本发明涉及植物遗传学和基因工程领域。具体而言,本发明涉及一种基因编辑方法在水稻中的应用,特别涉及通过编辑水稻osmatl基因能够创制水稻单倍体诱导系,能够应用于水稻育种以及在水稻中进行基因编辑。
背景技术:
杂交优势在农业生产上有很广泛的应用,通过将两个亲本自交系进行杂交得到的杂一代杂交种,将其用于生产可以获得远高于亲本的产量和品质。在大规模生产中农民需要品质高度均一的种子来保证产出,种子公司要提供品质均一的杂交种就需要高度纯合的亲本自交系。传统遗传育种不断地通过杂交方式改良亲本系,将更多更优良的性状整合到亲本系中,在这过程中也导致亲本的遗传背景发生重组,大量位点变得杂合,需要通过后续不断地回交或自交来固定遗传背景,然后才能用于制种。为了获得纯合的亲本自交系,育种实践中通常需要6次或者更多次回交或自交使遗传背景接近纯合,周期漫长。育种家迫切希望有方法可以缩短育种过程。
在二倍体型被子植物整个生活史中,配子体是单倍体而体细胞是二倍体。花粉是单倍体型雄配子,由一个大的营养细胞和两个精细胞组成,在花药中发育成熟。雌配子在胚珠中发育成熟,成熟胚囊中含有一个卵细胞、两个助细胞、三个反足细胞及一个含有两个极核的中央细胞。在受精过程中花粉在亲和的柱头上萌发形成花粉管,花粉管根据雌蕊的结构并感受雌蕊分泌的化学物质来定向,沿着花柱迅速伸长,准确地经过胚珠的珠孔进入胚囊。在接触到胚囊的某一个助细胞之后,花粉管迅速破裂,释放出两个精细胞,两个精细胞穿过凋亡助细胞的细胞质分别与卵细胞和中央细胞融合形成受精卵和胚乳,这种受精方式称为双受精。如果双受精失败,则可以有另一个花粉管再次与残留的助细胞作用完成受精。处于单倍体状态的配子细胞经过染色体加倍过程可以转变成二倍体细胞,这样所有的遗传位点都变成纯合而被固定下来。这种方法称为双单倍体技术。植物学家希望充分了解双受精过程,直接或间接利用单倍型的配子体,通过染色体加倍来产生双单倍体,缩短育种周期,比如首先获得杂交亲本的单倍体,然后通过自然或者人工染色体加倍过程,如秋水仙素处理等方法,一代即可获得纯合的自交系。双单倍体使得重组群体可以以纯合的形式被快速筛选。简而言之,双单倍体技术是改良作物整合新性状的最便捷的方式。
利用双单倍体技术的前提条件是获得单倍体。获得植物单倍体的方法主要包括两大类,一类是组织培养,如小孢子培养、大孢子培养等。组织培养的方法大多与基因型密切相关,在很多作物中都有报道,但是成本高昂,而且尽管文献报道了很多,但是在很多作物中并没有成熟可靠的体系。另一类是基于种子的在体单倍体诱导方法。这是一种在胚胎早期发育过程中来自诱导系一方的染色体发生丢失的特殊现象,包括远缘杂交或者与单倍体诱导系进行种内杂交,通常都是诱导系的基因组丢失而只保留另一方的遗传物质。基于种子的在体诱导方式则通常与基因型无关。这种现象在自然界很少见,在玉米、高粱、大麦、小麦等作物中有报道,但在水稻自然资源中还没有被发现。
玉米单倍体诱导系的使用和研究时间比较长,体系也比较完善。玉米单倍体诱导系stock6在1959年首次报道,在其自交群体中或者作为父本产生的杂交群体中可以达到2~3%的自身或母本单倍体。正常发育的胚乳是保证单倍体种子存活的关键。2017年三个独立团队先后分别发表了对玉米中导致单倍体产生的效应基因的研究成果(kelliher2017;liu2017;gilles2017),在玉米matrilineal基因靠近末端的位置有4个核苷酸的插入,导致移码突变,直接导致后代中产生单倍体。玉米matrilineal基因是一种磷脂酶,仅在精细胞中特异表达。matrilineal基因的发现为单倍体诱导系的推广应用奠定了基础,也为在其它作物(如水稻)中建立相应的单倍体诱导系统提供了理论支持。
基因编辑技术近年来发展迅速,大量高效编辑系统被开发出来。这些系统都基于人工设计的序列特异性核酸酶(sequence-specificnucleases,ssns)来对特定位置的基因组dna进行切割,如早期的锌指核酸酶(zincfingernuclease,zfn)、类转录激活效应因子核酸酶(transcriptionactivator-likeeffectornuclease,talen)、大范围核酸酶(meganuclease,mn)以及2012年开始兴起的规律成簇间隔短回文重复及其相关系统(clusteredregularlyinterspacedshortpalindromicrepeats/crispr-associated9,crispr/cas9)。crispr/cas9系统由于其操作方便而日益占据主导地位。基因编辑技术作为一种新型基因工程技术在生物技术领域,包括农业生物技术领域有着广泛应用。
技术实现要素:
本发明的发明人首次在水稻中应用基因编辑技术创制了水稻单倍体诱导系,并在3个不同水稻品系中检测了单倍体诱导效果,由此完成了本发明。
本发明涉及的水稻单倍体诱导系,用其花粉作为供体,与任何其它品种的母本水稻,包括自交系、杂交系或其它水稻品系,进行杂交,母本上结出的种子会包含正常的二倍体、只含有母本染色体的单倍体、以及或者其它倍性的种子。单倍体的种子可以通过细胞学检测、表型分析或者分子标记检测与二倍体种子区分,萌发后的单倍体种子可以培养成单倍体植株,经过自然加倍或者人工处理,如秋水仙素处理,转变成为双单倍体植株并可以生产纯合的自交系种子。
水稻单倍体诱导系对水稻育种具有很大大促进作用,可以降低水稻单倍体获得的技术难度和成本,显著缩短纯合自交系的选育周期。
水稻单倍体诱导系可以应用于其它品系的基因编辑,不需要对目标品系进行转化,只需要对单倍体诱导系进行操作,并可以应用于多个不同品系的编辑,且后代不携带转基因成分,可以显著降低育种成本,尤其对于遗传转化困难的品系有重大意义。
本申请首次报道了在水稻中创造单倍体诱导系的方法,并在3个不同水稻品系中成功测试了诱导单倍体的应用。
发明的有益效果
本研究中,发明人首次利用crispr/cas9技术通过编辑水稻内源osmatl基因,成功地创制了水稻单倍体诱导系,具有开创性的意义。野生型水稻osmatl基因基因组核苷酸序列和氨基酸序列分别如seqidno:3和seqidno:4所示。本发明中的水稻单倍体诱导系可以在不同水稻品系中作为父本诱导母本产生单倍体。数据显示,在不同品系中单倍体诱导率可以达到或超过5%。
下面通过若干附图和实施例,对本发明所涉及的技术方案做进一步的详细描述。
附图说明
图1为载体23843图谱。
图2为载体23844图谱。
图3为载体23845图谱。
图4为载体23846图谱。
图5为e0水稻编辑效率统计。
图6为e0水稻编辑植株自交结实率。
图7为e1自交群体中单倍体和二倍体的表现差异。
图8为e1水稻单倍体诱导系049a在2个不同水稻品系中诱导单倍体的效率。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1.材料与方法
材料
植物材料:籼稻自交系ir58025b,rl73,tb12,mn024529。
方法
植物生长条件:本发明中所涉及的水稻植株在温室中的培养条件为11小时光照(32摄氏度)13小时黑暗(22摄氏度),温室湿度控制在60~80%。
引物和探针:本发明中使用的克隆测序扩增引物以及实时荧光定量pcr引物和探针均利用美国appliedbiosystem公司的primerexpress3.0软件设计,并由invitrogen公司合成,序列信息见序列表(seqidno:9-33)。
实施例2.crispr/cas9编辑载体的构建
改进版的cas9在羧基端融合了核定位信号(cbcas9nu-02),并根据玉米中表达模式进行密码子优化,这种优化形式经过测试同样适用于水稻,以花椰菜花叶病毒35s启动子驱动cas9表达。改进并优化的cas9的核酸序列和氨基酸序列分别如seqidno:1和seqidno:2所示。籼稻自交系ir58025b中osmatl基因序列由内部基因组测序数据获得,并经过再次克隆测序验证。osmatl基因的编辑位点设计基于两种方案,一种是在靠近基因末端的位置(4号外显子区域)产生移码突变以产生与玉米天然单倍体诱导系中类似的突变类型,另一种方案是在尽量靠近基因前端的位置(1号外显子区域)产生突变造成完全基因敲除的效果。引导rna选择ngg上游临近的20个核苷酸,将核心12个核苷酸序列(紧邻ngg的12个核苷酸)连同ngg共15个核苷酸在内部数据库中进行blast分析,兼顾已有基因组信息的多个水稻基因组,包括籼稻和粳稻,确保在水稻基因组中只有唯一的精确匹配。同样的序列分别设计了单组分导向rna和双组分导向rna,分别用水稻u3和u6启动子驱动引导rna和骨架rna表达。单组分导向rna指将crrna和tracrrna通过一小段核苷酸序列连接成为一个整体分子,双组分导向rna指仍然保留crrna和tracrrna两个分子分离的结构,但是根据原始的crrna和tracrrna结构通过优化核苷酸序列增强二者的结合能力。玉米泛素启动子ubi驱动的磷酸甘露糖异构酶(pmi)作为转基因筛选标记。最终选定的引导rna及其单组分或双组分骨架由genscript公司(www.genscript.com)提供全序列合成。构建载体及鉴定的一般方法为本领域技术人员所熟知。具体序列信息参见序列表(其中crrna的骨架序列和tracrrna的序列分别如seqidno:7和seqidno:8所示;并且通过将seqidno:5或seqidno:6与seqidno:7直接相连就得到完整的crrna的序列)。载体图谱见附图1-4。
最后选定的由引导rna所靶向的靶位点序列如下:
载体23843/23844(靶向1号外显子):tgcagtcgaagtaatcggcg(seqidno:5)
载体23845/23846(靶向4号外显子):cgagaccggcaggtacgtcg(seqidno:6)。
按照这样的方式,获得了4种编辑载体,即载体23843、23844、23845和23846。在这些载体中,载体23843和23844靶向1号外显子,但是载体23843涉及单组分导向rna和载体23844涉及双组分导向rna;载体23845和23846靶向4号外显子,但是载体23845涉及单组分导向rna和载体23846涉及双组分导向rna。
实施例3.水稻ir58025b的遗传转化
将构建好的编辑载体转化农杆菌eha101并做鉴定,方法为本领域技术人员所熟知。由籼稻品种ir58025b种子诱导而来的胚性愈伤组织,与含有编辑载体的农杆菌eha101共同孵育,经过筛选再生等一系列步骤完成遗传转化。具体的流程包括,水稻愈伤组织与农杆菌共培养3天后,将愈伤组织转移至含有300毫克每升timentin的恢复培养基上恢复培养4~5天,之后将愈伤组织转移至含有300毫克每升timentin的筛选培养基上筛选4周,然后将抗性愈伤转移至再生培养基上培养4周,最后将再生水稻小苗转移至无筛选的生根培养基上继续培养10天。水稻转化过程更详细的方法及各种培养基成分可参考gui2014。
水稻愈伤和植株的生长条件如下:愈伤诱导25天,愈伤预培养7天,恢复培养5天,筛选培养28天,以上所有处理均在暗室进行;再生培养28天,生根培养10天在6000lux光照条件下,光周期为16小时光照8小时黑暗。温度始终控制在30摄氏度。
实施例4.osmatl基因编辑水稻e0转化事件的筛选和鉴定
经过遗传转化直接产生的转化事件为e0代编辑植株,其自交产生的后代为e1代,以后世代依次类推。在生根过程中对小苗取样进行第一次转基因检测。植物基因组dna的提取采用promega植物基因组提取试剂盒(货号:ff3761),每株植物取约10~20mg的叶片放入含有钢珠的96孔深孔板中,在-80℃超低温冰箱中放置30分钟以上,然后机械震荡破碎。具体提取操作步骤详见试剂盒说明书。提取的dna溶于100微升双蒸水中。dna样品用高通量实时荧光定量pcr(taqman)方法进行检测(具体操作参见taqman实验手册),同时检测突变位点的情况和转基因拷贝数的情况,低拷贝的转基因植株,并且至少1条染色体发生突变的植株为候选编辑植株,送至温室进一步培养,鉴定繁种。在植株恢复正常生长后一段时间,重新取样用二次taqman进行编辑位点确认。经过taqman确认含有目标区域突变的植株进一步用pcr测序进行基因型分析,确定具体的突变类型。具体的,在靶位点上下游约300bp的位置设计一对pcr扩增引物,扩增目标区域跨越切割位点,使用高保真dna聚合酶kod-plus-neo(toyobo)进行扩增,扩增片段切胶回收后克隆到peasy(peasy-bluntzerocloningkit,transgen)载体上并转化大肠杆菌。具体操作步骤参考产品说明。随机挑选10个独立的大肠杆菌菌落进行测序(lifetechnologies)。测序结果在vectornti中与参考序列进行比较,确定突变类型。如果10个克隆中有测序失败的情况则重新挑选克隆加测反应。如果所有克隆测序结果一致且与野生型序列不同,则该植株为纯合型编辑植株;如果测序克隆中包含一种突变类型和野生型,则该植株为杂合型编辑植株;如果测序克隆中包含两种突变类型且无野生型,则该植株为双等位基因型编辑植株;如果测序克隆中包含三种及以上突变类型或野生型,则该植株为嵌合型编辑植株。最终选出的纯合型或双等位基因型编辑植株作为进一步实验的材料。通过以上步骤得出转化效率(即转基因阳性率)平均约为40%,单组分系统编辑效率平均约为80%,双组分系统编辑效率平均约为15%。平均纯合型编辑植株比例约为36%。taqman和pcr测序引物具体序列见序列表。各载体最终e0编辑植株突变情况统计见附图5。
扩增引物列表:
exon1-f:gcgttcctgcaaatcacagtgacta(seqidno:9)
exon1-r:gatcgaagagaggagatcgaattcg(seqidno:10)
exon4-f:taagttcctggtgctgtccgtgg(seqidno:11)
exon4-r:tacagttactaacgcttgcacgcca(seqidno:12)。
实施例5.e1水稻编辑植株的基因型及表型鉴定
来自于4个载体的100多个独立e0转化事件在温室中进行自交繁种。除少数个体外,大部分收获到500~1500粒e1种子。在e1种子收获时,对e0编辑植株和同期种植的野生型ir58025b植株进行结实率分析。将主分蘖剪下,单独统计主分蘖上饱满籽粒和空壳的数量,用以下公式计算结实率:
结实率=饱满籽粒数量/(饱满籽粒数量+空壳数量)×100%。
通过统计不同载体e0编辑植株的结实率和编辑类型,发现在所有4个载体中,不含有野生型等位基因的植株结实率都显著低于野生型ir58025b(见附图6)。
e1水稻编辑植株的观察表型主要关注已知的与单倍体诱导系相关联的形状,如结实率,同时兼顾基本的农艺性状,如株高、分蘖数、叶长叶宽。株高在灌浆期进行测量,测量地上部分高度,从土壤表层到主茎上最高的穗子的自然距离。每个编辑事件随机取5棵植物用于测量株高和分蘖数,同时每棵植物随机取3片完全展开的真叶统计叶长度和叶宽度。统计发现所有e1代自交群体中分离出单倍体与群体中的二倍体均存在显著差异,株高、叶长叶宽均更下,分蘖数更少(见附图7)。
花粉活力通过碘化钾染色和萌发实验进行检测。收集上午9点左右自然开裂的花药释放出的新鲜花粉,加入碘化钾染色液,室温15分钟后在显微镜下观察花粉染色情况,统计被染色花粉的比例。在萌发实验中,2毫升花粉萌发液和新鲜花粉在培养皿中孵育,避光保湿,室温5至30分钟,然后在显微镜下观察花粉萌发情况并统计萌发率。统计发现e1编辑植株的花粉与野生型ir58025b的花粉在活力和萌发率上均没有明显差异。
每个独立编辑事件播种150~300粒种子,除去未萌发的种子,所有成活的植株均取样做倍性检测。植株体细胞倍性检测由北京市农林科学院蔬菜中心提供服务,采用bdfacscalibur流式细胞仪。检测样品为四叶期新鲜植物叶片,样品制备参照检测仪器供应商sysmex-partec提供的标准样品制备流程。制备好的样品用partec
e0单倍体诱导率=e1群体中单倍体数量/e1群体数量×100%。
单倍体诱导系127a和003d用载体23843来获得,单倍体诱导系068a用载体23844来获得,单倍体诱导系049a和011a用载体23845来获得,和单倍体诱导系288a用载体23846来获得。与这些单倍体诱导系中相同的基因突变既可以由单组分载体获得也可以由靶位点相同的双组分载体获得,其诱导效果视为等同。这些单倍体诱导系中的突变型osmatl基因的核酸序列如seqidno:34~41中任一个所示。
实施例6.水稻单倍体诱导系与不同品系的杂交
种植e1水稻编辑种子作为父本,苗期用前述taqman方法确定基因型,只保留不含有转基因成分的纯合突变植株用于杂交实验。播种不同批次的母本水稻,包括籼稻自交系rl73、tb12和mn024529,选择花期适合于杂交的批次用于实验。具体的,实验前一天把即将开花的母本进行修剪准备,只保留第二天会开花的分蘖,将多余的分蘖和会遮挡小花的叶片剪掉。实验当天8点开始处理母本,将颖壳已打开花药已伸出的小花去除,43摄氏度水浴5分钟,灭活花粉并促使颖壳打开。将处理完的母本与即将散粉的父本靠近,保证父本穗位高于母本,用纸袋将父母本分蘖包在一起,等到父本自然散粉,抖动父本让尽可能多的母本接受花粉。母本可以在连续3天内保持柱头活力,在接触花粉的情况下持续授粉。3天过后将母本转移至别处直至收获f1种子。特别的,对于细胞质型雄性不育系tb12,仅需温水浴促使颖壳打开,其余操作相同。更特别的,对于自交系rl73,由于其材料特殊性,高于43摄氏度的水温会严重影响雌蕊活性,而低于43摄氏度的水温不足以对花粉充分灭活,所以在42.5摄氏度的水浴处理之后,需要人工再将花药及未打开的小花去除,然后用于杂交。
实施例7.水稻单倍体诱导系在不同品系中诱导单倍体效果的检测
每个杂交组合播种150~300粒f1种子,除去未萌发的种子,所有成活的植株均取样做倍性检测。检测结果表明,来源于载体23845的单倍体诱导系049a在tb12中的单倍体诱导率可以达到5%,而在另两个测试品种中诱导率偏低,说明单倍体诱导系的诱导率依赖于基因型。水稻单倍体诱导系与不同水稻品系杂交的单倍体诱导率见附图8。
单倍体诱导率=f1群体中单倍体数量/f1群体数量×100%。
以上结果表明,本发明了首次提供了应用crispr/cas9技术在水稻中创制单倍体诱导系的方法,创制了若干水稻单倍体诱导系,并且可以在不同水稻品系中诱导单倍体产生,为在水稻中大规模生产单倍体提供了一种新的选择。
最后应予说明,尽管本发明的具体实施方式已经得到详细的描述,以上实施例仅用以说明本发明的技术方案而非限制,本领域技术人员将会理解,根据已经公开的所有教导,可以对那些细节进行各种修改或等同替换,这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
参考文献
[1]kelliher,t.,starr,d.,richbourg,l.,chintamanani,s.,delzer,b.,nuccio,m.l.,green,j.,chen,z.,mccuiston,j.,wang,w.etal.matrilineal,asperm-specificphospholipase,triggersmaizehaploidinduction.nature542,105-109(2017)
[2]gilles,l.m.,khaled,a.,laffaire,j.-b.,chaignon,s.,gendrot,g.,laplaige,j.,bergès,h.,beydon,g.,bayle,v.,barret,p.etal.lossofpollen-specificphospholipasenotlikedadtriggersgynogenesisinmaize.emboj.36:707-717(2017)
[3]liu,c.,li,x.,meng,d.,zhong,y.,chen,c.,dong,x.,xu,x.,chen,b.,li,w.,li,l.etal.a4-bpinsertionatzmpla1encodingaputativephospholipaseagenerateshaploidinductioninmaize.mol.plant10:520-522(2017)
[4]wang,y.,geng,l.,yuan,m.,wei,j.,jin,c.,li,m.,yu,k.,zhang,y.,jin,h.,wang,e.etal.deletionofatargetgeneinindicariceviacrispr/cas9.plantcellrep.36:1333-1343(2017)
[5]gui,h.,li,x.,liu,y.,li,x.therelationshipbetweenpmi(mana)geneexpressionandoptimalselectionpressureinindicaricetransformation.plantcellrep.33:1081-1090(2014).
序列表
seqidno:1
改进并优化的cas9核酸序列:
atggacaagaagtacagcatcggcctggacatcggcaccaacagcgtgggctgggccgtgatcaccgacgagtacaaggtgccgagcaagaagttcaaggtgctgggcaacaccgacaggcacagcatcaagaagaacctgatcggcgccctgctgttcgacagcggcgagaccgccgaggccaccaggctgaagaggaccgccaggaggaggtacaccaggaggaagaacaggatctgctacctgcaggagatcttcagcaacgagatggccaaggtggacgacagcttcttccacaggctggaggagagcttcctggtggaggaggacaagaagcacgagaggcacccgatcttcggcaacatcgtggacgaggtggcctaccacgagaagtacccgaccatctaccacctgaggaagaagctggtggacagcaccgacaaggccgacctgaggctgatctacctggccctggcccacatgatcaagttcaggggccacttcctgatcgagggcgacctgaacccggacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggagaacccgatcaacgccagcggcgtggacgccaaggccatcctgagcgccaggctgagcaagagcaggaggctggagaacctgatcgcccagctgccgggcgagaagaagaacggcctgttcggcaacctgatcgccctgagcctgggcctgaccccgaacttcaagagcaacttcgacctggccgaggacgccaagctgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgttcctggccgccaagaacctgagcgacgccatcctgctgagcgacatcctgagggtgaacaccgagatcaccaaggccccgctgagcgccagcatgatcaagaggtacgacgagcaccaccaggacctgaccctgctgaaggccctggtgaggcagcagctgccggagaagtacaaggagatcttcttcgaccagagcaagaacggctacgccggctacatcgacggcggcgccagccaggaggagttctacaagttcatcaagccgatcctggagaagatggacggcaccgaggagctgctggtgaagctgaacagggaggacctgctgaggaagcagaggaccttcgacaacggcagcatcccgcaccagatccacctgggcgagctgcacgccatcctgaggaggcaggaggacttctacccgttcctgaaggacaacagggagaagatcgagaagatcctgaccttccgcatcccgtactacgtgggcccgctggccaggggcaacagcaggttcgcctggatgaccaggaagagcgaggagaccatcaccccgtggaacttcgaggaggtggtggacaagggcgccagcgcccagagcttcatcgagaggatgaccaacttcgacaagaacctgccgaacgagaaggtgctgccgaagcacagcctgctgtacgagtacttcaccgtgtacaacgagctgaccaaggtgaagtacgtgaccgagggcatgaggaagccggccttcctgagcggcgagcagaagaaggccatcgtggacctgctgttcaagaccaacaggaaggtgaccgtgaagcagctgaaggaggactacttcaagaagatcgagtgcttcgacagcgtggagatcagcggcgtggaggacaggttcaacgccagcctgggcacctaccacgacctgctgaagatcatcaaggacaaggacttcctggacaacgaggagaacgaggacatcctggaggacatcgtgctgaccctgaccctgttcgaggacagggagatgatcgaggagaggctgaagacctacgcccacctgttcgacgacaaggtgatgaagcagctgaagaggaggaggtacaccggctggggcaggctgagcaggaagctgatcaacggcatcagggacaagcagagcggcaagaccatcctggacttcctgaagagcgacggcttcgccaacaggaacttcatgcagctgatccacgacgacagcctgaccttcaaggaggacatccagaaggcccaggtgagcggccagggcgacagcctgcacgagcacatcgccaacctggccggcagcccggccatcaagaagggcatcctgcagaccgtgaaggtggtggacgagctggtgaaggtgatgggcaggcacaagccggagaacatcgtgatcgagatggccagggagaaccagaccacccagaagggccagaagaacagcagggagaggatgaagaggatcgaggagggcatcaaggagctgggcagccagatcctgaaggagcacccggtggagaacacccagctgcagaacgagaagctgtacctgtactacctgcagaacggcagggacatgtacgtggaccaggagctggacatcaacaggctgagcgactacgacgtggaccacatcgtgccgcagagcttcctgaaggacgacagcatcgacaacaaggtgctgaccaggagcgacaagaacaggggcaagagcgacaacgtgccgagcgaggaggtggtgaagaagatgaaaaactactggaggcagctgctgaacgccaagctgatcacccagaggaagttcgacaacctgaccaaggccgagaggggcggcctgagcgagctggacaaggccggcttcattaaaaggcagctggtggagaccaggcagatcaccaagcacgtggcccagatcctggacagcaggatgaacaccaagtacgacgagaacgacaagctgatcagggaggtgaaggtgatcaccctgaagagcaagctggtgagcgacttcaggaaggacttccagttctacaaggtgagggagatcaataattaccaccacgcccacgacgcctacctgaacgccgtggtgggcaccgccctgattaaaaagtacccgaagctggagagcgagttcgtgtacggcgactacaaggtgtacgacgtgaggaagatgatcgccaagagcgagcaggagatcggcaaggccaccgccaagtacttcttctacagcaacatcatgaacttcttcaagaccgagatcaccctggccaacggcgagatcaggaagaggccgctgatcgagaccaacggcgagaccggcgagatcgtgtgggacaagggcagggacttcgccaccgtgaggaaggtgctgtccatgccgcaggtgaacatcgtgaagaagaccgaggtgcagaccggcggcttcagcaaggagagcatcctgccgaagaggaacagcgacaagctgatcgccaggaagaaggactgggatccgaagaagtacggcggcttcgacagcccgaccgtggcctacagcgtgctggtggtggccaaggtggagaagggcaagagcaagaagctgaagagcgtgaaggagctggtgggcatcaccatcatggagaggagcagcttcgagaagaacccagtggacttcctggaggccaagggctacaaggaggtgaagaaggacctgatcattaaactgccgaagtacagcctgttcgagctggagaacggcaggaagaggatgctggccagcgccggcgagctgcagaagggcaacgagctggccctgccgagcaagtacgtgaacttcctgtacctggccagccactacgagaagctgaagggcagcccggaggacaacgagcagaagcagctgttcgtggagcagcacaagcactacctggacgagatcatcgagcagatcagcgagttcagcaagagggtgatcctggccgacgccaacctggacaaggtgctgagcgcctacaacaagcacagggacaagccgatcagggagcaggccgagaacatcatccacctgttcaccctgaccaacctgggcgccccggccgccttcaagtacttcgacaccaccatcgacaggaagaggtacaccagcaccaaggaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagaccaggatcgacctgagccagctgggcggcgacagcagcccgccgaagaagaagaggaaggtgagctggaaggacgccagcggctggagcaggatgtga
seqidno:2
改进并优化的cas9氨基酸序列:
mdkkysigldigtnsvgwavitdeykvpskkfkvlgntdrhsikknligallfdsgetaeatrlkrtarrrytrrknricylqeifsnemakvddsffhrleesflveedkkherhpifgnivdevayhekyptiyhlrkklvdstdkadlrliylalahmikfrghfliegdlnpdnsdvdklfiqlvqtynqlfeenpinasgvdakailsarlsksrrlenliaqlpgekknglfgnlialslgltpnfksnfdlaedaklqlskdtydddldnllaqigdqyadlflaaknlsdaillsdilrvnteitkaplsasmikrydehhqdltllkalvrqqlpekykeiffdqskngyagyidggasqeefykfikpilekmdgteellvklnredllrkqrtfdngsiphqihlgelhailrrqedfypflkdnrekiekiltfripyyvgplargnsrfawmtrkseetitpwnfeevvdkgasaqsfiermtnfdknlpnekvlpkhsllyeyftvyneltkvkyvtegmrkpaflsgeqkkaivdllfktnrkvtvkqlkedyfkkiecfdsveisgvedrfnaslgtyhdllkiikdkdfldneenediledivltltlfedremieerlktyahlfddkvmkqlkrrrytgwgrlsrklingirdkqsgktildflksdgfanrnfmqlihddsltfkediqkaqvsgqgdslhehianlagspaikkgilqtvkvvdelvkvmgrhkpeniviemarenqttqkgqknsrermkrieegikelgsqilkehpventqlqneklylyylqngrdmyvdqeldinrlsdydvdhivpqsflkddsidnkvltrsdknrgksdnvpseevvkkmknywrqllnaklitqrkfdnltkaergglseldkagfikrqlvetrqitkhvaqildsrmntkydendklirevkvitlksklvsdfrkdfqfykvreinnyhhahdaylnavvgtalikkypklesefvygdykvydvrkmiakseqeigkatakyffysnimnffkteitlangeirkrplietngetgeivwdkgrdfatvrkvlsmpqvnivkktevqtggfskesilpkrnsdkliarkkdwdpkkyggfdsptvaysvlvvakvekgkskklksvkelvgitimerssfeknpvdfleakgykevkkdliiklpkyslfelengrkrmlasagelqkgnelalpskyvnflylashyeklkgspedneqkqlfveqhkhyldeiieqisefskrviladanldkvlsaynkhrdkpireqaeniihlftltnlgapaafkyfdttidrkrytstkevldatlihqsitglyetridlsqlggdssppkkkrkvswkdasgwsrm
seqidno:3
水稻osmatl基因基因组核苷酸序列:
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttcagctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtgcgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcacgccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatccacaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgccggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccggcgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcctcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccgacgcgcgcctcgccgattacttcgactgcatcgccgggaccagcaccggcggcctcatcaccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaaccgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaattcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgattgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaagatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggacaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccggcaggtacgtcgaggtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacatagttaatatttcaaattaaaaattcacattatatatat
seqidno:4
水稻osmatl基因氨基酸序列:
maasyscrrtceacstramagcvvgepasapgqrvtllaidgggirglipgtilaflearlqeldgpdarladyfdciagtstgglitamlaapgdhgrplfaasdinrfyldngplifpqkrcgmaaamaaltrpryngkylqgkirkmlgetrvrdtltnvviptfdvrllqptifstydaksmplknallsdicistsaaptylpahcfqttddatgkvrefdlidggvaannptmvamtqitkkimvkdkeelypvkpsdcgkflvlsvgtgstsdqgmytarqcsrwgivrwlrnkgmapiidifmaassdlvdihaavmfqslhsdgdylriqdntlhgdaatvdaatrdnmralvgigermlaqrvsrvnvetgryvevpgagsnadalrgfarqlseerrarlgrrnacggggegepsgvackr
靶位点序列:
seqidno:5
载体23843/23844(靶向1号外显子):tgcagtcgaagtaatcggcg
seqidno:6
载体23845/23846(靶向4号外显子):cgagaccggcaggtacgtcg
双组分导向rna:
seqidno:7
crrna的骨架序列:gttttagagctatgctgttttgaatggtcccaaaac
seqidno:8
tracrrna的序列:
ggaaccattcaaaacagcatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc
克隆测序扩增引物序列:
seqidno:9
exon1-f:gcgttcctgcaaatcacagtgacta
seqidno:10
exon1-r:gatcgaagagaggagatcgaattcg
seqidno:11
exon4-f:taagttcctggtgctgtccgtgg
seqidno:12
exon4-r:tacagttactaacgcttgcacgcca
实时荧光定量pcr的引物和探针序列:
seqidno:13
lb-f:ggcacaaaatcaccactcgat
seqidno:14
lb-r:ccgtgcagtcgatgataagct
seqidno:15
lb-probe:caggcagcccatcagaattaattctcatgtt
seqidno:16
rb-f:agggattttggtcatgagattatca
seqidno:17
rb-r:gcatgccaaccacaggaatta
seqidno:18
rb-probe:aaggatcttcacctagatccttttgatccgga
seqidno:19
spec-f:ttgttcactacgtgaaaggcga
seqidno:20
spec-r:gggtacggagatccactagagct
seqidno:21
spec-probe:accaaagtagtcggcaaa
seqidno:22
pmi-f:ccctatccccgttgacgac
seqidno:23
pmi-r:ctgatagtggtctccttgtcgct
seqidno:24
pmi-probe:ttcgccttcagcctgcacgacct
seqidno:25
cas9-f:ttgtgctgctccacgaaca
seqidno:26
cas9-r:gccagccactacgagaagct
seqidno:27
cas9-probe:ctgcttctgctcgttgtcctccgg
seqidno:28
osmatl-1-f:tgcaggagctggatggcc
seqidno:29
osmatl-1-r:gtgctggtcccggcg
seqidno:30
osmatl-1-probe:cagtcgaagtaatcggcgag
seqidno:31
osmatl-4-f:cgcagcgggtgtcga
seqidno:32
osmatl-4-r:cgtcggcgttgctgc
seqidno:33
osmatl-4-probe:cacctcgacgtacctg
部分诱导系中osmatl基因序列:
seqidno:34
osmatl-049a-1突变型核酸序列:
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttcagctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtgcgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcacgccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatccacaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgccggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccggcgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcctcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccgacgcgcgcctcgccgattacttcgactgcatcgccgggaccagcaccggcggcctcatcaccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaaccgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaattcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgattgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaagatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggacaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccggcaggtacgatcgaggtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacatagttaatatttcaaattaaaaattcacattatatatat
seqidno:35
osmatl-049a-2突变型核酸序列:
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttcagctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtgcgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcacgccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatccacaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgccggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccggcgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcctcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccgacgcgcgcctcgccgattacttcgactgcatcgccgggaccagcaccggcggcctcatcaccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaaccgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaattcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgattgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaagatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggacaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccggcaggtacgctcgaggtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacatagttaatatttcaaattaaaaattcacattatatatat
seqidno:36
osmatl-011a突变型核酸序列:
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttcagctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtgcgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcacgccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatccacaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgccggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccggcgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcctcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccgacgcgcgcctcgccgattacttcgactgcatcgccgggaccagcaccggcggcctcatcaccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaaccgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaattcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgattgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaagatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggacaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccggcaggtactcgaggtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacatagttaatatttcaaattaaaaattcacattatatatat
seqidno:37
osmatl-127a-1突变型核酸序列:
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttcagctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtgcgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcacgccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatccacaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgccggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccggcgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcctcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccgaccagcaccggcggcctcatcaccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaaccgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaattcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgattgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaagatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggacaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccggcaggtacgtcgaggtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacatagttaatatttcaaattaaaaattcacattatatatat
seqidno:38
osmatl-127a-2突变型核酸序列:
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttcagctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtgcgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcacgccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatccacaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgccggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccggcgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcctcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccgacgcgcgcctcgctcttcgactgcatcgccgggaccagcaccggcggcctcatcaccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaaccgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaattcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgattgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaagatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggacaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccggcaggtacgtcgaggtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacatagttaatatttcaaattaaaaattcacattatatatat
seqidno:39
osmatl-003d突变型核酸序列:
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttcagctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtgcgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcacgccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatccacaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgccggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccggcgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcctcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccgacgcatcgccgggaccagcaccggcggcctcatcaccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaaccgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaattcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgattgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaagatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggacaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccggcaggtacgtcgaggtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacatagttaatatttcaaattaaaaattcacattatatatat
seqidno:40
osmatl-288a突变型核酸序列:
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttcagctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtgcgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcacgccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatccacaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgccggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccggcgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcctcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccgacgcgcgcctcgccgattacttcgactgcatcgccgggaccagcaccggcggcctcatcaccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaaccgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaattcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgattgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaagatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggacaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccggctcgaggtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacatagttaatatttcaaattaaaaattcacattatatatat
seqidno:41
osmatl-068a突变型核酸序列:
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttcagctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtgcgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcacgccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatccacaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgccggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccggcgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcctcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccgacgcgcgcctcgcattacttcgactgcatcgccgggaccagcaccggcggcctcatcaccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaaccgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaattcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgattgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaagatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggacaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccggcaggtacgtcgaggtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacatagttaatatttcaaattaaaaattcacattatatatat
序列表
<110>先正达参股股份有限公司(syngentaparticipationsag),
先正达生物科技(中国)有限公司(syngentabiotechnologychinaco.,ltd)
<120>一种用基因编辑创制的水稻单倍体诱导系及其创制方法和应用
<130>81612-cn-reg-org-nat-1
<141>2018-07-06
<160>41
<170>siposequencelisting1.0
<210>1
<211>4170
<212>dna
<213>人工序列
<220>
<223>改进并优化的cas9核酸序列
<400>1
atggacaagaagtacagcatcggcctggacatcggcaccaacagcgtgggctgggccgtg60
atcaccgacgagtacaaggtgccgagcaagaagttcaaggtgctgggcaacaccgacagg120
cacagcatcaagaagaacctgatcggcgccctgctgttcgacagcggcgagaccgccgag180
gccaccaggctgaagaggaccgccaggaggaggtacaccaggaggaagaacaggatctgc240
tacctgcaggagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagg300
ctggaggagagcttcctggtggaggaggacaagaagcacgagaggcacccgatcttcggc360
aacatcgtggacgaggtggcctaccacgagaagtacccgaccatctaccacctgaggaag420
aagctggtggacagcaccgacaaggccgacctgaggctgatctacctggccctggcccac480
atgatcaagttcaggggccacttcctgatcgagggcgacctgaacccggacaacagcgac540
gtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggagaacccg600
atcaacgccagcggcgtggacgccaaggccatcctgagcgccaggctgagcaagagcagg660
aggctggagaacctgatcgcccagctgccgggcgagaagaagaacggcctgttcggcaac720
ctgatcgccctgagcctgggcctgaccccgaacttcaagagcaacttcgacctggccgag780
gacgccaagctgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcc840
cagatcggcgaccagtacgccgacctgttcctggccgccaagaacctgagcgacgccatc900
ctgctgagcgacatcctgagggtgaacaccgagatcaccaaggccccgctgagcgccagc960
atgatcaagaggtacgacgagcaccaccaggacctgaccctgctgaaggccctggtgagg1020
cagcagctgccggagaagtacaaggagatcttcttcgaccagagcaagaacggctacgcc1080
ggctacatcgacggcggcgccagccaggaggagttctacaagttcatcaagccgatcctg1140
gagaagatggacggcaccgaggagctgctggtgaagctgaacagggaggacctgctgagg1200
aagcagaggaccttcgacaacggcagcatcccgcaccagatccacctgggcgagctgcac1260
gccatcctgaggaggcaggaggacttctacccgttcctgaaggacaacagggagaagatc1320
gagaagatcctgaccttccgcatcccgtactacgtgggcccgctggccaggggcaacagc1380
aggttcgcctggatgaccaggaagagcgaggagaccatcaccccgtggaacttcgaggag1440
gtggtggacaagggcgccagcgcccagagcttcatcgagaggatgaccaacttcgacaag1500
aacctgccgaacgagaaggtgctgccgaagcacagcctgctgtacgagtacttcaccgtg1560
tacaacgagctgaccaaggtgaagtacgtgaccgagggcatgaggaagccggccttcctg1620
agcggcgagcagaagaaggccatcgtggacctgctgttcaagaccaacaggaaggtgacc1680
gtgaagcagctgaaggaggactacttcaagaagatcgagtgcttcgacagcgtggagatc1740
agcggcgtggaggacaggttcaacgccagcctgggcacctaccacgacctgctgaagatc1800
atcaaggacaaggacttcctggacaacgaggagaacgaggacatcctggaggacatcgtg1860
ctgaccctgaccctgttcgaggacagggagatgatcgaggagaggctgaagacctacgcc1920
cacctgttcgacgacaaggtgatgaagcagctgaagaggaggaggtacaccggctggggc1980
aggctgagcaggaagctgatcaacggcatcagggacaagcagagcggcaagaccatcctg2040
gacttcctgaagagcgacggcttcgccaacaggaacttcatgcagctgatccacgacgac2100
agcctgaccttcaaggaggacatccagaaggcccaggtgagcggccagggcgacagcctg2160
cacgagcacatcgccaacctggccggcagcccggccatcaagaagggcatcctgcagacc2220
gtgaaggtggtggacgagctggtgaaggtgatgggcaggcacaagccggagaacatcgtg2280
atcgagatggccagggagaaccagaccacccagaagggccagaagaacagcagggagagg2340
atgaagaggatcgaggagggcatcaaggagctgggcagccagatcctgaaggagcacccg2400
gtggagaacacccagctgcagaacgagaagctgtacctgtactacctgcagaacggcagg2460
gacatgtacgtggaccaggagctggacatcaacaggctgagcgactacgacgtggaccac2520
atcgtgccgcagagcttcctgaaggacgacagcatcgacaacaaggtgctgaccaggagc2580
gacaagaacaggggcaagagcgacaacgtgccgagcgaggaggtggtgaagaagatgaaa2640
aactactggaggcagctgctgaacgccaagctgatcacccagaggaagttcgacaacctg2700
accaaggccgagaggggcggcctgagcgagctggacaaggccggcttcattaaaaggcag2760
ctggtggagaccaggcagatcaccaagcacgtggcccagatcctggacagcaggatgaac2820
accaagtacgacgagaacgacaagctgatcagggaggtgaaggtgatcaccctgaagagc2880
aagctggtgagcgacttcaggaaggacttccagttctacaaggtgagggagatcaataat2940
taccaccacgcccacgacgcctacctgaacgccgtggtgggcaccgccctgattaaaaag3000
tacccgaagctggagagcgagttcgtgtacggcgactacaaggtgtacgacgtgaggaag3060
atgatcgccaagagcgagcaggagatcggcaaggccaccgccaagtacttcttctacagc3120
aacatcatgaacttcttcaagaccgagatcaccctggccaacggcgagatcaggaagagg3180
ccgctgatcgagaccaacggcgagaccggcgagatcgtgtgggacaagggcagggacttc3240
gccaccgtgaggaaggtgctgtccatgccgcaggtgaacatcgtgaagaagaccgaggtg3300
cagaccggcggcttcagcaaggagagcatcctgccgaagaggaacagcgacaagctgatc3360
gccaggaagaaggactgggatccgaagaagtacggcggcttcgacagcccgaccgtggcc3420
tacagcgtgctggtggtggccaaggtggagaagggcaagagcaagaagctgaagagcgtg3480
aaggagctggtgggcatcaccatcatggagaggagcagcttcgagaagaacccagtggac3540
ttcctggaggccaagggctacaaggaggtgaagaaggacctgatcattaaactgccgaag3600
tacagcctgttcgagctggagaacggcaggaagaggatgctggccagcgccggcgagctg3660
cagaagggcaacgagctggccctgccgagcaagtacgtgaacttcctgtacctggccagc3720
cactacgagaagctgaagggcagcccggaggacaacgagcagaagcagctgttcgtggag3780
cagcacaagcactacctggacgagatcatcgagcagatcagcgagttcagcaagagggtg3840
atcctggccgacgccaacctggacaaggtgctgagcgcctacaacaagcacagggacaag3900
ccgatcagggagcaggccgagaacatcatccacctgttcaccctgaccaacctgggcgcc3960
ccggccgccttcaagtacttcgacaccaccatcgacaggaagaggtacaccagcaccaag4020
gaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagaccaggatc4080
gacctgagccagctgggcggcgacagcagcccgccgaagaagaagaggaaggtgagctgg4140
aaggacgccagcggctggagcaggatgtga4170
<210>2
<211>1389
<212>prt
<213>人工序列
<220>
<223>改进并优化的cas9氨基酸序列
<400>2
metasplyslystyrserileglyleuaspileglythrasnserval
151015
glytrpalavalilethraspglutyrlysvalproserlyslysphe
202530
lysvalleuglyasnthrasparghisserilelyslysasnleuile
354045
glyalaleuleupheaspserglygluthralaglualathrargleu
505560
lysargthralaargargargtyrthrargarglysasnargilecys
65707580
tyrleuglngluilepheserasnglumetalalysvalaspaspser
859095
phephehisargleuglugluserpheleuvalglugluasplyslys
100105110
hisgluarghisproilepheglyasnilevalaspgluvalalatyr
115120125
hisglulystyrprothriletyrhisleuarglyslysleuvalasp
130135140
serthrasplysalaaspleuargleuiletyrleualaleualahis
145150155160
metilelyspheargglyhispheleuilegluglyaspleuasnpro
165170175
aspasnseraspvalasplysleupheileglnleuvalglnthrtyr
180185190
asnglnleupheglugluasnproileasnalaserglyvalaspala
195200205
lysalaileleuseralaargleuserlysserargargleugluasn
210215220
leuilealaglnleuproglyglulyslysasnglyleupheglyasn
225230235240
leuilealaleuserleuglyleuthrproasnphelysserasnphe
245250255
aspleualagluaspalalysleuglnleuserlysaspthrtyrasp
260265270
aspaspleuaspasnleuleualaglnileglyaspglntyralaasp
275280285
leupheleualaalalysasnleuseraspalaileleuleuserasp
290295300
ileleuargvalasnthrgluilethrlysalaproleuseralaser
305310315320
metilelysargtyraspgluhishisglnaspleuthrleuleulys
325330335
alaleuvalargglnglnleuproglulystyrlysgluilephephe
340345350
aspglnserlysasnglytyralaglytyrileaspglyglyalaser
355360365
glnglugluphetyrlyspheilelysproileleuglulysmetasp
370375380
glythrglugluleuleuvallysleuasnarggluaspleuleuarg
385390395400
lysglnargthrpheaspasnglyserileprohisglnilehisleu
405410415
glygluleuhisalaileleuargargglngluaspphetyrprophe
420425430
leulysaspasnargglulysileglulysileleuthrpheargile
435440445
protyrtyrvalglyproleualaargglyasnserargphealatrp
450455460
metthrarglysserglugluthrilethrprotrpasnphegluglu
465470475480
valvalasplysglyalaseralaglnserpheilegluargmetthr
485490495
asnpheasplysasnleuproasnglulysvalleuprolyshisser
500505510
leuleutyrglutyrphethrvaltyrasngluleuthrlysvallys
515520525
tyrvalthrgluglymetarglysproalapheleuserglyglugln
530535540
lyslysalailevalaspleuleuphelysthrasnarglysvalthr
545550555560
vallysglnleulysgluasptyrphelyslysileglucyspheasp
565570575
servalgluileserglyvalgluaspargpheasnalaserleugly
580585590
thrtyrhisaspleuleulysileilelysasplysasppheleuasp
595600605
asnglugluasngluaspileleugluaspilevalleuthrleuthr
610615620
leuphegluaspargglumetileglugluargleulysthrtyrala
625630635640
hisleupheaspasplysvalmetlysglnleulysargargargtyr
645650655
thrglytrpglyargleuserarglysleuileasnglyileargasp
660665670
lysglnserglylysthrileleuasppheleulysseraspglyphe
675680685
alaasnargasnphemetglnleuilehisaspaspserleuthrphe
690695700
lysgluaspileglnlysalaglnvalserglyglnglyaspserleu
705710715720
hisgluhisilealaasnleualaglyserproalailelyslysgly
725730735
ileleuglnthrvallysvalvalaspgluleuvallysvalmetgly
740745750
arghislysprogluasnilevalileglumetalaarggluasngln
755760765
thrthrglnlysglyglnlysasnserarggluargmetlysargile
770775780
glugluglyilelysgluleuglyserglnileleulysgluhispro
785790795800
valgluasnthrglnleuglnasnglulysleutyrleutyrtyrleu
805810815
glnasnglyargaspmettyrvalaspglngluleuaspileasnarg
820825830
leuserasptyraspvalasphisilevalproglnserpheleulys
835840845
aspaspserileaspasnlysvalleuthrargserasplysasnarg
850855860
glylysseraspasnvalproserglugluvalvallyslysmetlys
865870875880
asntyrtrpargglnleuleuasnalalysleuilethrglnarglys
885890895
pheaspasnleuthrlysalagluargglyglyleusergluleuasp
900905910
lysalaglypheilelysargglnleuvalgluthrargglnilethr
915920925
lyshisvalalaglnileleuaspserargmetasnthrlystyrasp
930935940
gluasnasplysleuilearggluvallysvalilethrleulysser
945950955960
lysleuvalseraspphearglysasppheglnphetyrlysvalarg
965970975
gluileasnasntyrhishisalahisaspalatyrleuasnalaval
980985990
valglythralaleuilelyslystyrprolysleuglusergluphe
99510001005
valtyrglyasptyrlysvaltyraspvalarglysmetilealalys
101010151020
sergluglngluileglylysalathralalystyrphephetyrser
1025103010351040
asnilemetasnphephelysthrgluilethrleualaasnglyglu
104510501055
ilearglysargproleuilegluthrasnglygluthrglygluile
106010651070
valtrpasplysglyargaspphealathrvalarglysvalleuser
107510801085
metproglnvalasnilevallyslysthrgluvalglnthrglygly
109010951100
pheserlysgluserileleuprolysargasnserasplysleuile
1105111011151120
alaarglyslysasptrpaspprolyslystyrglyglypheaspser
112511301135
prothrvalalatyrservalleuvalvalalalysvalglulysgly
114011451150
lysserlyslysleulysservallysgluleuvalglyilethrile
115511601165
metgluargserserpheglulysasnprovalasppheleugluala
117011751180
lysglytyrlysgluvallyslysaspleuileilelysleuprolys
1185119011951200
tyrserleuphegluleugluasnglyarglysargmetleualaser
120512101215
alaglygluleuglnlysglyasngluleualaleuproserlystyr
122012251230
valasnpheleutyrleualaserhistyrglulysleulysglyser
123512401245
progluaspasngluglnlysglnleuphevalgluglnhislyshis
125012551260
tyrleuaspgluileilegluglnileserglupheserlysargval
1265127012751280
ileleualaaspalaasnleuasplysvalleuseralatyrasnlys
128512901295
hisargasplysproilearggluglnalagluasnileilehisleu
130013051310
phethrleuthrasnleuglyalaproalaalaphelystyrpheasp
131513201325
thrthrileasparglysargtyrthrserthrlysgluvalleuasp
133013351340
alathrleuilehisglnserilethrglyleutyrgluthrargile
1345135013551360
aspleuserglnleuglyglyaspserserproprolyslyslysarg
136513701375
lysvalsertrplysaspalaserglytrpserargmet
13801385
<210>3
<211>2323
<212>dna
<213>水稻
<220>
<223>水稻osmatl基因基因组核苷酸序列
<400>3
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttca60
gctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtg120
cgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcac180
gccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatcc240
acaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgcc300
ggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccgg360
cgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcc420
tcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccg480
acgcgcgcctcgccgattacttcgactgcatcgccgggaccagcaccggcggcctcatca540
ccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatca600
accgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcga660
attcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcg720
tggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgag780
gtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcg840
cgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccat900
cttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctga960
ttgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctc1020
aagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctcccc1080
gcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatc1140
gacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaa1200
gatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttc1260
gcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaagg1320
acaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccg1380
tgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtggg1440
ggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcgg1500
ccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacg1560
gcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccg1620
ccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcggg1680
tgtcgagggtcaacgtcgagaccggcaggtacgtcgaggtgcccggcgccggcagcaacg1740
ccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtc1800
ggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgtt1860
agtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttac1920
ctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtg1980
cgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagc2040
acaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaat2100
atgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacact2160
ataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtga2220
ttgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggat2280
aacatagttaatatttcaaattaaaaattcacattatatatat2323
<210>4
<211>432
<212>prt
<213>水稻
<220>
<223>水稻osmatl基因氨基酸序列
<400>4
metalaalasertyrsercysargargthrcysglualacysserthr
151015
argalametalaglycysvalvalglygluproalaseralaprogly
202530
glnargvalthrleuleualaileaspglyglyglyileargglyleu
354045
ileproglythrileleualapheleuglualaargleuglngluleu
505560
aspglyproaspalaargleualaasptyrpheaspcysilealagly
65707580
thrserthrglyglyleuilethralametleualaalaproglyasp
859095
hisglyargproleuphealaalaseraspileasnargphetyrleu
100105110
aspasnglyproleuilepheproglnlysargcysglymetalaala
115120125
alametalaalaleuthrargproargtyrasnglylystyrleugln
130135140
glylysilearglysmetleuglygluthrargvalargaspthrleu
145150155160
thrasnvalvalileprothrpheaspvalargleuleuglnprothr
165170175
ilepheserthrtyraspalalyssermetproleulysasnalaleu
180185190
leuseraspilecysileserthrseralaalaprothrtyrleupro
195200205
alahiscyspheglnthrthraspaspalathrglylysvalargglu
210215220
pheaspleuileaspglyglyvalalaalaasnasnprothrmetval
225230235240
alametthrglnilethrlyslysilemetvallysasplysgluglu
245250255
leutyrprovallysproseraspcysglylyspheleuvalleuser
260265270
valglythrglyserthrseraspglnglymettyrthralaarggln
275280285
cysserargtrpglyilevalargtrpleuargasnlysglymetala
290295300
proileileaspilephemetalaalaserseraspleuvalaspile
305310315320
hisalaalavalmetpheglnserleuhisseraspglyasptyrleu
325330335
argileglnaspasnthrleuhisglyaspalaalathrvalaspala
340345350
alathrargaspasnmetargalaleuvalglyileglygluargmet
355360365
leualaglnargvalserargvalasnvalgluthrglyargtyrval
370375380
gluvalproglyalaglyserasnalaaspalaleuargglypheala
385390395400
argglnleuserglugluargargalaargleuglyargargasnala
405410415
cysglyglyglyglygluglygluproserglyvalalacyslysarg
420425430
<210>5
<211>20
<212>dna
<213>水稻
<220>
<223>1号外显子靶位点序列
<400>5
tgcagtcgaagtaatcggcg20
<210>6
<211>20
<212>dna
<213>水稻
<220>
<223>4号外显子靶位点序列
<400>6
cgagaccggcaggtacgtcg20
<210>7
<211>36
<212>dna
<213>人工序列
<220>
<223>crrna的骨架序列
<400>7
gttttagagctatgctgttttgaatggtcccaaaac36
<210>8
<211>79
<212>dna
<213>人工序列
<220>
<223>tracrrna的序列
<400>8
ggaaccattcaaaacagcatagcaagttaaaataaggctagtccgttatcaacttgaaaa60
agtggcaccgagtcggtgc79
<210>9
<211>25
<212>dna
<213>人工序列
<220>
<223>扩增引物exon1-f
<400>9
gcgttcctgcaaatcacagtgacta25
<210>10
<211>25
<212>dna
<213>人工序列
<220>
<223>扩增引物exon1-r
<400>10
gatcgaagagaggagatcgaattcg25
<210>11
<211>23
<212>dna
<213>人工序列
<220>
<223>扩增引物exon4-f
<400>11
taagttcctggtgctgtccgtgg23
<210>12
<211>25
<212>dna
<213>人工序列
<220>
<223>扩增引物exon4-r
<400>12
tacagttactaacgcttgcacgcca25
<210>13
<211>21
<212>dna
<213>人工序列
<220>
<223>引物lb-f
<400>13
ggcacaaaatcaccactcgat21
<210>14
<211>21
<212>dna
<213>人工序列
<220>
<223>引物lb-r
<400>14
ccgtgcagtcgatgataagct21
<210>15
<211>31
<212>dna
<213>人工序列
<220>
<223>探针lb-probe
<400>15
caggcagcccatcagaattaattctcatgtt31
<210>16
<211>25
<212>dna
<213>人工序列
<220>
<223>引物rb-f
<400>16
agggattttggtcatgagattatca25
<210>17
<211>21
<212>dna
<213>人工序列
<220>
<223>引物rb-r
<400>17
gcatgccaaccacaggaatta21
<210>18
<211>32
<212>dna
<213>人工序列
<220>
<223>探针rb-probe
<400>18
aaggatcttcacctagatccttttgatccgga32
<210>19
<211>22
<212>dna
<213>人工序列
<220>
<223>引物spec-f
<400>19
ttgttcactacgtgaaaggcga22
<210>20
<211>23
<212>dna
<213>人工序列
<220>
<223>引物spec-r
<400>20
gggtacggagatccactagagct23
<210>21
<211>18
<212>dna
<213>人工序列
<220>
<223>探针spec-probe
<400>21
accaaagtagtcggcaaa18
<210>22
<211>19
<212>dna
<213>人工序列
<220>
<223>引物pmi-f
<400>22
ccctatccccgttgacgac19
<210>23
<211>23
<212>dna
<213>人工序列
<220>
<223>引物pmi-r
<400>23
ctgatagtggtctccttgtcgct23
<210>24
<211>23
<212>dna
<213>人工序列
<220>
<223>探针pmi-probe
<400>24
ttcgccttcagcctgcacgacct23
<210>25
<211>19
<212>dna
<213>人工序列
<220>
<223>引物cas9-f
<400>25
ttgtgctgctccacgaaca19
<210>26
<211>20
<212>dna
<213>人工序列
<220>
<223>引物cas9-r
<400>26
gccagccactacgagaagct20
<210>27
<211>24
<212>dna
<213>人工序列
<220>
<223>探针cas9-probe
<400>27
ctgcttctgctcgttgtcctccgg24
<210>28
<211>18
<212>dna
<213>人工序列
<220>
<223>引物osmatl-1-f
<400>28
tgcaggagctggatggcc18
<210>29
<211>15
<212>dna
<213>人工序列
<220>
<223>引物osmatl-1-r
<400>29
gtgctggtcccggcg15
<210>30
<211>20
<212>dna
<213>人工序列
<220>
<223>探针osmatl-1-probe
<400>30
cagtcgaagtaatcggcgag20
<210>31
<211>15
<212>dna
<213>人工序列
<220>
<223>引物osmatl-4-f
<400>31
cgcagcgggtgtcga15
<210>32
<211>15
<212>dna
<213>人工序列
<220>
<223>引物osmatl-4-r
<400>32
cgtcggcgttgctgc15
<210>33
<211>16
<212>dna
<213>人工序列
<220>
<223>探针osmatl-4-probe
<400>33
cacctcgacgtacctg16
<210>34
<211>2324
<212>dna
<213>人工序列
<220>
<223>osmatl-049a-1突变型核酸序列
<400>34
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttca60
gctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtg120
cgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcac180
gccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatcc240
acaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgcc300
ggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccgg360
cgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcc420
tcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccg480
acgcgcgcctcgccgattacttcgactgcatcgccgggaccagcaccggcggcctcatca540
ccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatca600
accgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcga660
attcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcg720
tggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgag780
gtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcg840
cgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccat900
cttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctga960
ttgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctc1020
aagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctcccc1080
gcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatc1140
gacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaa1200
gatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttc1260
gcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaagg1320
acaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccg1380
tgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtggg1440
ggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcgg1500
ccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacg1560
gcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccg1620
ccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcggg1680
tgtcgagggtcaacgtcgagaccggcaggtacgatcgaggtgcccggcgccggcagcaac1740
gccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggt1800
cggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgt1860
tagtaactgtacacgcatcatgctgacgcgatcttttttatttttcttttttttttttta1920
cctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgt1980
gcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcag2040
cacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaa2100
tatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacac2160
tataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtg2220
attgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacatagga2280
taacatagttaatatttcaaattaaaaattcacattatatatat2324
<210>35
<211>2324
<212>dna
<213>人工序列
<220>
<223>osmatl-049a-2突变型核酸序列
<400>35
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttca60
gctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtg120
cgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcac180
gccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatcc240
acaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgcc300
ggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccgg360
cgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcc420
tcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccg480
acgcgcgcctcgccgattacttcgactgcatcgccgggaccagcaccggcggcctcatca540
ccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatca600
accgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcga660
attcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcg720
tggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgag780
gtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcg840
cgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccat900
cttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctga960
ttgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctc1020
aagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctcccc1080
gcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatc1140
gacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaa1200
gatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttc1260
gcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaagg1320
acaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccg1380
tgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtggg1440
ggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcgg1500
ccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacg1560
gcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccg1620
ccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcggg1680
tgtcgagggtcaacgtcgagaccggcaggtacgctcgaggtgcccggcgccggcagcaac1740
gccgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggt1800
cggcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgt1860
tagtaactgtacacgcatcatgctgacgcgatcttttttatttttcttttttttttttta1920
cctttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgt1980
gcgtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcag2040
cacaaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaa2100
tatgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacac2160
tataaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtg2220
attgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacatagga2280
taacatagttaatatttcaaattaaaaattcacattatatatat2324
<210>36
<211>2322
<212>dna
<213>人工序列
<220>
<223>osmatl-011a突变型核酸序列
<400>36
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttca60
gctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtg120
cgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcac180
gccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatcc240
acaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgcc300
ggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccgg360
cgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcc420
tcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccg480
acgcgcgcctcgccgattacttcgactgcatcgccgggaccagcaccggcggcctcatca540
ccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatca600
accgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcga660
attcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcg720
tggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgag780
gtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcg840
cgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccat900
cttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctga960
ttgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctc1020
aagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctcccc1080
gcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatc1140
gacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaa1200
gatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttc1260
gcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaagg1320
acaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccg1380
tgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtggg1440
ggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcgg1500
ccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacg1560
gcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccg1620
ccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcggg1680
tgtcgagggtcaacgtcgagaccggcaggtactcgaggtgcccggcgccggcagcaacgc1740
cgacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcg1800
gcgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgtta1860
gtaactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacc1920
tttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgc1980
gtataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagca2040
caaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaata2100
tgccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacacta2160
taaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgat2220
tgaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggata2280
acatagttaatatttcaaattaaaaattcacattatatatat2322
<210>37
<211>2285
<212>dna
<213>人工序列
<220>
<223>osmatl-127a-1突变型核酸序列
<400>37
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttca60
gctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtg120
cgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcac180
gccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatcc240
acaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgcc300
ggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccgg360
cgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcc420
tcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccg480
accagcaccggcggcctcatcaccgccatgctcgccgcgcccggcgaccacggccgcccg540
ctcttcgccgccagcgacatcaaccgcttctacctcgacaacggcccactcatcttccca600
caaaagtaactgatcacctcgaattcgatctcctctcttcgatctctgcattatttgatt660
tgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggcatggcggcggcca720
tggcggcgctgacgaggccgaggtacaacggcaagtacctgcaggggaagatcaggaaga780
tgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatccccacgttcgacg840
tcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcgttgattccatccg900
cattggcgttggaatcagctgattgtttgattgatcgaacaattgatcggttaaaatttt960
gcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatctgcatcagcacatc1020
cgcggcgccgacctacctccccgcgcactgcttccagaccaccgacgacgccaccggcaa1080
ggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacccggtaactaatca1140
atcaagcaatccatcaaacgaagatccacatgtgcattcctgtggtacaaatgctgatcg1200
atcgatggatggatcgattttcgcgagaacgtacagacgatggtggccatgacgcagatc1260
accaagaagataatggtgaaggacaaggaggagctgtacccggtaaagccgtcggactgc1320
ggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggaccaggggatgtacacg1380
gcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaaggggatggcgccc1440
atcatcgacatcttcatggcggccagctccgacctcgtcgacatccacgccgccgtcatg1500
ttccagtcgctgcacagcgacggcgactacctccgcatccaggacaacacgctccacggc1560
gacgccgccacggtggacgccgccaccagggacaacatgcgggcgctcgtcgggatcggc1620
gagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccggcaggtacgtcgag1680
gtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccaggcagctctccgag1740
gagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcggcgaaggagagccc1800
agcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctgacgcgatctttttt1860
atttttctttttttttttttacctttctagcggacatggggaataacaagacgtgacagt1920
agtgcaatcggtttgtaacgtgcgtataccaacattgatccatttcttcatcacagtttc1980
agttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagtcgtacacaacgta2040
caaaaatggattatctataaatatgccgccattttttttaaaataaaacggtaaaatcat2100
ttggcatgtatttatttacactataaactctcacatgtttttatggcaactttatatatt2160
tacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccatttttttaaagcag2220
aaccaatagcaacacataggataacatagttaatatttcaaattaaaaattcacattata2280
tatat2285
<210>38
<211>2318
<212>dna
<213>人工序列
<220>
<223>osmatl-127a-2突变型核酸序列
<400>38
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttca60
gctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtg120
cgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcac180
gccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatcc240
acaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgcc300
ggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccgg360
cgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcc420
tcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccg480
acgcgcgcctcgctcttcgactgcatcgccgggaccagcaccggcggcctcatcaccgcc540
atgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaaccgc600
ttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaattcg660
atctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtggcg720
tggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggtaca780
acggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcgaca840
cgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatcttct900
ccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgattgtt960
tgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaagaa1020
cgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgcgca1080
ctgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcgacgg1140
cggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaagatcc1200
acatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgcgag1260
aacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggacaag1320
gaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtgggc1380
accgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtgggggatc1440
gtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggccagc1500
tccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggcgac1560
tacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgccacc1620
agggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtgtcg1680
agggtcaacgtcgagaccggcaggtacgtcgaggtgcccggcgccggcagcaacgccgac1740
gcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcga1800
aacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaa1860
ctgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttc1920
tagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtat1980
accaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaa2040
cacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgcc2100
gccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaa2160
ctctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaa2220
atagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacat2280
agttaatatttcaaattaaaaattcacattatatatat2318
<210>39
<211>2298
<212>dna
<213>人工序列
<220>
<223>osmatl-003d突变型核酸序列
<400>39
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttca60
gctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtg120
cgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcac180
gccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatcc240
acaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgcc300
ggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccgg360
cgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcc420
tcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccg480
acgcatcgccgggaccagcaccggcggcctcatcaccgccatgctcgccgcgcccggcga540
ccacggccgcccgctcttcgccgccagcgacatcaaccgcttctacctcgacaacggccc600
actcatcttcccacaaaagtaactgatcacctcgaattcgatctcctctcttcgatctct660
gcattatttgatttgattggggattgtgggcggcgtggcgtggcgtccaggaggtgcggc720
atggcggcggccatggcggcgctgacgaggccgaggtacaacggcaagtacctgcagggg780
aagatcaggaagatgctgggcgagacgagggtgcgcgacacgctgacgaacgtcgtcatc840
cccacgttcgacgtcaggctgctccagccaaccatcttctccacatacgacgtgcgtgcg900
ttgattccatccgcattggcgttggaatcagctgattgtttgattgatcgaacaattgat960
cggttaaaattttgcaggcgaagagcatgccgctcaagaacgcgctcctctccgacatct1020
gcatcagcacatccgcggcgccgacctacctccccgcgcactgcttccagaccaccgacg1080
acgccaccggcaaggtccgcgagttcgacctcatcgacggcggcgtcgccgccaacaacc1140
cggtaactaatcaatcaagcaatccatcaaacgaagatccacatgtgcattcctgtggta1200
caaatgctgatcgatcgatggatggatcgattttcgcgagaacgtacagacgatggtggc1260
catgacgcagatcaccaagaagataatggtgaaggacaaggaggagctgtacccggtaaa1320
gccgtcggactgcggtaagttcctggtgctgtccgtgggcaccgggtcgacgtcggacca1380
ggggatgtacacggcgaggcagtgctcgcggtgggggatcgtccggtggctgcgcaacaa1440
ggggatggcgcccatcatcgacatcttcatggcggccagctccgacctcgtcgacatcca1500
cgccgccgtcatgttccagtcgctgcacagcgacggcgactacctccgcatccaggacaa1560
cacgctccacggcgacgccgccacggtggacgccgccaccagggacaacatgcgggcgct1620
cgtcgggatcggcgagcggatgctggcgcagcgggtgtcgagggtcaacgtcgagaccgg1680
caggtacgtcgaggtgcccggcgccggcagcaacgccgacgcgctgaggggcttcgccag1740
gcagctctccgaggagaggagggcgaggctaggtcggcgaaacgcctgcggcggcggcgg1800
cgaaggagagcccagcggcgtggcgtgcaagcgttagtaactgtacacgcatcatgctga1860
cgcgatcttttttatttttctttttttttttttacctttctagcggacatggggaataac1920
aagacgtgacagtagtgcaatcggtttgtaacgtgcgtataccaacattgatccatttct1980
tcatcacagtttcagttcatcagtatccgcgcagcacaaacacgtacacgtacgtacagt2040
cgtacacaacgtacaaaaatggattatctataaatatgccgccattttttttaaaataaa2100
acggtaaaatcatttggcatgtatttatttacactataaactctcacatgtttttatggc2160
aactttatatatttacgctgttaaatcaaaagtgattgaaatagggctaaaagcagccat2220
ttttttaaagcagaaccaatagcaacacataggataacatagttaatatttcaaattaaa2280
aattcacattatatatat2298
<210>40
<211>2316
<212>dna
<213>人工序列
<220>
<223>osmatl-288a突变型核酸序列
<400>40
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttca60
gctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtg120
cgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcac180
gccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatcc240
acaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgcc300
ggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccgg360
cgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcc420
tcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccg480
acgcgcgcctcgccgattacttcgactgcatcgccgggaccagcaccggcggcctcatca540
ccgccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatca600
accgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcga660
attcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcg720
tggcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgag780
gtacaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcg840
cgacacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccat900
cttctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctga960
ttgtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctc1020
aagaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctcccc1080
gcgcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatc1140
gacggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaa1200
gatccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttc1260
gcgagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaagg1320
acaaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccg1380
tgggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtggg1440
ggatcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcgg1500
ccagctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacg1560
gcgactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccg1620
ccaccagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcggg1680
tgtcgagggtcaacgtcgagaccggctcgaggtgcccggcgccggcagcaacgccgacgc1740
gctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcggcgaaa1800
cgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttagtaact1860
gtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacctttcta1920
gcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcgtatac1980
caacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcacaaaca2040
cgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatatgccgc2100
cattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactataaact2160
ctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgattgaaat2220
agggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataacatag2280
ttaatatttcaaattaaaaattcacattatatatat2316
<210>41
<211>2321
<212>dna
<213>人工序列
<220>
<223>osmatl-068a突变型核酸序列
<400>41
gggtgagtgactggagagccgttggttgggcacttgcggttgatctggtccgttgcttca60
gctcagagctctcacccgtctcttccaccaaagccgtgtgcccgtgtgggtgtgggtgtg120
cgcgcacactgcgctcgagcgcccgccaaaaatagttcgccgtagctcaaaaacgctcac180
gccgcgcgttcctgcaaatcacagtgactagtgacaaacgatcgatcgatccctccatcc240
acaaaccctcctcgatctcatcttccttcgtctcgtcaatggcggcgagctactcgtgcc300
ggcggacatgcgaggcgtgcagcacgagggcgatggccgggtgcgtggtgggcgagccgg360
cgtcggcgccggggcagcgggtgacgttgctggcgatcgacggcggcggcatcaggggcc420
tcatcccgggcaccatcctcgccttcctcgaggccaggctgcaggagctggatggccccg480
acgcgcgcctcgcattacttcgactgcatcgccgggaccagcaccggcggcctcatcacc540
gccatgctcgccgcgcccggcgaccacggccgcccgctcttcgccgccagcgacatcaac600
cgcttctacctcgacaacggcccactcatcttcccacaaaagtaactgatcacctcgaat660
tcgatctcctctcttcgatctctgcattatttgatttgattggggattgtgggcggcgtg720
gcgtggcgtccaggaggtgcggcatggcggcggccatggcggcgctgacgaggccgaggt780
acaacggcaagtacctgcaggggaagatcaggaagatgctgggcgagacgagggtgcgcg840
acacgctgacgaacgtcgtcatccccacgttcgacgtcaggctgctccagccaaccatct900
tctccacatacgacgtgcgtgcgttgattccatccgcattggcgttggaatcagctgatt960
gtttgattgatcgaacaattgatcggttaaaattttgcaggcgaagagcatgccgctcaa1020
gaacgcgctcctctccgacatctgcatcagcacatccgcggcgccgacctacctccccgc1080
gcactgcttccagaccaccgacgacgccaccggcaaggtccgcgagttcgacctcatcga1140
cggcggcgtcgccgccaacaacccggtaactaatcaatcaagcaatccatcaaacgaaga1200
tccacatgtgcattcctgtggtacaaatgctgatcgatcgatggatggatcgattttcgc1260
gagaacgtacagacgatggtggccatgacgcagatcaccaagaagataatggtgaaggac1320
aaggaggagctgtacccggtaaagccgtcggactgcggtaagttcctggtgctgtccgtg1380
ggcaccgggtcgacgtcggaccaggggatgtacacggcgaggcagtgctcgcggtggggg1440
atcgtccggtggctgcgcaacaaggggatggcgcccatcatcgacatcttcatggcggcc1500
agctccgacctcgtcgacatccacgccgccgtcatgttccagtcgctgcacagcgacggc1560
gactacctccgcatccaggacaacacgctccacggcgacgccgccacggtggacgccgcc1620
accagggacaacatgcgggcgctcgtcgggatcggcgagcggatgctggcgcagcgggtg1680
tcgagggtcaacgtcgagaccggcaggtacgtcgaggtgcccggcgccggcagcaacgcc1740
gacgcgctgaggggcttcgccaggcagctctccgaggagaggagggcgaggctaggtcgg1800
cgaaacgcctgcggcggcggcggcgaaggagagcccagcggcgtggcgtgcaagcgttag1860
taactgtacacgcatcatgctgacgcgatcttttttatttttctttttttttttttacct1920
ttctagcggacatggggaataacaagacgtgacagtagtgcaatcggtttgtaacgtgcg1980
tataccaacattgatccatttcttcatcacagtttcagttcatcagtatccgcgcagcac2040
aaacacgtacacgtacgtacagtcgtacacaacgtacaaaaatggattatctataaatat2100
gccgccattttttttaaaataaaacggtaaaatcatttggcatgtatttatttacactat2160
aaactctcacatgtttttatggcaactttatatatttacgctgttaaatcaaaagtgatt2220
gaaatagggctaaaagcagccatttttttaaagcagaaccaatagcaacacataggataa2280
catagttaatatttcaaattaaaaattcacattatatatat2321