua180

uccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgc240

aaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucu300

ccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugcacau360

ccugacgcgucggucagcagccaaacaaucauaggaaaugaaccgccgggaagcgccgaa420

uucauaucgaucggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaac480

gaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucg540

cgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgg600

uccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucaua660

ggaaaugaaccgaucgaauaaagguaccuguggggucauuuuuauuuauccucaucguca720

acaacaacuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuccu780

gcccccucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauagguag840

augaaauuaucccguguccgguccugauuccccgcaugcccugcacauccugacgcgucg900

gucagcagccaaacaaucauaggaaaugaaccccacauuuuaccgguaauaccggggggu960

cauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaaga1020

augucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcga1080

ggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgca1140

ugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaacccccc1200

ggaagcuuuccggaagagcuagcuuuuauuuuuuaucuucuccuuuccuuaaucucggau1260

uaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaa1320

aaaagcgcgaauuccgaucuuuuauuuuuuaucuucuccuuuccuuaaucucggauuauc1380

auuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaag1440

aucguauccgguaccugugguuuuauuuuuuaucuucuccuuuccuuaaucucggauuau1500

cauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaa1560

ccacaccuccaccggugggccguuuuauuuuuuaucuucuccuuuccuuaaucucggauu1620

aucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaa1680

aacggcccaagcuugacuccuauagugucaccuaaaugucuagauacuaagggagucuug1740

c1741

<210>86

<211>1517

<212>rna

<213>人工序列

<220>

<223>包括结构域d3和d2和间隔子的多聚体(d3)4_(d2)4rna

<400>86

ggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccacca60

agaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucaugg120

cgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauucccc180

gcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccg240

ccgggaagcgccgaauucauaucgaucggucauuuuuauuuauccucaucgucaacaaca300

acuaccgacaacaacgaaacaccaccaagaaugucaauccgcaaggguguuccugccccc360

ucgacgcgccugucgcgauccucauggcgaggaccgcgaucuccguauagguagaugaaa420

uuaucccguguccgguccugauuccccgcaugcccugcacauccugacgcgucggucagc480

agccaaacaaucauaggaaaugaaccgaucgaauaaagguaccuguggggucauuuuuau540

uuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaagaaugucaauc600

cgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcgaggaccgcga660

ucuccguauagguagaugaaauuaucccguguccgguccugauuccccgcaugcccugca720

cauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccccacauuuuaccg780

guaauaccggggggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaac840

gaaacaccaccaagaaugucaauccgcaaggguguuccugcccccucgacgcgccugucg900

cgauccucauggcgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgg960

uccugauuccccgcaugcccugcacauccugacgcgucggucagcagccaaacaaucaua1020

ggaaaugaaccccccggaagcuuuccggaagagcuagcuuuuauuuuuuaucuucuccuu1080

uccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaaca1140

agaggguaaaaagaaaaaagcgcgaauuccgaucuuuuauuuuuuaucuucuccuuuccu1200

uaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaagag1260

gguaaaaagaaaaagaucguauccgguaccugugguuuuauuuuuuaucuucuccuuucc1320

uuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaaga1380

ggguaaaaagaaaaaccacaccuccaccggugggccguuuuauuuuuuaucuucuccuuu1440

ccuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaa1500

gaggguaaaaagaaaaa1517

<210>87

<211>1356

<212>rna

<213>人工序列

<220>

<223>只包括结构域d3和d2但没有任何间隔子的多聚体(d3)4_(d2)4rna核心

<400>87

ggucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccacca60

agaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucaugg120

cgaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauucccc180

gcaugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccg240

gucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaa300

gaaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggc360

gaggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccg420

caugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccgg480

ucauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaag540

aaugucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcg600

aggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgc660

augcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccggu720

cauuuuuauuuauccucaucgucaacaacaacuaccgacaacaacgaaacaccaccaaga780

augucaauccgcaaggguguuccugcccccucgacgcgccugucgcgauccucauggcga840

ggaccgcgaucuccguauagguagaugaaauuaucccguguccgguccugauuccccgca900

ugcccugcacauccugacgcgucggucagcagccaaacaaucauaggaaaugaaccuuuu960

auuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacg1020

aaucgcagaugauaaacaagaggguaaaaagaaaaauuuuauuuuuuaucuucuccuuuc1080

cuuaaucucggauuaucauuucccucuccuaccuaccacgaaucgcagaugauaaacaag1140

aggguaaaaagaaaaauuuuauuuuuuaucuucuccuuuccuuaaucucggauuaucauu1200

ucccucuccuaccuaccacgaaucgcagaugauaaacaagaggguaaaaagaaaaauuuu1260

auuuuuuaucuucuccuuuccuuaaucucggauuaucauuucccucuccuaccuaccacg1320

aaucgcagaugauaaacaagaggguaaaaagaaaaa1356

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