CN1325959B - 来自基因簇的基因 - Google Patents
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Abstract
ML-236B为HMG-CoA还原酶的抑制剂并用于制备另一抑制剂-帕伐他丁。使用编码与存在于产ML-236B的微生物中的聚酮化合物合成酶簇有关的基因的多核苷酸,可以提高使用这种微生物的ML-236B的制备。
Description
本发明涉及一基因簇,尤其是来自基因簇的基因。
更具体地说,本发明涉及多核苷酸,如DNA,当将其导入产ML-236B的微生物中时它加速HMG-CoA还原酶抑制剂,ML-236B在该产ML-236B的微生物中的生物合成。本发明还涉及插入所述多核苷酸的载体、由所述载体转化的宿主细胞、由所述载体表达的蛋白质、一种使用所述多核苷酸和/或蛋白质生产ML-236B的方法,该方法包括从所述宿主细胞培养物中回收ML-236B,并且本发明还涉及其它相关方面。
帕伐他丁为HMG-CoA还原酶抑制剂。帕伐他丁钠已用于治疗血脂过多或高脂血症,并具有能降低血清胆固醇的有用药理效应。使用嗜碳酸气链霉菌通过微生物转化由柑桔青霉产生的ML-236B可以获得帕伐他丁[记载于Endo,A.等人的“抗生素杂志”29,1346(1976);Matsuoka,T.等人的“欧洲生物化学杂志”184,707(1989)和JP-昭57-2240A]。
已显示,ML-236B,一种帕伐他丁前体,以及洛伐他丁,一种HMG-CoA抑制剂,都具有部分相同结构。它们都是由聚酮化合物经生物合成的[记载于Moore,R.N.等人的“美国化学学会杂志”107,3694(1985);Shiao,M.和Don,H.S.的“中国科学院院报”11,23(1987)]。
聚酮化合物为由低分子量羧酸如乙酸、丙酸、丁酸等连续缩合反应产生的β-酮碳链衍生的化合物。根据每个β-酮羰基的缩合或还原的路径可以获得不同结构[记载于Hopwood,D.A.和Sherman,D.H.的“基因工程年鉴”24,37-66(1990);Hutchinson,C.R.和Fujii,I.“微生物学年鉴”49,201-238(1995)]。
有利于聚酮化合物合成的聚酮化合物合成酶(本文后面称之为PKS)为已知存在于丝状真菌和细菌中的酶。已使用分子生物学技术对这些丝状真菌酶进行了研究[如记载于Feng,G.H.和Leonard,T.J.的“细菌学杂志”177,6246(1995);Takano,Y.等人的“分子基因的基因工程”249,162(1995)]。在为产生洛伐他丁微生物的土曲霉中,已对与洛伐他丁生物合成有关的PKS基因进行了分析[记载于在日本的国际申请公开号平9-504436A并参见相应的WO9512661,它要求保护编码三醇聚酮化合物合成酶的DNA]。
与生物合成丝状真菌的二级代谢物有关的基因经常在基因组上形成一簇。在生物合成聚酮化合物的路径中,已知存在参与所述路径的基因簇。在生物合成黄曲霉毒素(它是由黄曲霉和寄生曲霉产生的聚酮化合物)时,已知编码参与所述生物合成的酶蛋白质(例如PKS)的基因形成簇结构。进行基因组分析并将每个微生物中参与生物合成黄曲霉毒素的基因进行比较[参见Yu,J.等人的“环境微生物应用”61,2365(1995)]。已报道,参与生物合成由构巢曲霉产生的柄曲霉素的基因在其基因组上形成约60kb连续区域的簇结构[记载于Brown,D.W.等人的“美国国家科学院院报”93,1418(1996)]。
已研究了在洛伐他丁合成过程中通过辅助蛋白对聚酮化合物合成酶活性的调节[参见Kennedy,J.等人的“科学”第284卷,1368(1999)]。
然而,时至今日,对ML-236B的生物合成和调节该合成的因子的分子生物学分析还不够。本发明打算解决该问题。
根据本发明,提供了一种适合用于加速ML-236B的生物合成的多核苷酸。
该多核苷酸典型地为编码包括氨基酸序列SEQ ID NO 38、42、44、46、48或50或者由这些序列组成的蛋白质的多核苷酸。还提供了编码具有至少一个缺失、添加、替代或改变的修饰氨基酸序列的该多核苷酸变体。
序列表构成本专利说明书的一部分。为了帮助理解,我们列出了以下所列序列的表格。
SEQ ID NO 特性
1 pML48插入片段
2 与SEQ ID NO1互补
3 实施例4PCR引物
4 实施例4的PCR引物
5 实施例8中5′-RACE的寡核苷酸DNA(1)
6 实施例8中5′-RACE的寡核苷酸DNA(1)
7 实施例8中5′-RACE的寡核苷酸DNA(1)
8 实施例8中5′-RACE的寡核苷酸DNA(1)
9 实施例8中5′-RACE的寡核苷酸DNA(1)
10 实施例8中5′-RACE的寡核苷酸DNA(1)
11 实施例8中5′-RACE的寡核苷酸DNA(2)
12 实施例8中5′-RACE的寡核苷酸DNA(2)
13 实施例8中5′-RACE的寡核苷酸DNA(2)
14 实施例8中5′-RACE的寡核苷酸DNA(2)
15 实施例8中5′-RACE的寡核苷酸DNA(2)
16 实施例8中5′-RACE的寡核苷酸DNA(2)
17 实施例8中5′-端cDNA片段
18 实施例8中5′-端cDNA片段
19 实施例8中5′-端cDNA片段
20 实施例8中5′-端cDNA片段
21 实施例8中5′-端cDNA片段
22 实施例8中5′-端cDNA片段
23 实施例8中3′-RACE的寡核苷酸DNA(3)
24 实施例8中3′-RACE的寡核苷酸DNA(3)
25 实施例8中3′-RACE的寡核苷酸DNA(3)
26 实施例8中3′-RACE的寡核苷酸DNA(3)
27 实施例8中3′-RACE的寡核苷酸DNA(3)
28 实施例8中3′-RACE的寡核苷酸DNA(3)
29 实施例8中3′-端cDNA片段
30 实施例8中3′-端cDNA片段
31 实施例8中3′-端cDNA片段
32 实施例8中3′-端cDNA片段
33 实施例8中3′-端cDNA片段
34 实施例8中3′-端cDNA片段
35 实施例9中的RT-PCR引物
36 实施例9中的RT-PCR引物
37 mlcE;cDNA核苷酸序列和推断氨基酸序列
38 推断mlcE多肽
39 实施例12中的RT-PCR引物
40 实施例12中的RT-PCR引物
41 mlcR;cDNA核苷酸序列和推断氨基酸序列
42 推断mlcR多肽
43 mlcA;cDNA核苷酸序列和推断氨基酸序列
44 推断mlcA多肽
45 mlcB;cDNA核苷酸序列和推断氨基酸序列
46 推断mlcB多肽
47 mlcC;cDNA核苷酸序列和推断氨基酸序列
48 推断mlcC多肽
49 mlcD;cDNA核苷酸序列和推断氨基酸序列
50 推断mlcD多肽
51 实施例17中的RT-PCR引物
52 实施例17中的RT-PCR引物
53 实施例17中的RT-PCR引物
54 实施例17中的RT-PCR引物
55 实施例17中的RT-PCR引物
56 实施例17中的RT-PCR引物
57 实施例17中的RT-PCR引物
58 实施例17中的RT-PCR引物
59 实施例17中的RT-PCR引物
60 实施例17中的RT-PCR引物
61 实施例17中的RT-PCR引物
62 实施例17中的RT-PCR引物
编码SEQ ID NO 38、42、44、46、48或50的氨基酸序列的多核苷酸可以为cDNA、基因组DNA或mRNA。将编码这六个序列的每个基因组DNA分别称之为结构基因mlcE、mlcR、mlcA、mlcB、mlcC和mlcD。不依赖这些赋值,我们相信,结构基因编码具有以下功能的蛋白质:
mlcA 聚酮化合物合成酶
mlcB 聚酮化合物合成酶
mlcC P450一氧合酶
mlcD HMG-CoA还原酶
mlcE 外排泵
mlcR 转录因子
我们已发现,导入mlcE或相应于mlcE的cDNA可以加速ML-236B的生物合成,导入mlcR或相应于mlcR的cDNA可以加速ML-236B的生物合成。而且,mlcR刺激mlcA-D的转录表达。通过基因破坏研究显示,在ML-236B的生产中单独地或共同地包括mlcA、B、C和D。
通过天然或人工改变获得的mlcA、B和/或C变体可用于生产ML-236B衍生物,包括例如帕伐他丁或洛伐他丁的抑制素。在这方面,使用这些变体以及仅一步发酵步骤就可以生产帕伐他丁,而不需目前使用嗜碳酸气链霉菌将ML-236B经微生物转换成帕伐他丁。
一优选的多核苷酸包括一含有SEQ ID NO 37或者含有能够加速ML-236B生物合成的其突变体或变体的序列。这种DNA多核苷酸可由转化的大肠杆菌pSAKexpE SANK 72499(FERM BP-7005)获得。
另一优选的多核苷酸包括一含有SEQ ID NO 41或者含有能够加速ML-236B生物合成的其突变体或变体的序列。这种DNA多核苷酸可从转化的大肠杆菌pSAKexpR SANK 72599(FERM BP-7006)获得。
本发明的多核苷酸可用于与一个或多个多核苷酸可操作地组合。优选组合适合用于提高产ML-236B的微生物中的ML236B产量。
这些组合的例子包括SEQ ID NO 37或其具有相似功能的变体与一个或多个选自SEQ ID NO 37本身、41、43、45、47或49的序列组合;以及SEQ ID NO41或其具有相似功能的变体与一个或多个选自SEQ ID NO 37、41本身、43、45、47或49的组合。
一方面,该多核苷酸优选为编码包括氨基酸序列SEQ ID NO 38、42、44、46、48或50或由这些氨基酸序列组成并且单独或与SEQ ID NO 37、SEQ ID NO41的多核苷酸或其具有相似功能的变体结合能加速ML-236B的生物合成的蛋白质的多核苷酸。
本发明还延伸到能够在严格条件下与本发明的多核苷酸杂交的多核苷酸。这些多核苷酸延伸到当导入产ML-236B的微生物中时适合加速产ML-236B的微生物中的ML-236B的生物合成的多核苷酸。
该多核苷酸典型地为DNA、cDNA或基因组DNA或RNA,并且可以为有义或反义。该多核苷酸典型地为纯化多核苷酸,例如没有其他细胞组分的多核苷酸。
本发明延伸至编码图示SEQ ID NO 38、42、44、46、48或50的氨基酸序列的多核苷酸变体,其中一个或多个核苷酸已改变。这些变化可以自然发生,并且可以在丰余或简并的遗传密码三联体内进行。这些简并地改变的多核苷酸因此编码相同氨基酸序列。在这些多核苷酸变体中,我们包括具有外显子和内含子的基因组DNA,而不是简单的cDNA序列。
本发明还延伸至编码图示SEQ ID NO 38、42、44、46、48或50的氨基酸序列的多核苷酸变体,它们编码具有至少一个缺失、添加、替代或改变的修饰氨基酸序列。因此,本发明延伸至编码比图示序列所编码的序列长度短、长或与之相等的氨基酸序列的图示序列的多核苷酸变体。优选这些变体多肽保留了加速ML-236B合成的能力,并优选具有与引起变体序列的亲代序列的活性基本上相似或者比之更好的活性。
这些多核苷酸变体保留了与亲代一定程度的同一性。该同一性适宜地为至少60%、至少80%、至少90%或至少95%或100%。变体的同一性优选由计算机软件来评定,例如使用进行同源性检索的算法的BLAST程序。
一方面,本发明的优选多核苷酸为选自以下的DNA:
(a)包括序列表中SEQ ID NO 37的第1个核苷酸至第1662个核苷酸中所示一个或多个核苷酸序列的DNA,并且它的特征在于当将其导入所述产ML-236B的微生物中时在该产ML-236B的微生物中加速ML-236B的生物合成;
(b)在严格条件下与(a)中所述DNA杂交的DNA,并且它的特征在于当将其导入所述产ML-236B的微生物中时在该产ML-236B的微生物中加速ML-236B的生物合成;
(c)包括序列表中SEQ ID NO 41的第1个核苷酸至第1380个核苷酸中所示一个或多个核苷酸序列的DNA,并且它的特征在于当将其导入所述产ML-236B的微生物中时在该产ML-236B的微生物中加速ML-236B的生物合成;
(d)在严格条件下与(c)中所述DNA杂交的DNA,并且它的特征在于当将其导入所述产ML-236B的微生物中时在该产ML-236B的微生物中加速ML-236B的生物合成。
本发明的多核苷酸在产ML-236B的微生物中加速ML-236B的生物合成。产ML-236B的微生物的例子包括青霉种,例如柑桔青霉、短密青霉[记载于Brown,A.G.等人的“帕金化学学会杂志-1”1165(1976)]、圆弧青霉[记载于Doss,S.L.等人的“国家产品杂志”49,357(1986)]等。其它例子包括:真青霉种M6603[记载于Endo,A.等人的“东京抗生素杂志”39,1609(1986)]、绿色拟青霉FERMP-6236[记载于JP-昭58-98092A]、拟青霉种M2016[记载于Endo,A.等人的“东京抗生素杂志”39,1609(1986)]、长臂木霉M6735[记载于Endo,A.等人的“东京抗生素杂志”39,1609(1986)]、金精隐壳霉(Hypomyceschrysospermus)IFO7798[记载于Endo,A.等人的“东京抗生素杂志”39,1609(1986)]、胶霉种YJ-9515[记载于WO9806867]、绿色木霉IFO5836[记载于JP-昭62-19159B]、网状孢子真青霉IFO9022[记载于JP-昭62-19159B]或任意其它适宜生物体。
在这些产ML-236B的微生物中,优选柑桔青霉,并且更优选柑桔青霉菌株SANK 13380。根据微生物保藏的布达佩斯条约,柑桔青霉SANK 13380菌株于1992年12月22日以保藏号FERM BP-4129保藏在工业科技署的生命科学和技术研究院。产ML-236B的微生物的例子还包括从天然源分离的那些以及天然或人工突变的那些。
本发明还提供了含有本发明多核苷酸的载体。例如由大肠杆菌pSAKexpESANK 72499(FERM BP-7005)或大肠杆菌pSAKexpR SANK 72599(FERM BP-7006)获得的载体。本发明的这些载体包括表达载体。
还提供了由本发明载体转化的宿主细胞,包括产ML-236B的微生物。本发明的宿主细胞包括柑桔青霉和大肠杆菌,例如大肠杆菌pSAKexpE SANK72499(FERM BP-7005)或大肠杆菌pSAKexpR SANK 72599(FERM BP-7006)。
此外,本发明延伸至由本发明多核苷酸编码的多肽。本发明多肽的例子包括序列SEQ ID NO 38或42、或具有与SEQ ID NO 38或42的一定程度同一性且能加速产ML236B的微生物中ML236B产生的其变体。其它多肽是由本发明的其它多核苷酸序列编码的那些,以及保留一定程度同一性的变体。
多肽变体与SEQ ID NO 38或42的同一性适宜地为至少80%、至少90%或至少95%或100%。变体的同一性优选由计算机软件评定,例如使用进行同源性检索的算法的BLAST程序。
本发明的多肽包括SEQ ID NO 38或42或变体的更短或更长序列.更短多肽包括SEQ ID NO 38、42或其变体的部分氨基酸序列,并且优选保留了加速ML236B生物合成的能力。更长多肽包括SEQ ID NO 38、42或其变体的全部或部分氨基酸序列,并且优选保留了加速ML236B生物合成的能力。更长多肽包括融合蛋白,例如Fc-融合蛋白。
本发明的多肽包括具有序列SEQ ID NO 38、SEQ ID NO 42、SEQ ID NO 44、SEQ ID NO 46、SEQ ID NO 48之一、或具有相似功能的其变体。本发明还提供了这些多肽的抗体。本发明既提供了多克隆抗体又提供了单克隆抗体。
所述抗体可用于调控ML-236B的生成并产生ML-236B的衍生物如包括帕伐他丁和洛伐他丁的抑制素。并且,所述抗体可优选用于ML-236B生物合成和其调控机制的分析。这种分析可用于调控ML-236B生成及产生ML-236B的衍生物。
具有本发明载体的本发明的宿主细胞可用于一种生产ML-236B的方法中,包括培养这种宿主细胞,然后从该培养物中回收ML-236B。在一种方法中,该载体包括mlcE或mlcR,不包括如mlcA、mlcB、mlcC或mlcD的其它基因。
通过本发明的方法的生产可以在没有相应于SEQ ID NO 44、SEQ ID NO46、SEQ ID NO 48或SEQ ID NO 50的重组mlcA、mlcB、mlcC和/或mlcD(多肽)时进行。
下面将详细描述本发明。
本发明的发明人已克隆出含有参与柑桔青霉中ML-236B的生物合成的基因组DNA。该基因组DNA以下称之为与ML-236B生物合成有关的基因组DNA,并由产ML-236B的微生物的基因组DNA文库克隆。该基因组DNA经分析,发现所述基因组DNA上的结构基因,然后使用含有柑桔青霉的mRNA的总RNA作为模板通过反转录-聚合酶链反应(以下称之为“RT-PCR”)获得相应于所述结构基因的cDNA。发现,当由含有所述cDNA的重组DNA载体转化产ML-236B的微生物时在该产ML-236B的微生物中的ML-236B的生物合成加速。
本发明尤其涉及cDNA(以下称之为加速ML-236B生物合成的cDNA),当将其导入所述产ML-236B的微生物中时它加速该产ML-236B的微生物中的ML-236B的生物合成。
本发明的加速ML-236B生物合成的多核苷酸,例如加速ML-236B生物合成的cDNA,例如包括:
(I)使用参与ML-236B的生物合成并存在于产ML-236B的微生物中的基因组DNA中的结构基因的转录产物(信使RNA,以下称之为mRNA)作为模板通过合成获得的DNA;
(II)DNA(I)与使用DNA(I)作为第一链合成的第二链DNA缔合形成的双链DNA;
(III)双链DNA(II)例如通过克隆等方法复制或扩增形成的双链DNA;
(IV)可以在严格条件下于上述DNA或mRNA杂交的DNA。
DNA(IV)可以是本文中任意结构基因序列所示的那些,例如序列表中SEQID NO 37的第1个核苷酸至第1662个核苷酸或者SEQ ID NO 41中第1个核苷酸至第1380个核苷酸,其中一个或多个核苷酸任选地经替换、缺失和/或添加,并且当将其导入产ML-236B的微生物中时它能加速该产ML-236B的微生物中的ML-236B的生物合成。
当两个单链核酸杂交时,它们在彼此互补或高度互补的区域形成双链分子,并且“严格条件”适宜地是指杂交溶液为6xSSC的情况[1xSSC为150mM NaCl和15mM柠檬酸钠的组合物],并且杂交温度为55℃。
加速ML-236B生物合成的cDNA例如可以通过从产ML-236B的微生物的cDNA文库中分离含该cDNA的克隆.或者,可以使用以与ML-236B生物合成有关的基因组DNA的核苷酸序列和产ML-236B的微生物的mRNA或总RNA为基础设计的一对引物进行RT-PCR.
产ML-236B的微生物为固有地具有产生ML-236B的能力的微生物。正如前面所述的,产ML-236B的微生物的例子包括青霉种,例如柑桔青霉、短密青霉、圆弧青霉等。并且其它例子包括:真青霉种M6603、绿色拟青霉FERMP-6236、拟青霉种M2016、长臂木霉M6735、金精隐壳霉IFO7798、胶霉种YJ-9515、绿色木霉IFO5836、网状孢子真青霉IFO9022或任意其它适宜生物体。
在这些产ML-236B的微生物中,优选柑桔青霉,并且更优选柑桔青霉菌株SANK 13380。根据微生物保藏的布达佩斯条约,柑桔青霉SANK 13380菌株于1992年12月22日以保藏号FERM BP-4129保藏在工业科技署的生命科学和技术研究院。产ML-236B的微生物的例子还包括从天然源分离的那些以及天然或人工突变的那些。
可以用适宜探针筛选产ML-236B的微生物的基因组DNA文库来获得与ML-236B生物合成有关的基因组DNA。适宜地以预测对ML-236B生物合成起作用的DNA序列为基础设计该探针,适宜地来源于丝状真菌。
对创建基因组DNA文库的方法的选择没有限制,可以使用任意适宜方法,优选为构建真核生物的基因组DNA文库的常规方法。其例子包括Maniatis等人的方法[Maniatis,T等人的“分子克隆”,实验室手则,第2版,冷泉港实验室,纽约冷泉港(1989)]。其它适宜的方法在本领域中为已知。
概括地,可以从所述产ML-236B的微生物的培养物中回收细胞,物理破碎这些细胞,提取存在于其核中的DNA并将所述DNA提纯,获得来自产ML-236B的微生物的基因组DNA。
可以在适合特定产ML-236B的微生物的条件下培养产ML-236B的微生物。例如,将细胞接种于MBG3-8培养基[组成:7%(w/v)甘油、3%(w/v)葡萄糖、1%(w/v)大豆粉、1%(w/v)蛋白胨(由Kyokuto Seivaku Kogyo公司生产)、1%(w/v)玉米浆(由Honen公司生产)、0.5%(w/v)硝酸钠、0.1%(w/v)硫酸镁七水合物(pH6.5)]中,并在22-28℃下摇动培养3-7天,可以将柑桔青霉(一种优选的产ML-236B的微生物)培养。通过将熔融的PGA琼脂培养基[组成:200g/L马铃薯浸出物、15%(w/v)甘油、2%(w/v)琼脂]倒入试管,并以一角度使琼脂固化,从而可以制备贮藏细菌用的斜面。然后使用铂针将柑桔青霉接种于该斜面中,接着在22-28℃下培养7-15天。通过将该斜面贮藏在0-4℃下可以在该斜面上连续地保持以这种方式生长的微生物或细菌。
通过离心可以回收在液体培养基中培养的产ML-236B的微生物的细胞,使用刮刀等从固体培养基刮下可以回收在固体培养基上培养的细胞。
在用液氮等将细胞冷冻之后,使用研棒和研钵研磨这些细胞可以将这些细胞物理破碎。使用例如十二烷基硫酸钠(下文称之为SDS)的表面活性剂或其它适宜的表面活性剂可以提取破碎细胞核心中的DNA。提取的基因组DNA适宜地用苯酚-氯仿处理以除去蛋白质,并通过乙醇沉淀以沉淀物回收。
通过适宜限制酶消化使所得基因组DNA片段化。对可用于限制消化的限制酶没有限制,一般优选可以获得的限制酶。其例子包括Sau3AI。其它适宜的酶在本领域中为已知。经过消化的DNA然后经过凝胶电泳,并从该凝胶中回收具有适宜大小的基因组DNA。DNA片段的大小没有特别的限制,但是优选20kb或之上。
同样,对用于构建基因组DNA文库的DNA载体没有限制,只要该载体具有在通过该载体转化的宿主细胞中复制所必需的DNA序列。适宜载体的例子包括质粒载体、噬菌体载体、粘粒载体、BAC载体等,优选粘粒载体。该DNA载体优选为表达载体。更优选地,该DNA载体包括在通过该载体转化的宿主细胞上赋予选择性表型的DNA或核苷酸序列。
该DNA载体适宜地为能用于克隆和表达的载体。优选地,该载体为能用于转化一个以上微生物宿主的穿梭载体。该穿梭载体适宜地具有能在宿主细胞中复制的DNA序列,并优选能在来自不同微生物组如细菌和真菌的大量不同宿主细胞中复制的序列。而且,该穿梭载体优选含有一能向一定范围的不同宿主细胞,例如来自不同微生物组的细胞提供选择性表型的DNA序列。
微生物组和通过该穿梭载体转化的宿主细胞的组合的选择没有特别限制,只要微生物组之一可用于克隆,另一具有产ML-236B的能力。这种组合例如可以是细菌与丝状真菌的组合,酵母与丝状真菌的组合,细菌与丝状真菌的组合为优选。对细菌的选择没有特别限制,只要它一般能用于生物工程,例如大肠杆菌、枯草芽胞杆菌等。优选大肠杆菌,更优选大肠杆菌XL1-Blue MR。相似地,对酵母种没有限制,只要它一般能用于生物工程,例如酿酒酵母等。丝状真菌的例子包括上述产ML-236B的微生物。微生物的其它适宜例子在本领域中为已知。
在本发明中,微生物组可选自细菌、丝状真菌和酵母。
上述穿梭载体的例子包括具有用于选择表型和cos位点的适宜标记基因的粘粒载体。其它适宜的载体在本领域中为已知。优选的载体为pSAKcos1,它是将来自粘粒载体pWE15(由STRATAGENE生产)的cos位点插入质粒pSAK333中构建的,含有大肠杆菌潮霉素B磷酸转移酶基因序列[记载于JP-平3-262486A]。构建pSAKcos1的一种方法示于图1中。本发明不限于该载体。
通过将穿梭载体引入宿主细胞可以制备基因组DNA文库,所述载体含有来自产ML-236B的微生物的基因组DNA片段。所用宿主细胞优选为大肠杆菌,更优选为大肠杆菌XL1-Blue MR。当宿主细胞为大肠杆菌时,可以通过体外包装进行引入。在本发明中,转化还包括通过体外包装引入外来DNA,并且转化的细胞还包括通过体外包装引入外来DNA的细胞。
可以使用抗体或核酸探针对基因组文库进行筛选以鉴定所需的克隆,优选使用核酸探针。优选以聚酮化合物生物合成有关的基因或DNA的核苷酸序列(优选由丝状真菌获得的序列)为基础制备该核酸探针。对特定基因的选择没有限制,只要其包含在聚酮化合物的生物合成中,并且其核苷酸序列为已知。这些基因的例子包括黄曲霉和寄生曲霉的黄曲霉毒素PKS基因、构巢曲霉的柄曲霉素PKS基因等。
例如通过合成含有上述已知基因组DNA序列部分的寡核苷酸探针、或者使用聚合酶链反应[下文称之为“PCR”,记载于Saiki,R.K.倒入的“科学”239,487(1988)]并使用基因组DNA为模板通过制备寡核苷酸引物并扩增靶DNA、或者使用mRNA为模板通过RT-PCR,可以获得适宜的核酸探针。获得这些探针的其它适宜方法在本领域中众所周知。
可以由产ML-236B的微生物例如使用PCR或RT-PCR获得核酸探针.优选以与聚酮化合物生物合成有关的基因的核苷酸序列(该核苷酸序列为已知)为基础进行用于PCR或RT-PCR的引物(下文称之为“用于PCR的引物)的设计.优选该基因为黄曲霉、寄生曲霉的黄曲霉毒素PKS基因、或者构巢曲霉的柄曲霉素PKS基因.
用于PCR的引物经适宜设计以含有编码在PKS基因中高度保守的氨基酸序列的核苷酸序列。鉴定相应于给定氨基酸序列的核苷酸序列的方法包括以宿主细胞的密码子使用为基础的推断法和使用多密码子制备混合寡核苷酸序列的方法(下文称之为“简并寡核苷酸”)。在后面的情况下,通过向其核苷酸序列引入次黄嘌呤可以降低寡核苷酸的多重性。
用于PCR的引物可以包括经设计与模板链退火的核苷酸序列,该引物与添加的5序列相连。对这种添加的5′核苷酸序列的选择没有特别限制,只要该引物可用于PCR或RT-PCR。这种添加的5′序列例如可以为易于PCT产物克隆操作的核苷酸序列。这种核苷酸序列例如可以为限制酶切割位点或含有限制酶切割位点的核苷酸序列。
而且,在设计用于PCR的引物时,优选鸟嘌呤(G)和胞嘧啶(C)碱基的总数为碱基总数的40-60%。而且,优选给定引物几乎没有或者没有自退火,在成对引物情况下,优选在引物之间几乎没有或者没有退火。
组成用于PCR的引物的核苷酸的数量没有特别限制,只要它可用于PCR。该数量的下限一般为10-14个核苷酸,上限为40-60个核苷酸。优选地,引物为14-40个寡核苷酸长。
用于PCR的引物优选为DNA。引物中的核苷酸可以为脱氧腺嘌呤、脱氧胞嘧啶、脱氧胸腺嘧啶和脱氧鸟嘌呤,以及其它脱氧肌苷。在用于PCR的引物的5′-端的核苷酸的5′-位置适宜为一羟基或者通过酯键与一磷酸相连的羟基。
可以通过常用于合成核酸的方法,例如亚磷酰胺法,合成用于PCR的引物。可优选将自动化DNA合成器用于该方法。
来自产ML-236B的微生物的基因组DNA和mRNA可分别用作PCR或RT-PCR的模板。总RNA也可代替mRNA用作RT-PCR的模板。
PCR产物或RT-PCR产物可通过插入适宜DNA载体克隆。对用于该克隆步骤的DNA载体的选择一般没有限制。易于PCR和RT-PCR产物克隆的试剂盒可商购获得。例如,Orignal TA Cloning Kit(由Invitrogen生产;使用pCR2.1为DNA载体)适宜用于这种克隆。
为了获得经过克隆的PCR产物,将含有所需PCR产物的质粒的转化过的宿主细胞培养,然后将这些质粒从这些细胞中提取并提纯。然后从所得质粒中回收插入的DNA片段。
在适合宿主细胞的条件下将转化过的宿主细胞进行适宜培养。优选的宿主细胞,大肠杆菌,可以在LB培养基[1%(w/v)胰蛋白胨、0.5%(w/v)酵母浸出物、0.5%(w/v)氯化钠]中在30-37℃下摇动培养18小时至2天。
通过回收宿主细胞并使质粒分离成不含其它细胞组分如基因组DNA或宿主蛋白,可以制备来自转化过的宿主细胞的培养物的质粒。按照Maniatis的碱性法[Maniatis,T等人的“分子克隆”,实验室手则,第2版,冷泉港实验室,纽约冷泉港(1989)]可以制备来自大肠杆菌培养物的质粒DNA。用于获得具有高纯度质粒的试剂盒可商购获得。优选Plasmid Mini Kit[由QIAGEN AG生产]。而且,用于大量生产质粒的试剂盒可商购获得。优选Plasmid Maxi Kit(由QIAGEN AG生产)。
在DNA样品适当稀释之后,可以在260nm的波长下通过测定吸光度,并以吸光度OD260为1的溶液含有50μg/ml DNA为基础计算来确定所得质粒DNA的浓度(记载于Maniatis等人的,参见上面).
由波长在280和260nm下的吸光度的比可以计算DNA的纯度(记载于Maniatis等人的,参见上面)。
标记核酸探针的方法通常分为放射性标记和非放射性标记。对用于放射性标记的放射性核苷酸的选择一般没有限制,并且例如可以为32P、35S、14C等。优选在标记中使用32P。对用于非放射性标记的试剂的选择一般也没有限制,只要它可常用于标记核酸,并且例如可以为地高辛配基、生物素等,优选地高辛配基。
核酸探针的标记方法一般也没有限制。优选常用方法,例如将探针插入使用标记的核苷酸底物通过PCR或RT-PCR获得的产物中的方法、切口平移法、使用随机引物、末端标记法以及使用标记过的核苷酸底物合成寡核苷酸DNA的方法。适宜方法可以根据核酸探针的种类从这些方法中选择。
通过与所述产ML-236B的微生物的基因组DNA的Southern印迹杂交,可以证实产ML-236B的微生物的基因组中存在的核苷酸序列与特定核酸探针的核苷酸序列相同。
按照Maniatis的方法(记载于Maniatis等人的,参见上面)可以进行Southern印迹杂交。
如上所述制备的标记过的核酸探针可用于筛选基因组DNA文库。对筛选方法的选择没有特别限制,只要它一般适宜基因克隆,但优选该克隆杂交法[记载于Maniatis等人的,参见上面]。
将用于菌落杂交的菌落在适合宿主细胞的条件下进行适宜培养。可以将一种优选宿主——大肠杆菌通过在LB琼脂培养基[1%(w/w)胰蛋白胨、0.5%(w/v)酵母浸出物、0.5%(w/v)氯化钠、1.5%(w/v)琼脂糖]中在30-37℃下培养18小时至2天进行培养。
通常通过从阳性克隆的培养物中提取质粒并将其提纯来制备来自通过菌落杂交获得的阳性克隆的重组DNA载体。
根据微生物保藏的布达佩斯条约,根据本发明获得的代表阳性克隆的转化大肠杆菌菌株,大肠杆菌pML48 SANK71199于1999年7月7日保藏在工业科技署的生命科学和技术研究院,并且记录的登记号为FERM BP-6780。
由大肠杆菌pML48SANK71199携带的典型DNA载体被命名为pML48。
通过测定重组DNA载体插入片段的核苷酸序列、Southern印迹杂交或该插入片段表达以确定功能,可以适宜地评价证实存在于阳性克隆中的重组DNA载体含有与ML-236B生物合成有关的基因组DNA。
可以根据Maxam和Gilbert化学修饰技术[记载于Maxam,A.M.M.和Gilbert,W.的“酶学方法”65,499(1980)]或双脱氧链终止法[记载于Messing,J.和Vieira,J.的“基因学”19,269(1982)]测定DNA的核苷酸序列。其它适宜方法在本领域中众所周知。如上所述,用于测定核苷酸序列的质粒DNA优选为高纯度样品。
pML48插入片段的核苷酸序列示于序列表的SEQ ID NO 1。在序列表的SEQID NO 2中所示的核苷酸序列与SEQ ID NO 1所示的核苷酸序列完全互补。通常,在一个种中基因组DNA的核苷酸序列能够具有遗传多态性,即同种异型差异。而且,在DNA克隆和测序过程中,已知核苷酸替代或其它改变可以一定频率发生。因此,本发明中与ML-236B生物合成有关的基因组DNA还包括可与序列表的SEQ ID NO 1或2的第1个核苷酸至第34203个核苷酸的DNA杂交的基因组DNA和其它DNA.优选基因组或其它DNA可以在严格条件下与序列表的SEQ ID NO 1或2的第1个核苷酸至第34203个核苷酸的DNA杂交,其中一个或多个核苷酸被替代、缺失和/或添加。此外,这些杂交基因组或其它DNA可以包括来源于除柑桔青霉SANK13380的产ML-236B的微生物的DNA。优选当将其导入产ML-236B的微生物中时它们能提高ML-236B的产量。
适宜地根据以下方法1)-3)分析与ML-236B生物合成有关的基因组DNA。
1)用基因分析软件分析
可以使用寻找基因的程序(下文称之为“GRAIL”)和检索同源序列的程序(BLASTN和BLASTX)定位基因组DNA内的基因。
GRAIL为检索基因组DNA中的结构基因的程序:通过将基因组序列分成7个评价基因序列外观的参数,使用神经网络法整合结果[记载于Uberbacher,E.C.和Mural,R.J.的“美国国家科学院院报”88,11261(1991)]。例如可以使用ApoComGRAIL Toolkit[由Apocom公司生产]。
BLAST为使用进行核苷酸序列和氨基酸序列同源性检索的算法的程序[记载于Altschul,S.F.、Madden,T.L.等人“核酸检索”25,3389(1997)]。
通过将DNA序列分成适宜长度并使用BLASTN进行遗传数据库的同源性检索,可以预测在样品基因组DNA序列上结构基因的定位和方向。在待测DNA序列上结构基因的定位和方向还可以通过将细分的基因组DNA序列翻译成6个翻译片段(三个在有义链上,其它三个在反义链上)并使用BLASTX在肽数据库中进行所得氨基酸序列的同源性检索来进行预测。
在真核生物体中基因组DNA中的结构基因的编码区有时与内含子断裂。为了分析具有这些缺口的结构基因,用于含缺口序列的BLAST程序更有效,优选有缺口的BLAST程序(安装于BLAST2:WISCONSIN GCG包10.0版)。
2)根据Northern印迹杂交法分析
可以使用Northern印迹杂交法研究通过段1)中所述分析方法预测的结构基因的表达。
适宜地,来自产ML-236B的微生物的总RNA从微生物培养物中获得。优选产ML-236B的微生物柑桔青霉的培养物可通过以下获得:将来自斜面的所述微生物接种到MGB3-8培养基中,接着摇动培养,在22-28℃下培养1-4天。
对从产ML-236B的微生物中提取RNA的方法的选择没有限制,优选硫氰酸胍-热酚法、硫氰酸胍-盐酸胍法等。用于制备高纯度总RNA的可商购获得的试剂盒的例子包括RNeasy Plant Mini Kit(由Qiagen AG生产)。而且,通过将总RNA涂敷到寡(dT)柱上并回收吸附在该柱中的组分可以获得mRNA。
可以与上述Southern印迹杂交法相似的方式向膜转移RNA、制备探针、杂交并监测信号。
3)转录物的5′-端和3′-端的分析
可以按照“RACE”(快速扩增cDNA末端)法分析每个转录物的5′-端和3′-端。RACE为使用以mRNA为模板的RT-PCR获得在基因的5′-端或3′-端含有已知核苷酸区和未知区的cDNA的方法[记载于Frohman,M.A.的“酶学法”218,340(1998)]。
可以按照以下方法进行5′-RACE。使用mRNA为模板根据反转录酶反应合成cDNA的第一链。作为引物,使用对核苷酸序列的已知部分经设计的反义寡核苷酸(1)。使用末端脱氧核苷酸转移酶将一均聚核苷酸链(由一种碱组成)加到该cDNA第一链的3′-端。然后,使用cDNA的第一链作模板通过PCR将5′-端区域中的双链cDNA扩增。为了扩增,使用2个引物;来自含有与均聚序列互补的序列的有义链的DNA寡核苷酸以及在反义链上且在寡核苷酸DNA(1)的3′-端侧的寡核苷酸(2)[记载于Frohman,M.A.的“酶学法”218,340(1993)]。有义5′-RACE的试剂盒可商购获得,适宜地为用于快速扩增cDNA端的5′RACE系统,2.0版(由GIBCO公司生产)。
3′RACE为一种使用存在于mRNA的3′-端的多腺苷酸区的方法。具体地说,使用mRNA为模板以及寡d(T)衔接子为引物通过反转录酶反义合成cDNA的第一链。然后,使用该cDNA的第一链为模板通过PCR在3′-端扩增双链cDNA。作为引物,使用在有义链上根据有义链的核苷酸序列的已知部分经设计的DNA寡核苷酸(3)以及反义链上的寡d(T)衔接子。用于3′RACE的试剂盒可商购获得,适宜地有Ready-To-Go T-primed First-Strand Kit(Phramacia公司)。
在以感兴趣核苷酸序列的已知部分为基础的引物设计中,优选将上面1)和2)的分析结果用于该RACE步骤。
使用上面1)-3)中的分析方法,可以推断结构基因在基因组DNA序列上的方向,转录起始位点位于结构基因中的定位、翻译起始密码子和翻译终止密码子位于其中的位置。以上述信息为基础,可以获得每个结构基因及其cDNA,即加速ML-236B生物合成的cDNA。
假设在根据本发明获得的重组DNA载体pML48中6个结构基因存在于该插入的序列上。它们被分别命名为mlcA、mlcB、mlcC、mlcD、mlcE和mlcR。其中,假设在序列表的SEQ ID NO 2所示的核苷酸序列上mlcA、mlcB、mlcE和mlcR具有编码区。假设在序列表的SEQ ID NO 1所示的核苷酸序列上mlcC和mlcD具有编码区。
获得相应于上述结构基因的特定加速ML-236B生物合成的cDNA的方法的例子包括:使用基于每个结构基因的序列及其侧翼DNA设计的引物用RT-PCR克隆,使用基于已知核苷酸序列设计的适宜DNA探针由cDNA文库克隆。其它适宜方法在本领域中为公众所知。为了在功能上表达根据这些方法获得的cDNA,优选获得全长cDNA。
下面解释使用RT-PCR获得加速ML-236B生物合成的cDNA的方法。
用于RT-PCR并用于获得加速ML-236B生物合成的cDNA的一对引物需要经设计,以便它选择性地与每个模板链退火,从而获得cDNA。然而,用于RT-PCR的引物与每对模板链完全互补并不是必要的,只要它们满足上述条件。用于RT-PCR能与反义链退火的适宜引物(下文称之为“有义引物”)为与一对反义链完全互补的有义引物(下文称之为“未替代的有义引物”)或者为与一对反义链不完全互补的有义引物(下文称之为“部分替代的有义引物”)。用于RT-PCR且能与有义链退火的其它适宜引物(下文称之为“反义引物”)为与一对有义链完全互补的反义引物(下文称之为“未替代的反义引物”)或者为与一对有义链不完全互补的反义引物(下文称之为“部分替代的反义引物”)。
有义引物经适宜设计,以便使用其获得的RT-PCR产物在翻译起始的起始位置含有密码子ATG.该RT-PCR产物适宜地还仅在具有原始ATG起始位点的读框中含有正确翻译终止密码子,并且没有其它(假)翻译终止位点.本发明预测的这些结构基因的翻译起始密码子的位置示于用于定位于序列表的SEQID NO 1和SEQ ID NO 2中的基因的表5中。
未替代的有义引物的5′-端适宜地为翻译起始密码子ATG的核苷酸“A”或者存在于其5′-端的碱基。
部分替代的有义引物选择性地与序列表的SEQ ID NO 1或SEQ ID NO 2中的特定区退火,序列表的SEQ ID NO 2的核苷酸序列与序列表SEQ ID NO 2的核苷酸序列完全互补。
当部分替代的有义引物含有存在于翻译起始密码子ATG的3′-端的核苷酸序列时,在该区域它适宜地不含在与该ATG相同的读框中为终止密码子(TAA、TAG或TGA)的核苷酸序列。
部分替代的有义引物可以含有核苷酸“A”、核苷酸序列“AT”或“ATG”(下文称之为“核苷酸或核苷酸序列m′”),它们与翻译起始密码子的核苷酸“A”、核苷酸序列“AT”或“ATG”(下文称之为“核苷酸或核苷酸序列m”)相对应。当该核苷酸m′为与序列“m”的“A”相对应的“A”时,我们优选m′“A”位于部分替代的有义引物的3′-端。类似地,当m′为“AT”时,我们优选该m′“AT”序列位于部分替代的有义引物的3′-端。当该核苷酸或核苷酸序列m为相应于m′“ATG”的“ATG”时,我们优选这些为引物中的3′-ATG的三核苷酸不是终止密码子。换句话说,对三核苷酸而言,其中在3′-端方向从m′“ATG”的A数起5′-端核苷酸为第(3xn+1)个核苷酸(n代表≥1的整数),该三核苷酸的核苷酸序列优选既不是TAA、TAG,也不是TGA。上述引物可用于获得在相应于用作RT-PCR模板的mRNA的翻译起始密码子的位置具有甲硫氨酸密码子的cDNA。
当部分替代的有义引物的3′-端为核苷酸位置(3xn+1)时,在使用部分替代的有义引物作为一个引物并且产ML-236B的微生物的RNA或mRNA为模板获得的RT-PCR产物中,或者在使用基因组DNA或cDNA为模板获得的PCR产物中,从该位置开始的该三核苷酸最好不是TAA、TAG或TGA。在3′-端方向从翻译起始密码子“ATG”的“A”计数该核苷酸位置,其中“n”代表≥1的整数。
当部分替代的有义引物的3′-端为核苷酸位置(3xn+2)时,位置3xn+2为中心核苷酸的三联体最好不是上面获得的PCR或RT-PCR产物的序列TAA、TAG或TGA。
当部分替代的有义引物的3′-端为核苷酸位置(3xn+3)时,位置(3xn+3)为该3′核苷酸的三联体最好不是序列TAA、TAG或TGA。
对有义引物的要求如上面讨论的。
设计一反义引物,以便当与有义引物一起配对时,编码每个结构基因(mlcA、mlcB、mlcC、mlcD、mlcE和mlcR)的cDNA可以使用RT-PCR在与相应肽的N-端至C-端相同的方向扩增。
对未替代的反义引物的选择没有限制,只要它为具有与位于cDNA的翻译终止位点区的核苷酸序列互补的核苷酸序列的反义引物。然而,优选具有与翻译终止密码子的3′端的碱基互补的5′-端碱基的引物,或者具有在所示引物碱基的5′-端的碱基的引物。更优选含有三个与翻译终止密码子互补的碱基的引物。表8-10显示了每个结构基因的翻译终止密码子,与该翻译终止密码子互补的序列、在每个结构基因编码的肽的C-端的氨基酸残基、编码该氨基酸残基的核苷酸序列、及其在SEQ ID NO 1或SEQ ID NO 2的位置。
部分替代的反义引物选择性地与序列表的SEQ ID NO 1或SEQ ID NO 2的核苷酸序列中的特定区域退火.
上面是对反义引物的要求。
可以向部分替代的有义引物和部分替代的反义引物的5′-端插入适宜的核苷酸序列,只要满足上述要求。对这种核苷酸序列的选择没有特别限制,只要该引物可用于PCR。适宜序列的例子包括便于PCR产物克隆的核苷酸序列,例如限制酶切割位点和含有适宜限制酶切割位点的核苷酸序列。
此外,根据上述说明并根据用于PCR的引物的常规设计,将该有义引物和反义引物适宜地设计。
如上所述,来自产ML-236B的微生物的mRNA或总RNA可用作RT-PCR的模板。在本发明中,相应于结构基因mlcE的加速ML-236B生物合成的cDNA是通过设计和合成适宜扩增pML48插入片段序列中结构基因mlcE的所有编码区的一对引物,然后使用SANK13380的总RNA作模板[序列表中核苷酸序列SDQ ID NO 35和36分别代表的引物]进行RT-PCR获得的。
使用序列表的核苷酸序列SEQ ID NO 39和40所代表的引物以相似方式获得相应于结构基因mlcR的加速ML-236B生物合成的cDNA。
如上所述,该RT-PCR产物可以通过插入到适宜DNA载体中经克隆。对用于这种克隆的DNA载体的选择没有限制,它适宜地为常用于DNA片段克隆的DNA载体。易于进行RT-PCR产物克隆的试剂盒可商购获得。并优选Original TA Cloning Kit[由Invitrogen生产:使用pCR2.1作DNA载体]。
通过将cDNA克隆到适合在产ML-236B的微生物中功能性表达的DNA载体中,可以获得在产ML-236B的微生物中使用上面方法获得的加速ML-236B生物合成的cDNA的功能性表达的证明。然后用重组DNA载体转化适合的细胞,并将转化细胞的ML-236B生物合成能力与未转化的宿主细胞比较。如果在转化细胞中功能性地表达加速ML-236B生物合成的cDNA,那么与宿主细胞的ML-236B生物合成能力相比,转化细胞的ML-236B生物合成能力得到提高。
对适合在产ML-236B的微生物中表达的DNA载体[下文称之为功能性表达载体]的选择没有特别限制,只要它可用于转化产ML-236B的微生物并且可以功能性地表达在该生物体中由加速ML-236B生物合成的cDNA编码的多肽。优选该载体在宿主细胞中稳定,并且具有能在宿主细胞中复制的核苷酸序列。
用于功能性表达的载体可以含有一个以上加速ML-236B生物合成的cDNA,例如相应于结构基因mlcE和/或mlcR的cDNA。
除导入产ML-236B的微生物中时加速ML-236B生物合成且相应于结构基因mlcE和/或mlcR的cDNA之外,用于功能性表达的载体可以含有一种以上的DNA。这种DNA的例子包括:相应于结构基因mlcA、mlcB、mlcC或mlcD的cDNA、与ML-236B生物合成有关的基因组DNA、编码本发明的加速ML-236B生物合成的cDNA的表达调节因子的DNA等。
用于功能性表达的载体优选包括向宿主细胞中的质粒提供选择性表型的核苷酸序列,并优选为穿梭载体。
而且,该选择性表型可以为药物抗性表型等,优选抗抗生素,更优选抗氨苄西林或者抗潮霉素B。
当表达载体为穿梭载体时,该载体适宜地含有允许该载体在一微生物组的宿主细胞中复制的核苷酸序列,以及由该载体插入片段编码的多肽在另一宿主细胞类型中表达所必需的核苷酸序列。优选该载体向转化的不同微生物组的每个宿主细胞提供不同选择性表型。微生物组组合的要求与用于本说明书中所述与ML-236B生物合成有关的DNA的克隆和表达的穿梭载体的要求相似。
在本发明中,适宜的穿梭载体DNA载体为pSAK700,它是由来源于存在于DNA载体pSAK333的构巢曲霉(记载于JP-平3-262486A)的3-磷酸甘油酯激酶(下文称之为“pgk”)、用于插入外来基因的衔接子和存在于该DNA的pgk终止子按该顺序(参见图4)组合构建的。
通过将相应于上述结构基因mlcE的cDNA插入上述的表达载体中,可以在产ML-236B的微生物中表达多肽。在本发明中,已通过将相应于结构基因mlcE的cDNA插入pSAK700的衔接子位点获得重组cDNA表达载体pSAKexpE。在pSAKexpE中插入的序列,即相应于结构基因mlcE的cDNA的核苷酸序列示于序列表的SEQ ID NO 37中。类似地,已通过将相应于结构基因mlcR的cDNA插入pSAK700的衔接子位点获得重组cDNA表达载体pSAKexpR。在pSAKexpR中插入的序列,即相应于结构基因mlcR的cDNA的核苷酸序列示于序列表的SEQ ID NO 41中。
大肠杆菌pSAKexpE SANK 72499,为由pSAKexpE转化的大肠杆菌菌株,根据微生物保藏的布达佩斯条约,2000年1月25日以保藏号FERM BP-7005保藏在工业科技署的生命科学和技术研究院。大肠杆菌pSAKexpR SANK72599,为由pSAKexpR转化的大肠杆菌菌株,根据微生物保藏的布达佩斯条约,2000年1月25日以保藏号FERM BP-7006保藏在工业科技署的生命科学和技术研究院。
可以根据宿主细胞适当地选择适合的转化方法,从而获得加速ML-236B生物合成的cDNA的表达、与ML-236B生物合成有关的基因组DNA或其片段。柑桔青霉(一种优选的产ML-236B的微生物)的转化可以通过以下进行:由柑桔青霉孢子制备原生质体,然后将重组DNA载体导入该原生质体[记载于Nara,F.等人的“当前基因技术”23,28(1993)]。
适宜地将来自柑桔青霉培养物斜面的孢子接种到PGA琼脂培养基的平皿上并在22-28℃下培养10-14天。然后从该平皿收获这些孢子,并将1×107-1×109个孢子接种到50-100ml YPL-20培养基[组成:0.1%(w/v)酵母浸出物(由Difco公司生产)、0.5%(w/v)聚胨(由Nihon Seiyaku公司生产)、20%(w/v)乳糖,pH5.0]中,然后在22-28℃下培养18小时-2天。从培养物中回收萌发孢子,并用细胞壁降解酶处理,从而获得原生质体。对细胞壁降解酶的选择没有特别限制,只要其可降解柑桔青霉的细胞壁,并且对微生物没有有害影响。其例子包括:酶解酶、壳质酶等。
在适宜条件下将含有加速ML-236B生物合成的cDNA的重组DNA载体和产ML-236B的微生物或其原生质体混合使重组DNA载体导入所述原生质体中,从而提供转化体。
在适合每个宿主细胞的条件下对产ML-236B的微生物的转化体进行适宜培养.柑桔青霉,一种优选的产ML-236B的微生物,的培养可以通过以下进行:在适宜再生细胞壁的条件下培养预先转化的原生质体,然后培养.即,可以将柑桔青霉的转化原生质体加入VGS中间层琼脂培养基[组成:Vogel最小化培养基、2%(w/v)葡萄糖、1M葡糖醇、2%(w/v)琼脂].然后将该VGS中间层琼脂夹在VGS下层琼脂培养基[组成:Vogel最小化培养基、2%(w/v)葡萄糖、1M葡糖醇、2.7%(w/v)琼脂]和含800μg/ml潮霉素B的VGS上层琼脂培养基[组成:Vogel最小化培养基、2%(w/v)葡萄糖、1M葡糖醇、1.5%(w/v)琼脂]之间,然后在22-28℃下培养7-15天.所得菌株在PGA培养基上在22-28℃下温育进行传代培养.用铂针将该菌株接种到由PGA培养基制成的斜面上,在22-28℃下培养10-14天,然后在0-4℃下保藏.
如上所述,通过将从所述斜面获得且具有再生细胞壁的柑桔青霉转化体接种到MBG 3-8培养基中,接着摇动在22-28℃下培养7-12天,可以有效地生产ML-236B。作为宿主的柑桔青霉可以在液体培养基中培养,也可以产生ML-236B。
通过将用于提纯天然产物的不同方法组合,可以提纯来自产ML-236B的微生物的转化体的培养物的ML-236B。对这些方法的选择没有特别限制,例如可以是通过离心、过滤分离固体和液体、用碱或酸处理、用有机溶剂提取、溶解、如吸附色谱法、分配色谱法等的色谱法、以及结晶法等。ML-236B可以为羟酸或内酯形式,它们可以相互转换。羟酸可转变成其更稳定的盐。使用这些物理性能,可以获得ML-236B羟酸形式(下文称之为羟酸)、ML-236B羟酸的盐(下文称之为羟酸盐)或者ML-236B内酯形式(下文称之为内酯)。
为了将环打开并转变成羟酸盐,该培养物在高温或室温下经过碱水解,然后反应溶液经酸化,接着过滤。滤液用有机溶剂提取,使其与水分离,从而得到为自由羟酸的想要产物。对有机溶剂的选择没有特别限制。其例子包括:脂族烃如己烷、庚烷等;芳族烃如苯、甲苯等;卤代烃如二氯甲烷、氯仿等;醚如二乙醚等;酯如甲酸乙酯、乙酸乙酯等;或由两种或多种溶剂组成的混合物。
可以将该自由羟酸溶于如氢氧化钠的碱金属盐水溶液中以羟酸盐形式获得想要化合物。
而且,可以通过在脱水的有机溶剂中加热自由羟酸或者通过其它适宜方法以内酯获得想要化合物。
可以使用柱色谱法等提纯和分离因此获得的自由羟酸、羟酸或内酯。用于色谱法的柱的载体没有特别限制。其例子包括:Sephadex LH-20(由Pharmacia公司生产)、Diaion HP-20(由Mitsubishi Kagaku公司生产)、硅胶、逆相载体等,优选C18系列载体。
对ML-236B的定量方法的选择没有特别限制,优选常用于定量有机化合物的方法。其例子包括:逆相高效液相色谱法(下文称之为“逆相HPLC”)等。按照逆相HPLC的定量如下:通过使产ML-236B的微生物的培养物经过碱水解,使用C18柱使水溶性部分经过逆相HPLC,测定UV吸收,将吸收值转换成ML-236B的量。对C18柱的选择没有特别限制,液相用于常规逆相HPLC的C18柱。其例子包括:SSC-ODS-262(直径6mm,长100mm,由Senshu Kagaku公司生产)等。对用于移动相的溶剂的选择没有特别限制,只要它为常用于逆相HPLC的溶剂。例如它为75%(w/v)甲醇-0.1%(w/v)三乙胺-0.1%(w/v)乙酸等。当在室温下将ML-236B加入SSC-ODS-262柱时,在使用75%(w/v)甲醇-0.1%(w/v)三乙胺-0.1%(w/v)乙酸作为以2ml/min的速度的移动相时,4.0分钟之后将ML-236B冲洗。使用用于HPLC的UV检测器测定ML-236B。用于UV测定的吸收波长为220-280nm,优选220-260nm,更优选236nm。
提供了含有使用本发明获得的ML-236B和药用载体的药用组合物。
还提供了含有由使用本发明获得的ML-236B转变的帕伐他丁和药用载体的药用组合物.
本发明的药用组合物可以为常规以及与ML-236B或帕伐他丁的现有制剂所用的相同。
治疗方法也为本发明的一部分,使用这些化合物或组合物治疗高脂血和其它病症。
现在参照以下附图和实施例更详细地描述本发明。这些实施例为描述性,并不用于限制本发明。
图1为DNA载体pSAKcos1的结构的图示图;
图2为pML48的插入序列的结构基因分析的结果;
图3显示了pML48的插入序列的Northern印迹杂交。
图4为cDNA表达载体pSAK700的结构的图示图;和
图5显示了用于在pSAKexpR转化体中转录mlcA-E和R的RT-PCR分析。
图5显示了用于在pSAKexpE转化体中转录mlcE的RT-PCR分析。
实施例1:pSAKcos1载体的构建
来源于大肠杆菌(JP-平3-262486A)的含有潮霉素B磷酸转移酶基因(下文称之为“HPT”)的质粒pSAK333用限制酶BamHI(由日本Takara Shuzo有限公司生产)消化,经处理形成具有T4DNA聚合酶(由日本Takara Shuzo有限公司生产)的平端。
如上面获得的DNA片段使用DNA连接试剂盒Ver.2(由日本Takara Shuzo有限公司生产)自连接成环状,然后用其转化大肠杆菌的感受态细胞JM109(由日本Takara Shuzo有限公司生产)。从转化大肠杆菌中选择具有缺失BamHI位点的质粒的菌株,并命名为pSAK360。
pSAK360用限制酶PvuII消化,然后用碱性磷酸酶处理,产生在5′-端去磷酸化的片段。由粘粒载体pWE15(由STRATAGENE生产)获得含有cos位点的SalI-ScaI片段(约3kb)并经处理形成具有T4DNA聚合酶的平端。接着将其连接到pSAK360的PvuII位点上。JM109用该DNA转化。从转化大肠杆菌中选择质粒中在PvuII位点插入SalI-ScaI片段(约3kb)的这些菌株,并将这些菌株所载有的质粒命名为pSAKcos1。pSAKcos1含有用于限制酶BamHI、EcoRI和NotI的切割位点,每个位点来源于pWE15。pSAKcos1具有一抗氨苄西林的基因和抗潮霉素的基因作为选择标记。
在以下实施例中,当将大肠杆菌用作宿主时,通过将40μg/ml氨苄西林(氨苄西林:由Sigma公司生产)加入相关培养基中进行pSAKcos1转化体、或者含外来基因插入片段的pSAKcos1的转化体的选择。当柑桔青霉SANK13380用作宿主时,通过将200μg/ml潮霉素(潮霉素B:由Sigma公司生产)加入相关培养基中进行pSAKcos1转化体、或者含外来基因插入片段的pSAKcos1的转化体的选择。
pSAKcos1的构建方法示于图1中。
实施例2:制备柑桔青霉SANK13380的基因组DNA
1)柑桔青霉SANK13380的培养物
在PGA琼脂培养基的斜面上进行柑桔青霉SANK13380的种子培养。即,使用铂针向琼脂接种柑桔青霉SANK13380,并在26℃下保持14天。将该斜面保持在4℃下。
通过液体充气培养进行主培养。将来自5mm2的上述斜面的细胞接种在于500ml锥形瓶中的50ml MBG3-8培养基中,在26℃下边以210rpm摇动培养5天。
2)由柑桔青霉SANK13380制备基因组DNA
在室温下将步骤1)中获得的培养物在10000xG下离心10分钟,并将细胞收获。在用干冰冷却的研钵中将3g(净重)细胞破碎,使其为粉末状。将这些破碎的细胞放在填充有20ml 62.5mM EDTA-2Na(由Wako Pure Chemical Industries有限公司生产)-5%(w/v)SDS-50mM Tris-HCl(由Wako Pure Chemical Industries有限公司生产)缓冲液(pH8.0)的离心管中,缓慢混合,然后在0℃下静置1小时。向其中添加10ml用10mM Tris-HCl-0.1mM EDTA.2Na(pH8.0,下文称之为“TE”)饱和的苯酚,然后将该混合物在50℃下缓慢搅拌1小时。
在室温下以10000×G离心10分钟之后,将15ml上层(水相)放入另一离心管中。向该溶液中加入0.5倍体积的TE饱和的苯酚和0.5倍体积的氯仿溶液。将该混合物搅拌2分钟,在室温下以10000×G离心10分钟(下文称之为“苯酚氯仿提取液”)。向10ml上层(水相)中加入10ml 8M乙酸铵(pH7.5)和25ml 2-丙醇(由Wako Pure Chemical Industries有限公司生产),接着在80℃下冷却15分钟,并在4℃下以10000×G离心10分钟。
沉淀之后,将沉淀物溶于5ml的TE中,之后向其中加入20μl的10mg/ml核糖核酸酶A(由Sigma公司生产)和250U核糖核酸酶T1(由GIBCO公司生产),接着在37℃下培养20分钟。将20ml 2-丙醇加入其中,缓慢混合。接着,将几缕基因组DNA汇集在巴氏滴管的尖端,并溶于1ml TE中。
接下来,将0.1倍体积的3M乙酸钠(pH6.5)和2.5倍体积的乙醇加入该DNA溶液中。将该溶液在-80℃下冷却15分钟,然后在4℃下以10000×G离心5分钟(下文称之为“乙醇沉淀”)。所得沉淀物溶于200μl的TE中,即为基因组DNA部分。
实施例3:柑桔青霉SANK13380的基因组DNA文库的制备
1)基因组DNA片段的制备
将0.25U的Sau3AI(日本Takara Shuzo有限公司)加入100μl的实施例2中获得的柑桔青霉SANK13380的基因组DNA(50μg)的水溶液中。间隔10、30、60、90和120秒后,取出20μl混合物样品,向每个样品中加入0.5M EDTA(pH8.0),使限制酶反应终止。通过琼脂糖凝胶电泳将所得部分消化过的DNA片段分离,回收含有30kb以上DNA片段的琼脂糖凝胶。
将所回收的凝胶细碎,放入Ultra Free C3离心过滤装置(由日本Milipore公司生产)中。在-80℃下将该凝胶冷却15分钟直至冷冻,然后通过在37℃下培养10分钟将该凝胶熔化。以5000×G离心5分钟,从而提取DNA。该DNA经过苯酚-氯仿提取和乙醇沉淀。所得沉淀物溶于少量且适量的TE中。
2)DNA载体pSAKcos1的预处理
将pSAKcos1用限制酶BamHI(日本Takara Shuzo有限公司)消化,然后在65℃下经碱性磷酸酶(日本Takara Shuzo有限公司)处理30分钟。所得反应溶液经过苯酚-氯仿提取和乙醇沉淀。所得沉淀溶于少量TE中。
3)连接和体外包装
将上面步骤1)中所述的基因组DNA片段(2μg和经过上述预处理的pSAKcos1(1μg)混合,然后使用DNA连接试剂盒Ver.2(日本Takara Shuzo有限公司)在16℃下连接16小时。所得反应液经过苯酚-氯仿提取和乙醇沉淀。所得沉淀物溶于5μl的TE中。使用GIGAPAK II Gold试剂盒(由STRATAGENE公司生产)将该连接产物溶液经过体外包装,从而提供含有重组DNA载体的大肠杆菌转化体。将3ml LB培养基倒在形成了大肠杆菌转化体菌落的平皿上,然后使用细胞刮棒回收在平皿上的菌落(称之为“回收液1”)。再用3ml LB培养基将该平皿冲洗,并回收细胞(称之为“回收液2”)。将甘油加入回收液1和2的混合物中,使最终浓度达到18%(称之为大肠杆菌细胞液),将其保藏在-80℃下作为柑桔青霉SANK13380的基因组DNA文库。
实施例4:使用柑桔青霉SANK13380的基因组DNA作模板通过PCR扩增PKS基因片段
1)用于PCR的引物的设计和合成
以黄曲霉PKS基因的氨基酸序列(记载于Brown,D.W.等人的“美国国家科学院院报”93,1418(1996))为基础,设计和合成序列表的SEQ ID NO 3和4中所示的简并引物。根据亚磷酰胺法进行该合成。
序列表的SEQ ID NO 3:
gayacngcntgyasttc
序列表的SEQ ID NO 4:
tcnccnknrcwgtgncc
在SEQ ID NO 3和4的核苷酸序列中,n代表肌苷(次黄嘌呤),y代表t或c,s代表g或c,k代表g或t,r代表g或a,w代表a或t。
2)通过PCR扩增DNA片段
制备50μl反应液,它含有用于上述步骤1中所述PCR的引物(每个100pmol)、实施例2中获得的柑桔青霉SANK13380的基因组DNA(500ng)、0.2mM dATP、0.2mM dCTP、0.2mM dGTP、0.2mM dTTP、50mM氯化钾、2mM氯化镁和1.25U Ex.Tac DNA聚合酶(日本Takara Shuzo有限公司)。该溶液经过由以下三个连续步骤组成的反应循环:在94℃下1分钟、在58℃下2分钟和70℃下3分钟。重复该循环30次,从而将该DNA片段扩增。使用TaKaRaPCRThermal Cycler MP TP 3000(由日本Takara Shuzo有限公司生产)进行PCR。
扩增的DNA片段经过琼脂糖凝胶电泳,然后将含有大小约为1.0-2.0kb的DNA片段的琼脂糖回收。从该凝胶中回收DNA,并经过苯酚-氯仿提取和乙醇沉淀。所得沉淀物溶于少量TE中。
3)连接和转化
使用TA克隆系统pCR2.1(由Invitrogen公司生产)将步骤2)中获得的DNA片段连接到质粒pCR2.1上,该质粒是以试剂盒的一部分提供的。该质粒转化成大肠杆菌JM109,从而提供转化体。
从所得转化体中选出几个菌落,并按照Maniatis等人的方法[记载于Maniatis,T等人的“分子克隆”,实验室手则,第2版,冷泉港实验室,纽约冷泉港(1989)]将其培养。即,将每个菌落接种于24ml含有2ml LB培养基的试管中,并在37℃下摇动培养18小时。
根据碱性法[记载于Maniatis,T等人的,参见上面]由培养物制备重组DNA载体.即,在室温和10000×G下将1.5ml培养液离心2分钟.然后由沉淀物回收细胞.向这些细胞中加入100μl的50mM葡萄糖溶液、25mM Tris-HCl、10mMEDTA(pH8.0),从而形成一悬浮液。向其中加入20μl的0.2N氢氧化钠-1%(w/v)SDS。将该悬浮液缓慢搅拌至微生物裂解。然后加入150μl的3M乙酸钾-11.5%(w/v)乙酸,使所有蛋白质变性,接着在室温及10000xG下离心10分钟。回收上层液。将上层液经过苯酚-氯仿提取和乙醇沉淀。所得沉淀物溶于50μl含有40μg/ml核糖核酸酶A(由Sigma公司生产)的TE中。
每个重组DNA载体用限制酶消化,并经过电泳。使用电泳时对所有插入片段具有不同消化图案的DNA测序器(377型:由日本Perkin Elmer生产)测定在这些重组DNA载体中的DNA插入片段的核苷酸序列。
以这种方式鉴定菌株为含有由柑桔青霉获得的PKS片段的重组DNA载体。
实施例5:柑桔青霉SANK13380的基因组Southern印迹杂交
1)电泳并转移到膜上
将实施例2中获得的柑桔青霉SANK13380的基因组DNA(10μg)用限制酶EcoRI、SalI、HindIII或Sacl(都是由日本Takara Shuzo有限公司生产的)消化,然后经过琼脂糖凝胶电泳。该凝胶是使用琼脂糖L03“TAKARA”(日本TakaraShuzo有限公司)制备的。电泳之后,将该凝胶浸泡在0.25N盐酸(由Wako Pure化学工业有限公司生产)中并在缓慢摇动下在室温下培养10分钟。将该凝胶转移到0.4N氢氧化钠(由Wako Pure化学工业有限公司生产)中,在室温下温和培养30分钟。使用Maniatis等人的碱性转移法(参见上面),将凝胶中的DNA转移到尼龙膜HybondTM-N+(由Amersham公司生产)上,并在其上固定。将该膜用2×SSC(1×SSC含有150mM NaCl、15mM柠檬酸钠)洗涤,然后经空气干燥。
2)杂交和信号的测定
将步骤1)中获得的膜与作为探针的实施例4中获得的PKS基因片段杂交。
作为探针,将1μg实施例4中获得的PKS基因插入片段用DIG DNA标记试剂盒(由Boeringer-Mannheim生产)标记并煮沸10分钟,然后在使用前快速冷却。
将步骤1)中所述的膜浸泡在杂交液(DIG Easy Hyb:由Boeringer-Mannheim生产)中,然后在20rpm的摇动下在42℃预杂交2小时。然后,将上述标记的探针加入杂交液中,使用Multishaker Oven HB(由TAITEC公司生产)在20rpm的摇动下在42℃杂交18小时。然后将经过杂交的膜使用2×SSC在室温下冲洗3次,每次20分钟,使用0.1xSSC在55℃下冲洗2次,每次30分钟。
将冲洗过的膜用用于核酸的DIG发光检测试剂盒(由Boeringer-Mannheim生产)处理并将其曝光于X射线胶卷(Lumifilm,由Boeringer-Mannheim生产)上。使用Fuji医用胶卷处理器FPM 800A(由Fuji胶卷公司生产)进行曝光。
结果证明,实施例4中获得的PKS基因片段存在于柑桔青霉的基因组上。
实施例6:使用PKS基因片段作探针筛选柑桔青霉SANK13380的基因组DNA文库
使用菌落杂交法克隆含有PKS基因的基因组DNA片段。
1)膜的制备
将以柑桔青霉SANK13380的基因组DNA文库(实施例3中所述)保藏的大肠杆菌细胞液稀释并涂布在LB琼脂培养基平皿上,以使每个平皿中可以生长5000-10000个菌落。将该平皿在26℃下保持18小时,并在4℃下冷却1小时。将HybondTM-N+膜(由Amersham公司生产)放在平皿上,并与其接触1分钟.将粘附有菌落的膜小心地从平皿重取出.将已与菌落接触的表面朝上并浸泡在200ml的1.5M氯化钠、0.5M Tris-HCl、1mM EDTA(pH7.5)溶液中持续3分钟,浸泡2次,然后用400ml的2×SSC冲洗。冲洗过的膜经空气干燥30分钟。
2)杂交
将实施例4中获得的PKS基因插入片段DNA(1μg)用作探针。使用DIGDNA标记试剂盒(由Boeringer-Mannheim生产)标记该DNA并煮沸10分钟,在使用前快速冷却。
将步骤1)中所述的膜浸泡在杂交液(DIG Easy Hyb:由Boeringer-Mannheim生产)中,然后在20rpm的摇动下在42℃预杂交2小时。然后,将上述标记的探针加入杂交液中,使用Multishaker Oven HB(由TAITEC公司生产)在20rpm的摇动下在42℃杂交18小时。然后将经过杂交的膜使用2×SSC在室温下冲洗3次,每次20分钟,使用0.1xSSC在68℃下冲洗2次,每次30分钟。
将冲洗过的膜用用于核酸的DIG发光检测试剂盒(由Boeringer-Mannheim生产)处理并将其曝光于X射线胶卷(Lumifilm,由Boeringer-Mannheim生产)上。使用Fuji医用胶卷处理器FPM 800A(由Fuji胶卷公司生产)进行曝光。
将上面步骤1)和2)称之为筛选。
将在第一次筛选时检测为阳性信号的平皿上菌落刮下并回收悬浮于LB培养基中的细胞。然后,将细胞适当稀释并涂布在适宜平皿上。接着,进行第二次筛选,以提纯阳性克隆。
在本实施例中获得的阳性克隆,即转化大肠杆菌,大肠杆菌pML48SANK71199菌株,根据微生物保藏的布达佩斯条约,于1999年7月7日以保藏号FERM BP-6780保藏在工业科技署的生命科学和技术研究院。
实施例7:分析重组DNA载体pML48(1)的插入序列
以实施例4中所述的相似方式培养实施例6中获得的大肠杆菌pML48SANK71199菌株并由该培养物制备重组DNA载体。
获得的DNA载体命名为pML48。与ML-236B生物合成有关的基因组DNA的pML48的插入片段用各种限制酶消化,并将所得片段亚克隆到pUC119(由日本Takara Shuzo有限公司生产)中。使用所得亚克隆作为探针,通过与实施例5中所述的相似的方法进行Southern印迹杂交。即,通过用各种限制酶消化pML48获得的产物经过电泳,将这些DNA转移到一膜上,并经过杂交。结果,使用本领域的标准技术制备pML48插入序列的限制酶切割图。
使用377型DNA测序器(由日本Perkin Elmer有限公司生产)测定每个亚克隆插入序列的核苷酸序列,接着测定pML48的整个核苷酸序列。
pML48的插入序列由总共34203个碱基组成。
pML48插入序列的核苷酸序列记载于序列表的SEQ ID NO 1和2中。序列表的SEQ ID NO 1和2中所述的序列彼此完全互补。
使用基因检索程序GRAIL(ApoCom GRAIL Toolkit:由Apocom公司生产)和同源性检索程序BLAST(有缺口的BLAST(BLAST2):安装于WISCONSIN GCG包10.0版中)分析pML48插入序列中结构基因的存在。
结果,预测在pML48的插入序列中存在6个不同结构基因,分别将其命名为mlcA、mlcB、mlcC、mlcD、mlcE和mlcR。而且,预测mlcA、mlcB、mlcE和mlcR在序列表的SEQ ID NO 2的核苷酸序列中具有编码区,并且mlcC和mlcD在序列表的SEQ ID NO 1的核苷酸序列中具有编码区。还预测了该插入序列中每个假定结构基因的相关位置和长度。
本实施例的结果示于图2中。每个箭头表示每个结构基因在pML48插入物上的位置、方向和相对大小。指向左边的箭头说明在ID SEQ NO 2上存在结构基因(mlcA、B、E或R)的编码区。指向右边的箭头说明在ID SEQ NO 1上存在结构基因(mlcC或D)的编码区。
实施例8:分析重组DNA载体pML48(2)的插入序列
通过Northern印迹杂交分析在实施例7中预测存在的结构基因的表达。分析5′-端和3′-端区域。
1)柑桔青霉SANK13380的总RNA的制备
将来自柑桔青霉SANK13380斜面培养物(实施例2中所述)的5mm2的细胞接种在于100ml锥形瓶的10ml MGB3-8培养基中,并在26℃下摇动培养3天。
使用异硫氰酸胍法用RNeasy Plant Mini Kit(由Qiagen AG生产)由该培养物制备总RNA。即,在室温下以5000×G将该培养物离心10分钟,回收细胞。接着,用液氮将2g(湿重)细胞冷冻,然后在研钵中破碎成粉末。将破碎的细胞悬浮在4ml裂解缓冲液中(含在试剂盒中)。将450μl悬浮液倒入试剂盒中所含的10个QIAshredder自旋柱每一个中,然后在室温下以1000×G离心10分钟。将所得的每个洗脱液回收,向其中加入225μl乙醇,然后将其加入试剂盒中所含的RNA小自旋柱中。用冲洗试剂盒中所含物质的缓冲液冲洗柱,接着用50μl无核糖核酸酶的蒸馏水洗脱每个柱中所吸附物。将该洗脱液作为总RNA部分。
2)Northern印迹杂交
通过将2.25μl含20μg柑桔青霉SANK13380的总RNA的水溶液加入1μl10xMOPS(组成:200mM 3-吗啉基丙烷磺酸、50mM乙酸钠、10mM EDTA.2Na;pH7.0;在高压锅中在121℃下灭菌20分钟之后使用;由Dojinkagaku Laboratory有限公司生产)、1.75μl甲醛和5μl甲酰胺中,接着混合,生产一RNA样品。将该RNA样品在65℃下保持10分钟,然后在冰水中快速冷却,并经过琼脂糖凝胶电泳。用于电泳的凝胶的制备如下:将10ml 10xMOPS和1g琼脂糖L03“TAKARA”(由日本Takara Shuzo有限公司生产)与72ml二碳酸二乙酯处理水(由Sigma公司生产)混合,加热使该琼脂糖溶解,然后冷却,接着加入18ml甲醛。使用1xMOPS(用10倍水稀释10xMOPS制成)作为缓冲液样品。将凝胶中的RNA转移到10xSSC中的HybondTM-N+(由Amersham公司生产)上。
将通过用下表1所示的限制酶1和2消化pML48的插入序列获得的DNA片段a、b、c、d和e用作探针。在图3的上方格中显示了每个探针在pML48上的位置。
表1
用于Northern印迹杂交的探针
探针 | 限制酶1 | 限制酶位点的核苷酸号* | 限制酶2 | 限制酶位点的核苷酸号* |
a | EcoRI | 6319-6324 | EcoRI | 15799-15804 |
探针 | 限制酶1 | 限制酶位点的核苷酸号* | 限制酶2 | 限制酶位点的核苷酸号* |
b | BamHI | 16793-16798 | PstI | 18164-18169 |
c | KpnI | 26025-26030 | BamHI | 27413-27418 |
d | SalI | 28691-28696 | SalI | 29551-29556 |
e | HindIII | 33050-33055 | SacI | 34039-34044 |
*每个核苷酸号存在于序列表的SEQ ID NO 1上
按照实施例5中所述的Southern印迹杂交进行探针的标记、杂交和信号的检测。
实施例的结果示于图3的下面方格中。
每个信号显示了存在于每个探针的核苷酸序列同源的转录产物。
结果暗示,预测存在于本实施例中的pML48的插入序列中的结构基因,即mlcA、mlcB、mlcC、mlcD、mlcE和mlcR被转录到柑桔青霉SANK13380中。
每个信号的位置未显示转录产物的相关大小。
3)按照5′RACE测定5′-端序列
使用快速扩增cDNA端的5′RACE系统,2.0版(由GIBCO公司生产)获得含有每个结构基因的5′-端区域的cDNA。
生产两种反义寡核苷酸DNA。如实施例7的结果和本实施例的第2)项所预测的,该设计以推定在pML48的插入序列的编码区中和每个结构基因的5′-端附近的核苷酸序列为基础。
表2显示了以每个结构基因的3′-端的核苷酸序列为基础设计的反义寡核苷酸DNA(1)的核苷酸序列。表3显示了以每个结构基因的5′-端的核苷酸序列为基础设计的反义寡核苷酸DNA(2)的核苷酸序列。
表2:用于按照5′RACE测定5′-端序列的寡核苷酸DNA(1)
基因 | 序列表的SEQ IDNO | 核苷酸序列 |
mlcA | SEQ ID NO 5 | Gcatgttcaatttgctctc |
mlcB | SEQ ID NO 6 | Ctggatcagactttctgc |
mlcC | SEQ ID NO 7 | Gtcgcagtagcatgggcc |
mlcD | SEQ ID NO 8 | Gtcagagtgatgctcttctc |
mlcE | SEQ ID NO 9 | Gttgagaggattgtgagggc |
基因 | 序列表的SEQ IDNO | 核苷酸序列 |
mlcR | SEQ ID NO 10 | Ttgcttgtgttggattgtc |
表3:用于按照5′RACE测定5′-端序列的寡核苷酸DNA(2)
基因 | 序列表的SEQ IDNO | 核苷酸序列 |
mlcA | SEQ ID NO 11 | Catggtactctcgcccgttc |
mlcB | SEQ ID NO 12 | Ctccccagtacgtaagctc |
mlcC | SEQ ID NO 13 | Ccataatgagtgtgactgttc |
mlcD | SEQ ID NO 14 | Gaacatctgcatccccgtc |
mlcE | SEQ ID NO 15 | Ggaaggcaaagaaagtgtac |
mlcR | SEQ ID NO 16 | Agattcattgctgttggcatc |
使用寡核苷酸DNA(1)作引物,并使用柑桔青霉SANK13380的总RNA作模板按照反转录反应合成cDNA第一链。即,将24μl的含有1μg总RNA、2.5pmol寡核苷酸DNA(1)和1μl SUPER SCRIPTTM II反转录酶(含在试剂盒中)的反应混合物在16℃下培养1小时,将该反应产物添加到试剂盒中所含的GLASSMAX自旋柱体,从而提纯cDNA的第一链。
使用试剂盒中所含的末端脱氧核糖核苷酸转移酶将多胞嘧啶链加入cDNA第一链的3′-端。
将50μl的含有已加入3′-端多胞嘧啶链的cDNA的第一链的反应混合物与40pmol寡核苷酸DNA(2)和40pmol Abriged锚定引物(试剂盒中所含的)混合,然后在94℃下培养2分钟。然后将94℃下30秒、55℃下30秒和72℃下2分钟的培养循环重复35次,接着在72℃下培养5分钟,在4℃下培养18小时。所得产物经过琼脂糖凝胶电泳,并从该凝胶中回收DNA。产物通过苯酚-氯仿提取和乙醇沉淀提纯,并以与使用pCR2.1的实施例4中所述方法相似的方式克隆。
上述操作为5′-RACE。
测定含有5′-端的cDNA片段的核苷酸序列,并预测转录起始点和翻译起始密码子的位置。
表4显示了描述相应于通过5′RACE获得的每个结构基因的5′-端cDNA片段的核苷酸序列SEQ ID NO。表5显示了SEQ ID NO,其中每个结构基因存在转录起始点和翻译起始点,并描述了转录起始点和翻译起始点的位置。
表4:显示5′-端cDNA片段的核苷酸序列的SEQ ID NO
基因 | 序列表的SEQ ID NO |
mlcA | SEQ ID NO 17 |
mlcB | SEQ ID NO 18 |
mlcC | SEQ ID NO 19 |
mlcD | SEQ ID NO 20 |
mlcE | SEQ ID NO 21 |
mlcR | SEQ ID NO 22 |
表5:每个基因的转录起始点和翻译起始点的位置
*序列表的SEQ ID NO 1和2的核苷酸序列彼此完全互补。
4)按照3′RACE测定3′-端序列
使用Ready To Go:T-引发的第一链试剂盒(由Pharmacia公司生产)获得含有每个结构基因的3′-端的cDNA。
由实施例7的结果和本实施例的第2)项预测,生产假设在编码区中和在pML48的插入序列的每个结构基因的3′-端附近的一种有义寡核苷酸DNA(3)。
对每个结构基因所产生的寡核苷酸DNA(3)的核苷酸序列示于表6中。
表6:按照3′RACE用于测定3′-端序列的寡核苷酸DNA(3)
基因 | 序列表的SEQ ID NO | 核苷酸序列 |
mlcA | SEQ ID NO23 | Atcataccatcttcaacaac |
mlcB | SEQ ID NO 24 | Gctagaataggttacaagcc |
mlcC | SEQ ID NO 25 | Acattgccaggcacccagac |
mlcD | SEQ ID NO 26 | Caacgcccaagctgccaatc |
mlcE | SEQ ID NO 27 | Gtcttttcctactatctacc |
mlcR | SEQ ID NO 28 | Ctttcccagctgctactatc |
使用NotI-d(T)18引物(试剂盒中所含的)以及柑桔青霉SANK13380(1μg)的总RNA作模板通过反转录反应合成cDNA的第一链。
将100μl的含有cDNA的第一链的反应混合物、40pmol寡核苷酸DNA(3)和NotI-d(T)18引物(试剂盒中所含的)在94℃下保持2分钟。将94℃下30秒、55℃下30秒和72℃下2分钟的培养循环重复35次,接着在72℃下培养5分钟,在4℃下培养18小时。所得产物经过琼脂糖凝胶电泳,然后从该凝胶中回收DNA。产物通过苯酚-氯仿提取和乙醇沉淀提纯,并以与使用pCR2.1的实施例4中所述方法相似的方式克隆。
上述操作为3′-RACE。
测定3′-端的cDNA的核苷酸序列,并预测翻译终止密码子的位置。
表7显示了描述相应于通过3′RACE获得的每个结构基因的3′-端cDNA片段的核苷酸序列的序列表的SEQ ID NO。表8显示了以序列表的SEQ ID NO 1和2为基础的翻译终止密码子和该密码子的位置。
表7:显示3′-端cDNA片段的核苷酸序列的SEQ ID NO
基因 | 序列表的SEQ ID NO |
mlcA | SEQ ID NO 29 |
mlcB | SEQ ID NO 30 |
mlcC | SEQ ID NO 31 |
mlcD | SEQ ID NO 32 |
mlcE | SEQ ID NO 33 |
mlcR | SEQ ID NO 34 |
表8:每个结构基因的翻译终止密码子和该翻译终止密码子的位置
基因 | 翻译终止密码子 | 存在翻译终止密码子的SEQ ID NO | 翻译终止密码子在SEQ IDNO 1或SEQ ID NO 2中的核苷酸号 |
mlcA | tag | SEQ ID NO 2 | 32723-32725 |
mlcB | taa | SEQ ID NO 2 | 19840-19842 |
mlcC | taa | SEQ ID NO 1 | 13479-13481 |
mlcD | tga | SEQ ID NO 1 | 27890-27892 |
mlcE | tga | SEQ ID NO 2 | 5730-5732 |
mlcR | tag | SEQ ID NO 2 | 1915-1917 |
*序列表的SEQ ID NO 1和2的核苷酸序列彼此完全互补。
表9显示了预测由每个结构基因编码的多肽的C-端氨基酸残基,编码该氨基酸残基的三核苷酸的核苷酸序列和该三核苷酸的位置。
表9:由每个结构基因编码的多肽的C-端氨基酸残基
基因 | C-端氨基酸残基 | 编码氨基酸的三核苷酸的核苷酸序列 | 存在三核苷酸的SEQ ID | 三核苷酸在SEQID NO 1或2中的核苷酸号 |
mlcA | 丙氨酸 | gcc | SEQ ID NO 2 | 32720-32722 |
mlcB | 丝氨酸 | agt | SEQ ID NO 2 | 19837-19839 |
mlcC | 半胱氨酸 | tgc | SEQ ID NO 1 | 13476-13478 |
mlcD | 精氨酸 | cgc | SEQ ID NO 1 | 27887-27889 |
mlcE | 丙氨酸 | gct | SEQ ID NO 2 | 5727-5729 |
mlcR | 丙氨酸 | gct | SEQ ID NO 2 | 1912-1914 |
*序列表的SEQ ID NO 1和2的核苷酸序列彼此完全互补。
表10汇集了与表8中所示翻译终止密码子互补的序列,存在该互补序列的SEQ ID以及该互补序列的位置。
表10:与每个结构基因的翻译终止密码子互补的序列
基因 | 与翻译终止密码子互补的序列 | 存在互补序列的SEQ ID NO | 该互补序列在SEQ ID NO 1或SEQ ID NO 2中的核苷酸号 |
mlcA | cta | SEQ ID NO 1 | 1479-1481 |
mlcB | tta | SEQ ID NO 1 | 14362-14364 |
mlcC | tta | SEQ ID NO 2 | 20723-20725 |
mlcD | tca | SEQ ID NO 2 | 6312-6314 |
mlcE | tca | SEQ ID NO 1 | 28472-28474 |
mlcR | cta | SEQ ID NO 1 | 32287-32289 |
*序列表的SEQ ID NO 1和2的核苷酸序列彼此完全互补。
如上所述,确定了每个结构基因的位置、其方向及其位置。以上述信息为基础,可以获得每个结构基因的转录产物和翻译产物。
实施例9:获得相应于结构基因mlcE的cDNA
1)制备总RNA
按照实施例8的方法制备柑桔青霉的总RNA。
2)设计引物
为了获得相应于实施例8中测得的结构基因mlcE的全长cDNA,设计并合成以下引物:
有义引物5′-gttaacatgtcagaacctctaccccc-3′(参见序列表的SEQ ID 35);和反义引物5′-aatatttcaagcatcagtctcaggcac-3′(参见序列表的SEQ ID 36)。
这些引物分别由结构基因mlcE的5′-端上游区上的序列和3′-端下游区的序列获得。按照亚磷酰胺法进行合成。
3)RT-PCR
为了获得编码mlcE基因产物的全长cDNA,使用Takara RNA LA PCR试剂盒(AWV)1.1版。
具体地说,将20μl的含有1μg总RNA、2.5pmol随机9聚引物(试剂盒中所含的)和1μl反转录酶(试剂盒中所含的)的反应混合物在42℃下培养30分钟,制得cDNA的第一链。然后通过在99℃下加热5分钟使反转录酶失活。
将100μl的含有所有量的cDNA的第一链的反应混合物(上面)、40pmol有义引物和40pmol反义引物的第二个反应混合物在94℃下培养2分钟。将94℃下30秒、60℃下30秒和72℃下2分钟的培养循环重复30次,接着在72℃下培养5分钟,在4℃下培养18小时。所得产物经过琼脂糖凝胶电泳,并从该凝胶中回收DNA。产物通过苯酚-氯仿提取和乙醇沉淀提纯,并以与使用pCR2.1的实施例4中所述方法相似的方式转化大肠杆菌感受态细胞JM109菌株(由日本Takara Shuzo有限公司生产)。从转化的大肠杆菌中选择载有带DNA片段的质粒的菌株,并将该菌株所载的质粒命名为pCRexpE。
测定所得重组DNA载体pCRexpE的插入DNA的核苷酸序列。该插入DNA含有相应于结构基因mlcE的全长cDNA。其核苷酸序列以及由该核苷酸序列推断的肽的氨基酸序列示于序列表的SEQ ID NO 37和/或SEQ ID NO 38中。
mlcE(多肽)的最近已知序列为与洛伐他丁的生物合成有关的基因簇上的ORF10,具有70%的同一性。
实施例10:构建表达载体pSAK700
使用实施例1中所述的载体pSAK333和pSAK360构建cDNA表达载体pSAK700。
pSAK333用限制酶BamH I和Hind III(由日本Takara Shuzo有限公司生产)消化,然后经过琼脂糖凝胶电泳。从该凝胶中回收一4.1kb片段,并用T4-DNA聚合酶(由日本Takara Shuzo有限公司生产)使该DNA端平头。
使用DNA连接试剂盒Ver.2(由日本Takara Shuzo有限公司生产)将EcoRI-NotI-BamHI衔接子(由日本Takara Shuzo有限公司生产)连接到上述DNA片段上。用该连接的DNA将大肠杆菌感受态细胞JM109菌株(由日本Takara Shuzo有限公司生产)转化。从转化的大肠杆菌中选择载有带该衔接子的质粒的菌株,并将该菌株所载的质粒命名为pSAK410。
pSAK360用限制酶Puv II和Ssp I消化,并经过电泳。从该凝胶中回收含有3-磷酸甘油酯激酶(下文称之为“pgk”)基因的启动子和终止子和来源于大肠杆菌的HPT的DNA片段(约2.9kb)。
使用DNA连接试剂盒Ver.2(由日本Takara Shuzo有限公司生产)将回收的上述DNA片段连接到pSAK410的Pvu II位点上。用该连接的DNA将大肠杆菌感受态细胞JM109菌株转化。从转化的大肠杆菌中选择载有带该DNA片段的质粒的菌株,并将该菌株所载的质粒命名为pSAK700。
pSAK700的构建示于图4。
pSAK700具有酶BamHI和NotI的单一限制酶位点。pSAK700还具有抗氨苄西林的基因(下文称之为“Amp”)和抗潮霉素的基因HTP作为选择标记。在以下实施例中,当使用大肠杆菌作为宿主时,通过将40μg/ml氨苄西林加到相应培养基中对由pSAK700或含有外来DNA的pSAK700转化的细胞进行选择。当柑桔青霉SANK13380用作宿主时,通过将200μg/ml潮霉素加到相应培养基中对由pSAK700或含有外来DNA的pSAK700转化的细胞进行选择。
实施例11:构建cDNA表达载体pSAKexpE
在有限制酶HpaI和SspI(由日本Takara Shuzo有限公司生产)的情况下将实施例9中获得的重组DNA载体pCRexpE在37℃下反应2小时,并使反应产物经过琼脂糖凝胶电泳。从该凝胶回收含有约1.7kb mlcE全长cDNA的带。
在pSAK700与限制酶Notl(由日本Takara Shuzo有限公司生产)在37℃下反应1小时之后,用T4DNA聚合酶(日本Takara Shuzo有限公司)在37℃下持续5分钟使该载体的末端平头。然后,该载体经过苯酚氯仿提取和乙醇沉淀。将该沉淀物DNA溶于少量TE中。向其中加入碱性磷酸酶并在65℃下培养30分钟。使用DNA连接试剂盒Ver.2(由日本Takara Shuzo有限公司生产)将如上所述制备的pSAK700连接到步骤1)中获得的1.7kb的DNA片段上.用该连接的DNA将大肠杆菌感受态细胞JM109菌株转化.获得由cDNA表达载体转化的大肠杆菌菌株.
根据微生物保藏的布达佩斯条约,将本实施例中获得的转化的大肠杆菌(命名为大肠杆菌pSAKexpE SANK 72499)于2000年1月25日以保藏号FERM
BP-7005保藏在工业科技署的生命科学和技术研究院。
实施例12:获得相应于结构基因mlcR的cDNA
1)制备总RNA
按照实施例8的方法制备柑桔青霉的总RNA。
2)设计引物
为了获得相应于实施例8中测得的结构基因mlcR的全长cDNA,设计并合成以下引物:
有义引物5′-ggatccatgtccctgccgcatgcaacgattc-3′(参见序列表的SEQ ID 39);和反义引物5′-ggatccctaagcaatattgtgtttcttcgc-3′(参见序列表的SEQ ID 40)。
这些引物分别由结构基因mlcR的5′-端上游区上的序列和3′-端下游区的序列获得。按照亚磷酰胺法进行合成。
3)RT-PCR
为了获得编码mlcR基因产物的全长cDNA,使用Takara RNA LA PCR试剂盒(AWV)1.1版。
具体地说,将20μl的含有1μg总RNA、2.5pmol随机9聚引物(试剂盒中所含的)和1μl反转录酶(试剂盒中所含的)的反应混合物在42℃下培养30分钟,制得cDNA的第一链。然后通过在99℃下加热5分钟使反转录酶失活。
将100μl的含有所有量的cDNA的第一链的反应混合物(上面)、40pmol有义引物和40pmol反义引物的第二个反应混合物在94℃下培养2分钟。将94℃下30秒、60℃下30秒和72℃下2分钟的培养循环重复30次,接着在72℃下培养5分钟,在4℃下培养18小时。所得产物经过琼脂糖凝胶电泳,并从该凝胶中回收DNA。产物通过苯酚-氯仿提取和乙醇沉淀提纯,并以与使用pCR2.1的实施例4中所述方法相似的方式转化大肠杆菌感受态细胞JM 109菌株(由日本Takara Shuzo有限公司生产)。从转化的大肠杆菌中选择载有带DNA片段的质粒的菌株,并将该菌株所载的质粒命名为pCRexpR。
测定所得重组DNA载体pCRexpR的插入DNA的核苷酸序列。该插入DNA含有相应于结构基因mlcR的全长cDNA。其核苷酸序列以及由该核苷酸序列推断的肽的氨基酸序列示于序列表的SEQ ID NO 41和/或SEQ ID NO 42中。
mlcR(多肽)的最近已知序列为与洛伐他丁的生物合成有关的基因簇上的lovE,具有34%的相同性。
实施例13:构建cDNA表达载体pSAKexpR
在有限制酶BamHI(由日本Takara Shuzo有限公司生产)的情况下将实施例12中获得的重组DNA载体pCRexpR在37℃下反应2小时,并使反应产物经过琼脂糖凝胶电泳。从该凝胶回收含有约1.4kb mlcR全长cDNA的带。
在pSAK700与限制酶BamHI(由日本Takara Shuzo有限公司生产)在37℃下反应1小时之后,加入碱性磷酸酶(由日本Takara Shuzo有限公司生产)并在65℃下反应30分钟。使用DNA连接试剂盒Ver.2(由日本Takara Shuzo有限公司生产)将如上所述用BamHI消化的pSAK700连接到步骤1)中获得的1.4kb的DNA片段上.用该连接的DNA将大肠杆菌感受态细胞JM109菌株转化.获得由cDNA表达载体转化的大肠杆菌菌株.
根据微生物保藏的布达佩斯条约,将本实施例中获得的转化的大肠杆菌(命名为大肠杆菌pSAKexpR SANK 72599)于2000年1月25日以保藏号FERMBP-7006保藏在工业科技署的生命科学和技术研究院。
实施例14:转化产ML-236B的微生物
1)制备原生质体
将来自柑桔青霉SANK 13380菌株的培养物斜面的孢子接种到PGA琼脂培养基上,然后在26℃下培养14天。然后从该培养物中回收柑桔青霉SANK 13380菌株的孢子,并将1×108个孢子接种到80ml YPL-20培养基中,在26℃下培养1天。通过显微镜下观察证实孢子发芽之后,在室温下以5000×G将发芽的孢子离心10分钟,并回收沉淀物。
孢子用无菌水冲洗3次,形成原生质体。即,将200mg酶解酶20T(由SeikagakuKogyo公司生产)和100mg壳质酶(由Sigma公司生产)溶于10ml 0.55M氯化镁溶液中,在室温下以5000×G离心10分钟。所得上层液用作酶溶液。将20ml该酶溶液和0.5g(湿重)发芽孢子放入100ml锥形瓶中并在30℃下缓慢摇动培养60分钟。使用显微镜证实发芽的孢子变成原生质体之后,将反应液通过3G-2玻璃滤器(由HARIO公司生产)过滤。滤液在室温下以1000×G离心10分钟,然后回收沉淀物原生质体。
2)转化
将步骤1)中获得的原生质体用30ml 0.55M氯化镁冲洗2次,用30ml由0.55M氯化镁、50mM氯化钙和10mM 3-吗啉基丙烷磺酸酯组成的溶液(pH6.3或更低,下文称之为MCM溶液)冲洗1次。然后将原生质体悬浮于100μl的4%(w/v)聚乙二醇8000、10mM 3-吗啉基丙烷磺酸酯、0.0025%(w/v)肝素(由Sigma公司生产)、50mM氯化镁的溶液(pH6.3或更低,下文称之为“转化液”)中。
将96μl含有约5×107个原生质体和10μl含有120μg pSAKexpE或pSAKexpR的TE混合,在冰上静置30分钟。向其中加入1.2ml的20%(w/v)聚乙二醇、50mM氯化镁、10mM 3-吗啉基丙烷磺酸的溶液(pH6.3)。然后将该液体缓慢移液,在室温下静置20分钟。向其中加入10ml MCM溶液,接着缓慢混合,在室温下以1000×G离心10分钟。从沉淀物中回收转化的原生质体。
3)转化原生质体的细胞壁的再生
将步骤2)中获得的转化原生质体悬浮于5ml液体VGS中间层琼脂培养基中,并放在10ml固体VGS下层琼脂培养基平皿上。在26℃下将该平皿培养1天,之后在每个平皿的上面放10ml含有5mg潮霉素B的液体VGS上层琼脂培养基(潮霉素的最终浓度为200μg/ml)。在26℃下培养14天之后,将两个菌株(即由pSAKexpE或pSAKexpR转化的原生质体获得的那些菌株)在含有200μg/ml PGA琼脂培养基上传代培养,并在用PGA琼脂培养基制备的斜面上亚培养,在26℃下培养14天。
将这些斜面保藏在4℃下。
测定实施例1:比较转化的菌株和原始菌株中的ML-236B生物合成能力
将实施例14中获得的转化菌株和柑桔青霉SANK13380培养并测定每个培养物中的ML-236B的量。
将涉及柑桔青霉SANK13380的来自实施例14所述培养转化菌株的斜面和来自实施例2中所述的斜面的5mm2接种物孢子培养。将细胞接种到在100ml锥形瓶中的10ml MBG3-8培养基中。然后在24℃下摇动培养2天,接着加入3.5ml 50%(w/v)甘油溶液。然后,在24℃下连续摇动培养10天。
向10ml该培养物中加入50ml 0.2N氢氧化钠,接着在26℃下摇动培养1小时。在室温下将该培养物以3000×G离心2分钟。回收1ml上层液,用9ml 75%甲醇混合,并进行HPLC。
使用SSC-ODS-262(直径为6mm,长100mm,由Senshu Kagaku有限公司生产)作为HPLC柱,使用75%(w/v)甲醇-0.1%(w/v)三乙胺-0.1%(w/v)乙酸作为移动相。在室温下以2ml/min的流速进行冲洗。在这些条件下,加入到该柱之后,将ML-236B冲洗4分钟。在236nm的吸收波长下用UV检测器进行测定。
在8个pSAKexpE转化菌株中的3个菌株的ML-236B生物合成能力增加。与原始菌株相比,这些菌株的ML-236B生物合成能力平均高10%。在例如单孢子处理等的传代培养之后,这3个菌株中也能稳定地保持ML-236B生物合成能力。这些结果说明,pSAKexpE的插入片段为加速ML-236B生物合成的cDNA。
在这些pSAKexpR转化菌株中有5个菌株的ML-236B生物合成能力增加。与原始菌株相比,这些菌株的ML-236B生物合成能力平均高15%。在例如单孢子处理等的传代培养之后,这5个菌株中也能稳定地保持ML-236B生物合成能力。这些结果说明,pSAKexpE的插入片段为加速ML-236B生物合成的cDNA。
因此,按照本发明从产ML-236B的微生物中获得的加速ML-236B生物合成的cDNA当导入产ML-236B的微生物中时加速了该产ML-236B的微生物的ML-236B生物合成。
实施例15:相应于结构基因mlc A-D的cDNA的序列测定
测定相应于结构基因mlc A的cDNA的序列。
用TAKARA LA PCR试剂盒ver1.1(Takara Shuzo有限公司)合成第一链cDNA。为了扩增cDNA的全部或部分区域,使用该第一链cDNA为模板并使用几对不同的寡核苷酸作引物进行几次PCR。
使用Big Dye Primer/终止循环测序试剂盒和ABI Prism 377序列(PE应用生物系统)将94℃下30秒、60℃下30秒和72℃下5分钟的循环重复30次。
将每个反应产物分别插入质粒pCR2.1。
获得每个重组质粒的大肠杆菌转化体。
测定由所述转化体获得的重组质粒的每个插入片段的核苷酸序列。
在比较上述几个RT-PCR产物和结构基因mlcA的核苷酸序列的基础上测定外显子和内含子的序列。
然后,测定相应于结构基因mlc A的cDNA的序列(SEQ ID NO 43)。推测由所述cDNA编码的多肽的相应氨基酸序列(SEQ ID NO 44),并且在同源性检索基础上使用该氨基酸序列推定该多肽的功能。
mlc A(多肽)的最近已知序列为与洛伐他丁生物合成有关的基因簇上的LNKS(lovB),具有60%同一性。
以相似方式,测定相应于结构基因mlcB的cDNA的序列(SEQ ID NO 45).推测由所述cDNA编码的多肽的相应氨基酸序列(SEQ ID NO 46),并且在同源性检索基础上使用该氨基酸序列推定该多肽的功能。
mlc B(多肽)的最近已知序列为与洛伐他丁生物合成有关的基因簇上的LDKS(lovF),具有61%同一性。
相似地,测定相应于结构基因mlc C的cDNA的序列(SEQ ID NO 47)。推测由所述cDNA编码的多肽的相应氨基酸序列(SEQ ID NO 48),并且在同源性检索基础上使用该氨基酸序列推定该多肽的功能。
mlc C(多肽)的最近已知序列为与洛伐他丁生物合成有关的基因簇上的lovA,具有72%同一性。
而且,测定相应于结构基因mlc D的cDNA的序列(SEQ ID NO 49)。推测由所述cDNA编码的多肽的相应氨基酸序列(SEQ ID NO 50),并且在同源性检索基础上使用该氨基酸序列推定该多肽的功能。
mlc D(多肽)的最近已知序列为与洛伐他丁生物合成有关的基因簇上的ORF8,具有63%同一性。
测定在SEQ ID NO 1或SEQ ID NO 2上的每个结构基因的外显子的位置,如下:
表11:mlcA-D的外显子在pML48插入片段中的位置
测定的SEQ ID NO 1或SEQ ID NO 2上每个结构基因的转录终止位点的位置如下:
表12:结构基因mlc A-E和R的转录终止位点在pML48插入片段中的位置
基因 | 存在转录终止位点的SEQ ID NO | 转录终止位点在SEQ ID NO 1或SEQ IDNO 2中的核苷酸号 |
mlcA | SEQ ID NO 2 | 32910 |
mlcB | SEQ ID NO 2 | 20052 |
mlcC | SEQ ID NO 1 | 13606 |
mlcD | SEQ ID NO 1 | 28130 |
mlcE | SEQ ID NO 2 | 5814 |
mlcR | SEQ ID NO 2 | 1918 |
实施例16:基因破坏的研究
使用同源重组经定点诱变破坏柑桔青霉的结构基因mlc A、B或D。
使用质粒pSAK333构建用于获得结构基因mlcA遭破坏的柑桔青霉突变体的重组质粒。
将在该pML48插入片段上的mlcA基因座的4.1-kb内KanI片段回收、提纯、用DNA Blunting Kit(Takara Shuzo有限公司)平端化并连接到PuuII消化过的pSAK333上。所得质粒命名为pdismlcA。
由pdismlcA转化柑桔青霉SANK 13380。
对pdismlcA转化体的基因组DNA进行Southern杂交,以证实结构基因mlcA已破坏。
所得mlcA已破坏的突变体不再产生ML-236B或其前体。
使用质粒pSAK333构建用于获得结构基因mlcB遭破坏的柑桔青霉突变体的重组质粒。
将在该pML48插入片段上的mlcA基因座的1.4-Kb PsI-BamHI片段回收、提纯、用DNA Blunting Kit(Takara Shuzo有限公司)平端化并与PuuII消化过的pSAK333相连。所得质粒命名为pdismlcB。
由pdismlcB转化柑桔青霉SANK 13380。
对pdismlcB转化体的基因组DNA进行Southern杂交,以证实结构基因mlcB已破坏。
所得mlcB已破坏的突变体不产生ML-236B,但产生ML-236A或ML-236B的前体。
使用质粒pSAK333构建用于获得结构基因mlcD遭破坏的柑桔青霉突变体的重组质粒。
将在该pML48插入片段上的mlcD基因座的1.4-Kb KpnI-BamHI片段回收、提纯、用DNA Blunting Kit(Takara Shuzo有限公司)平端化并与PuuII消化过的pSAK333相连。所得质粒命名为pdismlcD。
由pdismlcD转化柑桔青霉SANK 13380。
对pdismlcD转化体的基因组DNA进行Southern杂交,以证实结构基因mlcD已破坏。
由所得mlcD已破坏的突变体产生的ML-236B的量为未转化的对照宿主的30%。
实施例17:在pSAKexpR转化体中的mlc R的功能分析
将实施例12中获得的两个pSAKexpR转化体分别命名为TR1和TR2,并且将未转化的宿主细胞——柑桔青霉SANK13380接种于MBG3-8培养基中,单独如实施例8中所述进行培养。
从实施例8中所述的每个培养物中提取总RNA。
使用所述总RNA为模板并以结构基因mlc A、B、C、D、E或R的核苷酸序列为基础设计的一对寡核苷酸为引物进行RT-PCR。
表13:用于RT-PCR的引物对的核苷酸序列
T-PCR的靶 | 引物1 | SEQID NO | 引物2 | SEQ IDNO |
mlcA | 5′-gcaagctctgctaccagcac-3′ | 51 | 5′-ctaggccaacttcagagccg-3′ | 52 |
mlcB | 5′-agtcatgcaggatctgggtc-3′ | 53 | 5′-gcagacacatcggtgaagtc-3′ | 54 |
mlcC | 5′-aaaccgcacctgtctattcc-3′ | 55 | 5′-ctttgtggttggatgcatac-3′ | 56 |
mlcD | 5′-cgctctatcatttcgaggac-3′ | 57 | 5′-tcaatagacggcatggagac-3′ | 58 |
mlcE | 5′-atgtcagaacctctaccccc-3′ | 59 | 5′-tcaagcatcagtctcaggca-3′ | 60 |
mlcR | 5′-atgtccctgccgcatgcaac-3′ | 61 | 5′-ctaagcaatattgtgtttct-3′ | 62 |
图5示出了未转化的柑桔青霉13380和两个命名为TR1、TR2的转化体的RT-PCR分析的结果。
在pSAKexpR转化体培养的第一、第二和第三天使结构基因mlcA、B、C、D和R表达。
与之相反,仅在未转化的宿主细胞培养的第三天使所有这些结构基因表达。
在结构基因mlcE表达时在pSAKexpR转化体和未转化的宿主细胞之间没有差异。
结果暗示,由相应于结构基因mlcR的cDNA编码的蛋白质诱导位于与ML-236B生物合成有关的基因簇中其它一些结构基因(例如mlc A、B、C、D)转录。
实施例18:在pSAKexpE转化体中的mlc E的功能分析
将实施例12中获得的pSAKexpE转化体命名为TE1,并且将其未转化的宿主细胞——柑桔青霉SANK13380接种于MBG3-8培养基中,并如实施例8中所述单独进行培养。
从实施例8中所述的每个培养物中提取总RNA。
使用所述总RNA为模板并以结构基因mlc A、B、C、D、E或R的核苷酸序列为基础设计的一对寡核苷酸为引物进行RT-PCR。用于本实施例的引物与上面实施例的表中的那些相同。
图6示出了未转化的柑桔青霉13380和命名为TE1的转化体的RT-PCR分析的结果.
在pSAKexpE转化体培养的第一、第二和第三天使结构基因mlcE表达。
与之相反,仅在未转化的宿主细胞培养的第三天使结构基因mlcE表达。
另一方面,在结构基因mlc A、B、C、D和R表达时在pSAKexpE转化体和未转化的宿主细胞之间没有差异(数据未显示)。
结果暗示,由相应于结构基因mlcE的cDNA编码的蛋白质不依赖于结构基因mlc A、B、C、D和R而加速ML-236B生物合成。
序列表
<110>Sankyo有限公司
<120>来自基因簇的基因
<130>EPP83481
<150>JP 2000-116591
<151>2000-04-18
<150>JP 2000-117458
<151>2000-04-19
<160>62
<170>PatentIn version 3.0
<210>1
<211>34203
<212>DNA
<213>柑桔青霉
<400>1
gatcaatact acgtcgttgt tatttccttg tcagtaatga ctaacaaatt ccccagaaca 60
gacgaagtca cagctcacac cacaagagaa aatgagtcca gcgaggatta cagatttctc 120
gccaggcaaa ccgagaaaag ctctcttatg catccacggt gccgggtgct cagcagccat 180
attccgcgtc cagatctcta aactgcgcgt ggcgttgaaa aacgagtttg aattcgtata 240
tgcgaccgcg ccgtttagct ccagccccgg acccggcgtg cttcctgtct tccaaggcat 300
gggtccatac tacacctggt tccaaaagca tcatgacgcc gttacaaaca cgacaacccc 360
cacggtgggc gatagagtag cggctgtgat cgggcctgtg caaaagaccg tccaagattg 420
gtctataact aacccacagg cacccattgt cggcatagtg gccttctctg agggcgcatt 480
ggtcgccact ttgctgctcc atcaacagca aatgggaaaa ctgccatggt ttccgaaaat 540
gagcattgct gttttgattt gctgtttcta tagcgatgaa gccagagatt acatgagagc 600
cgaggcgcaa gacgacgacg acaagctaat aatcaacgtg ccgacactgc atcttcacgg 660
tcgtcaagat tttgctctcc aagggtcgag acagatggtt gaaacacatt acctgcctca 720
gaatgcagat gtactcgagt ttcagggaaa gcataatttt cccaacagac cgagtgatgt 780
ccaggagacg gtcaagcgct tccaacagct atatcaaaag gtcaagatgt caggttcatt 840
tgtctaggtg agacaacagg gtatatagca aggctctggc tctcatgcct agtccatacc 900
acatttttac tgaacaaatt tgaatagttc taatcttaca cggtttgaat gctcaccttc 960
caagggtgat ttagttatag tggtcgcgac catctcataa atatttcgtg aacatatttt 1020
ggatagatca tggaaggctc gttctgaaca ggcatgacag acatctaaaa ccactcgatc 1080
accacaacaa ggcactaaac cagtaactat ggaactattt gcaatggcgt cgaatttata 1140
tacaggatgg attgaaatca attccaagcc ttggaggttt caccttcctc acagagtctt 1200
tcgaaacgcg ctaccgaggt atatttatca ccgttacggt actctgaacc gcgctatcta 1260
acttgatgtt acgattgctg caataaagaa gagcaacgaa ggtagaagta attttgacaa 1320
agatacaaga cgaattcgct atttgtagat gaatatgcgt gtgtcaattg acgccgaatt 1380
caggatagat ttgccatctg ctctattgcc aatttctaat ccatctttat catgaacaac 1440
actcaaacca cacatctgaa ttcacggcgc tgaacgatct aggccaactt cagagccggg 1500
ttcatcgaga acatagtgag gattgaagaa aagtggtcta caaaggcctg agcgtgctca 1560
gggccataca gcgagctctg aagtttgaca tgaatgagtg ggtccttggt agggtcatcc 1620
cacatctcga gaacgatgtc ataaggagtg cgctcacggg aagcgagaac actcgtcatt 1680
ttggcattgc caattgagcc actctccgct tgaccctgct tgtaatcaaa gacagcctgg 1740
aacaaggggg cgtgtgtctg agtcttgggt tcctcgcctg aggtagggag attcaggcct 1800
agacagtcga ggatgacgcc atacggcacc cgcgcgtgtt gcatggcctc acgcacactg 1860
tccttggtgg ctacaaggtg ctcgccgaat gtcttgctgc cgacgaactc atcaaagcgc 1920
aggggaagca cgttagcgaa aaagcccatc gccgaaattt cttccatggt ggatcggttg 1980
gtttcggcga ggccgatggt tatgtctttg ctgccggtaa gacgcgccaa caaaacgtgg 2040
taggcggcca ggtagaactg catgggggtt gccttgtgct tgcggctccg ctctttgatt 2100
cggaaggcga ccatgggatc taaacgagca attgcttcat actgctgcca cgtgaatggc 2160
tgtatttgct gctgctctga attggcagca gggtcattga tcagattcat gatgggaagc 2220
acggttggcg cagatgacga gactttgcta tgcatggact tccagaacgc gatatcgtcc 2280
cccattcgcc cattttccag gttttcccgc tgttggacgg ctagatcaga gaattgggtc 2340
gatggtcgct gcattttcac cccgctgtaa atctgcccga tctcattgaa caggttttct 2400
gttgttgagc catcaccaac taatctgtgg tagccgatta ccaacaggtg gtcatctgtg 2460
ccccagtaga aatcaacgag tctgagagtg tcacctgtgg agatgctata gtttgtcttc 2520
tcgagtttcc ggtactcttc ctctgcctcc gcagcgttgt tcacctgaac aaagtgcact 2580
ctgttctccg ggttcttgag aaccacttgg acgggaccat ttaaatcgct gctatagtca 2640
tcgccagtaa caaagcacgt acggaagatc tcgtgacggc gcaatgaggc tttcagagcc 2700
cgcctcaacc ggtcgaggtc aatggtaccc ttcatgaaca tgccaatagt gttgttgaag 2760
atggtatgat cttttaccat ttgttgctgc ctccaggaat actcctggcc aagggacaac 2820
ctctcgcgac gaagaatctt acggcctccc tgctcattat cgtcctcttg ctcttcatcc 2880
tcttcggctg acgacgcatc tgtgctggta gcagagcttg cttcatcatg gctgtctgtt 2940
ggtgtcggag aagccccgct gtccgaggtt cccgtggaat caccaatttg caacagcagc 3000
ggaatggatg tagctgggag tcgggtggcc gcgtcgtcgg caagatcagc gacagaagca 3060
ccgccaagta ccctcaagag tgggaggtca aggtagagtt gctttgagaa ccatgagccg 3120
acagtcactg cacccaagga gtcgacacct tgatcaatga gaggaatggt tgggtccacg 3180
ctctccccgt ccgaaacttg gagggtaaca cggagtttct cagatagacc atctgcaact 3240
ttgttagttt gaactcgata tcaggaaacg catgagagat aacttaccaa tcacgatttg 3300
ccgaacttgg tctaaagttg ttgcttgttt gagctggtcg gcaatggagc ctttagaccc 3360
tgatccattg tcgccaccgt ctccgcgttg accgggaatt ttgaagtttc cgaaacgagg 3420
gtcgttgaag taaataattc gatcttgaag cgcagggtca agatctggga tacccgtggt 3480
aagctcaagg tccgccatgt caatgaccgt cttgcgctgt ggttgctgcc gggcacgctg 3540
gtcagacacg accgcttcgg cgaaaagcgt gtgcagctca tgctcttcaa ctgagtcaaa 3600
catgaaacgg atagcatcaa agtcctcctc catctcggcc ctcgtgacaa accctacacc 3660
gtaaacggca ccaatatcga tggttgatcc ctgtggttgt gcgttagtaa cttgacgtcg 3720
atgcatgata attcaggggt agaaaatacc gccaatcctc tggcgcaccg ttgctgggcc 3780
agagcctgta ggtaggcatt cgcagcgcca tagttggact ggccaggatt gccaataact 3840
gcaacaatgg acgaaaacat gatgaagaag tcgagcgcct tgctgcccgt ctgttcggag 3900
aaccgttcat gaagaatgcg tgctccttgt accttgggct tcaacaccat gtccatcatc 3960
tggtggtcca tgttcttcag catgacatcc tgcagcacca aaggcccgaa cgcgatgccg 4020
gcaacaggtg gcaacttcat atcgacaagc ttgccaaggc cagcatcgac tgaatcctca 4080
ttggcaacat ccctaaagaa agtaattgga taagtaaacg aggatgtggt agcaaggtgt 4140
gatgtgatat caatcaactt acattgacag aacggtgatg tcaccaccaa gtgcctccat 4200
gttggcgatc catttgggat caagtcgagg gttccggcta gtgagcacaa catggcgggc 4260
gccatgcaag atcatccagc gacagagaga gcgaccaagg tccccggtaa gaccaacaag 4320
caaatacgtc ttcttgttgg aaaataagtt accagagtcg atggggcaaa tcctagcgga 4380
cacctcattt tccttccagt cgatgacggt ggccagattg aagcgttggt cattgtggtt 4440
gacagagagc tgaccaggca agagaatttg tgtggctgta ataactttct cagtgtcgtc 4500
gacagtcgac gcagagacgg tattttttgc cattgccaca gagtgctcga ggattggaat 4560
atcctcaaca tgactaactt tgtatgtgga agctgtactt cggataagat agtcaccact 4620
gtacatgaag caactgggtg gtagcaactt ggccaaacgg ttggttatcc cggcagcagt 4680
ccggtcggta gacaagtcaa agaatgccat catgtttgtc ggcaggctgt gtttcagccg 4740
agcgtcggtt tccttggcat gtaatcggat ccaaggagcc ggaatagttt tgacgtcgga 4800
cagagttgtt gccaaatgaa cctgaacacc gtaggttttg gccgactcca gaattgcttt 4860
gacgcagaag attgggggct ccataatcag aattgatgca tcagagccaa aggactgagc 4920
gctagagaga attgtttcgg caaggagggc tgcagctgtg gacaacaaga aggaactatc 4980
ctcgccttcc gccatgttat cgggcagact atgcatgtag tttctcggta catgcagtat 5040
agatccattc ttctcagcca gggcgactac aggcacctca catgtattct ccagaatact 5100
gccctgcacg acatggaagt atccgagatg gcccacgcga attgcctggg gaagagcgta 5160
gcgaacacga acagttgctt ttccagcatg acgagcgtct tctaacgaat cacacgtctc 5220
ggttgactca agatagtaca tcgatgagga tgctcccctc gcctctttca gtgcaatggc 5280
cgtcttggac gaattaaagt taccgaaaat tggacgacga gacgagttca tacggtcgtt 5340
cctagcaata tcctgcttca aacgagggac ccaggcacga cccttgcacc agtacacttc 5400
gggctcatga gtccatgtta ttgattccaa aagctgatca tcgctctcct cgaagcgcaa 5460
aagttgctca acgaagaatt tggtgtctag gttctccaca gtatcgacat cgaagacgtg 5520
cgttcccaag tcagggttct cgagcttgat tgtcctcaac attccgatgg tgctggcctg 5580
gtggggatga tcaatccagg cattctctgt cagccacatc atgcgtccgg cgtagaagag 5640
aagagacttg actgcctcaa acttgtcctc ttcaaggttg caaaacactt catcatcaag 5700
ttccgagagg atgacaaaag tcgacttagg ctgcaaggcc gggtcgtcga gaacactttc 5760
cagccgcttg acggagtgga tgtgtctatg cggtagggca gctttcatgt cgttcaaaat 5820
gcgttcggtt tttgtcgatt cgccaccgat aaccactaat ggcgggtatg agtccttcaa 5880
tggagcagaa agtggatcat acaaacgctc aacggtggca tccacagcat gtgtactgaa 5940
gacagacggg atcaaatcat cctctcgatc aagtgtccga ctatcgacgc cagagaaccc 6000
aactctcttg agggtatgct cccattggtc aacggacccc gaggcactca aagcacgagt 6060
ttcgtcttct ccagtccatc gatcagcgaa aagcccagag atgaaggcga ggcgagcagg 6120
ctcgcgatgg gtgaccccga aagtaaccaa gtgaccaccc ggcttgagca aggaccttat 6180
gtgagccaat ttttcctcga agttggagct ggcatggagg acatcggatg caataatcag 6240
atcgtaggag tgaggcttga atccttgctc tgctgggctt ctgttgatgt ctagtgcctc 6300
aaactgcatg agaccgtcga attcggaaag ttgttcacgg gccttgccaa taacatccgc 6360
cgagatgtca gtgcaagtgt aactgttgaa accaagttga ggtgatgcaa gaacgcgctt 6420
cgtggcgatg cctgtaccca agcctaaaaa gcgaacgaca gattagcaaa ctgcctagtt 6480
acttacattt cagattcgac ttaccgatct caaggatatc aatggattgg tagcgatgag 6540
caatttggct aaccagatcc tgaacgacgt gtattgctga gccaaaggcg agcttgttgg 6600
tatagtactc ggtgaacaac ccatcgcggt tcatgatatc caaaggatcc ccgttcccgc 6660
gaacaattga aattaattct ttgcctaccc tttggatcag gcgcacatgt gggtgggacg 6720
agttgcttca agtaaaaggt taatataaaa gaatgaaaaa acacggaaca gctttgggtg 6780
tacctttcac acatttgctc aatgtgaaca gaagtgtcct cctcccaaga ctcctggtac 6840
cactgatggt ggccagcccg agcatcggcc tgaacctggt cacaccattc aatgtacttc 6900
tgggaatgga ggtcggcatt ttgacggtcg tcgggggtta tctgggctag gaaggatttg 6960
atgtagaagt aaacgattcg ctcgatggtc agaatgtcct ccttgtcccg agctatgatc 7020
aacgtcgcag ggtcctccag cagtttttcg ggcgtgaggg gtccccagac ccactttgcg 7080
aagattcggt ggtcggtcga agcagtcggg ggagagaaag gcttaaagac aatgttatca 7140
acttggaaaa gcgttgtctt ggtcgaatcg tacaccgtga tgtcgccgct caggaaatca 7200
cccttgtcgt gtgtgttgat tgtgtcaaac gcaagctcgg tttcaccaga attacccgcc 7260
gatatacaga gcgatggaat cagagtcact ctgtcaacgt gagtaggcac gtacaatgag 7320
cgtaggcgac gatctcctgg agaggaatac gctccaatga cagtctggaa cgcgatgtcc 7380
aggggcgctg ggtggagcaa gaggggctca ttgcgcaatt catccttaag tggaaggaaa 7440
gccaaggtgc cgctagcttt ggagtcggcc cttctcatgg tctgcaaacg acggaagtct 7500
ttgctgtagt catacccaag gaggtcaagt tcccgataga agaaatcgat gttgacattg 7560
ttcatctggg ggtactcttc ctcaggtggc ggcaaaagct gcgatgacgg tgatgcctcg 7620
ccaagggtta tgacgatttg gcctttggcg gatgtcgaaa gctcactctc ctttgccaga 7680
caggaatcaa taacaaattt gaccgtgact tggccatccg catcattgtc actggtgact 7740
tcggctgtca agttcagctc cacggaggtg ttttcatctt caaacacgat ggctttgttg 7800
atgctcatgt ccaagatttc caggagctga acttgggcgg cacgctcacc agccaccttc 7860
atggcagctt ccatggccat aattatgtac ccagcagcgg ggaacacagt ctggccttgt 7920
agcgcatgac cgtcgagcca ttccagatcc cggggcctga tgaagtttgt ccactggaag 7980
gtcgatgctg tgctgtaaga agaaagcttt ccaagcagaa gatggggcgc acctccacga 8040
agatgctggc gggtggagcg agattctgcc cagtattgac gagtatgatc ccaagagtat 8100
gtgggcaatg actttgacag gttttgaacg gcacgatcgg gccggacttg ttgtacgaag 8160
ccctcggcgt cgatactccg aactccgaaa cgctcccaaa tgtatcccag acctccagca 8220
aaagcgtcca catcgtcaac gtttcgtgcc aagcacccgg tatacggcag ctccacaccg 8280
gcaagagcat ccttgatggt ggctagacac ggacccttga gagcagggtg ggcgccaatt 8340
tcgatggcga cgtcgattag acgatgagtg atgactgctt tctgcacagc ctgcgagaac 8400
aagaccggag agacgagatt gtctttccaa taagcgggca tcacatcctg tacagtcatt 8460
tgcttgctgg tctcgtggac ggcagagaac caagcaacac tatcgttacc ttggccatcg 8520
gcaacagcac agtcgcactc cagcaatgcc ttgacatatg gagctgcgca tgggtgcatg 8580
tgatgcgaat ggtaggcctt gtcaactctc aagattctgg caaaagtgga ttcatcctcc 8640
aagacacctt caacgtgctg gatagcatcc atgtcgccgg agaaggtcac actatccggt 8700
gaattgctag cggcgacgca gacccgaccc tcaaaggctt cgagctcgca tagttccttt 8760
gcgtcatcgt acgacatacc tgccgctagc atagcgcctg tctggccgct tggagaagag 8820
gcatgctccg cggacacaac tccacgcaga tgcgcaatac ggatagcttg agtggcactg 8880
atgaatcctg ccgcaaaggc acaggcaatc tcacctgaac tgtggccgac aattgcactg 8940
aactcgatac cagctgcagc gagaagtcgg accagaacga tttgtacggc gcagcataga 9000
ggctgggaga agctggcgag tctgacgttt gaggcatccc cttcaagcat gagctggtca 9060
tacagtgtcc acgtaggccg atacttttca ggcagtgttt gcagtgaatt atccagctct 9120
tcgagaatgc ctctcacaaa tggcataccc accatgagct tcttcagcat gcccggccac 9180
tgtgcacctt ggccagtaaa gacacctagt acgcgagggt tgtcattcgc gtcggtgcgg 9240
aagtcggtga cgacctcacc gtccgcgatg gcagcctcca gtgccgcgcg ggctacttcc 9300
ttgttgtgtg ctgcaatcgc acgacggaag ggcaagatag accgtttctc aagtaaggta 9360
tatgcgatat catgcatgtc cacgtcatca tgcgtttcca gaaattggag catattttct 9420
agcgttgcct tcatggagcg ctgcgacttc gatgaaagca caaggggcaa gctgcatgca 9480
tctgcatctg aggtcacctc tgttaccact gctgtcggct tgtgtggagg agccatatac 9540
tcttcgataa tagcatgggc atttgtacca ccaaatcctg atgtgtttat atgtttagct 9600
aacttcactt tcgttctcaa gaagtgcagt tgaatcctta ccaaatgaat taacgctgac 9660
tctgcgaggc tgcccgggcg caacaatcgg ccattctgtg gcctccgttg caattttcaa 9720
gtgcgtatag aacggagcga cacggggact gatcttctca aacagcaggt ttggcgggat 9780
cacgccattt cgtacagcaa acgatgcctt cattaagccc gcaataccag cagtgccttc 9840
cgtgtgaccg agaactgtct tgatgctgcc gacaaaaagc tcatctttct cgccgtcgct 9900
gtcgattgtt ccatccttgt gtccgaagaa ggctgttgca atagcctcag cttcctgtgg 9960
gtcaccggct ggtgtaccag ttcctgggat cttcgtgtta gggagagaga gactttctgc 10020
aacttccata aggctgatac ttccagggaa taccacttac catgggcttc aaagaactgg 10080
cagcgttcct gggggttggt aatatcaaga ccagccttgg catatgtggc ccgaatgagg 10140
gcttcttgtg cgctatggtt tggcattgtg atacctgtcg ttcggccatc ttggttgata 10200
ccggtctctc ggataacaca ctcgatactg tccccgtcgc gcagtgcctg gctcagcgtt 10260
ttcaggacaa tagagcaaac accttcctaa aaagcagtta caggaggtca gtgccatctt 10320
gctttttttg aaaggaattg atgcattgtc aacttactcc tctggcatat ccatcggcag 10380
cagcatccca cattcgagat ctaccattgg gggacagcat gttcaatttg ctctccatta 10440
caaaggtcat ggggcccaat atcagattcg caccggctgc aaccgccatg gtactctcgc 10500
ccgttctaag ctgttggacg gccagatgca cggcagctaa ggatgaacta caggctgtgt 10560
cgatcgtcat ctgcagaatc agtcaggaat ctgtcagcac ttgacgaagt cgggctcgct 10620
caatgagtgg cactcacact cggcccatgc cagtcgaaga agtatgatac acggttggag 10680
gccacactga cagctacccc cgtggcagag tatgtaggaa tactatccaa ttcacgcgtc 10740
acgatagtct catagtcatg cgtcatcata ccgacgtaca cagcagtaga ggatccttga 10800
aggccttgga tccgtaggcc tgcgttggat acagcttcat agaccgtctc cagcagcagc 10860
ctttgctgtg ggtcaatcgt ttcggcctct ccagcttgga tgttgaagaa agaggcatca 10920
aaaccgcgta gatcctcctg cagcaagtat gcaaagggtg cgttcgtgcg cccggggtga 10980
gtgccatcgg ggctgtaaaa tgtatcgacg tcaaatctct ccttagggat cttggtctgt 11040
acatcccggg gctctttgag cagctcccaa agttttgatg gtgtgttgac accacctgga 11100
aaccgacaac cgcttcccac taccacaatt ggctcgtttg gatagttggc ttgatccata 11160
actgctgatc ctgtttttgg gcgataggat tgggattaaa ccttgtcttg cgtcagtaga 11220
tcttctcact gcatgccggg cacaacattt gttcttacag aatcgcagag ttgaatctct 11280
gagcgaacaa gccggccttg caaccgatac cgtcgttata tttacttgca cgtatcagta 11340
ctcatctaga ttcggacaat ttcaagatcc attctagtac tcaaatgccc ccacttccca 11400
gcaatgcaag ctcggcacct agcaaaccct cccggcgtca ttcggtgcac gaatagccat 11460
tcctccatac ggcgttattc ggtcacacga ggctgaatga atcaaacgtg aatatcaatt 11520
ggctgtatca aggtgaaacc gagtttttca ctcggattgt tcttgtgctg ctcggtgaag 11580
ctgctcctaa aggaaacaac cgaactgccc catccaggta aacttcgatt gggggggggg 11640
tttttttttt ttcaaggttg actggaagag tgctctcggc cacaaaatcc cagaagcatt 11700
agtgctgtta ttcgattata aaccgtcgca gcgctctcat tcttcgctct ttcttctttt 11760
ccactggtgt gcataggtcc tatctgtctc acgcaatgct cggccaggtt cttctgaccg 11820
tcgaatcgta ccaatgggta tcgacccctc aagcccttgt ggcggtcgca gtgcttctta 11880
gtctcatcgc ctaccgtttg cgggggcgcc agtccgaact gcaagtctat aatcccaaaa 11940
aatggtggga gttgacgacc atgagggcta ggcaggactt cgatacgtat ggtccgagct 12000
ggatcgaagc ttggttctcg aaaaacgaca agcccctgcg cttcattgtt gattccggct 12060
attgcaccat cctcccatcg tccatggccg acgagtttcg gaaaatcaaa gatatgtgca 12120
tgtacaagtt tttggcggat gtatgacctc tgaattttcc attgttgtaa ctcaatgacg 12180
tctctaagat tctgatgaat gtataggact ttcactctca tctccctgga ttcgacgggt 12240
tcaaggaaat ctgccaggat gcacatcttg tcaacaaagt tgttttgaac cagttacaaa 12300
cccaagcccc caagtacaca aagccattgg ctaccttggc cgacgctact attgccaagt 12360
tgttcggtaa aagcgagggt aagtgtcaat ttttctgtct tgagcattga gcctctggct 12420
gacataccgc gaatatacta gagtggcaaa ccgcacctgt ctattccaat ggattggacc 12480
ttgtcacacg aacagtcaca ctcattatgg tcggcgacaa aatctgccac aatgaggagt 12540
ggctggatat tgcaaagaac catgccgtga gtgtggcggt acaagctcgc caacttcgcg 12600
tatggcccat gctactgcga ccgctcgctc actggtttca accgcaagga cgcaaattgc 12660
gtgaccaagt gcgccgcgca cgaaagatca ttgatcctga gattcagcga cgacgtgctg 12720
aaaaggccgc atgtgtagcg aagggcgtgc agccgcccca gtacgtcgat accatgcaat 12780
ggtttgaaga caccgccgac ggccgctggt acgatgtggc gggtgctcag ctcgctatgg 12840
atttcgccgg catctacgcc tcgacggatc ttttcgtcgg tgcccttgtg gacattgcca 12900
ggcacccaga ccttattcag cctctccgcc aagagatccg cactgtaatc ggagaagggg 12960
gctggacgcc tgcctctctg ttcaagctga agctcctcga cagctgcatg aaagagacgc 13020
agcgaatcaa gccggtcgag tgcgccacta tgcgcagtac cgctctcaga gacatcactc 13080
tatccaatgg cctcttcatt cccaagggcg agttggccgc tgtggctgca gaccgcatga 13140
acaaccctga tgtgtgggaa aaccccgaaa attatgatcc ctaccgattt atgcgcatgc 13200
gcgaggatcc agacaaggcc ttcaccgctc aattggagaa taccaacggt gatcacatcg 13260
gcttcggctg gaacccacgc gcttgtcccg ggcggttctt cgcctcgaag gaaatcaaga 13320
ttctcctcgc tcatatactg attcagtatg atgtgaagcc tgtaccagga gacgatgaca 13380
aatactaccg tcacgctttt agcgttcgta tgcatccaac cacaaagctc atggtacgcc 13440
ggcgcaacga ggacatcccg ctccctcatg accggtgcta agatataaca cgcaaactaa 13500
aacaaatatg catccgtccc caggcttatt ccaatagttt ccgtcccaga gaaactaggt 13560
gctgtattag tcgagtaggt tagtaaaata aaacgcattt tattcgattg tgatgccttc 13620
tttgtaatcg aacgtggtgt agactttggc tatgtgcgag agacagaaac acagagagag 13680
agaagggaga gagtgtgtat tcctgctacg cagagcggcc atctgcttct ataccgccag 13740
ctacaccgcc acgtagggaa gtcggcagta atgaagcttt tctcccggta caatcaccga 13800
tctccccatt ctctcaggcg ttgactggcg cttacgatga cgagggctta ggctctgtta 13860
agtcttgatg ttcctactca acatccccga ctaggcgaaa gagaggacgg cgcaacgacg 13920
tggacacaag tactccctcc cgccttccga ctacatatcc acaatctgta cccactgccc 13980
gtgccaacgc ctttcgaccg ttcaacgcgc atttacaagg cttgcgggaa tcataatgga 14040
gagaaaaaga gagaactttt gacagtcaag cctccgaggt gctaagacag cttccctggt 14100
agtataaaaa gcattcactc ttccgacttc gagaacgagt gcacatgtgt actttgttgc 14160
ttctcagggc cactgtaatg gtatttcagg tatctctatt tactgctatc cagaagtcag 14220
gcattaaata gtcaggctca gcccaggctc gattcagatt ggattcaggc ttcagaccat 14280
ggccgctatg ctccttcgta ctatacctcc gtcgagctat acccgcttgg ccagacaaaa 14340
ggcttcactg aacccttcaa cttaactgca tttcgccaca actaactcga cgaggccggc 14400
gatggtgtta ccattcatga gctcaaagat cgacacatca acatggattt cagatgtgat 14460
ccagtttcga agttcaatgg cgacgagtga gtctacgccg acacctgcca ggtttttgga 14520
cgaggacatg tcgtcttctg ccagaccaaa cattcgcatc agcttttccg tcattgcttt 14580
gaggacgata gaaatggcct cgtcgtgaga ggtgaccctg cttagttggg cccgcacgcc 14640
atctggtcct tttttatgcg aagagacaaa ggattggtct gcatgaagga cttggcggta 14700
tttaagtccc acaaaccgct gttcctgtat ccagtttgcc tcggtccagt gagcacccgg 14760
ggatgtgttg attcctgtaa ccacagctgc gggaggtgat ggaaattgag gggaagaaca 14820
caggattgcc ttctccaaca catccatgac gtccttttca tgcataggct tgtaacctat 14880
tctagcgagc cggtcggcca caccacggcc agtttcagcc acgtatccaa cagacttgac 14940
catgcccaag tcaatggtga cagccggcat gccatgggct ctccggtggt gcgcaagtgc 15000
gtcctggaat gcaccagcag ctgcgtaatt ggcctggcct gccccaccca tgaccccaac 15060
aagggatgag agcatcacga agaagtcaac atcctgtgcg atcttgtgaa gataccaact 15120
accctgtact tttgggcgtg ttgctgcatt aaattcatcc aatgtcattc gcgatagaag 15180
cgcgtccttg agaaccatgg caccttgtat gatacctcga attggcggtg catgtgcttc 15240
ttcgcacaac cggagcacct tggtgacctg atcttgatct gagatgtcac atgcgtgtag 15300
atagacagcg cactgttgat tttgcaagct ggttatgaat ggactggcct ttgcacttct 15360
cgataggata atcaagtgct tcgcgccatg atcaacaagc cactgacaga tctgctttcc 15420
aattcccccc agcccaccag caactaggta agaactgtca ggcttcagct tcagcgagaa 15480
ccctccatcg ccgactggga ccagttcgtc cccagataca ttgaccacaa ctttgccaac 15540
atgctgacca ctctgcatcg tacggaaggc cttctcgatg tttgacaagg agtgctgctg 15600
gattggacca atcaagccaa tcgcttttgt ctcgaggagt tttgtgacat ggttcaacgc 15660
ttcggatact tcttcacttt tggctctttg ccacgagaga agatcaattg atgtgaaaga 15720
gacgtcccgg gtgaatggca gcatgtcaag tctgctgttt tgctccaggt ccttttttcc 15780
aatctcaaca aatctgccga attcggccat gcagtcaaag cttgcttgga ggagttgacc 15840
tgccaatgag tttagaacga catgaacgcc aagtccgccc gtgtaggctt tgatgccgtc 15900
gacgaataag tcattcctgc tcgagaagat atgatccgga ttgatgccga atttatcgcc 15960
gacaaagtca cgcttggctt gagttcccgc tgtgacgaag acctcggcac ccgcaagctg 16020
ggacaaaatg atcgctgctt gaccgacgcc tccagctcca ctgtggatca agactctttc 16080
gcctcgtcgt agctttgccg tggtataaag cgcaatatat gcggtagtga aagccagggg 16140
gaccgaagcg gcttctggga agcccatttc gtccggaata cggacgacat tagtgtacgg 16200
cgtctgtgtt ctggtcgccc aatggccttt cagtagtgca catacgcggt cccctaatct 16260
gaggccttgg ctagcggcag cagctccacc gagctttgtg atcactccgg cgcattcgaa 16320
gcccatcaca cggttggcct ccaattgacc catggcaacc atgacatccc gaaaattgag 16380
accgaaagct ttgggttcga tttctaccca atcatccgga agatccttgc cttcacgtcc 16440
ttcgtcgtct cgaaattgca gggagtctaa gagccctggc gtctcaacct ccatccgcag 16500
acgacgcccg ggttgctcga acggctgcag tgtgacctca accgcttctt ggtccttcca 16560
gtgcgggtca ttgaaaagtc gcggtacgtg gatgacgccg tttctctctg caaattcaaa 16620
ctccttgtct tcggaaaggt cgccgaggcg gccattgaag atattgcaga tagcatacag 16680
ggactcgtgg gtgtatgcgt ttcgagaagg atcgagatcc aacgatacat attccttccc 16740
gttattttcg ttgcggatgg tacgcagcag accaatatgt agagctttcc atggatcctc 16800
ggagctcatg gctgctcctc tagacaccca gagaagtgcg ttgcagttat tcagcatcgc 16860
ggtgatggat ttgaaggtct cgcttcccac ctctccaagg agcgaggact ccatttcccc 16920
aagaaaaatg catgtccttc cagtggtatc tacctcgccc agagcgttga tcgatgggct 16980
agaactggtc ttttcacaaa ttgctgcctg gagactttcc agccaagatg aaggaggtcg 17040
gagcgctccg tgcagcaaaa gcacctccga ttctgccact gtatccgggg ttgtattctc 17100
ttttctagcc gtcgatagca ttgtgctgat catgtaaaac tcatcgtctt cacaatcacg 17160
aacctccaat tccacaccgt tgaaaccgct cgtgtccaac atggtgttcc aaagatcggt 17220
agtgagcgat ggcgtcgact tccgctcagg ctcctcactg agccaccaac ctggcaacag 17280
tccgaaggta aagaacaaat cgagctgatc cctggtagtc tcaaccaaaa tcaagttgcc 17340
cccaggcttg agcaattttc gaacgttact cagtgttcgt ttcatgcatc gagttgcatg 17400
caggacctgg caagccacga ccacatcgta ggtggcacat tcaaaccctt gttgctcggg 17460
atcgctttca atatccaatt ttttgaaagt catcacgtct tgccaatccg caaattgctc 17520
acgcgccgac tcgaaaaacc cggcagacac atcggtgaag tcataacgat cgatcggctt 17580
ggtgtttccc aatgcattga caataagctt tgtgcagccg cccgtgcctc cgccaatctc 17640
caaaatgcga gaacgcgggt tcttgtgggc gcaaagtcgg atcagctcgc tggcttgtgc 17700
gtttgatcgg ctccatttga ttgcgttgac gtagtatctg cttagcagct gatcttgcat 17760
catcaactca agtggctctg tttcgcggcg tagcattgct attaactgag gtcctagacg 17820
agaaatcatc tcgccattga cgctttctcc agcgactctg gcctgtaggc atttcttctg 17880
ctcagcatcg tcacttagcc agtcgcaact ggctgggctg agcttgtttt gtctcgcaag 17940
gtccaattgg acattcatcc aatcgaaata cttctgaagg tggccatcca gatgttggat 18000
atcagaattt gtcaaatcag tgacagcctc ctgtataaag ttgatcgtgc atcttcggag 18060
gtccatcatg agttccgttt ctttcgtctc agcctcagtg ctcaactttt ctttgagcca 18120
agtggagtca cccaagctga tgtcaggggc ccaaacccag gagctgcagg catttcctgt 18180
gtcgttggag tctgactttt ggtcagagaa gctgcttcca accgactgga aaacaaggcc 18240
ttcaatctct atgactggga ttccgtccga gggagaagaa ccgctatcat agtcatcaaa 18300
cactgccaag tcggtagaga aggattgaga gttgcgatcc ttgatgctgg cctgtgcgtc 18360
cagagcatca ccagcctcca agtcagccag gctagaggat attttgacat ttcttagcct 18420
ccttggtacc atggccgttt tcatacgtgt tcccgcgtag ggtaacaccg tgtatgccgc 18480
ctggatcacc gagtccagag tagtaggatg gacgatgtgt cgattctcgt acgagtgagg 18540
catagccgag gcagtgtcag caatggaaaa tctgcaaaac gagccctgtc cattgttttg 18600
aattcgctga atgttctgaa aaatgggtcc gtggcatatc ccattcgcgt gtaaggactc 18660
ccagagatcg ttgggatcaa tgctccggtt atctgagcct agattcaacc tgcgtgaggc 18720
ttccacagtt gaacagtcaa ggtggcttct ttcgctctcc gaacgtatta atccggtgca 18780
gtgttctgtc caggtattat tttcgcccga aattgagtgc acagaaaatt gatgccagtt 18840
ctttgtgccg agggaccttt cctcacatga acggatcgtt aggcgcaggt caacctctgc 18900
ttctgcatca gcgggtatta tgagagcctg cgcgagttca acgtcacgca agttgtagtt 18960
gatgctagcc cccgcaactg gtgggcagac ttgtgaaaac ccctcgatgg ccatgctgat 19020
gaagccagct cccggaaaga tgatgctcga accaacgacg tgatctcgta tccatggaat 19080
atctgacaga cggagaacat gtttccattt aggcgcgaaa tgaggagaga gagattcccg 19140
tgagcctatc aaagtgtgag gcggatgggt tctctgtttg gactcacgac tgccgcgagg 19200
ctctctccaa taacgggttt ggtgattcca cgggtacgcc ggcaaatcgc tcagtacctt 19260
cactctgggc tcttttcttc catgaggaaa gtttatagcg tccattttga gcccataacc 19320
cttgcttatc aactccgtag cagcacgata cattgtctcc aacgagcttc tgccgcgaga 19380
aaggcaactg agatagttta tatctgttcc tttcagaccc agatcctgca tgacttggtt 19440
gattggacca ccaagcgctc cgtgaggccc tatttcaata atcacatcga cggctttctc 19500
tttggtgttg ggatcaaagc acatctcgcg gagtgaggac tcgaactcta ccggctgtag 19560
catactatcc atccagtgtg tgggatccaa tagcaattta agatcggtca tgcgactacc 19620
agtcttaggt gatgaatata atacaccctt tgaggtgtca gcattgggat tgtcgttgtt 19680
gttatccgag ttgaacagat ctctcagtga cgccccaaag gcatctgcca ttggtcgcat 19740
gtggcttgaa tggaaggctt cagtgacttt cagtttcctg gtaaagatgc catcggcgtg 19800
taacaacttt tcaagtttct cgattgcacc caaatctccc gacaccgtca cactacattg 19860
actgttgata catccaacca ccacacagcc gtcctcctgg ttgagacgcg aaatgtaaac 19920
attggtctca ctgcgaccaa gacccaccgc catcattcct cctttggctg ccaatgcggg 19980
cttgggctta gtggtcaata caccgcgtat ataagtgatc ccaatggccg accgcgcgga 20040
taaagcccca gctgcgtagg cagcagcagc ctctccactt gagtgactgg ttatccccgt 20100
tggccgaatt ccccatgacc aaaggagacg cacaagtgca atttggatag cggttgacag 20160
tggtagactg tattcggcat catttacccg agtcgtcagc tcatcacggt ggagctcctc 20220
tgtgcaattg aatgttagta cctcaagctt gatacagtat tacttttccc gggctcgcaa 20280
cttacccata aaattccaac tcgcgcccag ttgcttgatg tagccatcac attcaagaat 20340
cgcctgtttg aatactggga atgtattgac cagctctctg cccattgcat gccactgcgc 20400
cccctgaccg gtgaatacaa atccgagccg tactttctca ttcgctcgtt ttggttgatt 20460
ggactcatcg ctgagggcag aaacaaggcc gccaaggctg tctgctacat acactgacgt 20520
ccatggcaga atggaacggc gagagcctag tgtataggcg aggctggcga ggaagggttc 20580
cccgtcaatg tcagcgacgg atttaatgta gtctcgcagg cttgctatcg ttcgccgaca 20640
agcttgctcg tccttggcac gcacaacgta tatgcggctc tgtttggaac catcctcaac 20700
cctaccatgc tcagagttac cattgacatg cacttgatcc tctggcaggg ccaatgatgc 20760
gcgatcatat gattccaaaa tgacgtgagc attcgaacca ccaaagccga agttattgac 20820
agatgcgcga cgagtcccat ctttcacagg ccagtcttga gcagacatgg ggatctttga 20880
aacattaacc tttgaaacat ataactgaat ctgcgaatgc gcaaagcctt accttgatgt 20940
tcttttggtc aagcatcagc ttgctgttct tttgcaggaa ccgcgcatta gggggaatca 21000
agcccttctc caaggccaag gccaccttga ttatactggc caggccactg gcggcttctg 21060
tatggccaat atttgctttc acagagccaa ggtgcagagg atgtccttta aaagctgctg 21120
aaattgctga gatttcaagg gggtcaccag ttggtgttcc agttccgtgg gcctccacgt 21180
acgaggtcaa cgacatatct agcccagcct tatcgtaaca ctcctggatc agacttttct 21240
gcgccacatc actcggcgca gtaattgcgg gtgttttgcc atcctggttc agcgctgtct 21300
ctcgaatgac ggctcggata gggtcttggt ctcgcaacgc gttagggagg gcctttatta 21360
ccagagcggc aattccttcc ccgcgaccat atccattcgc tcgaggatca aaagagtacg 21420
agataccatc cggggacaaa aatctgtcat tgagcaacaa ggattgctta gttcaagact 21480
ctcgatctgg aatcttcttc ggaaaactca ccccaggttt gacatcgtaa caaaaacatc 21540
gggattgagc agaagatttg caccgataac gatggctgta tctgactccc cagtacgtaa 21600
gctctggcac gccaagtgca gtgcggtcaa tgtcgtcgaa caggccgtgt caaccgtcac 21660
gctgggacca cgtaagtcgt agaagtgtga tatccggttc gaaagcattg ttcctgagtt 21720
gccagttatg aaataacgcg gaactgtctc ggggtcacga ttgagcgaat cctgatagtc 21780
gtggtacatg acacccccaa acaccgacgt attagagcct gccataccat cgatggtgat 21840
accggctgga tgatggtcag tgacgtttgc ttacagtgag gatgacccac actacatacc 21900
actctccagc gattcgtaga ccacctcaag cataagccga tactgcggat ccatgcactg 21960
tccaatatta gatctctgcg tcccgggtta gatcaattga aataatcata cgctggcgac 22020
ctctgtggtc atgttgaaga acgcggcgtc aaataaagca ggatcctcgt cgatgaagtg 22080
tccacccttt acgtgggtct atccagtcat ccttggagtc agtaaccaag cttcagtgat 22140
gctcaaatct tgtgtcaaat attcaaaaca agatataaat gcatgcatgt tagatactca 22200
cggacccgac cctttcgcca ttcgggtggt atactcctct cacattgaat cgcgaggagg 22260
ggaccttaga ccaggcactg cctcctcttt caaccatttc ccaaagcttc tgtggactcg 22320
ttgcatctcc agcaaatcga catcccattc caactatggc aatgggcgtg gatgtgttag 22380
agcaagccga gcctgccatt gcggttgcgg ttgcggttgc ggttgcggtt gcggttacgg 22440
cgggggtatt gttcattcca acgttgtttc attgactgat atatcagtcg ccctggtgat 22500
aaaaccgttg atagtcttcc aacagtctac aggtccctgg catagctata gatgcataag 22560
ctgcccccga cacgtgattc atagttcggg gtttgttttc atcttggacg tgacacgata 22620
ttcgctctgt gcccatggga aaccccggac caccatgcta tgctcggggc aataccttag 22680
aggtaccggt tcgggaggca ttgtctgtcg tcacgataat cccgagtcaa aacgccgatg 22740
ggaaaccgtc gaacaagacg aaacaggtca ggccggccag gtagttttcg ggtataatgg 22800
aggctgtcag aatccgatac tccgtacaca gatgcgaaat acgcatacga gctatcaaac 22860
caaacgaatc caaaagcctt ggaaaagctt ggaaaggctt agtgggtaat cctgtcccaa 22920
ggtttgttga gggcctgagc gcagggtggg tcctgtaagc agttggtaat tcaatttcca 22980
acaatacaca atccccaaaa tttgcattat cggttgacta agacaagcaa acaaaatata 23040
tgcaggaagc gcaattcatc gcgagcaaac gatcatcatg agcatgtgac cctttcctct 23100
tttttctact tcggaaggcg gcatgatcat ctgtcagaac tcccaatcgg gagcaatacc 23160
ataccttacg gcaccccact cagacccatg cacaaagaaa atccatgcgc cgaatattga 23220
agccttggca acaaagcccc gtgtaactcc gaaggtatcc aaagaccgag agacgccgat 23280
ttgagagaca cgtacggagg tccacacaaa atgttcccga gtctatacac tatactccaa 23340
actgacttct tgtctacctg ggtatcttgt tcaggttgct gtttactgag ataaatgata 23400
ccgggggggg gggggggggg gggggttgac actggctttt cgtggacaga ataataccca 23460
tacatccctg cgtaagtagt cgtttcgaga agaatgtgtt tcgtggtgca ttactccgta 23520
ggcacaatat atttccattc ctcacgaagt ggcctcgtcc gggcgtgatc gatgcagctt 23580
gccgccccac caaaaaagga ccacaatacg agtcagatta gaaacgtcta acaggacgtc 23640
tatgtaagag gacgctcctt tgtatgtcgg atctaggcat gacaaaataa ctatacctag 23700
gtagtgttct gtcttattgg tcatttggcc tactttcgga acaatcttgg aagttcacat 23760
tcctaggtat cagggcaatt gattggtgtc cccagaattc ttttttctcg aataaaggat 23820
aaatttatgc ataaaaacct tggaaactga gcatagttat gagcacaaat actagttttc 23880
agtgcaattg gtcctactat cctttgcttg gtacccctta ccaattatac cctaggcagc 23940
agttgacacc ggtcatgaat ccattcataa aggtggacca gatgcaggga taaggaagcg 24000
aatctttccg ctgcctcagc ctcaggggcg cgcgccattt gttattttct tctactcatt 24060
tcccgtacct aggaactgtt cagttgtccc tcccaacccc ttgggccgaa caaccttcct 24120
ccaatctacg acggcagatt atacctaggc gcctaaccga ttaggttgct cattcgattt 24180
tggaggtatg cactttatct caagccctaa ttcccaattg aagtgctttt ccgtccccat 24240
ttgcagagct gactagattc ttttctcaga gactacctag ctataggtac cactccaagc 24300
tgtagcacag acctttcagc atggtcgctt cgttgctacc ctctcgcttt cgcggtaggg 24360
aatcaatgaa tcagcagcac cctctacgct cgggaaatcg ggcattgacc tccacactcc 24420
aatttctatc caaaacggcg tgtctacacc cgatccatac cgtttgcacc atagctattc 24480
tagctagtac cacatacgtt ggactactca aagacagctt cttccatggc cccgcaaacg 24540
ttgataaagc agaatggggc tctttggtcg aaggaagtcg aagcttgatc accggcccac 24600
agaatggctg gaagtggcag agcttcgacg gggatgcaga tgttctcgga gatttcaacc 24660
atcaagcact aatgaccttg gtattcccgg ggtcatatgg ggttgcatct caagcagcct 24720
caccattcct tgctcccctc cctgtgaacc tatctgtgat tgaccttccc tcaacgtcga 24780
gccctttaac cgcctattcg aaagataaag ttttcgcctt ctctgtggaa tacagcagcg 24840
cgccggaact cgtggctgct gttcaagaaa tccccaacaa cagtgccgac ctgaaattgc 24900
aggagacgca attgatcgag atggaacgcc agatgtggat catgaaggct gccagggctc 24960
acacaaaacg cagccttgct caatgggtgc acgatacctg gacagagtct cttgatctta 25020
tcaagagcgc tcaaacgctc gacgtggttg tcatggtgct aggttatata tcaatgcact 25080
tgactttcgt ctcactcttc ctcagcatga aaaaattggg atcgaaggtt tggctggcta 25140
caagcgtcct tttgtcgtca acatttgcct ttctcctcgg tctcgacgtg gccataagac 25200
taggggttcc gatgagcatg aggttgctat ccgaaggcct ccccttcttg gtggtgatcg 25260
ttggctttga gaagagcatc actctgacca gggctgtttt gtcctatgct gtgcagcacc 25320
gaaagcccca gaagatacag tctgaccagg gtagcgtgac agccattgct gaaagtacca 25380
tcaattacgc cgtacgaagc gccattcggg agaagggtta caatatcgtg tgccactacg 25440
tggtcgagat cctgctccta gttatcggtg ctgtcttagg catccaaggt gggctacagc 25500
acttctgtgt tctagctgca ttgatcctgt tctttgactg tctgctgctg tttacattct 25560
acactgcgat tctgtctatc aagctcgagg taaaccgcct caaacgtcat atcaacatgc 25620
ggtacgcgtt ggaagatgag ggtctcagtc agcggacggc ggagagtgtc gcgaccagca 25680
atgatgccca agacagtgca cgtacatatc tgtttggcaa tgatatgaaa ggcagcagtg 25740
ttccgaagtt caaattctgg atggtcgttg gtttccttat cgtcaacctc gtcaacatcg 25800
gctccaccct tttccaagcc tcttctagtg gatcgttgtc cagtatatca tcttggaccg 25860
aaagtctgag cggatcggcc attaaacccc cgcttgagcc cttcaaggta gctggaagtg 25920
gactagatga actacttttc caggcaagag ggcgcggtca atcgactatg gtcactgtcc 25980
tcgcccccat caagtacgaa ctagagtatc cttccattca ccgtggtacc tcgcagctac 26040
acgagtatgg agttggtgga aaaatggtcg gtagcctgct caccagcctg gaagatcccg 26100
tcctctccaa atgggtgttt gtggcacttg ccctaagtgt cgctctgaac agctatctgt 26160
tcaaggccgc cagactggga atcaaagatc ctaatctccc gagtcaccca gttgatccag 26220
ttgagcttga ccaggccgaa agcttcaacg ctgcccagaa ccagacccct cagattcaat 26280
caagtctcca agctcctcag accagagtgt tcactcctac caccaccgac agtgacagtg 26340
atgcctcatt agtcttaatt aaagcatctc taaaggtcac taagcgagca gaaggaaaga 26400
cagccactag tgaacttccc gtgtctcgca cacaaatcga actggacaat ttgctgaagc 26460
agaacacaat cagcgagttg aacgatgagg atgtcgttgc cttgtctttg cggggaaagg 26520
ttcccgggta tgccctagag aagagtctca aagactgcac tcgtgccgtc aaggttcgcc 26580
gctctatcat ttcgaggaca ccggctaccg cagagcttac aagtatgctg gagcactcga 26640
agctgccgta cgaaaactac gcctgggaac gcgtgctcgg tgcatgttgc gagaacgtta 26700
ttggctatat gccagtccct gttggcgtcg ccggtcctat tgttatcgac ggcaagagtt 26760
atttcattcc tatggcaacc accgagggcg tcctcgtcgc tagtgctagc cgtggcagta 26820
aggcaatcaa cctcggtggc ggtgccgtga cagtcctgac tggcgacggt atgacacgag 26880
gcccgtgtgt gaagtttgat gtccttgaac gagctggtgc tgctaagatc tggctcgatt 26940
cggacgtcgg ccagaccgta atgaaagaag ccttcaattc aaccagcaga tttgcgcgct 27000
tacaaagtat gcggacaact atcgccggta ctcacttata tattcgattt aagactacta 27060
ctggcgacgc tatgggaatg aatatgattt ctaagggcgt ggagcatgca ctgaatgtta 27120
tggcgacaga ggcaggtttc agcgatatga atattattac cctatcagga aattactgta 27180
cggataagaa accttcagct ttgaattgga tcgatggacg gggcaagggc attgtggccg 27240
aagccatcat accggcgaac gttgtcaggg atgtcttaaa gagcgatgtg gatagcatgg 27300
ttcagctcaa catatcgaaa aatctgattg ggtccgctat ggctggctca gttggcggct 27360
tcaacgccca agctgccaat cttgcggcag ccattttcat tgccacaggt caggatccgg 27420
cgcaagttgt ggagagcgct aactgcatca ctctcatgaa caagtaagtt gaaagcggcc 27480
gcttacttgg aaacattcac taatcctgtt tagtcttcgc ggatcgcttc aaatctctgt 27540
ctccatgccg tctattgagg ttggaacgtt gggcggtggt acgattctgg agccccaggg 27600
cgcaatgctt gacatgcttg gtgtccgcgg atcacacccg accactcccg gtgagaatgc 27660
acgtcaactt gcgcgcatca tcggaagcgc tgttttggct ggggagctct cgctatgtgc 27720
tgccctagcc gccggtcacc tggtcaaggc gcacatggcg cacaaccgtt ctgccccggc 27780
atcttcagcc ccttctcgaa gtgtctcccc gtcaggcgga accaggacag tccctgttcc 27840
taacaatgca ctgaggccga gtgctgcagc tactgatcgg gctcgacgct gattaggtcg 27900
gaatcttagg agcattccaa gctccgtacc ccctccagtg gattcattgc aggaggatca 27960
tattttttct cattggttgt tattgtcata attttcaaaa gcacaatgca atgagacagg 28020
caggtggtag agtgaacggc cagaaagggt atctcatgtt tatatgttgt tgaaatttac 28080
gatgcaagta gtagggaaga agaatatata aagagatggt ccttttccag agagtgttta 28140
ggtctgatcc ctcataatta tttaatgagt gaaagctttg ttcaagctat aacttactga 28200
gtaggttgaa tgttgatctg attcattcct gaggtatcag gattgatgcc tgaaacatca 28260
atcatccatt gtcagatgcc gtaactaact aactatgaat ctcaacatag ttatatgttg 28320
ccaatctagc cacggtgact agaaccttga gatggactta gactagacat gggtcgcggg 28380
caatgacata tagaatcttt gaaatcgaca ttaattaagt atgtggagat tctttgtgga 28440
ggcacggtaa tgtgtctatc tagcaacgcg gtcaagcatc agtctcaggc acagcccggg 28500
tgtcgttttt ggttgcaatc ttccgccatc ccattccaaa ggcaaacaca aacgtgcacg 28560
ccgtagctcc cactgctaag taaaaagtat gatcaacggc gagactgtaa gcttttacaa 28620
cccctggaag gttattcttg ctgaccacat ctctgaagcc agtcgcccct gctgccgtca 28680
cggcctgcgt gtcgacagtg ggcgcatact tgctcaggcc agttctcaaa ccggacccaa 28740
agacaaggtt agcaaagtcc aggaagagcg atcctccaaa cgtctgtcca aacacggcga 28800
gagaaattcc gagggcacct tgttcgggcg aaagcgtgct ttggatggcg atgataggct 28860
ggccattgag tattgatgtc agcgtctagc ggttgcatgc tcttcttgct ttgatacaaa 28920
gccgaaagcg tgagagatga tcaaaggttt catagcttac cgtttgcatg ccacaaccac 28980
gaccgaagcc cgcgataaat tggtacatga cccatttcac agttgatgta tggggctgga 29040
aggtggatac cagacctgcg cctatggcga cgagaacagc gctgcctagg gcccaaggca 29100
aatagtatcc tgtctttcca actggtgcgt catatgtcag tatacacgat atccaagccc 29160
gatgtcagac ggttgtggca agaaaggagc catagaaatg gacggggtgg agaaaaatgt 29220
gtacgcgagt ttcacttact tgcgaagcca gaaaccatag ccataatgac ttgtccaaga 29280
attccaggca acatgtacac accactcagt gtgggagaaa catccttcac agcctggaag 29340
tagatcggta gatagtagga aaagacaagc aaggagccag agaaaaagcc cataaataaa 29400
caagagcacc acacttgtcg tttaccagcc actgagccag gaatcatggc aacagcatcg 29460
ccaacatgac gctcccatag cacgaacgca atcagagcaa accctccgcc acagaacagg 29520
ccgatgatga cggaacttcg ccaggtgtag gtcgaccctc cccattctag tgcgagggaa 29580
atcatggttg cgaaggctgc aaagaccaca aagcctacaa ggtccagttt gcgaagtgtg 29640
gattttatgt tggccattgg tttgtcggtc gagagttcgc tgtccgtgga tgaaattcgg 29700
tcgggtatgg tgatgacgag aaggaggaat gcagcgacag cgccgatggg gagattgata 29760
taaaagcctg aattccaagt gagaacatgg acaacaatca taaaaaggcc aaaggtcaac 29820
atacaccatc gccaagtggc gtgttgagtg aaagcacctc cgagcagtgg tccacagaca 29880
atggcaatct gactaactga aaacatattg tcagacgacg aaccgttcgt ttggggtaca 29940
tcagatcttg agatgacata cgacccatca tcactccaat caaaacttca tatgcgaggt 30000
cagcgtgtac acggcaccca gcagacttcc aaaaatcggt tcccttacct ggttgcttgt 30060
gcttaggagc agctgttgag aggattgtga gggctccgtt gacaagacct gagcctccca 30120
ttccagcaac ggcccgccca acaatcaaca tggtggaaga tcttgcggca ccgcatagca 30180
ccgagcctag ttcaaaaata cagaggaagg caaagaaagt gtacttcaag cccaagagtg 30240
tatacaattt accggccagg ggctggagag cacagctaaa tatgatgtta gctaatctgt 30300
tcgtacaatg aacaaggtca aggagaacag agccatactt agccagaaga taagcactgc 30360
cgtaccaccc tacatcgttc agagagtgga actcgcttgt gatatgtggg attgcctgtg 30420
gctggagtca attgactgtg ctgcgctctg ttctgaggta gccaccatct taccgtgacg 30480
ataatggaca tatcaaggag catcaaaaat gctacgaaag taactgaagc aaccaccagc 30540
ccgagcttga ggcctgtgat gtgctgggac ttggactcag tcgcttcgag cgtgtcattt 30600
tgactttctt ccttctgtgg ccttggttcc ccttctttag ggggtagagg ttctgacatc 30660
gcgcaattcc ttccgacttt tgcttcaagg ggcggtgtga atctctactg cgcggcgctt 30720
ctatagtacc tgtgttttgg tgtatgaatg atctcgctct cgttgtttcg ttaaggtccg 30780
ctagcctgaa gtcagattga tggatgggga tcaggggaaa ttggcgacgt ctttaatttt 30840
gcttttcttt gttaccggaa gtgttgcggt attagcgtgt ctgggcttat ttacgacgca 30900
caagatgcat tgaactggcc ccactgctag atctcactag tattgtggtt gtaatttacc 30960
tatactccat attgactggg caggttttga acacaaccca caccccccca tactacacat 31020
tagttttgca tattttcctg ggggccaaaa aaaccccaaa aggcttcaat attttgcggc 31080
caatggagag tgtaactaat ttggcccaca ctccggtggt atcaatcgga tctcactgca 31140
tatatgatga aagcaagagg gggcaggaga tacgctcttt attggctgtc tgcgcgaagc 31200
tgggcaaatg caaataaaaa gacaaacaac cagctggaag accgggcgac aaacatggtt 31260
tacctaacac cctcgatccc aacaatgtgc atgttaatca atgtgctccg tggggagtat 31320
gaactataac atacgaagca gccattcatg tcaaaaaaaa aaccaggcga atgggcgtcg 31380
tcaacggttt cacataagta ctatattgta ctaactaccc gtgagactgg agagaacagt 31440
ctcgcgcgaa gaaacgataa gagcatcggt catatcggtc catctcggtc taagtgtatg 31500
agaatattcc gacgtgaatc catccgtcag tgatcaatgt ctccaagtaa ttcatcattt 31560
caattaccct cgctttactc cgtagaatac aagaccttac tagcgcaaac aagtgggggc 31620
taacggtgtg atctccttcc gttgcggccg ccacctcggt tccagccgta atacgacgac 31680
ccgtctatcg cgacccccta gccttggcca tttttggcgt tacagtaaag ctttggagag 31740
aaacgccaag ggaaaatgct agccaccaat tctataaatt actcttcaca tgcagctagt 31800
atcactggta agtctacggg gcacatgtaa aatttttatt actttctaat aatctttcca 31860
agttcttttc cacggggccc caatgcttaa aatactcaaa agacgtgaaa aacctgcaag 31920
ccgccagtga tatcacacgt aatgcctcaa cagcctgatt ccgagccatt atatgctgtt 31980
tgatgatctc aaattgagat ggcgagcgct ggatctggga aattggtagt gggattggta 32040
tagaaacgta agtgcagaag accatgtaat aagtacatat ggaggctatg tgatggcccg 32100
atctagtttc ttcaatatag cgctgggtat aaaaaaaagc aggggctttc tcagggtaat 32160
gtcgcagtct acaacgagtg gcgtccactg acagggaaag gcgagcgggg ctatgctacc 32220
ttcaatttcc atagaggggg gatgcaccat ctccgacaat ctatagttac tcaaacaggt 32280
acggtactaa gcaatattgt gtttcttcgc taatgcgaat atttccttat agcaacgtcg 32340
caacacattt atcgtcttcc ctgaggcctt tgttgacttg ggctcttcgt ctccggcttc 32400
gtcactccaa agcacagata ggagacgaga ggccggcgtt atggttttat tttcagcgcc 32460
aaggatttgc cacgatgtgc ttggcatatc tgataggacc tattccccct ctcccggtca 32520
gcgcattgct gatgtatgca agggaagaaa agactggtgg ttatcggtcc cacttactag 32580
acgaatagat gccgcagccc cgtgctcctg tgctatcccc aaagcagtct caatctcact 32640
caatagtcga aggcttacac gcaatgtcgt gcatgcagaa gataaggcgt gcatgaatgg 32700
gtcgagatgt gaaatgagct cgccgatatg aagattagag tgaaacgagg gaagtgcttc 32760
ggctcttcca ttgtcatttc tagtggttga gccagaccag taccaatcca ttcgtgtgct 32820
ttgcttttgt ccacaaggtt gggctttcat cacctcggat agtagcagct gggaaagtga 32880
tgtcatgatt ttgacagaca acatgtagca atgcaccgcc atgaacaagt tcttggtttg 32940
cagacaccca tctaacatgc tgctattgct gctcgtgatc acacgttctt gaagatgtag 33000
tagcaatcta ccaaaggcat tcaaaaagtc ccctatcggg tctaggaaga agctttagcg 33060
acaatcaaga ggcagtaaac aggcagaatt gaaaatctca cagcttaaaa ttttttgctt 33120
gggccattcc acagtcaccc cgtggagtat tacctctagg tcctgtgaca catccgacag 33180
actttcgaaa aggtctcgtt gcgtgttgct tgtgttggat tgtccggatg acgagttccc 33240
ctctacttcg aggtcaaaca gcgatggcga gacaggcgcc gttgcatcca aagggccttc 33300
aaagtcgtag cctagatctg gtatccccga agattcattg ctgttggcat cgtcgcgaaa 33360
tgtatttggc tgaggccagc cgccgggaaa cgactcggga tcatcaaagt tgattgatgt 33420
atcatagaat tgcagggttg ccgctgatgg ttctgataat gtttccttga gtgccgaggt 33480
gccaatatgc gtaggtggtg agcagtaagg tggaggagtc tctgccaatg atgagaagac 33540
cgtagaagat gtcgcggtca tcggttgtga ggtttctgtg gctcttgtag ttccagctgc 33600
ggcttcttta tgtaaattgc gcttgggtag cctttcgctg tacacacacc ttaatccggc 33660
ttgttgacaa cgttgacact gagcacggac taaattggca ttgctaccgg tacatttgag 33720
cttttgtgca tgacaccggt cacatgagcg tcgaaacgcg cgacggcgta ggttcgtcgg 33780
aatcgttgca tgcggcaggg acataattat tggattaaga tcaaataatg tgaggtgaga 33840
ctttgcatgt tcctggatct ttatgtattg gaattggaga gtaagctcgt gcaggagata 33900
agttcaggtc gtcttgctgg aagacttact aagttatatg caaacaagtg ttttcgagcg 33960
gacaccaaaa gccaatagtc ttactatgaa tgtcttttca gtcacccgga gaaatactct 34020
tagcctctgc tcttatgcga gctcatcaaa gctgggcata cataccccat ccagcgccac 34080
gtattacact agaaagagtt ctaaaagaaa tagattcggc cccccatctg gctatcatat 34140
atgccagatg aaatacctgt aacgtggggc ataaaaaggc aggctctagt ctaccagcag 34200
atc 34203
<210>2
<211>34203
<212>DNA
<213>柑桔青霉
<400>2
gatctgctgg tagactagag cctgcctttt tatgccccac gttacaggta tttcatctgg 60
catatatgat agccagatgg ggggccgaat ctatttcttt tagaactctt tctagtgtaa 120
tacgtggcgc tggatggggt atgtatgccc agctttgatg agctcgcata agagcagagg 180
ctaagagtat ttctccgggt gactgaaaag acattcatag taagactatt ggcttttggt 240
gtccgctcga aaacacttgt ttgcatataa cttagtaagt cttccagcaa gacgacctga 300
acttatctcc tgcacgagct tactctccaa ttccaataca taaagatcca ggaacatgca 360
aagtctcacc tcacattatt tgatcttaat ccaataatta tgtccctgcc gcatgcaacg 420
attccgacga acctacgccg tcgcgcgttt cgacgctcat gtgaccggtg tcatgcacaa 480
aagctcaaat gtaccggtag caatgccaat ttagtccgtg ctcagtgtca acgttgtcaa 540
caagccggat taaggtgtgt gtacagcgaa aggctaccca agcgcaattt acataaagaa 600
gccgcagctg gaactacaag agccacagaa acctcacaac cgatgaccgc gacatcttct 660
acggtcttct catcattggc agagactcct ccaccttact gctcaccacc tacgcatatt 720
ggcacctcgg cactcaagga aacattatca gaaccatcag cggcaaccct gcaattctat 780
gatacatcaa tcaactttga tgatcccgag tcgtttcccg gcggctggcc tcagccaaat 840
acatttcgcg acgatgccaa cagcaatgaa tcttcgggga taccagatct aggctacgac 900
tttgaaggcc ctttggatgc aacggcgcct gtctcgccat cgctgtttga cctcgaagta 960
gaggggaact cgtcatccgg acaatccaac acaagcaaca cgcaacgaga ccttttcgaa 1020
agtctgtcgg atgtgtcaca ggacctagag gtaatactcc acggggtgac tgtggaatgg 1080
cccaagcaaa aaattttaag ctgtgagatt ttcaattctg cctgtttact gcctcttgat 1140
tgtcgctaaa gcttcttcct agacccgata ggggactttt tgaatgcctt tggtagattg 1200
ctactacatc ttcaagaacg tgtgatcacg agcagcaata gcagcatgtt agatgggtgt 1260
ctgcaaacca agaacttgtt catggcggtg cattgctaca tgttgtctgt caaaatcatg 1320
acatcacttt cccagctgct actatccgag gtgatgaaag cccaaccttg tggacaaaag 1380
caaagcacac gaatggattg gtactggtct ggctcaacca ctagaaatga caatggaaga 1440
gccgaagcac ttccctcgtt tcactctaat cttcatatcg gcgagctcat ttcacatctc 1500
gacccattca tgcacgcctt atcttctgca tgcacgacat tgcgtgtaag ccttcgacta 1560
ttgagtgaga ttgagactgc tttggggata gcacaggagc acggggctgc ggcatctatt 1620
cgtctagtaa gtgggaccga taaccaccag tcttttcttc ccttgcatac atcagcaatg 1680
cgctgaccgg gagaggggga ataggtccta tcagatatgc caagcacatc gtggcaaatc 1740
cttggcgctg aaaataaaac cataacgccg gcctctcgtc tcctatctgt gctttggagt 1800
gacgaagccg gagacgaaga gcccaagtca acaaaggcct cagggaagac gataaatgtg 1860
ttgcgacgtt gctataagga aatattcgca ttagcgaaga aacacaatat tgcttagtac 1920
cgtacctgtt tgagtaacta tagattgtcg gagatggtgc atcccccctc tatggaaatt 1980
gaaggtagca tagccccgct cgcctttccc tgtcagtgga cgccactcgt tgtagactgc 2040
gacattaccc tgagaaagcc cctgcttttt tttataccca gcgctatatt gaagaaacta 2100
gatcgggcca tcacatagcc tccatatgta cttattacat ggtcttctgc acttacgttt 2160
ctataccaat cccactacca atttcccaga tccagcgctc gccatctcaa tttgagatca 2220
tcaaacagca tataatggct cggaatcagg ctgttgaggc attacgtgtg atatcactgg 2280
cggcttgcag gtttttcacg tcttttgagt attttaagca ttggggcccc gtggaaaaga 2340
acttggaaag attattagaa agtaataaaa attttacatg tgccccgtag acttaccagt 2400
gatactagct gcatgtgaag agtaatttat agaattggtg gctagcattt tcccttggcg 2460
tttctctcca aagctttact gtaacgccaa aaatggccaa ggctaggggg tcgcgataga 2520
cgggtcgtcg tattacggct ggaaccgagg tggcggccgc aacggaagga gatcacaccg 2580
ttagccccca cttgtttgcg ctagtaaggt cttgtattct acggagtaaa gcgagggtaa 2640
ttgaaatgat gaattacttg gagacattga tcactgacgg atggattcac gtcggaatat 2700
tctcatacac ttagaccgag atggaccgat atgaccgatg ctcttatcgt ttcttcgcgc 2760
gagactgttc tctccagtct cacgggtagt tagtacaata tagtacttat gtgaaaccgt 2820
tgacgacgcc cattcgcctg gttttttttt tgacatgaat ggctgcttcg tatgttatag 2880
ttcatactcc ccacggagca cattgattaa catgcacatt gttgggatcg agggtgttag 2940
gtaaaccatg tttgtcgccc ggtcttccag ctggttgttt gtctttttat ttgcatttgc 3000
ccagcttcgc gcagacagcc aataaagagc gtatctcctg ccccctcttg ctttcatcat 3060
atatgcagtg agatccgatt gataccaccg gagtgtgggc caaattagtt acactctcca 3120
ttggccgcaa aatattgaag ccttttgggg tttttttggc ccccaggaaa atatgcaaaa 3180
ctaatgtgta gtatgggggg gtgtgggttg tgttcaaaac ctgcccagtc aatatggagt 3240
ataggtaaat tacaaccaca atactagtga gatctagcag tggggccagt tcaatgcatc 3300
ttgtgcgtcg taaataagcc cagacacgct aataccgcaa cacttccggt aacaaagaaa 3360
agcaaaatta aagacgtcgc caatttcccc tgatccccat ccatcaatct gacttcaggc 3420
tagcggacct taacgaaaca acgagagcga gatcattcat acaccaaaac acaggtacta 3480
tagaagcgcc gcgcagtaga gattcacacc gccccttgaa gcaaaagtcg gaaggaattg 3540
cgcgatgtca gaacctctac cccctaaaga aggggaacca aggccacaga aggaagaaag 3600
tcaaaatgac acgctcgaag cgactgagtc caagtcccag cacatcacag gcctcaagct 3660
cgggctggtg gttgcttcag ttactttcgt agcatttttg atgctccttg atatgtccat 3720
tatcgtcacg gtaagatggt ggctacctca gaacagagcg cagcacagtc aattgactcc 3780
agccacaggc aatcccacat atcacaagcg agttccactc tctgaacgat gtagggtggt 3840
acggcagtgc ttatcttctg gctaagtatg gctctgttct ccttgacctt gttcattgta 3900
cgaacagatt agctaacatc atatttagct gtgctctcca gcccctggcc ggtaaattgt 3960
atacactctt gggcttgaag tacactttct ttgccttcct ctgtattttt gaactaggct 4020
cggtgctatg cggtgccgca agatcttcca ccatgttgat tgttgggcgg gccgttgctg 4080
gaatgggagg ctcaggtctt gtcaacggag ccctcacaat cctctcaaca gctgctccta 4140
agcacaagca accaggtaag ggaaccgatt tttggaagtc tgctgggtgc cgtgtacacg 4200
ctgacctcgc atatgaagtt ttgattggag tgatgatggg tcgtatgtca tctcaagatc 4260
tgatgtaccc caaacgaacg gttcgtcgtc tgacaatatg ttttcagtta gtcagattgc 4320
cattgtctgt ggaccactgc tcggaggtgc tttcactcaa cacgccactt ggcgatggtg 4380
tatgttgacc tttggccttt ttatgattgt tgtccatgtt ctcacttgga attcaggctt 4440
ttatatcaat ctccccatcg gcgctgtcgc tgcattcctc cttctcgtca tcaccatacc 4500
cgaccgaatt tcatccacgg acagcgaact ctcgaccgac aaaccaatgg ccaacataaa 4560
atccacactt cgcaaactgg accttgtagg ctttgtggtc tttgcagcct tcgcaaccat 4620
gatttccctc gcactagaat ggggagggtc gacctacacc tggcgaagtt ccgtcatcat 4680
cggcctgttc tgtggcggag ggtttgctct gattgcgttc gtgctatggg agcgtcatgt 4740
tggcgatgct gttgccatga ttcctggctc agtggctggt aaacgacaag tgtggtgctc 4800
ttgtttattt atgggctttt tctctggctc cttgcttgtc ttttcctact atctaccgat 4860
ctacttccag gctgtgaagg atgtttctcc cacactgagt ggtgtgtaca tgttgcctgg 4920
aattcttgga caagtcatta tggctatggt ttctggcttc gcaagtaagt gaaactcgcg 4980
tacacatttt tctccacccc gtccatttct atggctcctt tcttgccaca accgtctgac 5040
atcgggcttg gatatcgtgt atactgacat atgacgcacc agttggaaag acaggatact 5100
atttgccttg ggccctaggc agcgctgttc tcgtcgccat aggcgcaggt ctggtatcca 5160
ccttccagcc ccatacatca actgtgaaat gggtcatgta ccaatttatc gcgggcttcg 5220
gtcgtggttg tggcatgcaa acggtaagct atgaaacctt tgatcatctc tcacgctttc 5280
ggctttgtat caaagcaaga agagcatgca accgctagac gctgacatca atactcaatg 5340
gccagcctat catcgccatc caaagcacgc tttcgcccga acaaggtgcc ctcggaattt 5400
ctctcgccgt gtttggacag acgtttggag gatcgctctt cctggacttt gctaaccttg 5460
tctttgggtc cggtttgaga actggcctga gcaagtatgc gcccactgtc gacacgcagg 5520
ccgtgacggc agcaggggcg actggcttca gagatgtggt cagcaagaat aaccttccag 5580
gggttgtaaa agcttacagt ctcgccgttg atcatacttt ttacttagca gtgggagcta 5640
cggcgtgcac gtttgtgttt gcctttggaa tgggatggcg gaagattgca accaaaaacg 5700
acacccgggc tgtgcctgag actgatgctt gaccgcgttg ctagatagac acattaccgt 5760
gcctccacaa agaatctcca catacttaat taatgtcgat ttcaaagatt ctatatgtca 5820
ttgcccgcga cccatgtcta gtctaagtcc atctcaaggt tctagtcacc gtggctagat 5880
tggcaacata taactatgtt gagattcata gttagttagt tacggcatct gacaatggat 5940
gattgatgtt tcaggcatca atcctgatac ctcaggaatg aatcagatca acattcaacc 6000
tactcagtaa gttatagctt gaacaaagct ttcactcatt aaataattat gagggatcag 6060
acctaaacac tctctggaaa aggaccatct ctttatatat tcttcttccc tactacttgc 6120
atcgtaaatt tcaacaacat ataaacatga gatacccttt ctggccgttc actctaccac 6180
ctgcctgtct cattgcattg tgcttttgaa aattatgaca ataacaacca atgagaaaaa 6240
atatgatcct cctgcaatga atccactgga gggggtacgg agcttggaat gctcctaaga 6300
ttccgaccta atcagcgtcg agcccgatca gtagctgcag cactcggcct cagtgcattg 6360
ttaggaacag ggactgtcct ggttccgcct gacggggaga cacttcgaga aggggctgaa 6420
gatgccgggg cagaacggtt gtgcgccatg tgcgccttga ccaggtgacc ggcggctagg 6480
gcagcacata gcgagagctc cccagccaaa acagcgcttc cgatgatgcg cgcaagttga 6540
cgtgcattct caccgggagt ggtcgggtgt gatccgcgga caccaagcat gtcaagcatt 6600
gcgccctggg gctccagaat cgtaccaccg cccaacgttc caacctcaat agacggcatg 6660
gagacagaga tttgaagcga tccgcgaaga ctaaacagga ttagtgaatg tttccaagta 6720
agcggccgct ttcaacttac ttgttcatga gagtgatgca gttagcgctc tccacaactt 6780
gcgccggatc ctgacctgtg gcaatgaaaa tggctgccgc aagattggca gcttgggcgt 6840
tgaagccgcc aactgagcca gccatagcgg acccaatcag atttttcgat atgttgagct 6900
gaaccatgct atccacatcg ctctttaaga catccctgac aacgttcgcc ggtatgatgg 6960
cttcggccac aatgcccttg ccccgtccat cgatccaatt caaagctgaa ggtttcttat 7020
ccgtacagta atttcctgat agggtaataa tattcatatc gctgaaacct gcctctgtcg 7080
ccataacatt cagtgcatgc tccacgccct tagaaatcat attcattccc atagcgtcgc 7140
cagtagtagt cttaaatcga atatataagt gagtaccggc gatagttgtc cgcatacttt 7200
gtaagcgcgc aaatctgctg gttgaattga aggcttcttt cattacggtc tggccgacgt 7260
ccgaatcgag ccagatctta gcagcaccag ctcgttcaag gacatcaaac ttcacacacg 7320
ggcctcgtgt cataccgtcg ccagtcagga ctgtcacggc accgccaccg aggttgattg 7380
ccttactgcc acggctagca ctagcgacga ggacgccctc ggtggttgcc ataggaatga 7440
aataactctt gccgtcgata acaataggac cggcgacgcc aacagggact ggcatatagc 7500
caataacgtt ctcgcaacat gcaccgagca cgcgttccca ggcgtagttt tcgtacggca 7560
gcttcgagtg ctccagcata cttgtaagct ctgcggtagc cggtgtcctc gaaatgatag 7620
agcggcgaac cttgacggca cgagtgcagt ctttgagact cttctctagg gcatacccgg 7680
gaacctttcc ccgcaaagac aaggcaacga catcctcatc gttcaactcg ctgattgtgt 7740
tctgcttcag caaattgtcc agttcgattt gtgtgcgaga cacgggaagt tcactagtgg 7800
ctgtctttcc ttctgctcgc ttagtgacct ttagagatgc tttaattaag actaatgagg 7860
catcactgtc actgtcggtg gtggtaggag tgaacactct ggtctgagga gcttggagac 7920
ttgattgaat ctgaggggtc tggttctggg cagcgttgaa gctttcggcc tggtcaagct 7980
caactggatc aactgggtga ctcgggagat taggatcttt gattcccagt ctggcggcct 8040
tgaacagata gctgttcaga gcgacactta gggcaagtgc cacaaacacc catttggaga 8100
ggacgggatc ttccaggctg gtgagcaggc taccgaccat ttttccacca actccatact 8160
cgtgtagctg cgaggtacca cggtgaatgg aaggatactc tagttcgtac ttgatggggg 8220
cgaggacagt gaccatagtc gattgaccgc gccctcttgc ctggaaaagt agttcatcta 8280
gtccacttcc agctaccttg aagggctcaa gcgggggttt aatggccgat ccgctcagac 8340
tttcggtcca agatgatata ctggacaacg atccactaga agaggcttgg aaaagggtgg 8400
agccgatgtt gacgaggttg acgataagga aaccaacgac catccagaat ttgaacttcg 8460
gaacactgct gcctttcata tcattgccaa acagatatgt acgtgcactg tcttgggcat 8520
cattgctggt cgcgacactc tccgccgtcc gctgactgag accctcatct tccaacgcgt 8580
accgcatgtt gatatgacgt ttgaggcggt ttacctcgag cttgatagac agaatcgcag 8640
tgtagaatgt aaacagcagc agacagtcaa agaacaggat caatgcagct agaacacaga 8700
agtgctgtag cccaccttgg atgcctaaga cagcaccgat aactaggagc aggatctcga 8760
ccacgtagtg gcacacgata ttgtaaccct tctcccgaat ggcgcttcgt acggcgtaat 8820
tgatggtact ttcagcaatg gctgtcacgc taccctggtc agactgtatc ttctggggct 8880
ttcggtgctg cacagcatag gacaaaacag ccctggtcag agtgatgctc ttctcaaagc 8940
caacgatcac caccaagaag gggaggcctt cggatagcaa cctcatgctc atcggaaccc 9000
ctagtcttat ggccacgtcg agaccgagga gaaaggcaaa tgttgacgac aaaaggacgc 9060
ttgtagccag ccaaaccttc gatcccaatt ttttcatgct gaggaagagt gagacgaaag 9120
tcaagtgcat tgatatataa cctagcacca tgacaaccac gtcgagcgtt tgagcgctct 9180
tgataagatc aagagactct gtccaggtat cgtgcaccca ttgagcaagg ctgcgttttg 9240
tgtgagccct ggcagccttc atgatccaca tctggcgttc catctcgatc aattgcgtct 9300
cctgcaattt caggtcggca ctgttgttgg ggatttcttg aacagcagcc acgagttccg 9360
gcgcgctgct gtattccaca gagaaggcga aaactttatc tttcgaatag gcggttaaag 9420
ggctcgacgt tgagggaagg tcaatcacag ataggttcac agggagggga gcaaggaatg 9480
gtgaggctgc ttgagatgca accccatatg accccgggaa taccaaggtc attagtgctt 9540
gatggttgaa atctccgaga acatctgcat ccccgtcgaa gctctgccac ttccagccat 9600
tctgtgggcc ggtgatcaag cttcgacttc cttcgaccaa agagccccat tctgctttat 9660
caacgtttgc ggggccatgg aagaagctgt ctttgagtag tccaacgtat gtggtactag 9720
ctagaatagc tatggtgcaa acggtatgga tcgggtgtag acacgccgtt ttggatagaa 9780
attggagtgt ggaggtcaat gcccgatttc ccgagcgtag agggtgctgc tgattcattg 9840
attccctacc gcgaaagcga gagggtagca acgaagcgac catgctgaaa ggtctgtgct 9900
acagcttgga gtggtaccta tagctaggta gtctctgaga aaagaatcta gtcagctctg 9960
caaatgggga cggaaaagca cttcaattgg gaattagggc ttgagataaa gtgcatacct 10020
ccaaaatcga atgagcaacc taatcggtta ggcgcctagg tataatctgc cgtcgtagat 10080
tggaggaagg ttgttcggcc caaggggttg ggagggacaa ctgaacagtt cctaggtacg 10140
ggaaatgagt agaagaaaat aacaaatggc gcgcgcccct gaggctgagg cagcggaaag 10200
attcgcttcc ttatccctgc atctggtcca cctttatgaa tggattcatg accggtgtca 10260
actgctgcct agggtataat tggtaagggg taccaagcaa aggatagtag gaccaattgc 10320
actgaaaact agtatttgtg ctcataacta tgctcagttt ccaaggtttt tatgcataaa 10380
tttatccttt attcgagaaa aaagaattct ggggacacca atcaattgcc ctgataccta 10440
ggaatgtgaa cttccaagat tgttccgaaa gtaggccaaa tgaccaataa gacagaacac 10500
tacctaggta tagttatttt gtcatgccta gatccgacat acaaaggagc gtcctcttac 10560
atagacgtcc tgttagacgt ttctaatctg actcgtattg tggtcctttt ttggtggggc 10620
ggcaagctgc atcgatcacg cccggacgag gccacttcgt gaggaatgga aatatattgt 10680
gcctacggag taatgcacca cgaaacacat tcttctcgaa acgactactt acgcagggat 10740
gtatgggtat tattctgtcc acgaaaagcc agtgtcaacc cccccccccc cccccccccc 10800
cggtatcatt tatctcagta aacagcaacc tgaacaagat acccaggtag acaagaagtc 10860
agtttggagt atagtgtata gactcgggaa cattttgtgt ggacctccgt acgtgtctct 10920
caaatcggcg tctctcggtc tttggatacc ttcggagtta cacggggctt tgttgccaag 10980
gcttcaatat tcggcgcatg gattttcttt gtgcatgggt ctgagtgggg tgccgtaagg 11040
tatggtattg ctcccgattg ggagttctga cagatgatca tgccgccttc cgaagtagaa 11100
aaaagaggaa agggtcacat gctcatgatg atcgtttgct cgcgatgaat tgcgcttcct 11160
gcatatattt tgtttgcttg tcttagtcaa ccgataatgc aaattttggg gattgtgtat 11220
tgttggaaat tgaattacca actgcttaca ggacccaccc tgcgctcagg ccctcaacaa 11280
accttgggac aggattaccc actaagcctt tccaagcttt tccaaggctt ttggattcgt 11340
ttggtttgat agctcgtatg cgtatttcgc atctgtgtac ggagtatcgg attctgacag 11400
cctccattat acccgaaaac tacctggccg gcctgacctg tttcgtcttg ttcgacggtt 11460
tcccatcggc gttttgactc gggattatcg tgacgacaga caatgcctcc cgaaccggta 11520
cctctaaggt attgccccga gcatagcatg gtggtccggg gtttcccatg ggcacagagc 11580
gaatatcgtg tcacgtccaa gatgaaaaca aaccccgaac tatgaatcac gtgtcggggg 11640
cagcttatgc atctatagct atgccaggga cctgtagact gttggaagac tatcaacggt 11700
tttatcacca gggcgactga tatatcagtc aatgaaacaa cgttggaatg aacaataccc 11760
ccgccgtaac cgcaaccgca accgcaaccg caaccgcaac cgcaatggca ggctcggctt 11820
gctctaacac atccacgccc attgccatag ttggaatggg atgtcgattt gctggagatg 11880
caacgagtcc acagaagctt tgggaaatgg ttgaaagagg aggcagtgcc tggtctaagg 11940
tcccctcctc gcgattcaat gtgagaggag tataccaccc gaatggcgaa agggtcgggt 12000
ccgtgagtat ctaacatgca tgcatttata tcttgttttg aatatttgac acaagatttg 12060
agcatcactg aagcttggtt actgactcca aggatgactg gatagaccca cgtaaagggt 12120
ggacacttca tcgacgagga tcctgcttta tttgacgccg cgttcttcaa catgaccaca 12180
gaggtcgcca gcgtatgatt atttcaattg atctaacccg ggacgcagag atctaatatt 12240
ggacagtgca tggatccgca gtatcggctt atgcttgagg tggtctacga atcgctggag 12300
agtggtatgt agtgtgggtc atcctcactg taagcaaacg tcactgacca tcatccagcc 12360
ggtatcacca tcgatggtat ggcaggctct aatacgtcgg tgtttggggg tgtcatgtac 12420
cacgactatc aggattcgct caatcgtgac cccgagacag ttccgcgtta tttcataact 12480
ggcaactcag gaacaatgct ttcgaaccgg atatcacact tctacgactt acgtggtccc 12540
agcgtgacgg ttgacacggc ctgttcgacg acattgaccg cactgcactt ggcgtgccag 12600
agcttacgta ctggggagtc agatacagcc atcgttatcg gtgcaaatct tctgctcaat 12660
cccgatgttt ttgttacgat gtcaaacctg gggtgagttt tccgaagaag attccagatc 12720
gagagtcttg aactaagcaa tccttgttgc tcaatgacag atttttgtcc ccggatggta 12780
tctcgtactc ttttgatcct cgagcgaatg gatatggtcg cggggaagga attgccgctc 12840
tggtaataaa ggccctccct aacgcgttgc gagaccaaga ccctatccga gccgtcattc 12900
gagagacagc gctgaaccag gatggcaaaa cacccgcaat tactgcgccg agtgatgtgg 12960
cgcagaaaag tctgatccag gagtgttacg ataaggctgg gctagatatg tcgttgacct 13020
cgtacgtgga ggcccacgga actggaacac caactggtga cccccttgaa atctcagcaa 13080
tttcagcagc ttttaaagga catcctctgc accttggctc tgtgaaagca aatattggcc 13140
atacagaagc cgccagtggc ctggccagta taatcaaggt ggccttggcc ttggagaagg 13200
gcttgattcc ccctaatgcg cggttcctgc aaaagaacag caagctgatg cttgaccaaa 13260
agaacatcaa ggtaaggctt tgcgcattcg cagattcagt tatatgtttc aaaggttaat 13320
gtttcaaaga tccccatgtc tgctcaagac tggcctgtga aagatgggac tcgtcgcgca 13380
tctgtcaata acttcggctt tggtggttcg aatgctcacg tcattttgga atcatatgat 13440
cgcgcatcat tggccctgcc agaggatcaa gtgcatgtca atggtaactc tgagcatggt 13500
agggttgagg atggttccaa acagagccgc atatacgttg tgcgtgccaa ggacgagcaa 13560
gcttgtcggc gaacgatagc aagcctgcga gactacatta aatccgtcgc tgacattgac 13620
ggggaaccct tcctcgccag cctcgcctat acactaggct ctcgccgttc cattctgcca 13680
tggacgtcag tgtatgtagc agacagcctt ggcggccttg tttctgccct cagcgatgag 13740
tccaatcaac caaaacgagc gaatgagaaa gtacggctcg gatttgtatt caccggtcag 13800
ggggcgcagt ggcatgcaat gggcagagag ctggtcaata cattcccagt attcaaacag 13860
gcgattcttg aatgtgatgg ctacatcaag caactgggcg cgagttggaa ttttatgggt 13920
aagttgcgag cccgggaaaa gtaatactgt atcaagcttg aggtactaac attcaattgc 13980
acagaggagc tccaccgtga tgagctgacg actcgggtaa atgatgccga atacagtcta 14040
ccactgtcaa ccgctatcca aattgcactt gtgcgtctcc tttggtcatg gggaattcgg 14100
ccaacgggga taaccagtca ctcaagtgga gaggctgctg ctgcctacgc agctggggct 14160
ttatccgcgc ggtcggccat tgggatcact tatatacgcg gtgtattgac cactaagccc 14220
aagcccgcat tggcagccaa aggaggaatg atggcggtgg gtcttggtcg cagtgagacc 14280
aatgtttaca tttcgcgtct caaccaggag gacggctgtg tggtggttgg atgtatcaac 14340
agtcaatgta gtgtgacggt gtcgggagat ttgggtgcaa tcgagaaact tgaaaagttg 14400
ttacacgccg atggcatctt taccaggaaa ctgaaagtca ctgaagcctt ccattcaagc 14460
cacatgcgac caatggcaga tgcctttggg gcgtcactga gagatctgtt caactcggat 14520
aacaacaacg acaatcccaa tgctgacacc tcaaagggtg tattatattc atcacctaag 14580
actggtagtc gcatgaccga tcttaaattg ctattggatc ccacacactg gatggatagt 14640
atgctacagc cggtagagtt cgagtcctca ctccgcgaga tgtgctttga tcccaacacc 14700
aaagagaaag ccgtcgatgt gattattgaa atagggcctc acggagcgct tggtggtcca 14760
atcaaccaag tcatgcagga tctgggtctg aaaggaacag atataaacta tctcagttgc 14820
ctttctcgcg gcagaagctc gttggagaca atgtatcgtg ctgctacgga gttgataagc 14880
aagggttatg ggctcaaaat ggacgctata aactttcctc atggaagaaa agagcccaga 14940
gtgaaggtac tgagcgattt gccggcgtac ccgtggaatc accaaacccg ttattggaga 15000
gagcctcgcg gcagtcgtga gtccaaacag agaacccatc cgcctcacac tttgataggc 15060
tcacgggaat ctctctctcc tcatttcgcg cctaaatgga aacatgttct ccgtctgtca 15120
gatattccat ggatacgaga tcacgtcgtt ggttcgagca tcatctttcc gggagctggc 15180
ttcatcagca tggccatcga ggggttttca caagtctgcc caccagttgc gggggctagc 15240
atcaactaca acttgcgtga cgttgaactc gcgcaggctc tcataatacc cgctgatgca 15300
gaagcagagg ttgacctgcg cctaacgatc cgttcatgtg aggaaaggtc cctcggcaca 15360
aagaactggc atcaattttc tgtgcactca atttcgggcg aaaataatac ctggacagaa 15420
cactgcaccg gattaatacg ttcggagagc gaaagaagcc accttgactg ttcaactgtg 15480
gaagcctcac gcaggttgaa tctaggctca gataaccgga gcattgatcc caacgatctc 15540
tgggagtcct tacacgcgaa tgggatatgc cacggaccca tttttcagaa cattcagcga 15600
attcaaaaca atggacaggg ctcgttttgc agattttcca ttgctgacac tgcctcggct 15660
atgcctcact cgtacgagaa tcgacacatc gtccatccta ctactctgga ctcggtgatc 15720
caggcggcat acacggtgtt accctacgcg ggaacacgta tgaaaacggc catggtacca 15780
aggaggctaa gaaatgtcaa aatatcctct agcctggctg acttggaggc tggtgatgct 15840
ctggacgcac aggccagcat caaggatcgc aactctcaat ccttctctac cgacttggca 15900
gtgtttgatg actatgatag cggttcttct ccctcggacg gaatcccagt catagagatt 15960
gaaggccttg ttttccagtc ggttggaagc agcttctctg accaaaagtc agactccaac 16020
gacacagaaa atgcctgcag ctcctgggtt tgggcccctg acatcagctt gggtgactcc 16080
acttggctca aagaaaagtt gagcactgag gctgagacga aagaaacgga actcatgatg 16140
gacctccgaa gatgcacgat caactttata caggaggctg tcactgattt gacaaattct 16200
gatatccaac atctggatgg ccaccttcag aagtatttcg attggatgaa tgtccaattg 16260
gaccttgcga gacaaaacaa gctcagccca gccagttgcg actggctaag tgacgatgct 16320
gagcagaaga aatgcctaca ggccagagtc gctggagaaa gcgtcaatgg cgagatgatt 16380
tctcgtctag gacctcagtt aatagcaatg ctacgccgcg aaacagagcc acttgagttg 16440
atgatgcaag atcagctgct aagcagatac tacgtcaacg caatcaaatg gagccgatca 16500
aacgcacaag ccagcgagct gatccgactt tgcgcccaca agaacccgcg ttctcgcatt 16560
ttggagattg gcggaggcac gggcggctgc acaaagctta ttgtcaatgc attgggaaac 16620
accaagccga tcgatcgtta tgacttcacc gatgtgtctg ccgggttttt cgagtcggcg 16680
cgtgagcaat ttgcggattg gcaagacgtg atgactttca aaaaattgga tattgaaagc 16740
gatcccgagc aacaagggtt tgaatgtgcc acctacgatg tggtcgtggc ttgccaggtc 16800
ctgcatgcaa ctcgatgcat gaaacgaaca ctgagtaacg ttcgaaaatt gctcaagcct 16860
gggggcaact tgattttggt tgagactacc agggatcagc tcgatttgtt ctttaccttc 16920
ggactgttgc caggttggtg gctcagtgag gagcctgagc ggaagtcgac gccatcgctc 16980
actaccgatc tttggaacac catgttggac acgagcggtt tcaacggtgt ggaattggag 17040
gttcgtgatt gtgaagacga tgagttttac atgatcagca caatgctatc gacggctaga 17100
aaagagaata caaccccgga tacagtggca gaatcggagg tgcttttgct gcacggagcg 17160
ctccgacctc cttcatcttg gctggaaagt ctccaggcag caatttgtga aaagaccagt 17220
tctagcccat cgatcaacgc tctgggcgag gtagatacca ctggaaggac atgcattttt 17280
cttggggaaa tggagtcctc gctccttgga gaggtgggaa gcgagacctt caaatccatc 17340
accgcgatgc tgaataactg caacgcactt ctctgggtgt ctagaggagc agccatgagc 17400
tccgaggatc catggaaagc tctacatatt ggtctgctgc gtaccatccg caacgaaaat 17460
aacgggaagg aatatgtatc gttggatctc gatccttctc gaaacgcata cacccacgag 17520
tccctgtatg ctatctgcaa tatcttcaat ggccgcctcg gcgacctttc cgaagacaag 17580
gagtttgaat ttgcagagag aaacggcgtc atccacgtac cgcgactttt caatgacccg 17640
cactggaagg accaagaagc ggttgaggtc acactgcagc cgttcgagca acccgggcgt 17700
cgtctgcgga tggaggttga gacgccaggg ctcttagact ccctgcaatt tcgagacgac 17760
gaaggacgtg aaggcaagga tcttccggat gattgggtag aaatcgaacc caaagctttc 17820
ggtctcaatt ttcgggatgt catggttgcc atgggtcaat tggaggccaa ccgtgtgatg 17880
ggcttcgaat gcgccggagt gatcacaaag ctcggtggag ctgctgccgc tagccaaggc 17940
ctcagattag gggaccgcgt atgtgcacta ctgaaaggcc attgggcgac cagaacacag 18000
acgccgtaca ctaatgtcgt ccgtattccg gacgaaatgg gcttcccaga agccgcttcg 18060
gtccccctgg ctttcactac cgcatatatt gcgctttata ccacggcaaa gctacgacga 18120
ggcgaaagag tcttgatcca cagtggagct ggaggcgtcg gtcaagcagc gatcattttg 18180
tcccagcttg cgggtgccga ggtcttcgtc acagcgggaa ctcaagccaa gcgtgacttt 18240
gtcggcgata aattcggcat caatccggat catatcttct cgagcaggaa tgacttattc 18300
gtcgacggca tcaaagccta cacgggcgga cttggcgttc atgtcgttct aaactcattg 18360
gcaggtcaac tcctccaagc aagctttgac tgcatggccg aattcggcag atttgttgag 18420
attggaaaaa aggacctgga gcaaaacagc agacttgaca tgctgccatt cacccgggac 18480
gtctctttca catcaattga tcttctctcg tggcaaagag ccaaaagtga agaagtatcc 18540
gaagcgttga accatgtcac aaaactcctc gagacaaaag cgattggctt gattggtcca 18600
atccagcagc actccttgtc aaacatcgag aaggccttcc gtacgatgca gagtggtcag 18660
catgttggca aagttgtggt caatgtatct ggggacgaac tggtcccagt cggcgatgga 18720
gggttctcgc tgaagctgaa gcctgacagt tcttacctag ttgctggtgg gctgggggga 18780
attggaaagc agatctgtca gtggcttgtt gatcatggcg cgaagcactt gattatccta 18840
tcgagaagtg caaaggccag tccattcata accagcttgc aaaatcaaca gtgcgctgtc 18900
tatctacacg catgtgacat ctcagatcaa gatcaggtca ccaaggtgct ccggttgtgc 18960
gaagaagcac atgcaccgcc aattcgaggt atcatacaag gtgccatggt tctcaaggac 19020
gcgcttctat cgcgaatgac attggatgaa tttaatgcag caacacgccc aaaagtacag 19080
ggtagttggt atcttcacaa gatcgcacag gatgttgact tcttcgtgat gctctcatcc 19140
cttgttgggg tcatgggtgg ggcaggccag gccaattacg cagctgctgg tgcattccag 19200
gacgcacttg cgcaccaccg gagagcccat ggcatgccgg ctgtcaccat tgacttgggc 19260
atggtcaagt ctgttggata cgtggctgaa actggccgtg gtgtggccga ccggctcgct 19320
agaataggtt acaagcctat gcatgaaaag gacgtcatgg atgtgttgga gaaggcaatc 19380
ctgtgttctt cccctcaatt tccatcacct cccgcagctg tggttacagg aatcaacaca 19440
tccccgggtg ctcactggac cgaggcaaac tggatacagg aacagcggtt tgtgggactt 19500
aaataccgcc aagtccttca tgcagaccaa tcctttgtct cttcgcataa aaaaggacca 19560
gatggcgtgc gggcccaact aagcagggtc acctctcacg acgaggccat ttctatcgtc 19620
ctcaaagcaa tgacggaaaa gctgatgcga atgtttggtc tggcagaaga cgacatgtcc 19680
tcgtccaaaa acctggcagg tgtcggcgta gactcactcg tcgccattga acttcgaaac 19740
tggatcacat ctgaaatcca tgttgatgtg tcgatctttg agctcatgaa tggtaacacc 19800
atcgccggcc tcgtcgagtt agttgtggcg aaatgcagtt aagttgaagg gttcagtgaa 19860
gccttttgtc tggccaagcg ggtatagctc gacggaggta tagtacgaag gagcatagcg 19920
gccatggtct gaagcctgaa tccaatctga atcgagcctg ggctgagcct gactatttaa 19980
tgcctgactt ctggatagca gtaaatagag atacctgaaa taccattaca gtggccctga 20040
gaagcaacaa agtacacatg tgcactcgtt ctcgaagtcg gaagagtgaa tgctttttat 20100
actaccaggg aagctgtctt agcacctcgg aggcttgact gtcaaaagtt ctctcttttt 20160
ctctccatta tgattcccgc aagccttgta aatgcgcgtt gaacggtcga aaggcgttgg 20220
cacgggcagt gggtacagat tgtggatatg tagtcggaag gcgggaggga gtacttgtgt 20280
ccacgtcgtt gcgccgtcct ctctttcgcc tagtcgggga tgttgagtag gaacatcaag 20340
acttaacaga gcctaagccc tcgtcatcgt aagcgccagt caacgcctga gagaatgggg 20400
agatcggtga ttgtaccggg agaaaagctt cattactgcc gacttcccta cgtggcggtg 20460
tagctggcgg tatagaagca gatggccgct ctgcgtagca ggaatacaca ctctctccct 20520
tctctctctc tgtgtttctg tctctcgcac atagccaaag tctacaccac gttcgattac 20580
aaagaaggca tcacaatcga ataaaatgcg ttttatttta ctaacctact cgactaatac 20640
agcacctagt ttctctggga cggaaactat tggaataagc ctggggacgg atgcatattt 20700
gttttagttt gcgtgttata tcttagcacc ggtcatgagg gagcgggatg tcctcgttgc 20760
gccggcgtac catgagcttt gtggttggat gcatacgaac gctaaaagcg tgacggtagt 20820
atttgtcatc gtctcctggt acaggcttca catcatactg aatcagtata tgagcgagga 20880
gaatcttgat ttccttcgag gcgaagaacc gcccgggaca agcgcgtggg ttccagccga 20940
agccgatgtg atcaccgttg gtattctcca attgagcggt gaaggccttg tctggatcct 21000
cgcgcatgcg cataaatcgg tagggatcat aattttcggg gttttcccac acatcagggt 21060
tgttcatgcg gtctgcagcc acagcggcca actcgccctt gggaatgaag aggccattgg 21120
atagagtgat gtctctgaga gcggtactgc gcatagtggc gcactcgacc ggcttgattc 21180
gctgcgtctc tttcatgcag ctgtcgagga gcttcagctt gaacagagag gcaggcgtcc 21240
agcccccttc tccgattaca gtgcggatct cttggcggag aggctgaata aggtctgggt 21300
gcctggcaat gtccacaagg gcaccgacga aaagatccgt cgaggcgtag atgccggcga 21360
aatccatagc gagctgagca cccgccacat cgtaccagcg gccgtcggcg gtgtcttcaa 21420
accattgcat ggtatcgacg tactggggcg gctgcacgcc cttcgctaca catgcggcct 21480
tttcagcacg tcgtcgctga atctcaggat caatgatctt tcgtgcgcgg cgcacttggt 21540
cacgcaattt gcgtccttgc ggttgaaacc agtgagcgag cggtcgcagt agcatgggcc 21600
atacgcgaag ttggcgagct tgtaccgcca cactcacggc atggttcttt gcaatatcca 21660
gccactcctc attgtggcag attttgtcgc cgaccataat gagtgtgact gttcgtgtga 21720
caaggtccaa tccattggaa tagacaggtg cggtttgcca ctctagtata ttcgcggtat 21780
gtcagccaga ggctcaatgc tcaagacaga aaaattgaca cttaccctcg cttttaccga 21840
acaacttggc aatagtagcg tcggccaagg tagccaatgg ctttgtgtac ttgggggctt 21900
gggtttgtaa ctggttcaaa acaactttgt tgacaagatg tgcatcctgg cagatttcct 21960
tgaacccgtc gaatccaggg agatgagagt gaaagtccta tacattcatc agaatcttag 22020
agacgtcatt gagttacaac aatggaaaat tcagaggtca tacatccgcc aaaaacttgt 22080
acatgcacat atctttgatt ttccgaaact cgtcggccat ggacgatggg aggatggtgc 22140
aatagccgga atcaacaatg aagcgcaggg gcttgtcgtt tttcgagaac caagcttcga 22200
tccagctcgg accatacgta tcgaagtcct gcctagccct catggtcgtc aactcccacc 22260
attttttggg attatagact tgcagttcgg actggcgccc ccgcaaacgg taggcgatga 22320
gactaagaag cactgcgacc gccacaaggg cttgaggggt cgatacccat tggtacgatt 22380
cgacggtcag aagaacctgg ccgagcattg cgtgagacag ataggaccta tgcacaccag 22440
tggaaaagaa gaaagagcga agaatgagag cgctgcgacg gtttataatc gaataacagc 22500
actaatgctt ctgggatttt gtggccgaga gcactcttcc agtcaacctt gaaaaaaaaa 22560
aaaccccccc cccaatcgaa gtttacctgg atggggcagt tcggttgttt cctttaggag 22620
cagcttcacc gagcagcaca agaacaatcc gagtgaaaaa ctcggtttca ccttgataca 22680
gccaattgat attcacgttt gattcattca gcctcgtgtg accgaataac gccgtatgga 22740
ggaatggcta ttcgtgcacc gaatgacgcc gggagggttt gctaggtgcc gagcttgcat 22800
tgctgggaag tgggggcatt tgagtactag aatggatctt gaaattgtcc gaatctagat 22860
gagtactgat acgtgcaagt aaatataacg acggtatcgg ttgcaaggcc ggcttgttcg 22920
ctcagagatt caactctgcg attctgtaag aacaaatgtt gtgcccggca tgcagtgaga 22980
agatctactg acgcaagaca aggtttaatc ccaatcctat cgcccaaaaa caggatcagc 23040
agttatggat caagccaact atccaaacga gccaattgtg gtagtgggaa gcggttgtcg 23100
gtttccaggt ggtgtcaaca caccatcaaa actttgggag ctgctcaaag agccccggga 23160
tgtacagacc aagatcccta aggagagatt tgacgtcgat acattttaca gccccgatgg 23220
cactcacccc gggcgcacga acgcaccctt tgcatacttg ctgcaggagg atctacgcgg 23280
ttttgatgcc tctttcttca acatccaagc tggagaggcc gaaacgattg acccacagca 23340
aaggctgctg ctggagacgg tctatgaagc tgtatccaac gcaggcctac ggatccaagg 23400
ccttcaagga tcctctactg ctgtgtacgt cggtatgatg acgcatgact atgagactat 23460
cgtgacgcgt gaattggata gtattcctac atactctgcc acgggggtag ctgtcagtgt 23520
ggcctccaac cgtgtatcat acttcttcga ctggcatggg ccgagtgtga gtgccactca 23580
ttgagcgagc ccgacttcgt caagtgctga cagattcctg actgattctg cagatgacga 23640
tcgacacagc ctgtagttca tccttagctg ccgtgcatct ggccgtccaa cagcttagaa 23700
cgggcgagag taccatggcg gttgcagccg gtgcgaatct gatattgggc cccatgacct 23760
ttgtaatgga gagcaaattg aacatgctgt cccccaatgg tagatctcga atgtgggatg 23820
ctgctgccga tggatatgcc agaggagtaa gttgacaatg catcaattcc tttcaaaaaa 23880
agcaagatgg cactgacctc ctgtaactgc tttttaggaa ggtgtttgct ctattgtcct 23940
gaaaacgctg agccaggcac tgcgcgacgg ggacagtatc gagtgtgtta tccgagagac 24000
cggtatcaac caagatggcc gaacgacagg tatcacaatg ccaaaccata gcgcacaaga 24060
agccctcatt cgggccacat atgccaaggc tggtcttgat attaccaacc cccaggaacg 24120
ctgccagttc tttgaagccc atggtaagtg gtattccctg gaagtatcag ccttatggaa 24180
gttgcagaaa gtctctctct ccctaacacg aagatcccag gaactggtac accagccggt 24240
gacccacagg aagctgaggc tattgcaaca gccttcttcg gacacaagga tggaacaatc 24300
gacagcgacg gcgagaaaga tgagcttttt gtcggcagca tcaagacagt tctcggtcac 24360
acggaaggca ctgctggtat tgcgggctta atgaaggcat cgtttgctgt acgaaatggc 24420
gtgatcccgc caaacctgct gtttgagaag atcagtcccc gtgtcgctcc gttctatacg 24480
cacttgaaaa ttgcaacgga ggccacagaa tggccgattg ttgcgcccgg gcagcctcgc 24540
agagtcagcg ttaattcatt tggtaaggat tcaactgcac ttcttgagaa cgaaagtgaa 24600
gttagctaaa catataaaca catcaggatt tggtggtaca aatgcccatg ctattatcga 24660
agagtatatg gctcctccac acaagccgac agcagtggta acagaggtga cctcagatgc 24720
agatgcatgc agcttgcccc ttgtgctttc atcgaagtcg cagcgctcca tgaaggcaac 24780
gctagaaaat atgctccaat ttctggaaac gcatgatgac gtggacatgc atgatatcgc 24840
atatacctta cttgagaaac ggtctatctt gcccttccgt cgtgcgattg cagcacacaa 24900
caaggaagta gcccgcgcgg cactggaggc tgccatcgcg gacggtgagg tcgtcaccga 24960
cttccgcacc gacgcgaatg acaaccctcg cgtactaggt gtctttactg gccaaggtgc 25020
acagtggccg ggcatgctga agaagctcat ggtgggtatg ccatttgtga gaggcattct 25080
cgaagagctg gataattcac tgcaaacact gcctgaaaag tatcggccta cgtggacact 25140
gtatgaccag ctcatgcttg aaggggatgc ctcaaacgtc agactcgcca gcttctccca 25200
gcctctatgc tgcgccgtac aaatcgttct ggtccgactt ctcgctgcag ctggtatcga 25260
gttcagtgca attgtcggcc acagttcagg tgagattgcc tgtgcctttg cggcaggatt 25320
catcagtgcc actcaagcta tccgtattgc gcatctgcgt ggagttgtgt ccgcggagca 25380
tgcctcttct ccaagcggcc agacaggcgc tatgctagcg gcaggtatgt cgtacgatga 25440
cgcaaaggaa ctatgcgagc tcgaagcctt tgagggtcgg gtctgcgtcg ccgctagcaa 25500
ttcaccggat agtgtgacct tctccggcga catggatgct atccagcacg ttgaaggtgt 25560
cttggaggat gaatccactt ttgccagaat cttgagagtt gacaaggcct accattcgca 25620
tcacatgcac ccatgcgcag ctccatatgt caaggcattg ctggagtgcg actgtgctgt 25680
tgccgatggc caaggtaacg atagtgttgc ttggttctct gccgtccacg agaccagcaa 25740
gcaaatgact gtacaggatg tgatgcccgc ttattggaaa gacaatctcg tctctccggt 25800
cttgttctcg caggctgtgc agaaagcagt catcactcat cgtctaatcg acgtcgccat 25860
cgaaattggc gcccaccctg ctctcaaggg tccgtgtcta gccaccatca aggatgctct 25920
tgccggtgtg gagctgccgt ataccgggtg cttggcacga aacgttgacg atgtggacgc 25980
ttttgctgga ggtctgggat acatttggga gcgtttcgga gttcggagta tcgacgccga 26040
gggcttcgta caacaagtcc ggcccgatcg tgccgttcaa aacctgtcaa agtcattgcc 26100
cacatactct tgggatcata ctcgtcaata ctgggcagaa tctcgctcca cccgccagca 26160
tcttcgtgga ggtgcgcccc atcttctgct tggaaagctt tcttcttaca gcacagcatc 26220
gaccttccag tggacaaact tcatcaggcc ccgggatctg gaatggctcg acggtcatgc 26280
gctacaaggc cagactgtgt tccccgctgc tgggtacata attatggcca tggaagctgc 26340
catgaaggtg gctggtgagc gtgccgccca agttcagctc ctggaaatct tggacatgag 26400
catcaacaaa gccatcgtgt ttgaagatga aaacacctcc gtggagctga acttgacagc 26460
cgaagtcacc agtgacaatg atgcggatgg ccaagtcacg gtcaaatttg ttattgattc 26520
ctgtctggca aaggagagtg agctttcgac atccgccaaa ggccaaatcg tcataaccct 26580
tggcgaggca tcaccgtcat cgcagctttt gccgccacct gaggaagagt acccccagat 26640
gaacaatgtc aacatcgatt tcttctatcg ggaacttgac ctccttgggt atgactacag 26700
caaagacttc cgtcgtttgc agaccatgag aagggccgac tccaaagcta gcggcacctt 26760
ggctttcctt ccacttaagg atgaattgcg caatgagccc ctcttgctcc acccagcgcc 26820
cctggacatc gcgttccaga ctgtcattgg agcgtattcc tctccaggag atcgtcgcct 26880
acgctcattg tacgtgccta ctcacgttga cagagtgact ctgattccat cgctctgtat 26940
atcggcgggt aattctggtg aaaccgagct tgcgtttgac acaatcaaca cacacgacaa 27000
gggtgatttc ctgagcggcg acatcacggt gtacgattcg accaagacaa cgcttttcca 27060
agttgataac attgtcttta agcctttctc tcccccgact gcttcgaccg accaccgaat 27120
cttcgcaaag tgggtctggg gacccctcac gcccgaaaaa ctgctggagg accctgcgac 27180
gttgatcata gctcgggaca aggaggacat tctgaccatc gagcgaatcg tttacttcta 27240
catcaaatcc ttcctagccc agataacccc cgacgaccgt caaaatgccg acctccattc 27300
ccagaagtac attgaatggt gtgaccaggt tcaggccgat gctcgggctg gccaccatca 27360
gtggtaccag gagtcttggg aggaggacac ttctgttcac attgagcaaa tgtgtgaaag 27420
gtacacccaa agctgttccg tgttttttca ttcttttata ttaacctttt acttgaagca 27480
actcgtccca cccacatgtg cgcctgatcc aaagggtagg caaagaatta atttcaattg 27540
ttcgcgggaa cggggatcct ttggatatca tgaaccgcga tgggttgttc accgagtact 27600
ataccaacaa gctcgccttt ggctcagcaa tacacgtcgt tcaggatctg gttagccaaa 27660
ttgctcatcg ctaccaatcc attgatatcc ttgagatcgg taagtcgaat ctgaaatgta 27720
agtaactagg cagtttgcta atctgtcgtt cgctttttag gcttgggtac aggcatcgcc 27780
acgaagcgcg ttcttgcatc acctcaactt ggtttcaaca gttacacttg cactgacatc 27840
tcggcggatg ttattggcaa ggcccgtgaa caactttccg aattcgacgg tctcatgcag 27900
tttgaggcac tagacatcaa cagaagccca gcagagcaag gattcaagcc tcactcctac 27960
gatctgatta ttgcatccga tgtcctccat gccagctcca acttcgagga aaaattggct 28020
cacataaggt ccttgctcaa gccgggtggt cacttggtta ctttcggggt cacccatcgc 28080
gagcctgctc gcctcgcctt catctctggg cttttcgctg atcgatggac tggagaagac 28140
gaaactcgtg ctttgagtgc ctcggggtcc gttgaccaat gggagcatac cctcaagaga 28200
gttgggttct ctggcgtcga tagtcggaca cttgatcgag aggatgattt gatcccgtct 28260
gtcttcagta cacatgctgt ggatgccacc gttgagcgtt tgtatgatcc actttctgct 28320
ccattgaagg actcataccc gccattagtg gttatcggtg gcgaatcgac aaaaaccgaa 28380
cgcattttga acgacatgaa agctgcccta ccgcatagac acatccactc cgtcaagcgg 28440
ctggaaagtg ttctcgacga cccggccttg cagcctaagt cgacttttgt catcctctcg 28500
gaacttgatg atgaagtgtt ttgcaacctt gaagaggaca agtttgaggc agtcaagtct 28560
cttctcttct acgccggacg catgatgtgg ctgacagaga atgcctggat tgatcatccc 28620
caccaggcca gcaccatcgg aatgttgagg acaatcaagc tcgagaaccc tgacttggga 28680
acgcacgtct tcgatgtcga tactgtggag aacctagaca ccaaattctt cgttgagcaa 28740
cttttgcgct tcgaggagag cgatgatcag cttttggaat caataacatg gactcatgag 28800
cccgaagtgt actggtgcaa gggtcgtgcc tgggtccctc gtttgaagca ggatattgct 28860
aggaacgacc gtatgaactc gtctcgtcgt ccaattttcg gtaactttaa ttcgtccaag 28920
acggccattg cactgaaaga ggcgagggga gcatcctcat cgatgtacta tcttgagtca 28980
accgagacgt gtgattcgtt agaagacgct cgtcatgctg gaaaagcaac tgttcgtgtt 29040
cgctacgctc ttccccaggc aattcgcgtg ggccatctcg gatacttcca tgtcgtgcag 29100
ggcagtattc tggagaatac atgtgaggtg cctgtagtcg ccctggctga gaagaatgga 29160
tctatactgc atgtaccgag aaactacatg catagtctgc ccgataacat ggcggaaggc 29220
gaggatagtt ccttcttgtt gtccacagct gcagccctcc ttgccgaaac aattctctct 29280
agcgctcagt cctttggctc tgatgcatca attctgatta tggagccccc aatcttctgc 29340
gtcaaagcaa ttctggagtc ggccaaaacc tacggtgttc aggttcattt ggcaacaact 29400
ctgtccgacg tcaaaactat tccggctcct tggatccgat tacatgccaa ggaaaccgac 29460
gctcggctga aacacagcct gccgacaaac atgatggcat tctttgactt gtctaccgac 29520
cggactgctg ccgggataac caaccgtttg gccaagttgc taccacccag ttgcttcatg 29580
tacagtggtg actatcttat ccgaagtaca gcttccacat acaaagttag tcatgttgag 29640
gatattccaa tcctcgagca ctctgtggca atggcaaaaa ataccgtctc tgcgtcgact 29700
gtcgacgaca ctgagaaagt tattacagcc acacaaattc tcttgcctgg tcagctctct 29760
gtcaaccaca atgaccaacg cttcaatctg gccaccgtca tcgactggaa ggaaaatgag 29820
gtgtccgcta ggatttgccc catcgactct ggtaacttat tttccaacaa gaagacgtat 29880
ttgcttgttg gtcttaccgg ggaccttggt cgctctctct gtcgctggat gatcttgcat 29940
ggcgcccgcc atgttgtgct cactagccgg aaccctcgac ttgatcccaa atggatcgcc 30000
aacatggagg cacttggtgg tgacatcacc gttctgtcaa tgtaagttga ttgatatcac 30060
atcacacctt gctaccacat cctcgtttac ttatccaatt actttcttta gggatgttgc 30120
caatgaggat tcagtcgatg ctggccttgg caagcttgtc gatatgaagt tgccacctgt 30180
tgccggcatc gcgttcgggc ctttggtgct gcaggatgtc atgctgaaga acatggacca 30240
ccagatgatg gacatggtgt tgaagcccaa ggtacaagga gcacgcattc ttcatgaacg 30300
gttctccgaa cagacgggca gcaaggcgct cgacttcttc atcatgtttt cgtccattgt 30360
tgcagttatt ggcaatcctg gccagtccaa ctatggcgct gcgaatgcct acctacaggc 30420
tctggcccag caacggtgcg ccagaggatt ggcggtattt tctacccctg aattatcatg 30480
catcgacgtc aagttactaa cgcacaacca cagggatcaa ccatcgatat tggtgccgtt 30540
tacggtgtag ggtttgtcac gagggccgag atggaggagg actttgatgc tatccgtttc 30600
atgtttgact cagttgaaga gcatgagctg cacacgcttt tcgccgaagc ggtcgtgtct 30660
gaccagcgtg cccggcagca accacagcgc aagacggtca ttgacatggc ggaccttgag 30720
cttaccacgg gtatcccaga tcttgaccct gcgcttcaag atcgaattat ttacttcaac 30780
gaccctcgtt tcggaaactt caaaattccc ggtcaacgcg gagacggtgg cgacaatgga 30840
tcagggtcta aaggctccat tgccgaccag ctcaaacaag caacaacttt agaccaagtt 30900
cggcaaatcg tgattggtaa gttatctctc atgcgtttcc tgatatcgag ttcaaactaa 30960
caaagttgca gatggtctat ctgagaaact ccgtgttacc ctccaagttt cggacgggga 31020
gagcgtggac ccaaccattc ctctcattga tcaaggtgtc gactccttgg gtgcagtgac 31080
tgtcggctca tggttctcaa agcaactcta ccttgacctc ccactcttga gggtacttgg 31140
cggtgcttct gtcgctgatc ttgccgacga cgcggccacc cgactcccag ctacatccat 31200
tccgctgctg ttgcaaattg gtgattccac gggaacctcg gacagcgggg cttctccgac 31260
accaacagac agccatgatg aagcaagctc tgctaccagc acagatgcgt cgtcagccga 31320
agaggatgaa gagcaagagg acgataatga gcagggaggc cgtaagattc ttcgtcgcga 31380
gaggttgtcc cttggccagg agtattcctg gaggcagcaa caaatggtaa aagatcatac 31440
catcttcaac aacactattg gcatgttcat gaagggtacc attgacctcg accggttgag 31500
gcgggctctg aaagcctcat tgcgccgtca cgagatcttc cgtacgtgct ttgttactgg 31560
cgatgactat agcagcgatt taaatggtcc cgtccaagtg gttctcaaga acccggagaa 31620
cagagtgcac tttgttcagg tgaacaacgc tgcggaggca gaggaagagt accggaaact 31680
cgagaagaca aactatagca tctccacagg tgacactctc agactcgttg atttctactg 31740
gggcacagat gaccacctgt tggtaatcgg ctaccacaga ttagttggtg atggctcaac 31800
aacagaaaac ctgttcaatg agatcgggca gatttacagc ggggtgaaaa tgcagcgacc 31860
atcgacccaa ttctctgatc tagccgtcca acagcgggaa aacctggaaa atgggcgaat 31920
gggggacgat atcgcgttct ggaagtccat gcatagcaaa gtctcgtcat ctgcgccaac 31980
cgtgcttccc atcatgaatc tgatcaatga ccctgctgcc aattcagagc agcagcaaat 32040
acagccattc acgtggcagc agtatgaagc aattgctcgt ttagatccca tggtcgcctt 32100
ccgaatcaaa gagcggagcc gcaagcacaa ggcaaccccc atgcagttct acctggccgc 32160
ctaccacgtt ttgttggcgc gtcttaccgg cagcaaagac ataaccatcg gcctcgccga 32220
aaccaaccga tccaccatgg aagaaatttc ggcgatgggc tttttcgcta acgtgcttcc 32280
cctgcgcttt gatgagttcg tcggcagcaa gacattcggc gagcaccttg tagccaccaa 32340
ggacagtgtg cgtgaggcca tgcaacacgc gcgggtgccg tatggcgtca tcctcgactg 32400
tctaggcctg aatctcccta cctcaggcga ggaacccaag actcagacac acgccccctt 32460
gttccaggct gtctttgatt acaagcaggg tcaagcggag agtggctcaa ttggcaatgc 32520
caaaatgacg agtgttctcg cttcccgtga gcgcactcct tatgacatcg ttctcgagat 32580
gtgggatgac cctaccaagg acccactcat tcatgtcaaa cttcagagct cgctgtatgg 32640
ccctgagcac gctcaggcct ttgtagacca cttttcttca atcctcacta tgttctcgat 32700
gaacccggct ctgaagttgg cctagatcgt tcagcgccgt gaattcagat gtgtggtttg 32760
agtgttgttc atgataaaga tggattagaa attggcaata gagcagatgg caaatctatc 32820
ctgaattcgg cgtcaattga cacacgcata ttcatctaca aatagcgaat tcgtcttgta 32880
tctttgtcaa aattacttct accttcgttg ctcttcttta ttgcagcaat cgtaacatca 32940
agttagatag cgcggttcag agtaccgtaa cggtgataaa tatacctcgg tagcgcgttt 33000
cgaaagactc tgtgaggaag gtgaaacctc caaggcttgg aattgatttc aatccatcct 33060
gtatataaat tcgacgccat tgcaaatagt tccatagtta ctggtttagt gccttgttgt 33120
ggtgatcgag tggttttaga tgtctgtcat gcctgttcag aacgagcctt ccatgatcta 33180
tccaaaatat gttcacgaaa tatttatgag atggtcgcga ccactataac taaatcaccc 33240
ttggaaggtg agcattcaaa ccgtgtaaga ttagaactat tcaaatttgt tcagtaaaaa 33300
tgtggtatgg actaggcatg agagccagag ccttgctata taccctgttg tctcacctag 33360
acaaatgaac ctgacatctt gaccttttga tatagctgtt ggaagcgctt gaccgtctcc 33420
tggacatcac tcggtctgtt gggaaaatta tgctttccct gaaactcgag tacatctgca 33480
ttctgaggca ggtaatgtgt ttcaaccatc tgtctcgacc cttggagagc aaaatcttga 33540
cgaccgtgaa gatgcagtgt cggcacgttg attattagct tgtcgtcgtc gtcttgcgcc 33600
tcggctctca tgtaatctct ggcttcatcg ctatagaaac agcaaatcaa aacagcaatg 33660
ctcattttcg gaaaccatgg cagttttccc atttgctgtt gatggagcag caaagtggcg 33720
accaatgcgc cctcagagaa ggccactatg ccgacaatgg gtgcctgtgg gttagttata 33780
gaccaatctt ggacggtctt ttgcacaggc ccgatcacag ccgctactct atcgcccacc 33840
gtgggggttg tcgtgtttgt aacggcgtca tgatgctttt ggaaccaggt gtagtatgga 33900
cccatgcctt ggaagacagg aagcacgccg ggtccggggc tggagctaaa cggcgcggtc 33960
gcatatacga attcaaactc gtttttcaac gccacgcgca gtttagagat ctggacgcgg 34020
aatatggctg ctgagcaccc ggcaccgtgg atgcataaga gagcttttct cggtttgcct 34080
ggcgagaaat ctgtaatcct cgctggactc attttctctt gtggtgtgag ctgtgacttc 34140
gtctgttctg gggaatttgt tagtcattac tgacaaggaa ataacaacga cgtagtattg 34200
atc 34203
<210>3
<211>17
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>人工序列的说明:具有由黄曲霉PKS的氨基酸序列推断的
DNA序列的混合引物
<220>
<221>修饰碱基
<222>(6)..(6)
<223>i
<220>
<221>修饰碱基
<222>(9)..(9)
<223>i
<400>3
gayacngcnt gyasttc 17
<210>4
<211>17
<212>DNA
<213>人工序列
<220>
<221>misc_feature
<223>人工序列的说明:具有由黄曲霉PKS的氨基酸序列推断的
DNA序列的混合引物
<220>
<221>修饰碱基
<222>(3)..(3)
<223>i
<220>
<221>修饰碱基
<222>(6)..(6)
<223>i
<220>
<221>修饰碱基
<222>(8)..(8)
<223>i
<220>
<221>修饰碱基
<222>(15)..(15)
<223>i
<400>4
tcnccnknrc wgtgncc 17
<210>5
<211>19
<212>DNA
<213>柑桔青霉
<400>5
gcatgttcaa tttgctctc 19
<210>6
<211>19
<212>DNA
<213>柑桔青霉
<400>6
ctggatcaga cttttctgc 19
<210>7
<211>18
<212>DNA
<213>柑桔青霉
<400>7
gtcgcagtag catgggcc 18
<210>8
<211>20
<212>DNA
<213>柑桔青霉
<400>8
gtcagagtga tgctcttctc 20
<210>9
<211>20
<212>DNA
<213>柑桔青霉
<400>9
gttgagagga ttgtgagggc 20
<210>10
<211>19
<212>DNA
<213>柑桔青霉
<400>10
ttgcttgtgt tggattgtc 19
<210>11
<211>20
<212>DNA
<213>柑桔青霉
<400>11
catggtactc tcgcccgttc 20
<210>12
<211>19
<212>DNA
<213>柑桔青霉
<400>12
ctccccagta cgtaagctc 19
<210>13
<211>21
<212>DNA
<213>柑桔青霉
<400>13
ccataatgag tgtgactgtt c 21
<210>14
<211>19
<212>DNA
<213>柑桔青霉
<400>14
gaacatctgc atccccgtc 19
<210>15
<211>20
<212>DNA
<213>柑桔青霉
<400>15
ggaaggc aaa gaaagtgtac 20
<210>16
<211>21
<212>DNA
<213>柑桔青霉
<400>16
agattcattg ctgttggcat c 21
<210>17
<211>722
<212>DNA
<213>柑桔青霉
<400>17
ggccacgcgt cgactagtac gggggggggg gggggggggg gcttgttcgc tcagagattc 60
aactctgcga ttctgtttaa tcccaatcct atcgcccaaa aacaggatca gcagttatgg 120
atcaagccaa ctatccaaac gagccaattg tggtagtggg aagcggttgt cggtttccag 180
gtggtgtcaa cacaccatca aaactttggg agctgctcaa agagccccgg gatgtacaga 240
ccaagatccc taaggagaga tttgacgtcg atacatttta cagccccgat ggcactcacc 300
ccgggcgcac gaacgcaccc tttgcatact tgctgcagga ggatctacgc ggttttgatg 360
cctctttctt caacatccaa gctggagagg ccgaaacgat tgacccacag caaaggctgc 420
tgctggagac ggtctatgaa gctgtatcca acgcaggcct acggatccaa ggccttcaag 480
gatcctctac tgctgtgtac gtcggtatga tgacgcatga ctatgagact atcgtgacgc 540
gtgaattgga tagtattcct acatactctg ccacgggggt agctgtcagt gtggcctcca 600
accgtgtatc atacttcttc gactggcatg ggccgagtat gacgatcgac acagcctgta 660
gttcatcctt agctgccgtg catctggccg tccaacagct tagaacgggc gagagtacca 720
tg 722
<210>18
<211>760
<212>DNA
<213>柑桔青霉
<400>18
ggccacgcgt cgactagtac gggggggggg gggggggggg gactatcaac ggttttatca 60
ccagggcgac tgatatatca gtcaatgaaa caacgttgga atgaacaata cccccgccgt 120
aaccgcaacc gcaaccgcaa ccgcaaccgc aaccgcaatg gcaggctcgg cttgctctaa 180
cacatccacg cccattgcca tagttggaat gggatgtcga tttgctggag atgcaacgag 240
tccacagaag ctttgggaaa tggttgaaag aggaggcagt gcctggtcta aggtcccctc 300
ctcgcgattc aatgtgagag gagtatacca cccgaatggc gaaagggtcg ggtccaccca 360
cgtaaagggt ggacacttca tcgacgagga tcctgcttta tttgacgccg cgttcttcaa 420
catgaccaca gaggtcgcca gctgcatgga tccgcagtat cggcttatgc ttgaggtggt 480
ctacgaatcg ctggagagtg ccggtatcac catcgatggt atggcaggct ctaatacgtc 540
ggtgtttggg ggtgtcatgt accacgacta tcaggattcg ctcaatcgtg accccgagac 600
agttccgcgt tatttcataa ctggcaactc aggaacaatg ctttcgaacc ggatatcaca 660
cttctacgac ttacgtggtc ccagcgtgac ggttgacacg gcctgttcga cgacattgac 720
cgcactgcac ttggcgtgcc agagcttacg tactggggag 760
<210>19
<211>773
<212>DNA
<213>柑桔青霉
<400>19
ggccacgcgt cgactagtac gggggggggg ggtttttttt ttttcaaggt tgactggaag 60
agtgctctcg gccacaaaat cccagaagca ttagtgctgt tattcgatta taaaccgtcg 120
cagcgctctc attcttcgct ctttcttctt ttccactggt gtgcataggt cctatctgtc 180
tcacgcaatg ctcggccagg ttcttctgac cgtcgaatcg taccaatggg tatcgacccc 240
tcaagccctt gtggcggtcg cagtgcttct tagtctcatc gcctaccgtt tgcgggggcg 300
ccagtccgaa ctgcaagtct ataatcccaa aaaatggtgg gagttgacga ccatgagggc 360
taggcaggac ttcgatacgt atggtccgag ctggatcgaa gcttggttct cgaaaaacga 420
caagcccctg cgcttcattg ttgattccgg ctattgcacc atcctcccat cgtccatggc 480
cgacgagttt cggaaaatca aagatatgtg catgtacaag tttttggcgg atgactttca 540
ctctcatctc cctggattcg acgggttcaa ggaaatctgc caggatgcac atcttgtcaa 600
caaagttgtt ttgaaccagt tacaaaccca agcccccaag tacacaaagc cattggctac 660
cttggccgac gctactattg ccaagttgtt cggtaaaagc gaggagtggc aaaccgcacc 720
tgtctattcc aatggattgg accttgtcac acgaacagtc acactcatta tgg 773
<210>20
<211>527
<212>DNA
<213>柑桔青霉
<400>20
ggccacgcgt cgactagtac gggggggggg gtacctagga actgttcagt tgtccctccc 60
aaccccttgg gccgaacaac cttcctccaa tctacgacgg cagattatac ctaggcgcct 120
aaccgattag gttgctcatt cgattttgga gagactacct agctataggt accactccaa 180
gctgtagcac agacctttca gcatggtcgc ttcgttgcta ccctctcgct ttcgcggtag 240
ggaatcaatg aatcagcagc accctctacg ctcgggaaat cgggcattga cctccacact 300
ccaatttcta tccaaaacgg cgtgtctaca cccgatccat accgtttgca ccatagctat 360
tctagctagt accacatacg ttggactact caaagacagc ttcttccatg gccccgcaaa 420
cgttgataaa gcagaatggg gctctttggt cgaaggaagt cgaagcttga tcaccggccc 480
acagaatggc tggaagtggc agagcttcga cggggatgca gatgttc 527
<210>21
<211>522
<212>DNA
<213>柑桔青霉
<400>21
ggccacgcgt cgactagtac gggggggggg gggggggggg ggatccatca atctgacttc 60
aggctagcgg accttaacga aacaacgaga gcgagatcat tcatacacca aaacacaggt 120
actatagaag cgccgcgcag tagagattca caccgcccct tgaagcaaaa gtcggaagga 180
attgcgcgat gtcagaacct ctacccccta aagaagggga accaaggcca cagaaggaag 240
aaagtcaaaa tgacacgctc gaagcgactg agtccaagtc ccagcacatc acaggcctca 300
agctcgggct ggtggttgct tcagttactt tcgtagcatt tttgatgctc cttgatatgt 360
ccattatcgt cacggcaatc ccacatatca caagcgagtt ccactctctg aacgatgtag 420
ggtggtacgg cagtgcttat cttctggcta actgtgctct ccagcccctg gccggtaaat 480
tgtatacact cttgggcttg aagtacactt tctttgcctt cc 522
<210>22
<211>541
<212>DNA
<213>柑桔青霉
<400>22
ggccacgcgt cgactagtac gggggggggg ggctcacctc acattatttg atcttaatcc 60
aataattatg tccctgccgc atgcaacgat tccgacgaac ctacgccgtc gcgcgtttcg 120
acgctcatgt gaccggtgtc atgcacaaaa gctcaaatgt accggtagca atgccaattt 180
agtccgtgct cagtgtcaac gttgtcagca agccggatta aggtgtgtgt acagcgaaag 240
gctacccaag cgcaatttac ataaagaagc cgcagctgga actacaagag ccacagaaac 300
ctcacaaccg atgaccgcga catcttctac ggtcttctca tcattggcag agactcctcc 360
accttactgc tcaccaccta cgcatattgg cacctcggca ctcaaggaaa cattatcaga 420
accatcagcg gcaaccctgc aattctatga tacatcaatc aactttgatg atcccgagtc 480
gtttcccggc ggctggcctc agccaaatac atttcgcgac gatgccaaca gcaatgaatc 540
t 541
<210>23
<211>20
<212>DNA
<213>柑桔青霉
<400>23
atcataccat cttcaacaac 20
<210>24
<211>20
<212>DNA
<213>柑桔青霉
<400>24
gctagaatag gttacaagcc 20
<210>25
<211>20
<212>DNA
<213>柑桔青霉
<400>25
acattgccag gcacccagac 20
<210>26
<211>20
<212>DNA
<213>柑桔青霉
<400>26
caacgcccaa gctgccaatc 20
<210>27
<211>20
<212>DNA
<213>柑桔青霉
<400>27
gtcttttcct actatctacc 20
<210>28
<211>20
<212>DNA
<213>柑桔青霉
<400>28
ctttcccagc tgctactatc 20
<210>29
<211>1524
<212>DNA
<213>柑桔青霉
<400>29
aactggaaga attcgcggcc gcaggaattt tttttttttt tttttttcaa cgaaggtaga 60
agtaattttg acaaagatac aagacgaatt cgctatttgt agatgaatat gcgtgtgtca 120
attgaagccg aattcaggat agatttgcca tctgctctat tgccaatttc taatccatct 180
ttatcatgaa caacactcaa accacacatc tgaattcacg gcgctgaacg atctaggcca 240
acttcagagc cgggttcatc gagaacatag tgaggattga agaaaagtgg tctacaaagg 300
cctgagcgtg ctcagggcca tacagcgagc tctgaagttt gacatgaatg agtgggtcct 360
tggtagggtc atcccacatc tcgagaacga tgtcataagg agtgcgctca cgggaagcga 420
gaacactcgt cattttggca ttgccaattg agccactctc cgcttgaccc tgcttgtaat 480
caaagacagc ctggaacaag ggggcgtgtg tctgagtctt gggttcctcg cctgaggtag 540
ggagattcag gcctagacag tcgaggatga cgccatacgg cacccgcgcg tgttgcatgg 600
cctcacgcac actgtccttg gtggctacaa ggtgctcgcc gaatgtcttg ctgccgacga 660
actcatcaaa gcgcagggga agcacgttag cgaaaaagcc catcgccgaa atttcttcca 720
tggtggatcg gttggtttcg gcgaggccga tggttatgtc tttgctgccg gtaagacgcg 780
ccaacaaaac gtggtaggcg gccaggtaga actgcatggg ggttgccttg tgcttgcggc 840
tccgctcttt gattcggaag gcgaccatgg gatctaaacg agcaattgct tcatactgct 900
gccacgtgaa tggctgtatt tgctgctgct ctgaattggc agcagggtca ttgatcagat 960
tcatgatggg aagcacggtt ggcgcagatg acgagacttt gctatgcatg gacttccaga 1020
acgcgatatc gtcccccatt cgcccatttt ccaggttttc ccgctgttgg acggctagat 1080
cagagaattg ggtcgatggt cgctgcattt tcaccccgct gtaaatctgc ccgatctcat 1140
tgaacaggtt ttctgttgtt gagccatcac caactaatct gtggtagccg attaccaaca 1200
ggtggtcatc tgtgccccag tagaaatcaa cgagtctgag agtgtcacct gtggagatgc 1260
tatagtttgt cttctcgagt ttccggtact cttcctctgc ctccgcagcg ttgttcacct 1320
gaacaaagtg cactctgttc tccgggttct tgagaaccac ttggacggga ccatttaaat 1380
cgctgctata gtcatcgcca gtaacaaagc acgtacggaa gatctcgtga cggcgcaatg 1440
aggctttcag agcccgcctc aaccggtcga ggtcaatggt acccttcatg aacatgccaa 1500
tagtgttgtt gaagatggta tgat 1524
<210>30
<211>784
<212>DNA
<213>柑桔青霉
<400>30
aactggaaga attcgcggcc gcaggaattt tttttttttt tttttttttc tttgttgctt 60
ctcagggcca ctgtaatggt atttcaggta tctctattta ctgctatcca gaagtcaggc 120
attaaatagt caggctcagc ccaggctcga ttcagattgg attcaggctt cagaccatgg 180
ccgctatgct ccttcgtact atacctccgt cgagctatac ccgcttggcc agacaaaagg 240
cttcactgaa cccttcaact taactgcatt tcgccacaac taactcgacg aggccggcga 300
tggtgttacc attcatgagc tcaaagatcg acacatcaac atggatttca gatgtgatcc 360
agtttcgaag ttcaatggcg acgagtgagt ctacgccgac acctgccagg tttttggacg 420
aggacatgtc gtcttctgcc agaccaaaca ttcgcatcag cttttccgtc attgctttga 480
ggacgataga aatggcctcg tcgtgagagg tgaccctgct tagttgggcc cgcacgccat 540
ctggtccttt tttatgcgaa gagacaaagg attggtctgc atgaaggact tggcggtatt 600
taagtcccac aaaccgctgt tcctgtatcc agtttgcctc ggtccagtga gcacccgggg 660
atgtgttgat tcctgtaacc acagctgcgg gaggtgatgg aaattgaggg gaagaacaca 720
ggattgcctt ctccaacaca tccatgacgt ccttttcatg cataggcttg taacctattc 780
tagc 784
<210>31
<211>764
<212>DNA
<213>柑桔青霉
<400>31
aactggaaga attcgcggcc gcaggaattt tttttttttt tttttttttc gaataaaatg 60
cgttttattt tactaaccta ctcgactaat acagcaccta gtttctctgg gacggaaacc 120
attggaataa gcctggggac ggatgcatat ttgttttagt ttgcgtgtta tatcttagca 180
ccggtcatga gggagcggga tgtcctcgtt gcgccggcgt accatgagct ttgtggttgg 240
atgcatacga acgctaaaag cgtgacggta gtatttgtca tcgtctcctg gtacaggctt 300
cacatcatac tgaatcagta tatgagcgag gagaatcttg atttccttcg aggcgaagaa 360
ccgcccggga caagcgcgtg ggttccagcc gaagccgatg tgatcaccgt tggtattctc 420
caattgagcg gtgaaggcct tgtctggatc ctcgcgcatg cgcataaatc ggtagggatc 480
ataattttcg gggttttccc acacatcagg gttgttcatg cggtctgcag ccacagcggc 540
caactcgccc ttgggaatga agaggccatt ggatagagtg atgtctctga gagcggtact 600
gcgcatagtg gcgcactcga ccggcttgat tcgctgcgtc tctttcatgc agctgtcgag 660
gagcttcagc ttgaacagag aggcaggcgt ccagccccct tctccgatta cagtgcggat 720
ctcttggcgg agaggctgaa taaggtctgg gtgcctggca atgt 764
<210>32
<211>765
<212>DNA
<213>柑桔青霉
<400>32
aactggaaga attcgcggcc gcaggaattt tttttttttt ttttttctgg aaaaggacca 60
tctctttata tattcttctt ccctactact tgcatcgtaa atttcaacaa catataaaca 120
tgagataccc tttctggccg ttcactctac cacctgcctg tctcattgca ttgtgctttt 180
gaaaattatg acaataacaa ccaatgagaa aaaatatgat cctcctgcaa tgaatccact 240
ggagggggta cggagcttgg aatgctccta agattccgac ctaatcagcg tcgagcccga 300
tcagtagctg cagcactcgg cctcagtgca ttgttaggaa cagggactgt cctggttccg 360
cctgacgggg agacacttcg agaaggggct gaagatgccg gggcagaacg gttgtgcgcc 420
atgtgcgcct tgaccaggtg accggcggct agggcagcac atagcgagag ctccccagcc 480
aaaacagcgc ttccgatgat gcgcgcaagt tgacgtgcat tctcaccggg agtggtcggg 540
tgtgatccgc ggacaccaag catgtcaagc attgcgccct ggggctccag aatcgtacca 600
ccgcccaacg ttccaacctc aatagacggc atggagacag agatttgaag cgatccgcga 660
agattgttca tgagagtgat gcagttagcg ctctccacaa cttgcgccgg atcctgacct 720
gtggcaatga aaatggctgc cgcaagattg gcagcttggg cgttg 765
<210>33
<211>802
<212>DNA
<213>柑桔青霉
<400>33
aactggaaga attcgcggcc gcaggaattt tttttttttt tttttataga atctttgaaa 60
tcgacattaa ttaagtatgt ggagattctt tgtggaggca cggtaatgtg tctatctagc 120
aacgcggtca agcatcagtc tcaggcacag cccgggtgtc gtttttggtt gcaatcttcc 180
gccatcccat tccaaaggca aacacaaacg tgcacgccgt agctcccact gctaagtaaa 240
aagtatgatc aacggcgaga ctgtaagctt ttacaacccc tggaaggtta ttcttgctga 300
ccacatctct gaagccagtc gcccctgctg ccgtcacggc ctgcgtgtcg acagtgggcg 360
catacttgct caggccagtt ctcaaaccgg acccaaagac aaggttagca aagtccagga 420
agagcgatcc tccaaacgtc tgtccaaaca cggcgagaga aattccgagg gcaccttgtt 480
cgggcgaaag cgtgctttgg atggcgatga taggcgtttg catgccacaa ccacgaccga 540
agcccgcgat aaattggtac atgacccatt tcacagttga tgtatggggc tggaaggtgg 600
ataccagacc tgcgcctatg gcgacgagaa cagcgctgcc tagggcccaa ggcaaatagt 660
atcctgtctt tccaattgcg aagccagaaa ccatagccat aatgacttgt ccaagaattc 720
caggcaacat gtacacacca ctcagtgtgg gagaaacatc cttcacagcc tggaagtaga 780
tcggtagata gtaggaaaag ac 802
<210>34
<211>562
<212>DNA
<213>柑桔青霉
<400>34
aactggaaga attcgcggcc gcaggaattt tttttttttt ttttttttac taagcaatat 60
tgtgtttctt cgctaatgcg aatatttcct tatagcaacg tcgcaacaca tttatcgtct 120
tccctgaggc ctttgttgac ttgggctctt cgtctccggc ttcgtcactc caaagcacag 180
ataggagacg agaggccggc gttatggttt tattttcagc gccaaggatt tgccacgatg 240
tgcttggcat atctgatagg actagacgaa tagatgccgc agccccgtgc tcctgtgcta 300
tccccaaagc agtctcaatc ccactcaata gtcgaaggct tacacgcaat gtcgtgcatg 360
cagaagataa ggcgtgcatg aatgggtcga gatgtgaaat gagctcgccg atatgaagat 420
tagagtgaaa cgagggaagt gcttcggctc ttccattgtc atttctagtg gttgagccag 480
accagtacca atccattcgt gtgctttgct tttgtccaca aggttgggct ttcatcacct 540
cggatagtag cagctgggaa ag 562
<210>35
<211>26
<212>DNA
<213>柑桔青霉
<400>35
gttaacatgt cagaacctct accccc 26
<210>36
<211>27
<212>DNA
<213>柑桔青霉
<400>36
aatatttcaa gcatcagtct caggcac 27
<210>37
<211>1662
<212>DNA
<213>柑桔青霉
<220>
<221>CDS
<222>(1)..(1662)
<400>37
atg tca gaa cct cta ccc cct aaa gaa ggg gaa cca agg cca cag aag 48
Met Ser Glu Pro Leu Pro Pro Lys Glu Gly Glu Pro Arg Pro Gln Lys
1 5 10 15
gaa gaa agt caa aat gac acg ctc gaa gcg act gag tcc aag tcc cag 96
Glu Glu Ser Gln Asn Asp Thr Leu Glu Ala Thr Glu Ser Lys Ser Gln
20 25 30
cac atc aca ggc ctc aag ctc ggg ctg gtg gtt gct tca gtt act ttc 144
His Ile Thr Gly Leu Lys Leu Gly Leu Val Val Ala Ser Val Thr Phe
35 40 45
gta gca ttt ttg atg ctc ctt gat atg tcc att atc gtc acg gca atc 192
Val Ala Phe Leu Met Leu Leu Asp Met Ser Ile Ile Val Thr Ala Ile
50 55 60
cca cat atc aca agc gag ttc cac tct ctg aac gat gta ggg tgg tac 240
Pro His Ile Thr Ser Glu Phe His Ser Leu Asn Asp Val Gly Trp Tyr
65 70 75 80
ggc agt gct tat ctt ctg gct aac tgt gct ctc cag ccc ctg gcc ggt 288
Gly Ser Ala Tyr Leu Leu Ala Asn Cys Ala Leu Gln Pro Leu Ala Gly
85 90 95
aaa ttg tat aca ctc ttg ggc ttg aag tac act ttc ttt gcc ttc ctc 336
Lys Leu Tyr Thr Leu Leu Gly Leu Lys Tyr Thr Phe Phe Ala Phe Leu
100 105 110
tgt att ttt gaa cta ggc tcg gtg cta tgc ggt gcc gca aga tct tcc 384
Cys Ile Phe Glu Leu Gly Ser Val Leu Cys Gly Ala Ala Arg Ser Ser
115 120 125
acc atg ttg att gtt ggg cgg gcc gtt gct gga atg gga ggc tca ggt 432
Thr Met Leu Ile Val Gly Arg Ala Val Ala Gly Met Gly Gly Ser Gly
130 135 140
ctt gtc aac gga gcc ctc aca atc ctc tca aca gct gct cct aag cac 480
Leu Val Asn Gly Ala Leu Thr Ile Leu Ser Thr Ala Ala Pro Lys His
145 150 155 160
aag caa cca gtt ttg att gga gtg atg atg ggt ctt agt cag att gcc 528
Lys Gln Pro Val Leu Ile Gly Val Met Met Gly Leu Ser Gln Ile Ala
165 170 175
att gtc tgt gga cca ctg ctc gga ggt gct ttc act caa cac gcc act 576
Ile Val Cys Gly Pro Leu Leu Gly Gly Ala Phe Thr Gln His Ala Thr
180 185 190
tgg cga tgg tgc ttt tat atc aat ctc ccc atc ggc gct gtc gct gca 624
Trp Arg Trp Cys Phe Tyr Ile Asn Leu Pro Ile Gly Ala Val Ala Ala
195 200 205
ttc ctc ctt ctc gtc atc acc ata ccc gac cga att tca tcc acg gac 672
Phe Leu Leu Leu Val Ile Thr Ile Pro Asp Arg Ile Ser Ser Thr Asp
210 215 220
agc gaa ctc tcg acc gac aaa cca atg gcc aac ata aaa tcc aca ctt 720
Ser Glu Leu Ser Thr Asp Lys Pro Met Ala Asn Ile Lys Ser Thr Leu
225 230 235 240
cgc aaa ctg gac ctt gta ggc ttt gtg gtc ttt gca gcc ttc gca acc 768
Arg Lys Leu Asp Leu Val Gly Phe Val Val Phe Ala Ala Phe Ala Thr
245 250 255
atg att tcc ctc gca cta gaa tgg gga ggg tcg acc tac acc tgg cga 816
Met Ile Ser Leu Ala Leu Glu Trp Gly Gly Ser Thr Tyr Thr Trp Arg
260 265 270
agt tcc gtc atc atc ggc ctg ttc tgt ggc gga ggg ttt gct ctg att 864
Ser Ser Val Ile Ile Gly Leu Phe Cys Gly Gly Gly Phe Ala Leu Ile
275 280 285
gcg ttc gtg cta tgg gag cgt cat gtt ggc gat gct gtt gcc atg att 912
Ala Phe Val Leu Trp Glu Arg His Val Gly Asp Ala Val Ala Met Ile
290 295 300
cct ggc tca gtg gct ggt aaa cga caa gtg tgg tgc tct tgt tta ttt 960
Pro Gly Ser Val Ala Gly Lys Arg Gln Val Trp Cys Ser Cys Leu Phe
305 310 315 320
atg ggc ttt ttc tct ggc tcc ttg ctt gtc ttt tcc tac tat cta ccg 1008
Met Gly Phe Phe Ser Gly Ser Leu Leu Val Phe Ser Tyr Tyr Leu Pro
325 330 335
atc tac ttc cag gct gtg aag gat gtt tct ccc aca ctg agt ggt gtg 1056
Ile Tyr Phe Gln Ala Val Lys Asp Val Ser Pro Thr Leu Ser Gly Val
340 345 350
tac atg ttg cct gga att ctt gga caa gtc att atg gct atg gtt tct 1104
Tyr Met Leu Pro Gly Ile Leu Gly Gln Val Ile Met Ala Met Val Ser
355 360 365
ggc ttc gca att gga aag aca gga tac tat ttg cct tgg gcc cta ggc 1152
Gly Phe Ala Ile Gly Lys Thr Gly Tyr Tyr Leu Pro Trp Ala Leu Gly
370 375 380
agc gct gtt ctc gtc gcc ata ggc gca ggt ctg gta tcc acc ttc cag 1200
Ser Ala Val Leu Val Ala Ile Gly Ala Gly Leu Val Ser Thr Phe Gln
385 390 395 400
ccc cat aca tca act gtg aaa tgg gtc atg tac caa ttt atc gcg ggc 1248
Pro His Thr Ser Thr Val Lys Trp Val Met Tyr Gln Phe Ile Ala Gly
405 410 415
ttc ggt cgt ggt tgt ggc atg caa acg cct atc atc gcc atc caa agc 1296
Phe Gly Arg Gly Cys Gly Met Gln Thr Pro Ile Ile Ala Ile Gln Ser
420 425 430
acg ctt tcg ccc gaa caa ggt gcc ctc gga att tct ctc gcc gtg ttt 1344
Thr Leu Ser Pro Glu Gln Gly Ala Leu Gly Ile Ser Leu Ala Val Phe
435 440 445
gga cag acg ttt gga gga tcg ctc ttc ctg gac ttt gct aac ctt gtc 1392
Gly Gln Thr Phe Gly Gly Ser Leu Phe Leu Asp Phe Ala Asn Leu Val
450 455 460
ttt ggg tcc ggt ttg aga act ggc ctg agc aag tat gcg ccc act gtc 1440
Phe Gly Ser Gly Leu Arg Thr Gly Leu Ser Lys Tyr Ala Pro Thr Val
465 470 475 480
gac acg cag gcc gtg acg gca gca ggg gcg act ggc ttc aga gat gtg 1488
Asp Thr Gln Ala Val Thr Ala Ala Gly Ala Thr Gly Phe Arg Asp Val
485 490 495
gtc agc aag aat aac ctt cca ggg gtt gta aaa gct tac agt ctc gcc 1536
Val Ser Lys Asn Asn Leu Pro Gly Val Val Lys Ala Tyr Ser Leu Ala
500 505 510
gtt gat cat act ttt tac tta gca gtg gga gct acg gcg tgc acg ttt 1584
Val Asp His Thr Phe Tyr Leu Ala Val Gly Ala Thr Ala Cys Thr Phe
515 520 525
gtg ttt gcc ttt gga atg gga tgg cgg aag att gca acc aaa aac gac 1632
Val Phe Ala Phe Gly Met Gly Trp Arg Lys Ile Ala Thr Lys Asn Asp
530 535 540
acc cgg gct gtg cct gag act gat gct tga 1662
Thr Arg Ala Val Pro Glu Thr Asp Ala
545 550
<210>38
<211>553
<212>PRT
<213>柑桔青霉
<400>38
Met Ser Glu Pro Leu Pro Pro Lys Glu Gly Glu Pro Arg Pro Gln Lys
1 5 10 15
Glu Glu Ser Gln Asn Asp Thr Leu Glu Ala Thr Glu Ser Lys Ser Gln
20 25 30
His Ile Thr Gly Leu Lys Leu Gly Leu Val Val Ala Ser Val Thr Phe
35 40 45
Val Ala Phe Leu Met Leu Leu Asp Met Ser Ile Ile Val Thr Ala Ile
50 55 60
Pro His Ile Thr Ser Glu Phe His Ser Leu Asn Asp Val Gly Trp Tyr
65 70 75 80
Gly Ser Ala Tyr Leu Leu Ala Asn Cys Ala Leu Gln Pro Leu Ala Gly
85 90 95
Lys Leu Tyr Thr Leu Leu Gly Leu Lys Tyr Thr Phe Phe Ala Phe Leu
100 105 110
Cys Ile Phe Glu Leu Gly Ser Val Leu Cys Gly Ala Ala Arg Ser Ser
115 120 125
Thr Met Leu Ile Val Gly Arg Ala Val Ala Gly Met Gly Gly Ser Gly
130 135 140
Leu Val Asn Gly Ala Leu Thr Ile Leu Ser Thr Ala Ala Pro Lys His
145 150 155 160
Lys Gln Pro Val Leu Ile Gly Val Met Met Gly Leu Ser Gln Ile Ala
165 170 175
Ile Val Cys Gly Pro Leu Leu Gly Gly Ala Phe Thr Gln His Ala Thr
180 185 190
Trp Arg Trp Cys Phe Tyr Ile Asn Leu Pro Ile Gly Ala Val Ala Ala
195 200 205
Phe Leu Leu Leu Val Ile Thr Ile Pro Asp Arg Ile Ser Ser Thr Asp
210 215 220
Ser Glu Leu Ser Thr Asp Lys Pro Met Ala Asn Ile Lys Ser Thr Leu
225 230 235 240
Arg Lys Leu Asp Leu Val Gly Phe Val Val Phe Ala Ala Phe Ala Thr
245 250 255
Met Ile Ser Leu Ala Leu Glu Trp Gly Gly Ser Thr Tyr Thr Trp Arg
260 265 270
Ser Ser Val Ile Ile Gly Leu Phe Cys Gly Gly Gly Phe Ala Leu Ile
275 280 285
Ala Phe Val Leu Trp Glu Arg His Val Gly Asp Ala Val Ala Met Ile
290 295 300
Pro Gly Ser Val Ala Gly Lys Arg Gln Val Trp Cys Ser Cys Leu Phe
305 310 315 320
Met Gly Phe Phe Ser Gly Ser Leu Leu Val Phe Ser Tyr Tyr Leu Pro
325 330 335
Ile Tyr Phe Gln Ala Val Lys Asp Val Ser Pro Thr Leu Ser Gly Val
340 345 350
Tyr Met Leu Pro Gly Ile Leu Gly Gln Val Ile Met Ala Met Val Ser
355 360 365
Gly Phe Ala Ile Gly Lys Thr Gly Tyr Tyr Leu Pro Trp Ala Leu Gly
370 375 380
Ser Ala Val Leu Val Ala Ile Gly Ala Gly Leu Val Ser Thr Phe Gln
385 390 395 400
Pro His Thr Ser Thr Val Lys Trp Val Met Tyr Gln Phe Ile Ala Gly
405 410 415
Phe Gly Arg Gly Cys Gly Met Gln Thr Pro Ile Ile Ala Ile Gln Ser
420 425 430
Thr Leu Ser Pro Glu Gln Gly Ala Leu Gly Ile Ser Leu Ala Val Phe
435 440 445
Gly Gln Thr Phe Gly Gly Ser Leu Phe Leu Asp Phe Ala Asn Leu Val
450 455 460
Phe Gly Ser Gly Leu Arg Thr Gly Leu Ser Lys Tyr Ala Pro Thr Val
465 470 475 480
Asp Thr Gln Ala Val Thr Ala Ala Gly Ala Thr Gly Phe Arg Asp Val
485 490 495
Val Ser Lys Asn Asn Leu Pro Gly Val Val Lys Ala Tyr Ser Leu Ala
500 505 510
Val Asp His Thr Phe Tyr Leu Ala Val Gly Ala Thr Ala Cys Thr Phe
515 520 525
Val Phe Ala Phe Gly Met Gly Trp Arg Lys Ile Ala Thr Lys Asn Asp
530 535 540
Thr Arg Ala Val Pro Glu Thr Asp Ala
545 550
<210>39
<211>31
<212>DNA
<213>柑桔青霉
<400>39
ggatccatgt ccctgccgca tgcaacgatt c 31
<210>40
<211>30
<212>DNA
<213>柑桔青霉
<400>40
ggatccctaa gcaatattgt gtttcttcgc 30
<210>41
<211>1380
<212>DNA
<213>柑桔青霉
<220>
<221>CDS
<222>(1)..(1380)
<400>41
atg tcc ctg ccg cat gca acg att ccg acg aac cta cgc cgt cgc gcg 48
Met Ser Leu Pro His Ala Thr Ile Pro Thr Asn Leu Arg Arg Arg Ala
1 5 10 15
ttt cga cgc tca tgt gac cgg tgt cat gca caa aag ctc aaa tgt acc 96
Phe Arg Arg Ser Cys Asp Arg Cys His Ala Gln Lys Leu Lys Cys Thr
20 25 30
ggt agc aat gcc aat tta gtc cgt gct cag tgt caa cgt tgt caa caa 144
Gly Ser Asn Ala Asn Leu Val Arg Ala Gln Cys Gln Arg Cys Gln Gln
35 40 45
gcc gga tta agg tgt gtg tac agc gaa agg cta ccc aag cgc aat tta 192
Ala Gly Leu Arg Cys Val Tyr Ser Glu Arg Leu Pro Lys Arg Asn Leu
50 55 60
cat aaa gaa gcc gca gct gga act aca aga gcc aca gaa acc tca caa 240
His Lys Glu Ala Ala Ala Gly Thr Thr Arg Ala Thr Glu Thr Ser Gln
65 70 75 80
ccg atg acc gcg aca tct tct acg gtc ttc tca tca ttg gca gag act 288
Pro Met Thr Ala Thr Ser Ser Thr Val Phe Ser Ser Leu Ala Glu Thr
85 90 95
cct cca cct tac tgc tca cca cct acg cat att ggc acc tcg gca ctc 336
Pro Pro Pro Tyr Cys Ser Pro Pro Thr His Ile Gly Thr Ser Ala Leu
100 105 110
aag gaa aca tta tca gaa cca tca gcg gca acc ctg caa ttc tat gat 384
Lys Glu Thr Leu Ser Glu Pro Ser Ala Ala Thr Leu Gln Phe Tyr Asp
115 120 125
aca tca atc aac ttt gat gat ccc gag tcg ttt ccc ggc ggc tgg cct 432
Thr Ser Ile Asn Phe Asp Asp Pro Glu Ser Phe Pro Gly Gly Trp Pro
130 135 140
cag cca aat aca ttt cgc gac gat gcc aac agc aat gaa tct tcg ggg 480
Gln Pro Asn Thr Phe Arg Asp Asp Ala Asn Ser Asn Glu Ser Ser Gly
145 150 155 160
ata cca gat cta ggc tac gac ttt gaa ggc cct ttg gat gca acg gcg 528
Ile Pro Asp Leu Gly Tyr Asp Phe Glu Gly Pro Leu Asp Ala Thr Ala
165 170 175
cct gtc tcg cca tcg ctg ttt gac ctc gaa gta gag ggg aac tcg tca 576
Pro Val Ser Pro Ser Leu Phe Asp Leu Glu Val Glu Gly Asn Ser Ser
180 185 190
tcc gga caa tcc aac aca agc aac acg caa cga gac ctt ttc gaa agt 624
Ser Gly Gln Ser Asn Thr Ser Asn Thr Gln Arg Asp Leu Phe Glu Ser
195 200 205
ctg tcg gat gtg tca cag gac cta gag gta ata ctc cac ggg gtg act 672
Leu Ser Asp Val Ser Gln Asp Leu Glu Val Ile Leu His Gly Val Thr
210 215 220
gtg gaa tgg ccc aag caa aaa att tta agc tac ccg ata ggg gac ttt 720
Val Glu Trp Pro Lys Gln Lys Ile Leu Ser Tyr Pro Ile Gly Asp Phe
225 230 235 240
ttg aat gcc ttt ggt aga ttg cta cta cat ctt caa gaa cgt gtg atc 768
Leu Asn Ala Phe Gly Arg Leu Leu Leu His Leu Gln Glu Arg Val Ile
245 250 255
acg agc agc aat agc agc atg tta gat ggg tgt ctg caa acc aag aac 816
Thr Ser Ser Asn Ser Ser Met Leu Asp Gly Cys Leu Gln Thr Lys Asn
260 265 270
ttg ttc atg gcg gtg cat tgc tac atg ttg tct gtc aaa atc atg aca 864
Leu Phe Met Ala Val His Cys Tyr Met Leu Ser Val Lys Ile Met Thr
275 280 285
tca ctt tcc cag ctg cta cta tcc gag gtg atg aaa gcc caa cct tgt 912
Ser Leu Ser Gln Leu Leu Leu Ser Glu Val Met Lys Ala Gln Pro Cys
290 295 300
gga caa aag caa agc aca cga atg gat tgg tac tgg tct ggc tca acc 960
Gly Gln Lys Gln Ser Thr Arg Met Asp Trp Tyr Trp Ser Gly Ser Thr
305 310 315 320
act aga aat gac aat gga aga gcc gaa gca ctt ccc tcg ttt cac tct 1008
Thr Arg Asn Asp Asn Gly Arg Ala Glu Ala Leu Pro Ser Phe His Ser
325 330 335
aat ctt cat atc ggc gag ctc att tca cat ctc gac cca ttc atg cac 1056
Asn Leu His Ile Gly Glu Leu Ile Ser His Leu Asp Pro Phe Met His
340 345 350
gcc tta tct tct gca tgc acg aca ttg cgt gta agc ctt cga cta ttg 1104
Ala Leu Ser Ser Ala Cys Thr Thr Leu Arg Val Ser Leu Arg Leu Leu
355 360 365
agt gag att gag act gct ttg ggg ata gca cag gag cac ggg gct gcg 1152
Ser Glu Ile Glu Thr Ala Leu Gly Ile Ala Gln Glu His Gly Ala Ala
370 375 380
gca tct att cgt cta gtc cta tca gat atg cca agc aca tcg tgg caa 1200
Ala Ser Ile Arg Leu Val Leu Ser Asp Met Pro Ser Thr Ser Trp Gln
385 390 395 400
atc ctt ggc gct gaa aat aaa acc ata acg ccg gcc tct cgt ctc cta 1248
Ile Leu Gly Ala Glu Asn Lys Thr Ile Thr Pro Ala Ser Arg Leu Leu
405 410 415
tct gtg ctt tgg agt gac gaa gcc gga gac gaa gag ccc aag tca aca 1296
Ser Val Leu Trp Ser Asp Glu Ala Gly Asp Glu Glu Pro Lys Ser Thr
420 425 430
aag gcc tca ggg aag acg ata aat gtg ttg cga cgt tgc tat aag gaa 1344
Lys Ala Ser Gly Lys Thr Ile Asn Val Leu Arg Arg Cys Tyr Lys Glu
435 440 445
ata ttc gca tta gcg aag aaa cac aat att gct tag 1380
Ile Phe Ala Leu Ala Lys Lys His Asn Ile Ala
450 455
<210>42
<211>459
<212>PRT
<213>柑桔青霉
<400>42
Met Ser Leu Pro His Ala Thr Ile Pro Thr Asn Leu Arg Arg Arg Ala
1 5 10 15
Phe Arg Arg Ser Cys Asp Arg Cys His Ala Gln Lys Leu Lys Cys Thr
20 25 30
Gly Ser Asn Ala Asn Leu Val Arg Ala Gln Cys Gln Arg Cys Gln Gln
35 40 45
Ala Gly Leu Arg Cys Val Tyr Ser Glu Arg Leu Pro Lys Arg Asn Leu
50 55 60
His Lys Glu Ala Ala Ala Gly Thr Thr Arg Ala Thr Glu Thr Ser Gln
65 70 75 80
Pro Met Thr Ala Thr Ser Ser Thr Val Phe Ser Ser Leu Ala Glu Thr
85 90 95
Pro Pro Pro Tyr Cys Ser Pro Pro Thr His Ile Gly Thr Ser Ala Leu
100 105 110
Lys Glu Thr Leu Ser Glu Pro Ser Ala Ala Thr Leu Gln Phe Tyr Asp
115 120 125
Thr Ser Ile Asn Phe Asp Asp Pro Glu Ser Phe Pro Gly Gly Trp Pro
130 135 140
Gln Pro Asn Thr Phe Arg Asp Asp Ala Asn Ser Asn Glu Ser Ser Gly
145 150 155 160
Ile Pro Asp Leu Gly Tyr Asp Phe Glu Gly Pro Leu Asp Ala Thr Ala
165 170 175
Pro Val Ser Pro Ser Leu Phe Asp Leu Glu Val Glu Gly Asn Ser Ser
180 185 190
Ser Gly Gln Ser Asn Thr Ser Asn Thr Gln Arg Asp Leu Phe Glu Ser
195 200 205
Leu Ser Asp Val Ser Gln Asp Leu Glu Val Ile Leu His Gly Val Thr
210 215 220
Val Glu Trp Pro Lys Gln Lys Ile Leu Ser Tyr Pro Ile Gly Asp Phe
225 230 235 240
Leu Asn Ala Phe Gly Arg Leu Leu Leu His Leu Gln Glu Arg Val Ile
245 250 255
Thr Ser Ser Asn Ser Ser Met Leu Asp Gly Cys Leu Gln Thr Lys Asn
260 265 270
Leu Phe Met Ala Val His Cys Tyr Met Leu Ser Val Lys Ile Met Thr
275 280 285
Ser Leu Ser Gln Leu Leu Leu Ser Glu Val Met Lys Ala Gln Pro Cys
290 295 300
Gly Gln Lys Gln Ser Thr Arg Met Asp Trp Tyr Trp Ser Gly Ser Thr
305 310 315 320
Thr Arg Asn Asp Asn Gly Arg Ala Glu Ala Leu Pro Ser Phe His Ser
325 330 335
Asn Leu His Ile Gly Glu Leu Ile Ser His Leu Asp Pro Phe Met His
340 345 350
Ala Leu Ser Ser Ala Cys Thr Thr Leu Arg Val Ser Leu Arg Leu Leu
355 360 365
Ser Glu Ile Glu Thr Ala Leu Gly Ile Ala Gln Glu His Gly Ala Ala
370 375 380
Ala Ser Ile Arg Leu Val Leu Ser Asp Met Pro Ser Thr Ser Trp Gln
385 390 395 400
Ile Leu Gly Ala Glu Asn Lys Thr Ile Thr Pro Ala Ser Arg Leu Leu
405 410 415
Ser Val Leu Trp Ser Asp Glu Ala Gly Asp Glu Glu Pro Lys Ser Thr
420 425 430
Lys Ala Ser Gly Lys Thr Ile Asn Val Leu Arg Arg Cys Tyr Lys Glu
435 440 445
Ile Phe Ala Leu Ala Lys Lys His Asn Ile Ala
450 455
<210>43
<211>9099
<212>DNA
<213>柑桔青霉
<220>
<221>CDS
<222>(1)..(9099)
<400>43
atg gat caa gcc aac tat cca aac gag cca att gtg gta gtg gga agc 48
Met Asp Gln Ala Asn Tyr Pro Asn Glu Pro Ile Val Val Val Gly Ser
1 5 10 15
ggt tgt cgg ttt cca ggt ggt gtc aaa aca cca tca aaa ctt tgg gag 96
Gly Cys Arg Phe Pro Gly Gly Val Asn Thr Pro Ser Lys Leu Trp Glu
20 25 30
ctg ctc aaa gag ccc cgg gat gta cag acc aag atc cct aag gag aga 144
Leu Leu Lys Glu Pro Arg Asp Val Gln Thr Lys Ile Pro Lys Glu Arg
35 40 45
ttt gac gtc gat aca ttt tac agc ccc gat ggc act cac ccc ggg cgc 192
Phe Asp Val Asp Thr Phe Tyr Ser Pro Asp Gly Thr His Pro Gly Arg
50 55 60
acg aac gca ccc ttt gca tac ttg ctg cag gag gat cta cgc ggt ttt 240
Thr Asn Ala Pro Phe Ala Tyr Leu Leu Gln Glu Asp Leu Arg Gly Phe
65 70 75 80
gat gcc tct ttc ttc aac atc caa gct gga gag gcc gaa acg att gac 288
Asp Ala Ser Phe Phe Asn Ile Gln Ala Gly Glu Ala Glu Thr Ile Asp
85 90 95
cca cag caa agg ctg ctg ctg gag acg gtc tat gaa gct gta tcc aac 336
Pro Gln Gln Arg Leu Leu Leu Glu Thr Val Tyr Glu Ala Val Ser Asn
100 105 110
gca ggc cta cgg atc caa ggc ctt caa gga tcc tct act gct gtg tac 384
Ala Gly Leu Arg Ile Gln Gly Leu Gln Gly Ser Ser Thr Ala Val Tyr
115 120 125
gtc ggt atg atg acg cat gac tat gag act atc gtg acg cgt gaa ttg 432
Val Gly Met Met Thr His Asp Tyr Glu Thr Ile Val Thr Arg Glu Leu
130 135 140
gat agt att cct aca tac tct gcc acg ggg gta gct gtc agt gtg gcc 480
Asp Ser Ile Pro Thr Tyr Ser Ala Thr Gly Val Ala Val Ser Val Ala
145 150 155 160
tcc aac cgt gta tca tac ttc ttc gac tgg cat ggg ccg agt atg acg 528
Ser Asn Arg Val Ser Tyr Phe Phe Asp Trp His Gly Pro Ser Met Thr
165 170 175
atc gac aca gcc tgt agt tca tcc tta gct gcc gtg cat ctg gcc gtc 576
Ile Asp Thr Ala Cys Ser Ser Ser Leu Ala Ala Val His Leu Ala Val
180 185 190
caa cag ctt aga acg ggc gag agt acc atg gcg gtt gca gcc ggt gcg 624
Gln Gln Leu Arg Thr Gly Glu Ser Thr Met Ala Val Ala Ala Gly Ala
195 200 205
aat ctg ata ttg ggc ccc atg acc ttt gta atg gag agc aaa ttg aac 672
Asn Leu Ile Leu Gly Pro Met Thr Phe Val Met Glu Ser Lys Leu Asn
210 215 220
atg ctg tcc ccc aat ggt aga tct cga atg tgg gat gct gct gcc gat 720
Met Leu Ser Pro Asn Gly Arg Ser Arg Met Trp Asp Ala Ala Ala Asp
225 230 235 240
gga tat gcc aga gga gaa ggt gtt tgc tct att gtc ctg aaa acg ctg 768
Gly Tyr Ala Arg Gly Glu Gly Val Cys Ser Ile Val Leu Lys Thr Leu
245 250 255
agc cag gca ctg cgc gac ggg gac agt atc gag tgt gtt atc cga gag 816
Ser Gln Ala Leu Arg Asp Gly Asp Ser Ile Glu Cys Val Ile Arg Glu
260 265 270
acc ggt atc aac caa gat ggc cga acg aca ggt atc aca atg cca aac 864
Thr Gly Ile Asn Gln Asp Gly Arg Thr Thr Gly Ile Thr Met Pro Asn
275 280 285
cat agc gca caa gaa gcc ctc att cgg gcc aca tat gcc aag gct ggt 912
His Ser Ala Gln Glu Ala Leu Ile Arg Ala Thr Tyr Ala Lys Ala Gly
290 295 300
ctt gat att acc aac ccc cag gaa cgc tgc cag ttc ttt gaa gcc cat 960
Leu Asp Ile Thr Asn Pro Gln Glu Arg Cys Gln Phe Phe Glu Ala His
305 310 315 320
gga act ggt aca cca gcc ggt gac cca cag gaa gct gag gct att gca 1008
Gly Thr Gly Thr Pro Ala Gly Asp Pro Gln Glu Ala Glu Ala Ile Ala
325 330 335
aca gcc ttc ttc gga cac aag gat gga aca atc gac agc gac ggc gag 1056
Thr Ala Phe Phe Gly His Lys Asp Gly Thr Ile Asp Ser Asp Gly Glu
340 345 350
aaa gat gag ctt ttt gtc ggc agc atc aag aca gtt ctc ggt cac acg 1104
Lys Asp Glu Leu Phe Val Gly Ser Ile Lys Thr Val Leu Gly His Thr
355 360 365
gaa ggc act gct ggt att gcg ggc tta atg aag gca tcg ttt gct gta 1152
Glu Gly Thr Ala Gly Ile Ala Gly Leu Met Lys Ala Ser Phe Ala Val
370 375 380
cga aat ggc gtg atc ccg cca aac ctg ctg ttt gag aag atc agt ccc 1200
Arg Asn Gly Val Ile Pro Pro Asn Leu Leu Phe Glu Lys Ile Ser Pro
385 390 395 400
cgt gtc gct ccg ttc tat acg cac ttg aaa att gca acg gag gcc aca 1248
Arg Val Ala Pro Phe Tyr Thr His Leu Lys Ile Ala Thr Glu Ala Thr
405 410 415
gaa tgg ccg att gtt gcg ccc ggg cag cct cgc aga gtc agc gtt aat 1296
Glu Trp Pro Ile Val Ala Pro Gly Gln Pro Arg Arg Val Ser Val Asn
420 425 430
tca ttt gga ttt ggt ggt aca aat gcc cat gct att atc gaa gag tat 1344
Ser Phe Gly Phe Gly Gly Thr Asn Ala His Ala Ile Ile Glu Glu Tyr
435 440 445
atg gct cct cca cac aag ccg aca gca gtg gta aca gag gtg acc tca 1392
Met Ala Pro Pro His Lys Pro Thr Ala Val Val Thr Glu Val Thr Ser
450 455 460
gat gca gat gca tgc agc ttg ccc ctt gtg ctt tca tcg aag tcg cag 1440
Asp Ala Asp Ala Cys Ser Leu Pro Leu Val Leu Ser Ser Lys Ser Gln
465 470 475 480
cgc tcc atg aag gca acg cta gaa aat atg ctc caa ttt ctg gaa acg 1488
Arg Ser Met Lys Ala Thr Leu Glu Asn Met Leu Gln Phe Leu Glu Thr
485 490 495
cat gat gac gtg gac atg cat gat atc gca tat acc tta ctt gag aaa 1536
His Asp Asp Val Asp Met His Asp Ile Ala Tyr Thr Leu Leu Glu Lys
500 505 510
cgg tct atc ttg ccc ttc cgt cgt gcg att gca gca cac aac aag gaa 1584
Arg Ser Ile Leu Pro Phe Arg Arg Ala Ile Ala Ala His Asn Lys Glu
515 520 525
gta gcc cgc gcg gca ctg gag gct gcc atc gcg gac ggt gag gtc gtc 1632
Val Ala Arg Ala Ala Leu Glu Ala Ala Ile Ala Asp Gly Glu Val Val
530 535 540
acc gac ttc cgc acc gac gcg aat gac aac cct cgc gta cta ggt gtc 1680
Thr Asp Phe Arg Thr Asp Ala Asn Asp Asn Pro Arg Val Leu Gly Val
545 550 555 560
ttt act ggc caa ggt gca cag tgg ccg ggc atg ctg aag aag ctc atg 1728
Phe Thr Gly Gln Gly Ala Gln Trp Pro Gly Met Leu Lys Lys Leu Met
565 570 575
gtg ggt atg cca ttt gtg aga ggc att ctc gaa gag ctg gat aat tca 1776
Val Gly Met Pro Phe Val Arg Gly Ile Leu Glu Glu Leu Asp Asn Ser
580 585 590
ctg caa aca ctg cct gaa aag tat cgg cct acg tgg aca ctg tat gac 1824
Leu Gln Thr Leu Pro Glu Lys Tyr Arg Pro Thr Trp Thr Leu Tyr Asp
595 600 605
cag ctc atg ctt gaa ggg gat gcc tca aac gtc aga ctc gcc agc ttc 1872
Gln Leu Met Leu Glu Gly Asp Ala Ser Asn Val Arg Leu Ala Ser Phe
610 615 620
tcc cag cct cta tgc tgc gcc gta caa atc gtt ctg gtc cga ctt ctc 1920
Ser Gln Pro Leu Cys Cys Ala Val Gln Ile Val Leu Val Arg Leu Leu
625 630 635 640
gct gca gct ggt atc gag ttc agt gca att gtc ggc cac agt tca ggt 1968
Ala Ala Ala Gly Ile Glu Phe Ser Ala Ile Val Gly His Ser Ser Gly
645 650 655
gag att gcc tgt gcc ttt gcg gca gga ttc atc agt gcc act caa gct 2016
Glu Ile Ala Cys Ala Phe Ala Ala Gly Phe Ile Ser Ala Thr Gln Ala
660 665 670
atc cgt att gcg cat ctg cgt gga gtt gtg tcc gcg gag cat gcc tct 2064
Ile Arg Ile Ala His Leu Arg Gly Val Val Ser Ala Glu His Ala Ser
675 680 685
tct cca agc ggc cag aca ggc gct atg cta gcg gca ggt atg tcg tac 2112
Ser Pro Ser Gly Gln Thr Gly Ala Met Leu Ala Ala Gly Met Ser Tyr
690 695 700
gat gac gca aag gaa cta tgc gag ctc gaa gcc ttt gag ggt cgg gtc 2160
Asp Asp Ala Lys Glu Leu Cys Glu Leu Glu Ala Phe Glu Gly Arg Val
705 710 715 720
tgc gtc gcc gct agc aat tca ccg gat agt gtg acc ttc tcc ggc gac 2208
Cys Val Ala Ala Ser Asn Ser Pro Asp Ser Val Thr Phe Ser Gly Asp
725 730 735
atg gat gct atc cag cac gtt gaa ggt gtc ttg gag gat gaa tcc act 2256
Met Asp Ala Ile Gln His Val Glu Gly Val Leu Glu Asp Glu Ser Thr
740 745 750
ttt gcc aga atc ttg aga gtt gac aag gcc tac cat tcg cat cac atg 2304
Phe Ala Arg Ile Leu Arg Val Asp Lys Ala Tyr His Ser His His Met
755 760 765
cac cca tgc gca gct cca tat gtc aag gca ttg ctg gag tgc gac tgt 2352
His Pro Cys Ala Ala Pro Tyr Val Lys Ala Leu Leu Glu Cys Asp Cys
770 775 780
gct gtt gcc gat ggc caa ggt aac gat agt gtt gct tgg ttc tct gcc 2400
Ala Val Ala Asp Gly Gln Gly Asn Asp Ser Val Ala Trp Phe Ser Ala
785 790 795 800
gtc cac gag acc agc aag caa atg act gta cag gat gtg atg ccc gct 2448
Val His Glu Thr Ser Lys Gln Met Thr Val Gln Asp Val Met Pro Ala
805 810 815
tat tgg aaa gac aat ctc gtc tct ccg gtc ttg ttc tcg cag gct gtg 2496
Tyr Trp Lys Asp Asn Leu Val Ser Pro Val Leu Phe Ser Gln Ala Val
820 825 830
cag aaa gca gtc atc act cat cgt cta atc gac gtc gcc atc gaa att 2544
Gln Lys Ala Val Ile Thr His Arg Leu Ile Asp Val Ala Ile Glu Ile
835 840 845
ggc gcc cac cct gct ctc aag ggt ccg tgt cta gcc acc atc aag gat 2592
Gly Ala His Pro Ala Leu Lys Gly Pro Cys Leu Ala Thr Ile Lys Asp
850 855 860
gct ctt gcc ggt gtg gag ctg ccg tat acc ggg tgc ttg gca cga aac 2640
Ala Leu Ala Gly Val Glu Leu Pro Tyr Thr Gly Cys Leu Ala Arg Asn
865 870 875 880
gtt gac gat gtg gac gct ttt gct gga ggt ctg gga tac att tgg gag 2688
Val Asp Asp Val Asp Ala Phe Ala Gly Gly Leu Gly Tyr Ile Trp Glu
885 890 895
cgt ttc gga gtt cgg agt atc gac gcc gag ggc ttc gta caa caa gtc 2736
Arg Phe Gly Val Arg Ser Ile Asp Ala Glu Gly Phe Val Gln Gln Val
900 905 910
cgg ccc gat cgt gcc gtt caa aac ctg tca aag tca ttg ccc aca tac 2784
Arg Pro Asp Arg Ala Val Gln Asn Leu Ser Lys Ser Leu Pro Thr Tyr
915 920 925
tct tgg gat cat act cgt caa tac tgg gca gaa tct cgc tcc acc cgc 2832
Ser Trp Asp His Thr Arg Gln Tyr Trp Ala Glu Ser Arg Ser Thr Arg
930 935 940
cag cat ctt cgt gga ggt gcg ccc cat ctt ctg ctt gga aag ctt tct 2880
Gln His Leu Arg Gly Gly Ala Pro His Leu Leu Leu Gly Lys Leu Ser
945 950 955 960
tct tac agc aca gca tcg acc ttc cag tgg aca aac ttc atc agg ccc 2928
Ser Tyr Ser Thr Ala Ser Thr Phe Gln Trp Thr Asn Phe Ile Arg Pro
965 970 975
cgg gat ctg gaa tgg ctc gac ggt cat gcg cta caa ggc cag act gtg 2976
Arg Asp Leu Glu Trp Leu Asp Gly His Ala Leu Gln Gly Gln Thr Val
980 985 990
ttc ccc gct gct ggg tac ata att atg gcc atg gaa gct gcc atg aag 3024
Phe Pro Ala Ala Gly Tyr Ile Ile Met Ala Met Glu Ala Ala Met Lys
995 1000 1005
gtg gct ggt gag cgt gcc gcc caa gtt cag ctc ctg gaa atc ttg 3069
Val Ala Gly Glu Arg Ala Ala Gln Val Gln Leu Leu Glu Ile Leu
1010 1015 1020
gac atg agc atc aac aaa gcc atc gtg ttt gaa gat gaa aac acc 3114
Asp Met Ser Ile Asn Lys Ala Ile Val Phe Glu Asp Glu Asn Thr
1025 1030 1035
tcc gtg gag ctg aac ttg aca gcc gaa gtc acc agt gac aat gat 3159
Ser Val Glu Leu Asn Leu Thr Ala Glu Val Thr Ser Asp Asn Asp
1040 1045 1050
gcg gat ggc caa gtc acg gtc aaa ttt gtt att gat tcc tgt ctg 3204
Ala Asp Gly Gln Val Thr Val Lys Phe Val Ile Asp Ser Cys Leu
1055 1060 1065
gca aag gag agt gag ctt tcg aca tcc gcc aaa ggc caa atc gtc 3249
Ala Lys Glu Ser Glu Leu Ser Thr Ser Ala Lys Gly Gln Ile Val
1070 1075 1080
ata acc ctt ggc gag gca tca ccg tca tcg cag ctt ttg ccg cca 3294
Ile Thr Leu Gly Glu Ala Ser Pro Ser Ser Gln Leu Leu Pro Pro
1085 1090 1095
cct gag gaa gag tac ccc cag atg aac aat gtc aac atc gat ttc 3339
Pro Glu Glu Glu Tyr Pro Gln Met Asn Asn Val Asn Ile Asp Phe
1100 1105 1110
ttc tat cgg gaa ctt gac ctc ctt ggg tat gac tac agc aaa gac 3384
Phe Tyr Arg Glu Leu Asp Leu Leu Gly Tyr Asp Tyr Ser Lys Asp
1115 1120 1125
ttc cgt cgt ttg cag acc atg aga agg gcc gac tcc aaa gct agc 3429
Phe Arg Arg Leu Gln Thr Met Arg Arg Ala Asp Ser Lys Ala Ser
1130 1135 1140
ggc acc ttg gct ttc ctt cca ctt aag gat gaa ttg cgc aat gag 3474
Gly Thr Leu Ala Phe Leu Pro Leu Lys Asp Glu Leu Arg Asn Glu
1145 1150 1155
ccc ctc ttg ctc cac cca gcg ccc ctg gac atc gcg ttc cag act 3519
Pro Leu Leu Leu His Pro Ala Pro Leu Asp Ile Ala Phe Gln Thr
1160 1165 1170
gtc att gga gcg tat tcc tct cca gga gat cgt cgc cta cgc tca 3564
Val Ile Gly Ala Tyr Ser Ser Pro Gly Asp Arg Arg Leu Arg Ser
1175 1180 1185
ttg tac gtg cct act cac gtt gac aga gtg act ctg att cca tcg 3609
Leu Tyr Val Pro Thr His Val Asp Arg Val Thr Leu Ile Pro Ser
1190 1195 1200
ctc tgt ata tcg gcg ggt aat tct ggt gaa acc gag ctt gcg ttt 3654
Leu Cys Ile Ser Ala Gly Asn Ser Gly Glu Thr Glu Leu Ala Phe
1205 1210 1215
gac aca atc aac aca cac gac aag ggt gat ttc ctg agc ggc gac 3699
Asp Thr Ile Asn Thr His Asp Lys Gly Asp Phe Leu Ser Gly Asp
1220 1225 1230
atc acg gtg tac gat tcg acc aag aca acg ctt ttc caa gtt gat 3744
Ile Thr Val Tyr Asp Ser Thr Lys Thr Thr Leu Phe Gln Val Asp
1235 1240 1245
aac att gtc ttt aag cct ttc tct ccc ccg act gct tcg acc gac 3789
Asn Ile Val Phe Lys Pro Phe Ser Pro Pro Thr Ala Ser Thr Asp
1250 1255 1260
cac cga atc ttc gca aag tgg gtc tgg gga ccc ctc acg ccc gaa 3834
His Arg Ile Phe Ala Lys Trp Val Trp Gly Pro Leu Thr Pro Glu
1265 1270 1275
aaa ctg ctg gag gac cct gcg acg ttg atc ata gct cgg gac aag 3879
Lys Leu Leu Glu Asp Pro Ala Thr Leu Ile Ile Ala Arg Asp Lys
1280 1285 1290
gag gac att ctg acc atc gag cga atc gtt tac ttc tac atc aaa 3924
Glu Asp Ile Leu Thr Ile Glu Arg Ile Val Tyr Phe Tyr Ile Lys
1295 1300 1305
tcc ttc cta gcc cag ata acc ccc gac gac cgt caa aat gcc gac 3969
Ser Phe Leu Ala Gln Ile Thr Pro Asp Asp Arg Gln Asn Ala Asp
1310 1315 1320
ctc cat tcc cag aag tac att gaa tgg tgt gac cag gtt cag gcc 4014
Leu His Ser Gln Lys Tyr Ile Glu Trp Cys Asp Gln Val Gln Ala
1325 1330 1335
gat gct cgg gct ggc cac cat cag tgg tac cag gag tct tgg gag 4059
Asp Ala Arg Ala Gly His His Gln Trp Tyr Gln Glu Ser Trp Glu
1340 1345 1350
gag gac act tct gtt cac att gag caa atg tgt gaa agc aac tcg 4104
Glu Asp Thr 8er Val His Ile Glu Gln Met Cys Glu Ser Asn Ser
1355 1360 1365
tcc cac cca cat gtg cgc ctg atc caa agg gta ggc aaa gaa tta 4149
Ser His Pro His Val Arg Leu Ile Gln Arg Val Gly Lys Glu Leu
1370 1375 1380
att tca att gtt cgc ggg aac ggg gat cct ttg gat atc atg aac 4194
Ile Ser Ile Val Arg Gly Asn Gly Asp Pro Leu Asp Ile Met Asn
1385 1390 1395
cgc gat ggg ttg ttc acc gag tac tat acc aac aag ctc gcc ttt 4239
Arg Asp Gly Leu Phe Thr Glu Tyr Tyr Thr Asn Lys Leu Ala Phe
1400 1405 1410
ggc tca gca ata cac gtc gtt cag gat ctg gtt agc caa att gct 4284
Gly Ser Ala Ile His Val Val Gln Asp Leu Val Ser Gln Ile Ala
1415 1420 1425
cat cgc tac caa tcc att gat atc ctt gag atc ggc ttg ggt aca 4329
His Arg Tyr Gln Ser Ile Asp Ile Leu Glu Ile Gly Leu Gly Thr
1430 1435 1440
ggc atc gcc acg aag cgc gtt ctt gca tca cct caa ctt ggt ttc 4374
Gly Ile Ala Thr Lys Arg Val Leu Ala Ser Pro Gln Leu Gly Phe
1445 1450 1455
aac agt tac act tgc act gac atc tcg gcg gat gtt att ggc aag 4419
Asn Ser Tyr Thr Cys Thr Asp Ile Ser Ala Asp Val Ile Gly Lys
1460 1465 1470
gcc cgt gaa caa ctt tcc gaa ttc gac ggt ctc atg cag ttt gag 4464
Ala Arg Glu Gln Leu Ser Glu Phe Asp Gly Leu Met Gln Phe Glu
1475 1480 1485
gca cta gac atc aac aga agc cca gca gag caa gga ttc aag cct 4509
Ala Leu Asp Ile Asn Arg Ser Pro Ala Glu Gln Gly Phe Lys Pro
1490 1495 1500
cac tcc tac gat ctg att att gca tcc gat gtc ctc cat gcc agc 4554
His Ser Tyr Asp Leu Ile Ile Ala Ser Asp Val Leu His Ala Ser
1505 1510 1515
tcc aac ttc gag gaa aaa ttg gct cac ata agg tcc ttg ctc aag 4599
Ser Asn Phe Glu Glu Lys Leu Ala His Ile Arg Ser Leu Leu Lys
1520 1525 1530
ccg ggt ggt cac ttg gtt act ttc ggg gtc acc cat cgc gag cct 4644
Pro Gly Gly His Leu Val Thr Phe Gly Val Thr His Arg Glu Pro
1535 1540 1545
gct cgc ctc gcc ttc atc tct ggg ctt ttc gct gat cga tgg act 4689
Ala Arg Leu Ala Phe Ile Ser Gly Leu Phe Ala Asp Arg Trp Thr
1550 1555 1560
gga gaa gac gaa act cgt gct ttg agt gcc tcg ggg tcc gtt gac 4734
Gly Glu Asp Glu Thr Arg Ala Leu Ser Ala Ser Gly Ser Val Asp
1565 1570 1575
caa tgg gag cat acc ctc aag aga gtt ggg ttc tct ggc gtc gat 4779
Gln Trp Glu His Thr Leu Lys Arg Val Gly Phe Ser Gly Val Asp
1580 1585 1590
agt cgg aca ctt gat cga gag gat gat ttg atc ccg tct gtc ttc 4824
Ser Arg Thr Leu Asp Arg Glu Asp Asp Leu Ile Pro Ser Val Phe
1595 1600 1605
agt aca cat gct gtg gat gcc acc gtt gag cgt ttg tat gat cca 4869
Ser Thr His Ala Val Asp Ala Thr Val Glu Arg Leu Tyr Asp Pro
1610 1615 1620
ctt tct gct cca ttg aag gac tca tac ccg cca tta gtg gtt atc 4914
Leu Ser Ala Pro Leu Lys Asp Ser Tyr Pro Pro Leu Val Val Ile
1625 1630 1635
ggt ggc gaa tcg aca aaa acc gaa cgc att ttg aac gac atg aaa 4959
Gly Gly Glu Ser Thr Lys Thr Glu Arg Ile Leu Asn Asp Met Lys
1640 1645 1650
gct gcc cta ccg cat aga cac atc cac tcc gtc aag cgg ctg gaa 5004
Ala Ala Leu Pro His Arg His Ile His Ser Val Lys Arg Leu Glu
1655 1660 1665
agt gtt ctc gac gac ccg gcc ttg cag cct aag tcg act ttt gtc 5049
Ser Val Leu Asp Asp Pro Ala Leu Gln Pro Lys Ser Thr Phe Val
1670 1675 1680
atc ctc tcg gaa ctt gat gat gaa gtg ttt tgc aac ctt gaa gag 5094
Ile Leu Ser Glu Leu Asp Asp Glu Val Phe Cys Asn Leu Glu Glu
1685 1690 1695
gac aag ttt gag gca gtc aag tct ctt ctc ttc tac gcc gga cgc 5139
Asp Lys Phe Glu Ala Val Lys Ser Leu Leu Phe Tyr Ala Gly Arg
1700 1705 1710
atg atg tgg ctg aca gag aat gcc tgg att gat cat ccc cac cag 5184
Met Met Trp Leu Thr Glu Asn Ala Trp Ile Asp His Pro His Gln
1715 1720 1725
gcc agc acc atc gga atg ttg agg aca atc aag ctc gag aac cct 5229
Ala Ser Thr Ile Gly Met Leu Arg Thr Ile Lys Leu Glu Asn Pro
1730 1735 1740
gac ttg gga acg cac gtc ttc gat gtc gat act gtg gag aac cta 5274
Asp Leu Gly Thr His Val Phe Asp Val Asp Thr Val Glu Asn Leu
1745 1750 1755
gac acc aaa ttc ttc gtt gag caa ctt ttg cgc ttc gag gag agc 5319
Asp Thr Lys Phe Phe Val Glu Gln Leu Leu Arg Phe Glu Glu Ser
1760 1765 1770
gat gat cag ctt ttg gaa tca ata aca tgg act cat gag ccc gaa 5364
Asp Asp Gln Leu Leu Glu Ser Ile Thr Trp Thr His Glu Pro Glu
1775 1780 1785
gtg tac tgg tgc aag ggt cgt gcc tgg gtc cct cgt ttg aag cag 5409
Val Tyr Trp Cys Lys Gly Arg Ala Trp Val Pro Arg Leu Lys Gln
1790 1795 1800
gat att gct agg aac gac cgt atg aac tcg tct cgt cgt cca att 5454
Asp Ile Ala Arg Asn Asp Arg Met Asn Ser Ser Arg Arg Pro Ile
1805 1810 1815
ttc ggt aac ttt aat tcg tcc aag acg gcc att gca ctg aaa gag 5499
Phe Gly Asn Phe Asn Ser Ser Lys Thr Ala Ile Ala Leu Lys Glu
1820 1825 1830
gcg agg gga gca tcc tca tcg atg tac tat ctt gag tca acc gag 5544
Ala Arg Gly Ala Ser Ser Ser Met Tyr Tyr Leu Glu Ser Thr Glu
1835 1840 1845
acg tgt gat tcg tta gaa gac gct cgt cat gct gga aaa gca act 5589
Thr Cys Asp Ser Leu Glu Asp Ala Arg His Ala Gly Lys Ala Thr
1850 1855 1860
gtt cgt gtt cgc tac gct ctt ccc cag gca att cgc gtg ggc cat 5634
Val Arg Val Arg Tyr Ala Leu Pro Gln Ala Ile Arg Val Gly His
1865 1870 1875
ctc gga tac ttc cat gtc gtg cag ggc agt att ctg gag aat aca 5679
Leu Gly Tyr Phe His Val Val Gln Gly Ser Ile Leu Glu Asn Thr
1880 1885 1890
tgt gag gtg cct gta gtc gcc ctg gct gag aag aat gga tct ata 5724
Cys Glu Val Pro Val Val Ala Leu Ala Glu Lys Asn Gly Ser Ile
1895 1900 1905
ctg cat gta ccg aga aac tac atg cat agt ctg ccc gat aac atg 5769
Leu His Val Pro Arg Asn Tyr Met His Ser Leu Pro Asp Asn Met
1910 1915 1920
gcg gaa ggc gag gat agt tcc ttc ttg ttg tcc aca gct gca gcc 5814
Ala Glu Gly Glu Asp Ser Ser Phe Leu Leu Ser Thr Ala Ala Ala
1925 1930 1935
ctc ctt gcc gaa aca att ctc tct agc gct cag tcc ttt ggc tct 5859
Leu Leu Ala Glu Thr Ile Leu Ser Ser Ala Gln Ser Phe Gly Ser
1940 1945 1950
gat gca tca att ctg att atg gag ccc cca atc ttc tgc gtc aaa 5904
Asp Ala Ser Ile Leu Ile Met Glu Pro Pro Ile Phe Cys Val Lys
1955 1960 1965
gca att ctg gag tcg gcc aaa acc tac ggt gtt cag gtt cat ttg 5949
Ala Ile Leu Glu Ser Ala Lys Thr Tyr Gly Val Gln Val His Leu
1970 1975 1980
gca aca act ctg tcc gac gtc aaa act att ccg gct cct tgg atc 5994
Ala Thr Thr Leu Ser Asp Val Lys Thr Ile Pro Ala Pro Trp Ile
1985 1990 1995
cga tta cat gcc aag gaa acc gac gct cgg ctg aaa cac agc ctg 6039
Arg Leu His Ala Lys Glu Thr Asp Ala Arg Leu Lys His Ser Leu
2000 2005 2010
ccg aca aac atg atg gca ttc ttt gac ttg tct acc gac cgg act 6084
Pro Thr Asn Met Met Ala Phe Phe Asp Leu Ser Thr Asp Arg Thr
2015 2020 2025
gct gcc ggg ata acc aac cgt ttg gcc aag ttg cta cca ccc agt 6129
Ala Ala Gly Ile Thr Asn Arg Leu Ala Lys Leu Leu Pro Pro Ser
2030 2035 2040
tgc ttc atg tac agt ggt gac tat ctt atc cga agt aca gct tcc 6174
Cys Phe Met Tyr Ser Gly Asp Tyr Leu Ile Arg Ser Thr Ala Ser
2045 2050 2055
aca tac aaa gtt agt cat gtt gag gat att cca atc ctc gag cac 6219
Thr Tyr Lys Val Ser His Val Glu Asp Ile Pro Ile Leu Glu His
2060 2065 2070
tct gtg gca atg gca aaa aat acc gtc tct gcg tcg act gtc gac 6264
Ser Val Ala Met Ala Lys Asn Thr Val Ser Ala Ser Thr Val Asp
2075 2080 2085
gac act gag aaa gtt att aca gcc aca caa att ctc ttg cct ggt 6309
Asp Thr Glu Lys Val Ile Thr Ala Thr Gln Ile Leu Leu Pro Gly
2090 2095 2100
cag ctc tct gtc aac cac aat gac caa cgc ttc aat ctg gcc acc 6354
Gln Leu Ser Val Asn His Asn Asp Gln Arg Phe Asn Leu Ala Thr
2105 2110 2115
gtc atc gac tgg aag gaa aat gag gtg tcc gct agg att tgc ccc 6399
Val Ile Asp Trp Lys Glu Asn Glu Val Ser Ala Arg Ile Cys Pro
2120 2125 2130
atc gac tct ggt aac tta ttt tcc aac aag aag acg tat ttg ctt 6444
Ile Asp Ser Gly Asn Leu Phe Ser Asn Lys Lys Thr Tyr Leu Leu
2135 2140 2145
gtt ggt ctt acc ggg gac ctt ggt cgc tct ctc tgt cgc tgg atg 6489
Val Gly Leu Thr Gly Asp Leu Gly Arg Ser Leu Cys Arg Trp Met
2150 2155 2160
atc ttg cat ggc gcc cgc cat gtt gtg ctc act agc cgg aac cct 6534
Ile Leu His Gly Ala Arg His Val Val Leu Thr Ser Arg Asn Pro
2165 2170 2175
cga ctt gat ccc aaa tgg atc gcc aac atg gag gca ctt ggt ggt 6579
Arg Leu Asp Pro Lys Trp Ile Ala Asn Met Glu Ala Leu Gly Gly
2180 2185 2190
gac atc acc gtt ctg tca atg gat gtt gcc aat gag gat tca gtc 6624
Asp Ile Thr Val Leu Ser Met Asp Val Ala Asn Glu Asp Ser Val
2195 2200 2205
gat gct ggc ctt ggc aag ctt gtc gat atg aag ttg cca cct gtt 6669
Asp Ala Gly Leu Gly Lys Leu Val Asp Met Lys Leu Pro Pro Val
2210 2215 2220
gcc ggc atc gcg ttc ggg cct ttg gtg ctg cag gat gtc atg ctg 6714
Ala Gly Ile Ala Phe Gly Pro Leu Val Leu Gln Asp Val Met Leu
2225 2230 2235
aag aac atg gac cac cag atg atg gac atg gtg ttg aag ccc aag 6759
Lys Asn Met Asp His Gln Met Met Asp Met Val Leu Lys Pro Lys
2240 2245 2250
gta caa gga gca cgc att ctt cat gaa cgg ttc tcc gaa cag acg 6804
Val Gln Gly Ala Arg Ile Leu His Glu Arg Phe Ser Glu Gln Thr
2255 2260 2265
ggc agc aag gcg ctc gac ttc ttc atc atg ttt tcg tcc att gtt 6849
Gly Ser Lys Ala Leu Asp Phe Phe Ile Met Phe Ser Ser Ile Val
2270 2275 2280
gca gtt att ggc aat cct ggc cag tcc aac tat ggc gct gcg aat 6894
Ala Val Ile Gly Asn Pro Gly Gln Ser Asn Tyr Gly Ala Ala Asn
2285 2290 2295
gcc tac cta cag gct ctg gcc cag caa cgg tgc gcc aga gga ttg 6939
Ala Tyr Leu Gln Ala Leu Ala Gln Gln Arg Cys Ala Arg Gly Leu
2300 2305 2310
gcg gga tca acc atc gat att ggt gcc gtt tac ggt gta ggg ttt 6984
Ala Gly Ser Thr Ile Asp Ile Gly Ala Val Tyr Gly Val Gly Phe
2315 2320 2325
gtc acg agg gcc gag atg gag gag gac ttt gat gct atc cgt ttc 7029
Val Thr Arg Ala Glu Met Glu Glu Asp Phe Asp Ala Ile Arg Phe
2330 2335 2340
atg ttt gac tca gtt gaa gag cat gag ctg cac acg ctt ttc gcc 7074
Met Phe Asp Ser Val Glu Glu His Glu Leu His Thr Leu Phe Ala
2345 2350 2355
gaa gcg gtc gtg tct gac cag cgt gcc cgg cag caa cca cag cgc 7119
Glu Ala Val Val Ser Asp Gln Arg Ala Arg Gln Gln Pro Gln Arg
2360 2365 2370
aag acg gtc att gac atg gcg gac ctt gag ctt acc acg ggt atc 7164
Lys Thr Val Ile Asp Met Ala Asp Leu Glu Leu Thr Thr Gly Ile
2375 2380 2385
cca gat ctt gac cct gcg ctt caa gat cga att att tac ttc aac 7209
Pro Asp Leu Asp Pro Ala Leu Gln Asp Arg Ile Ile Tyr Phe Asn
2390 2395 2400
gac cct cgt ttc gga aac ttc aaa att ccc ggt caa cgc gga gac 7254
Asp Pro Arg Phe Gly Asn Phe Lys Ile Pro Gly Gln Arg Gly Asp
2405 2410 2415
ggt ggc gac aat gga tca ggg tct aaa ggc tcc att gcc gac cag 7299
Gly Gly Asp Asn Gly Ser Gly Ser Lys Gly Ser Ile Ala Asp Gln
2420 2425 2430
ctc aaa caa gca aca act tta gac caa gtt cgg caa atc gtg att 7344
Leu Lys Gln Ala Thr Thr Leu Asp Gln Val Arg Gln Ile Val Ile
2435 2440 2445
gat ggt cta tct gag aaa ctc cgt gtt acc ctc caa gtt tcg gac 7389
Asp Gly Leu Ser Glu Lys Leu Arg Val Thr Leu Gln Val Ser Asp
2450 2455 2460
ggg gag agc gtg gac cca acc att cct ctc att gat caa ggt gtc 7434
Gly Glu Ser Val Asp Pro Thr Ile Pro Leu Ile Asp Gln Gly Val
2465 2470 2475
gac tcc ttg ggt gca gtg act gtc ggc tca tgg ttc tca aag caa 7479
Asp Ser Leu Gly Ala Val Thr Val Gly Ser Trp Phe Ser Lys Gln
2480 2485 2490
ctc tac ctt gac ctc cca ctc ttg agg gta ctt ggc ggt gct tct 7524
Leu Tyr Leu Asp Leu Pro Leu Leu Arg Val Leu Gly Gly Ala Ser
2495 2500 2505
gtc gct gat ctt gcc gac gac gcg gcc acc cga ctc cca gct aca 7569
Val Ala Asp Leu Ala Asp Asp Ala Ala Thr Arg Leu Pro Ala Thr
2510 2515 2520
tcc att ccg ctg ctg ttg caa att ggt gat tcc acg gga acc tcg 7614
Ser Ile Pro Leu Leu Leu Gln Ile Gly Asp Ser Thr Gly Thr Ser
2525 2530 2535
gac agc ggg gct tct ccg aca cca aca gac agc cat gat gaa gca 7659
Asp Ser Gly Ala Ser Pro Thr Pro Thr Asp Ser His Asp Glu Ala
2540 2545 2550
agc tct gct acc agc aca gat gcg tcg tca gcc gaa gag gat gaa 7704
Ser Ser Ala Thr Ser Thr Asp Ala Ser Ser Ala Glu Glu Asp Glu
2555 2560 2565
gag caa gag gac gat aat gag cag gga ggc cgt aag att ctt cgt 7749
Glu Gln Glu Asp Asp Asn Glu Gln Gly Gly Arg Lys Ile Leu Arg
2570 2575 2580
cgc gag agg ttg tcc ctt ggc cag gag tat tcc tgg agg cag caa 7794
Arg Glu Arg Leu Ser Leu Gly Gln Glu Tyr Ser Trp Arg Gln Gln
2585 2590 2595
caa atg gta aaa gat cat acc atc ttc aac aac act att ggc atg 7839
Gln Met Val Lys Asp His Thr Ile Phe Asn Asn Thr Ile Gly Met
2600 2605 2610
ttc atg aag ggt acc att gac ctc gac cgg ttg agg cgg gct ctg 7884
Phe Met Lys Gly Thr Ile Asp Leu Asp Arg Leu Arg Arg Ala Leu
2615 2620 2625
aaa gcc tca ttg cgc cgt cac gag atc ttc cgt acg tgc ttt gtt 7929
Lys Ala Ser Leu Arg Arg His Glu Ile Phe Arg Thr Cys Phe Val
2630 2635 2640
act ggc gat gac tat agc agc gat tta aat ggt ccc gtc caa gtg 7974
Thr Gly Asp Asp Tyr Ser Ser Asp Leu Asn Gly Pro Val Gln Val
2645 2650 2655
gtt ctc aag aac ccg gag aac aga gtg cac ttt gtt cag gtg aac 8019
Val Leu Lys Asn Pro Glu Asn Arg Val His Phe Val Gln Val Asn
2660 2665 2670
aac gct gcg gag gca gag gaa gag tac cgg aaa ctc gag aag aca 8064
Asn Ala Ala Glu Ala Glu Glu Glu Tyr Arg Lys Leu Glu Lys Thr
2675 2680 2685
aac tat agc atc tcc aca ggt gac act ctc aga ctc gtt gat ttc 8109
Asn Tyr Ser Ile Ser Thr Gly Asp Thr Leu Arg Leu Val Asp Phe
2690 2695 2700
tac tgg ggc aca gat gac cac ctg ttg gta atc ggc tac cac aga 8154
Tyr Trp Gly Thr Asp Asp His Leu Leu Val Ile Gly Tyr His Arg
2705 2710 2715
tta gtt ggt gat ggc tca aca aca gaa aac ctg ttc aat gag atc 8199
Leu Val Gly Asp Gly Ser Thr Thr Glu Asn Leu Phe Asn Glu Ile
2720 2725 2730
ggg cag att tac agc ggg gtg aaa atg cag cga cca tcg acc caa 8244
Gly Gln Ile Tyr Ser Gly Val Lys Met Gln Arg Pro Ser Thr Gln
2735 2740 2745
ttc tct gat cta gcc gtc caa cag cgg gaa aac ctg gaa aat ggg 8289
Phe Ser Asp Leu Ala Val Gln Gln Arg Glu Asn Leu Glu Asn Gly
2750 2755 2760
cga atg ggg gac gat atc gcg ttc tgg aag tcc atg cat agc aaa 8334
Arg Met Gly Asp Asp Ile Ala Phe Trp Lys Ser Met His Ser Lys
2765 2770 2775
gtc tcg tca tct gcg cca acc gtg ctt ccc atc atg aat ctg atc 8379
Val Ser Ser Ser Ala Pro Thr Val Leu Pro Ile Met Asn Leu Ile
2780 2785 2790
aat gac cct gct gcc aat tca gag cag cag caa ata cag cca ttc 8424
Asn Asp Pro Ala Ala Asn Ser Glu Gln Gln Gln Ile Gln Pro Phe
2795 2800 2805
acg tgg cag cag tat gaa gca att gct cgt tta gat ccc atg gtc 8469
Thr Trp Gln Gln Tyr Glu Ala Ile Ala Arg Leu Asp Pro Met Val
2810 2815 2820
gcc ttc cga atc aaa gag cgg agc cgc aag cac aag gca acc ccc 8514
Ala Phe Arg Ile Lys Glu Arg Ser Arg Lys His Lys Ala Thr Pro
2825 2830 2835
atg cag ttc tac ctg gcc gcc tac cac gtt ttg ttg gcg cgt ctt 8559
Met Gln Phe Tyr Leu Ala Ala Tyr His Val Leu Leu Ala Arg Leu
2840 2845 2850
acc ggc agc aaa gac ata acc atc ggc ctc gcc gaa acc aac cga 8604
Thr Gly Ser Lys Asp Ile Thr Ile Gly Leu Ala Glu Thr Asn Arg
2855 2860 2865
tcc acc atg gaa gaa att tcg gcg atg ggc ttt ttc gct aac gtg 8649
Ser Thr Met Glu Glu Ile Ser Ala Met Gly Phe Phe Ala Asn Val
2870 2875 2880
ctt ccc ctg cgc ttt gat gag ttc gtc ggc agc aag aca ttc ggc 8694
Leu Pro Leu Arg Phe Asp Glu Phe Val Gly Ser Lys Thr Phe Gly
2885 2890 2895
gag cac ctt gta gcc acc aag gac agt gtg cgt gag gcc atg caa 8739
Glu His Leu Val Ala Thr Lys Asp Ser Val Arg Glu Ala Met Gln
2900 2905 2910
cac gcg cgg gtg ccg tat ggc gtc atc ctc gac tgt cta ggc ctg 8784
His Ala Arg Val Pro Tyr Gly Val Ile Leu Asp Cys Leu Gly Leu
2915 2920 2925
aat ctc cct acc tca ggc gag gaa ccc aag act cag aca cac gcc 8829
Asn Leu Pro Thr Ser Gly Glu Glu Pro Lys Thr Gln Thr His Ala
2930 2935 2940
ccc ttg ttc cag gct gtc ttt gat tac aag cag ggt caa gcg gag 8874
Pro Leu Phe Gln Ala Val Phe Asp Tyr Lys Gln Gly Gln Ala Glu
2945 2950 2955
agt ggc tca att ggc aat gcc aaa atg acg agt gtt ctc gct tcc 8919
Ser Gly Ser Ile Gly Asn Ala Lys Met Thr Ser Val Leu Ala Ser
2960 2965 2970
cgt gag cgc act cct tat gac atc gtt ctc gag atg tgg gat gac 8964
Arg Glu Arg Thr Pro Tyr Asp Ile Val Leu Glu Met Trp Asp Asp
2975 2980 2985
cct acc aag gac cca ctc att cat gtc aaa ctt cag agc tcg ctg 9009
Pro Thr Lys Asp Pro Leu Ile His Val Lys Leu Gln Ser Ser Leu
2990 2995 3000
tat ggc cct gag cac gct cag gcc ttt gta gac cac ttt tct tca 9054
Tyr Gly Pro Glu His Ala Gln Ala Phe Val Asp His Phe Ser Ser
3005 3010 3015
atc ctc act atg ttc tcg atg aac ccg gct ctg aag ttg gcc tag 9099
Ile Leu Thr Met Phe Ser Met Asn Pro Ala Leu Lys Leu Ala
3020 3025 3030
<210>44
<211>3032
<212>PRT
<213>柑桔青霉
<400>44
Met Asp Gln Ala Asn Tyr Pro Asn Glu Pro Ile Val Val Val Gly Ser
1 5 10 15
Gly Cys Arg Phe Pro Gly Gly Val Asn Thr Pro Ser Lys Leu Trp Glu
20 25 30
Leu Leu Lys Glu Pro Arg Asp Val Gln Thr Lys Ile Pro Lys Glu Arg
35 40 45
Phe Asp Val Asp Thr Phe Tyr Ser Pro Asp Gly Thr His Pro Gly Arg
50 55 60
Thr Asn Ala Pro Phe Ala Tyr Leu Leu Gln Glu Asp Leu Arg Gly Phe
65 70 75 80
Asp Ala Ser Phe Phe Asn Ile Gln Ala Gly Glu Ala Glu Thr Ile Asp
85 90 95
Pro Gln Gln Arg Leu Leu Leu Glu Thr Val Tyr Glu Ala Val Ser Asn
100 105 110
Ala Gly Leu Arg Ile Glu Gly Leu Gln Gly Ser Ser Thr Ala Val Tyr
115 120 125
Val Gly Met Met Thr His Asp Tyr Glu Thr Ile Val Thr Arg Glu Leu
130 135 140
Asp Ser Ile Pro Thr Tyr Ser Ala Thr Gly Val Ala Val Ser Val Ala
145 150 155 160
Ser Asn Arg Val Ser Tyr Phe Phe Asp Trp His Gly Pro Ser Met Thr
165 170 175
Ile Asp Thr Ala Cys Ser Ser Ser Leu Ala Ala Val His Leu Ala Val
180 185 190
Gln Gln Leu Arg Thr Gly Glu Ser Thr Met Ala Val Ala Ala Gly Ala
195 200 205
Asn Leu Ile Leu Gly Pro Met Thr Phe Val Met Glu Ser Lys Leu Asn
210 215 220
Met Leu Ser Pro Asn Gly Arg Ser Arg Met Trp Asp Ala Ala Ala Asp
225 230 235 240
Gly Tyr Ala Arg Gly Glu Gly Val Cys Ser Ile Val Leu Lys Thr Leu
245 250 255
Ser Gln Ala Leu Arg Asp Gly Asp Ser Ile Glu Cys Val Ile Arg Glu
260 265 270
Thr Gly Ile Asn Gln Asp Gly Arg Thr Thr Gly Ile Thr Met Pro Asn
275 280 285
His Ser Ala Gln Glu Ala Leu Ile Arg Ala Thr Tyr Ala Lys Ala Gly
290 295 300
Leu Asp Ile Thr Asn Pro Gln Glu Arg Cys Gln Phe Phe Glu Ala His
305 310 315 320
Gly Thr Gly Thr Pro Ala Gly Asp Pro Gln Glu Ala Glu Ala Ile Ala
325 330 335
Thr Ala Phe Phe Gly His Lys Asp Gly Thr Ile Asp Ser Asp Gly Glu
340 345 350
Lys Asp Glu Leu Phe Val Gly Ser Ile Lys Thr Val Leu Gly His Thr
355 360 365
Glu Gly Thr Ala Gly Ile Ala Gly Leu Met Lys Ala Ser Phe Ala Val
370 375 380
Arg Asn GIy Val Ile Pro Pro Asn Leu Leu Phe Glu Lys Ile Ser Pro
385 390 395 400
Arg Val Ala Pro Phe Tyr Thr His Leu Lys Ile Ala Thr Glu Ala Thr
405 410 415
Glu Trp Pro Ile Val Ala Pro Gly Gln Pro Arg Arg Val Ser Val Asn
420 425 430
Ser Phe Gly Phe Gly Gly Thr Asn Ala His Ala Ile Ile Glu Glu Tyr
435 440 445
Met Ala Pro Pro His Lys Pro Thr Ala Val Val Thr Glu Val Thr Ser
450 455 460
Asp Ala Asp Ala Cys Ser Leu Pro Leu Val Leu Ser Ser Lys Ser Gln
465 470 475 480
Arg Ser Met Lys Ala Thr Leu Glu Asn Met Leu Gln Phe Leu Glu Thr
485 490 495
His Asp Asp Val Asp Met His Asp Ile Ala Tyr Thr Leu Leu Glu Lys
500 505 510
Arg Ser Ile Leu Pro Phe Arg Arg Ala Ile Ala Ala His Asn Lys Glu
515 520 525
Val Ala Arg Ala Ala Leu Glu Ala Ala Ile Ala Asp Gly Glu Val Val
530 535 540
Thr Asp Phe Arg Thr Asp Ala Asn Asp Asn Pro Arg Val Leu Gly Val
545 550 555 560
Phe Thr Gly Gln Gly Ala Gln Trp Pro Gly Met Leu Lys Lys Leu Met
565 570 575
Val Gly Met Pro Phe Val Arg Gly Ile Leu Glu Glu Leu Asp Asn Ser
580 585 590
Leu Gln Thr Leu Pro Glu Lys Tyr Arg Pro Thr Trp Thr Leu Tyr Asp
595 600 605
Gln Leu Met Leu Glu Gly Asp Ala Ser Asn Val Arg Leu Ala Ser Phe
610 615 620
Ser Gln Pro Leu Cys Cys Ala Val Gln Ile Val Leu Val Arg Leu Leu
625 630 635 640
Ala Ala Ala Gly Ile Glu Phe Ser Ala Ile Val Gly His Ser Ser Gly
645 650 655
Glu Ile Ala Cys Ala Phe Ala Ala Gly Phe Ile Ser Ala Thr Gln Ala
660 665 670
Ile Arg Ile Ala His Leu Arg Gly Val Val Ser Ala Glu His Ala Ser
675 680 685
Ser Pro Ser Gly Gln Thr Gly Ala Met Leu Ala Ala Gly Met Ser Tyr
690 695 700
Asp Asp Ala Lys Glu Leu Cys Glu Leu Glu Ala Phe Glu Gly Arg Val
705 710 715 720
Cys Val Ala Ala Ser Asn Ser Pro Asp Ser Val Thr Phe Ser Gly Asp
725 730 735
Met Asp Ala Ile Gln His Val Glu Gly Val Leu Glu Asp Glu Ser Thr
740 745 750
Phe Ala Arg Ile Leu Arg Val Asp Lys Ala Tyr His Ser His His Met
755 760 765
His Pro Cys Ala Ala Pro Tyr Val Lys Ala Leu Leu Glu Cys Asp Cys
770 775 780
Ala Val Ala Asp Gly Gln Gly Asn Asp Ser Val Ala Trp Phe Ser Ala
785 790 795 800
Val His Glu Thr Ser Lys Gln Met Thr Val Gln Asp Val Met Pro Ala
805 810 815
Tyr Trp Lys Asp Asn Leu Val Ser Pro Val Leu Phe Ser Gln Ala Val
820 825 830
Gln Lys Ala Val Ile Thr His Arg Leu Ile Asp Val Ala Ile Glu Ile
835 840 845
Gly Ala His Pro Ala Leu Lys Gly Pro Cys Leu Ala Thr Ile Lys Asp
850 855 860
Ala Leu Ala Gly Val Glu Leu Pro Tyr Thr Gly Cys Leu Ala Arg Asn
865 870 875 880
Val Asp Asp Val Asp Ala Phe Ala Gly Gly Leu Gly Tyr Ile Trp Glu
885 890 895
Arg Phe Gly Val Arg Ser Ile Asp Ala Glu Gly Phe Val Gln Gln Val
900 905 910
Arg Pro Asp Arg Ala Val Gln Asn Leu Ser Lys Ser Leu Pro Thr Tyr
915 920 925
Ser Trp Asp His Thr Arg Gln Tyr Trp Ala Glu Ser Arg Ser Thr Arg
930 935 940
Gln His Leu Arg Gly Gly Ala Pro His Leu Leu Leu Gly Lys Leu Ser
945 950 955 960
Ser Tyr Ser Thr Ala Ser Thr Phe Gln Trp Thr Asn Phe Ile Arg Pro
965 970 975
Arg Asp Leu Glu Trp Leu Asp Gly His Ala Leu Gln Gly Gln Thr Val
980 985 990
Phe Pro Ala Ala Gly Tyr Ile Ile Met Ala Met Glu Ala Ala Met Lys
995 1000 1005
Val Ala Gly Glu Arg Ala Ala Gln Val Gln Leu Leu Glu Ile Leu
1010 1015 1020
Asp Met Ser Ile Asn Lys Ala Ile Val Phe Glu Asp Glu Asn Thr
1025 1030 1035
Ser Val Glu Leu Asn Leu Thr Ala Glu Val Thr Ser Asp Asn Asp
1040 1045 1050
Ala Asp Gly Gln Val Thr Val Lys Phe Val Ile Asp Ser Cys Leu
1055 1060 1065
Ala Lys Glu Ser Glu Leu Ser Thr Ser Ala Lys Gly Gln Ile Val
1070 1075 1080
Ile Thr Leu Gly Glu Ala Ser Pro Ser Ser Gln Leu Leu Pro Pro
1085 1090 1095
Pro Glu Glu Glu Tyr Pro Gln Met Asn Asn Val Asn Ile Asp Phe
1100 1105 1110
Phe Tyr Arg Glu Leu Asp Leu Leu Gly Tyr Asp Tyr Ser Lys Asp
1115 1120 1125
Phe Arg Arg Leu Gln Thr Met Arg Arg Ala Asp Ser Lys Ala Ser
1130 1135 1140
Gly Thr Leu Ala Phe Leu Pro Leu Lys Asp Glu Leu Arg Asn Glu
1145 1150 1155
Pro Leu Leu Leu His Pro Ala Pro Leu Asp Ile Ala Phe Gln Thr
1160 1165 1170
Val Ile Gly Ala Tyr Ser Ser Pro Gly Asp Arg Arg Leu Arg Ser
1175 1180 1185
Leu Tyr Val Pro Thr His Val Asp Arg Val Thr Leu Ile Pro Ser
1190 1195 1200
Leu Cys Ile Ser Ala Gly Asn Ser Gly Glu Thr Glu Leu Ala Phe
1205 1210 1215
Asp Thr Ile Asn Thr His Asp Lys Gly Asp Phe Leu Ser Gly Asp
1220 1225 1230
Ile Thr Val Tyr Asp Ser Thr Lys Thr Thr Leu Phe Gln Val Asp
1235 1240 1245
Asn Ile Val Phe Lys Pro Phe Ser Pro Pro Thr Ala Ser Thr Asp
1250 1255 1260
His Arg Ile Phe Ala Lys Trp Val Trp Gly Pro Leu Thr Pro Glu
1265 1270 1275
Lys Leu Leu Glu Asp Pro Ala Thr Leu Ile Ile Ala Arg Asp Lys
1280 1285 1290
Glu Asp Ile Leu Thr Ile Glu Arg Ile Val Tyr Phe Tyr Ile Lys
1295 1300 1305
Ser Phe Leu Ala Gln Ile Thr Pro Asp Asp Arg Gln Asn Ala Asp
1310 1315 1320
Leu His Ser Gln Lys Tyr Ile Glu Trp Cys Asp Gln Val Gln Ala
1325 1330 1335
Asp Ala Arg Ala Gly His His Gln Trp Tyr Gln Glu Ser Trp Glu
1340 1345 1350
Glu Asp Thr Ser Val His Ile Glu Gln Met Cys Glu Ser Asn Ser
1355 1360 1365
Ser His Pro His Val Arg Leu Ile Gln Arg Val Gly Lys Glu Leu
1370 1375 1380
Ile Ser Ile Val Arg Gly Asn Gly Asp Pro Leu Asp Ile Met Asn
1385 1390 1395
Arg Asp Gly Leu Phe Thr Glu Tyr Tyr Thr Asn Lys Leu Ala Phe
1400 1405 1410
Gly Ser Ala Ile His Val Val Gln Asp Leu Val Ser Gln Ile Ala
1415 1420 1425
His Arg Tyr Gln Ser Ile Asp Ile Leu Glu Ile Gly Leu Gly Thr
1430 1435 1440
Gly Ile Ala Thr Lys Arg Val Leu Ala Ser Pro Gln Leu Gly Phe
1445 1450 1455
Asn Ser Tyr Thr Cys Thr Asp Ile Ser Ala Asp Val Ile Gly Lys
1460 1465 1470
Ala Arg Glu Gln Leu Ser Glu Phe Asp Gly Leu Met Gln Phe Glu
1475 1480 1485
Ala Leu Asp Ile Asn Arg Ser Pro Ala Glu Gln Gly Phe Lys Pro
1490 1495 1500
His Ser Tyr Asp Leu Ile Ile Ala Ser Asp Val Leu His Ala Ser
1505 1510 1515
Ser Asn Phe Glu Glu Lys Leu Ala His Ile Arg Ser Leu Leu Lys
1520 1525 1530
Pro Gly Gly His Leu Val Thr Phe Gly Val Thr His Arg Glu Pro
1535 1540 1545
Ala Arg Leu Ala Phe Ile Ser Gly Leu Phe Ala Asp Arg Trp Thr
1550 1555 1560
Gly Glu Asp Glu Thr Arg Ala Leu Ser Ala Ser Gly Ser Val Asp
1565 1570 1575
Gln Trp Glu His Thr Leu Lys Arg Val Gly Phe Ser Gly Val Asp
1580 1585 1590
Ser Arg Thr Leu Asp Arg Glu Asp Asp Leu Ile Pro Ser Val Phe
1595 1600 1605
Ser Thr His Ala Val Asp Ala Thr Val Glu Arg Leu Tyr Asp Pro
1610 1615 1620
Leu Ser Ala Pro Leu Lys Asp Ser Tyr Pro Pro Leu Val Val Ile
1625 1630 1635
Gly Gly Glu Ser Thr Lys Thr Glu Arg Ile Leu Asn Asp Met Lys
1640 1645 1650
Ala Ala Leu Pro His Arg His Ile His Ser Val Lys Arg Leu Glu
1655 1660 1665
Ser Val Leu Asp Asp Pro Ala Leu Gln Pro Lys Ser Thr Phe Val
1670 1675 1680
Ile Leu Ser Glu Leu Asp Asp Glu Val Phe Cys Asn Leu Glu Glu
1685 1690 1695
Asp Lys Phe Glu Ala Val Lys Ser Leu Leu Phe Tyr Ala Gly Arg
1700 1705 1710
Met Met Trp Leu Thr Glu Asn Ala Trp Ile Asp His Pro His Gln
1715 1720 1725
Ala Ser Thr Ile Gly Met Leu Arg Thr Ile Lys Leu Glu Asn Pro
1730 1735 1740
Asp Leu Gly Thr His Val Phe Asp Val Asp Thr Val Glu Asn Leu
1745 1750 1755
Asp Thr Lys Phe Phe Val Glu Gln Leu Leu Arg Phe Glu Glu Ser
1760 1765 1770
Asp Asp Gln Leu Leu Glu Ser Ile Thr Trp Thr His Glu Pro Glu
1775 1780 1785
Val Tyr Trp Cys Lys Gly Arg Ala Trp Val Pro Arg Leu Lys Gln
1790 1795 1800
Asp Ile Ala Arg Asn Asp Arg Met Asn Ser Ser Arg Arg Pro Ile
1805 1810 1815
Phe Gly Asn Phe Asn Ser Ser Lys Thr Ala Ile Ala Leu Lys Glu
1820 1825 1830
Ala Arg Gly Ala Ser Ser Ser Met Tyr Tyr Leu Glu Ser Thr Glu
1835 1840 1845
Thr Cys Asp Ser Leu Glu Asp Ala Arg His Ala Gly Lys Ala Thr
1850 1855 1860
Val Arg Val Arg Tyr Ala Leu Pro Gln Ala Ile Arg Val Gly His
1865 1870 1875
Leu Gly Tyr Phe His Val Val Gln Gly Ser Ile Leu Glu Asn Thr
1880 1885 1890
Cys Glu Val Pro Val Val Ala Leu Ala Glu Lys Asn Gly Ser Ile
1895 1900 1905
Leu His Val Pro Arg Asn Tyr Met His Ser Leu Pro Asp Asn Met
1910 1915 1920
Ala Glu Gly Glu Asp Ser Ser Phe Leu Leu Ser Thr Ala Ala Ala
1925 1930 1935
Leu Leu Ala Glu Thr Ile Leu Ser Ser Ala Gln Ser Phe Gly Ser
1940 1945 1950
Asp Ala Ser Ile Leu Ile Met Glu Pro Pro Ile Phe Cys Val Lys
1955 1960 1965
Ala Ile Leu Glu Ser Ala Lys Thr Tyr Gly Val Gln Val His Leu
1970 1975 1980
Ala Thr Thr Leu Ser Asp Val Lys Thr Ile Pro Ala Pro Trp Ile
1985 1990 1995
Arg Leu His Ala Lys Glu Thr Asp Ala Arg Leu Lys His Ser Leu
2000 2005 2010
Pro Thr Asn Met Met Ala Phe Phe Asp Leu Ser Thr Asp Arg Thr
2015 2020 2025
Ala Ala Gly Ile Thr Asn Arg Leu Ala Lys Leu Leu Pro Pro Ser
2030 2035 2040
Cys Phe Met Tyr Ser Gly Asp Tyr Leu Ile Arg Ser Thr Ala Ser
2045 2050 2055
Thr Tyr Lys Val Ser His Val Glu Asp Ile Pro Ile Leu Glu His
2060 2065 2070
Ser Val Ala Met Ala Lys Asn Thr Val Ser Ala Ser Thr Val Asp
2075 2080 2085
Asp Thr Glu Lys Val Ile Thr Ala Thr Gln Ile Leu Leu Pro Gly
2090 2095 2100
Gln Leu Ser Val Asn His Asn Asp Gln Arg Phe Asn Leu Ala Thr
2105 2110 2115
Val Ile Asp Trp Lys Glu Asn Glu Val Ser Ala Arg Ile Cys Pro
2120 2125 2130
Ile Asp Ser Gly Asn Leu Phe Ser Asn Lys Lys Thr Tyr Leu Leu
2135 2140 2145
Val Gly Leu Thr Gly Asp Leu Gly Arg Ser Leu Cys Arg Trp Met
2150 2155 2160
Ile Leu His Gly Ala Arg His Val Val Leu Thr Ser Arg Asn Pro
2165 2170 2175
Arg Leu Asp Pro Lys Trp Ile Ala Asn Met Glu Ala Leu Gly Gly
2180 2185 2190
Asp Ile Thr Val Leu Ser Met Asp Val Ala Asn Glu Asp Ser Val
2195 2200 2205
Asp Ala Gly Leu Gly Lys Leu Val Asp Met Lys Leu Pro Pro Val
2210 2215 2220
Ala Gly Ile Ala Phe Gly Pro Leu Val Leu Gln Asp Val Met Leu
2225 2230 2235
Lys Asn Met Asp His Gln Met Met Asp Met Val Leu Lys Pro Lys
2240 2245 2250
Val Gln Gly Ala Arg Ile Leu His Glu Arg Phe Ser Glu Gln Thr
2255 2260 2265
Gly Ser Lys Ala Leu Asp Phe Phe Ile Met Phe Ser Ser Ile Val
2270 2275 2280
Ala Val Ile Gly Asn Pro Gly Gln Ser Asn Tyr Gly Ala Ala Asn
2285 2290 2295
Ala Tyr Leu Gln Ala Leu Ala Gln Gln Arg Cys Ala Arg Gly Leu
2300 2305 2310
Ala Gly Ser Thr Ile Asp Ile Gly Ala Val Tyr Gly Val Gly Phe
2315 2320 2325
Val Thr Arg Ala Glu Met Glu Glu Asp Phe Asp Ala Ile Arg Phe
2330 2335 2340
Met Phe Asp Ser Val Glu Glu His Glu Leu His Thr Leu Phe Ala
2345 2350 2355
Glu Ala Val Val Ser Asp Gln Arg Ala Arg Gln Gln Pro Gln Arg
2360 2365 2370
Lys Thr Val Ile Asp Met Ala Asp Leu Glu Leu Thr Thr Gly Ile
2375 2380 2385
Pro Asp Leu Asp Pro Ala Leu Gln Asp Arg Ile Ile Tyr Phe Asn
2390 2395 2400
Asp Pro Arg Phe Gly Asn Phe Lys Ile Pro Gly Gln Arg Gly Asp
2405 2410 2415
Gly Gly Asp Asn Gly Ser Gly Ser Lys Gly Ser Ile Ala Asp Gln
2420 2425 2430
Leu Lys Gln Ala Thr Thr Leu Asp Gln Val Arg Gln Ile Val Ile
2435 2440 2445
Asp Gly Leu Ser Glu Lys Leu Arg Val Thr Leu Gln Val Ser Asp
2450 2455 2460
Gly Glu Ser Val Asp Pro Thr Ile Pro Leu Ile Asp Gln Gly Val
2465 2470 2475
Asp Ser Leu Gly Ala Val Thr Val Gly Ser Trp Phe Ser Lys Gln
2480 2485 2490
Leu Tyr Leu Asp Leu Pro Leu Leu Arg Val Leu Gly Gly Ala Ser
2495 2500 2505
Val Ala Asp Leu Ala Asp Asp Ala Ala Thr Arg Leu Pro Ala Thr
2510 2515 2520
Ser Ile Pro Leu Leu Leu Gln Ile Gly Asp Ser Thr Gly Thr Ser
2525 2530 2535
Asp Ser Gly Ala Ser Pro Thr Pro Thr Asp Ser His Asp Glu Ala
2540 2545 2550
Ser Ser Ala Thr Ser Thr Asp Ala Ser Ser Ala Glu Glu Asp Glu
2555 2560 2565
Glu Gln Glu Asp Asp Asn Glu Gln Gly Gly Arg Lys Ile Leu Arg
2570 2575 2580
Arg Glu Arg Leu Ser Leu Gly Gln Glu Tyr Ser Trp Arg Gln Gln
2585 2590 2595
Gln Met Val Lys Asp His Thr Ile Phe Asn Asn Thr Ile Gly Met
2600 2605 2610
Phe Met Lys Gly Thr Ile Asp Leu Asp Arg Leu Arg Arg Ala Leu
2615 2620 2625
Lys Ala Ser Leu Arg Arg His Glu Ile Phe Arg Thr Cys Phe Val
2630 2635 2640
Thr Gly Asp Asp Tyr Ser Ser Asp Leu Asn Gly Pro Val Gln Val
2645 2650 2655
Val Leu Lys Asn Pro Glu Asn Arg Val His Phe Val Gln Val Asn
2660 2665 2670
Asn Ala Ala Glu Ala Glu Glu Glu Tyr Arg Lys Leu Glu Lys Thr
2675 2680 2685
Asn Tyr Ser Ile Ser Thr Gly Asp Thr Leu Arg Leu Val Asp Phe
2690 2695 2700
Tyr Trp Gly Thr Asp Asp His Leu Leu Val Ile Gly Tyr His Arg
2705 2710 2715
Leu Val Gly Asp Gly Ser Thr Thr Glu Asn Leu Phe Asn Glu Ile
2720 2725 2730
Gly Gln Ile Tyr Ser Gly Val Lys Met Gln Arg Pro Ser Thr Gln
2735 2740 2745
Phe Ser Asp Leu Ala Val Gln Gln Arg Glu Asn Leu Glu Asn Gly
2750 2755 2760
Arg Met Gly Asp Asp Ile Ala Phe Trp Lys Ser Met His Ser Lys
2765 2770 2775
Val Ser Ser Ser Ala Pro Thr Val Leu Pro Ile Met Asn Leu Ile
2780 2785 2790
Asn Asp Pro Ala Ala Asn Ser Glu Gln Gln Gln Ile Gln Pro Phe
2795 2800 2805
Thr Trp Gln Gln Tyr Glu Ala Ile Ala Arg Leu Asp Pro Met Val
2810 2815 2820
Ala Phe Arg Ile Lys Glu Arg Ser Arg Lys His Lys Ala Thr Pro
2825 2830 2835
Met Gln Phe Tyr Leu Ala Ala Tyr His Val Leu Leu Ala Arg Leu
2840 2845 2850
Thr Gly Ser Lys Asp Ile Thr Ile Gly Leu Ala Glu Thr Asn Arg
2855 2860 2865
Ser Thr Met Glu Glu Ile Ser Ala Met Gly Phe Phe Ala Asn Val
2870 2875 2880
Leu Pro Leu Arg Phe Asp Glu Phe Val Gly Ser Lys Thr Phe Gly
2885 2890 2895
Glu His Leu Val Ala Thr Lys Asp Ser Val Arg Glu Ala Met Gln
2900 2905 2910
His Ala Arg Val Pro Tyr Gly Val Ile Leu Asp Cys Leu Gly Leu
2915 2920 2925
Asn Leu Pro Thr Ser Gly Glu Glu Pro Lys Thr Gln Thr His Ala
2930 2935 2940
Pro Leu Phe Gln Ala Val Phe Asp Tyr Lys Gln Gly Gln Ala Glu
2945 2950 2955
Ser Gly Ser Ile Gly Asn Ala Lys Met Thr Ser Val Leu Ala Ser
2960 2965 2970
Arg Glu Arg Thr Pro Tyr Asp Ile Val Leu Glu Met Trp Asp Asp
2975 2980 2985
Pro Thr Lys Asp Pro Leu Ile His Val Lys Leu Gln Ser Ser Leu
2990 2995 3000
Tyr Gly Pro Glu His Ala Gln Ala Phe Val Asp His Phe Ser Ser
3005 3010 3015
Ile Leu Thr Met Phe Ser Met Asn Pro Ala Leu Lys Leu Ala
3020 3025 3030
<210>45
<211>7692
<212>DNA
<213>柑桔青霉
<220>
<221>CDS
<222>(1)..(7692)
<400>45
atg aac aat acc ccc gcc gta acc gca acc gca acc gca acc gca acc 48
Met Asn Asn Thr Pro Ala Val Thr Ala Thr Ala Thr Ala Thr Ala Thr
1 5 10 15
gca acc gca atg gca ggc tcg gct tgc tct aac aca tcc acg ccc att 96
Ala Thr Ala Met Ala Gly Ser Ala Cys Ser Asn Thr Ser Thr Pro Ile
20 25 30
gcc ata gtt gga atg gga tgt cga ttt gct gga gat gca acg agt cca 144
Ala Ile Val Gly Met Gly Cys Arg Phe Ala Gly Asp Ala Thr Ser Pro
35 40 45
cag aag ctt tgg gaa atg gtt gaa aga gga ggc agt gcc tgg tct aag 192
Gln Lys Leu Trp Glu Met Val Glu Arg Gly Gly Ser Ala Trp Ser Lys
50 55 60
gtc ccc tcc tcg cga ttc aat gtg aga gga gta tac cac ccg aat ggc 240
Val Pro Ser Ser Arg Phe Asn Val Arg Gly Val Tyr His Pro Asn Gly
65 70 75 80
gaa agg gtc ggg tcc acc cac gta aag ggt gga cac ttc atc gac gag 288
Glu Arg Val Gly Ser Thr His Val Lys Gly Gly His Phe Ile Asp Glu
85 90 95
gat cct gct tta ttt gac gcc gcg ttc ttc aac atg acc aca gag gtc 336
Asp Pro Ala Leu Phe Asp Ala Ala Phe Phe ASn Met Thr Thr Glu Val
100 105 110
gcc agc tgc atg gat ccg cag tat cgg ctt atg ctt gag gtg gtc tac 384
Ala Ser Cys Met Asp Pro Gln Tyr Arg Leu Met Leu Glu Val Val Tyr
115 120 125
gaa tcg ctg gag agt gcc ggt atc acc atc gat ggt atg gca ggc tct 432
Glu Ser Leu Glu Ser Ala Gly Ile Thr Ile Asp Gly Met Ala Gly Ser
130 135 140
aat acg tcg gtg ttt ggg ggt gtc atg tac cac gac tat cag gat tcg 480
Asn Thr Ser Val Phe Gly Gly Val Met Tyr His Asp Tyr Gln Asp Ser
145 150 155 160
ctc aat cgt gac ccc gag aca gtt ccg cgt tat ttc ata act ggc aac 528
Leu Asn Arg Asp Pro Glu Thr Val Pro Arg Tyr Phe Ile Thr Gly Asn
165 170 175
tca gga aca atg ctt tcg aac cgg ata tca cac ttc tac gac tta cgt 576
Ser Gly Thr Met Leu Ser Asn Arg Ile Ser His Phe Tyr Asp Leu Arg
180 185 190
ggt ccc agc gtg acg gtt gac acg gcc tgt tcg acg aca ttg acc gca 624
Gly Pro Ser Val Thr Val Asp Thr Ala Cys Ser Thr Thr Leu Thr Ala
195 200 205
ctg cac ttg gcg tgc cag agc tta cgt act ggg gag tca gat aca gcc 672
Leu His Leu Ala Cys Gln Ser Leu Arg Thr Gly Glu Ser Asp Thr Ala
210 215 220
atc gtt atc ggt gca aat ctt ctg ctc aat ccc gat gtt ttt gtt acg 720
Ile Val Ile Gly Ala Asn Leu Leu Leu Asn Pro Asp Val Phe Val Thr
225 230 235 240
atg tca aac ctg gga ttt ttg tcc ccg gat ggt atc tcg tac tct ttt 768
Met Ser Asn Leu Gly Phe Leu Ser Pro Asp Gly Ile Ser Tyr Ser Phe
245 250 255
gat cct cga gcg aat gga tat ggt cgc ggg gaa gga att gcc gct ctg 816
Asp Pro Arg Ala Asn Gly Tyr Gly Arg Gly Glu Gly Ile Ala Ala Leu
260 265 270
gta ata aag gcc ctc cct aac gcg ttg cga gac caa gac cct atc cga 864
Val Ile Lys Ala Leu Pro Asn Ala Leu Arg Asp Gln Asp Pro Ile Arg
275 280 285
gcc gtc att cga gag aca gcg ctg aac cag gat ggc aaa aca ccc gca 912
Ala Val Ile Arg Glu Thr Ala Leu Asn Gln Asp Gly Lys Thr Pro Ala
290 295 300
att act gcg ccg agt gat gtg gcg cag aaa agt ctg atc cag gag tgt 960
Ile Thr Ala Pro Ser Asp Val Ala Gln Lys Ser Leu Ile Gln Glu Cys
305 310 315 320
tac gat aag gct ggg cta gat atg tcg ttg acc tcg tac gtg gag gcc 1008
Tyr Asp Lys Ala Gly Leu Asp Met Ser Leu Thr Ser Tyr Val Glu Ala
325 330 335
cac gga act gga aca cca act ggt gac ccc ctt gaa atc tca gca att 1056
His Gly Thr Gly Thr Pro Thr Gly Asp Pro Leu Glu Ile Ser Ala Ile
340 345 350
tca gca gct ttt aaa gga cat cct ctg cac ctt ggc tct gtg aaa gca 1104
Ser Ala Ala Phe Lys Gly His Pro Leu His Leu Gly Ser Val Lys Ala
355 360 365
aat att ggc cat aca gaa gcc gcc agt ggc ctg gcc agt ata atc aag 1152
Asn Ile Gly His Thr Glu Ala Ala Ser Gly Leu Ala Ser Ile Ile Lys
370 375 380
gtg gcc ttg gcc ttg gag aag ggc ttg att ccc cct aat gcg cgg ttc 1200
Val Ala Leu Ala Leu Glu Lys Gly Leu Ile Pro Pro Asn Ala Arg Phe
385 390 395 400
ctg caa aag aac agc aag ctg atg ctt gac caa aag aac atc aag atc 1248
Leu Gln Lys Asn Ser Lys Leu Met Leu Asp Gln Lys Asn Ile Lys Ile
405 410 415
ccc atg tct gct caa gac tgg cct gtg aaa gat ggg act cgt cgc gca 1296
Pro Met Ser Ala Gln Asp Trp Pro Val Lys Asp Gly Thr Arg Arg Ala
420 425 430
tct gtc aat aac ttc ggc ttt ggt ggt tcg aat gct cac gtc att ttg 1344
Ser Val Asn Asn Phe Gly Phe Gly Gly Ser Asn Ala His Val Ile Leu
435 440 445
gaa tca tat gat cgc gca tca ttg gcc ctg cca gag gat caa gtg cat 1392
Glu Ser Tyr Asp Arg Ala Ser Leu Ala Leu Pro Glu Asp Gln Val His
450 455 460
gtc aat ggt aac tct gag cat ggt agg gtt gag gat ggt tcc aaa cag 1440
Val Asn Gly Asn Ser Glu His Gly Arg Val Glu Asp Gly Ser Lys Gln
465 470 475 480
agc cgc ata tac gtt gtg cgt gcc aag gac gag caa gct tgt cgg cga 1488
Ser Arg Ile Tyr Val Val Arg Ala Lys Asp Glu Gln Ala Cys Arg Arg
485 490 495
acg ata gca agc ctg cga gac tac att aaa tcc gtc gct gac att gac 1536
Thr Ile Ala Ser Leu Arg Asp Tyr Ile Lys Ser Val Ala Asp Ile Asp
500 505 510
ggg gaa ccc ttc ctc gcc agc ctc gcc tat aca cta ggc tct cgc cgt 1584
Gly Glu Pro Phe Leu Ala Ser Leu Ala Tyr Thr Leu Gly Ser Arg Arg
515 520 525
tcc att ctg cca tgg acg tca gtg tat gta gca gac agc ctt ggc ggc 1632
Ser Ile Leu Pro Trp Thr Ser Val Tyr Val Ala Asp Ser Leu Gly Gly
530 535 540
ctt gtt tct gcc ctc agc gat gag tcc aat caa cca aaa cga gcg aat 1680
Leu Val Ser Ala Leu Ser Asp Glu Ser Asn Gln Pro Lys Arg Ala Asn
545 550 555 560
gag aaa gta cgg ctc gga ttt gta ttc acc ggt cag ggg gcg cag tgg 1728
Glu Lys Val Arg Leu Gly Phe Val Phe Thr Gly Gln Gly Ala Gln Trp
565 570 575
cat gca atg ggc aga gag ctg gtc aat aca ttc cca gta ttc aaa cag 1776
His Ala Met Gly Arg Glu Leu Val Asn Thr Phe Pro Val Phe Lys Gln
580 585 590
gcg att ctt gaa tgt gat ggc tac atc aag caa ctg ggc gcg agt tgg 1824
Ala Ile Leu Glu Cys Asp Gly Tyr Ile Lys Gln Leu Gly Ala Ser Trp
595 600 605
aat ttt atg gag gag ctc cac cgt gat gag ctg acg act cgg gta aat 1872
Asn Phe Met Glu Glu Leu His Arg Asp Glu Leu Thr Thr Arg Val Asn
610 615 620
gat gcc gaa tac agt cta cca ctg tca acc gct atc caa att gca ctt 1920
Asp Ala Glu Tyr Ser Leu Pro Leu Ser Thr Ala Ile Gln Ile Ala Leu
625 630 635 640
gtg cgt ctc ctt tgg tca tgg gga att cgg cca acg ggg ata acc agt 1968
Val Arg Leu Leu Trp Ser Trp Gly Ile Arg Pro Thr Gly Ile Thr Ser
645 650 655
cac tca agt gga gag gct gct gct gcc tac gca gct ggg gct tta tcc 2016
His Ser Ser Gly Glu Ala Ala Ala Ala Tyr Ala Ala Gly Ala Leu Ser
660 665 670
gcg cgg tcg gcc att ggg atc act tat ata cgc ggt gta ttg acc act 2064
Ala Arg Ser Ala Ile Gly Ile Thr Tyr Ile Arg Gly Val Leu Thr Thr
675 680 685
aag ccc aag ccc gca ttg gca gcc aaa gga gga atg atg gcg gtg ggt 2112
Lys Pro Lys Pro Ala Leu Ala Ala Lys Gly Gly Met Met Ala Val Gly
690 695 700
ctt ggt cgc agt gag acc aat gtt tac att tcg cgt ctc aac cag gag 2160
Leu Gly Arg Ser Glu Thr Asn Val Tyr Ile Ser Arg Leu Asn Gln Glu
705 710 715 720
gac ggc tgt gtg gtg gtt gga tgt atc aac agt caa tgt agt gtg acg 2208
Asp Gly Cys Val Val Val Gly Cys Ile Asn Ser Gln Cys Ser Val Thr
725 730 735
gtg tcg gga gat ttg ggt gca atc gag aaa ctt gaa aag ttg tta cac 2256
Val Ser Gly Asp Leu Gly Ala Ile Glu Lys Leu Glu Lys Leu Leu His
740 745 750
gcc gat ggc atc ttt acc agg aaa ctg aaa gtc act gaa gcc ttc cat 2304
Ala Asp Gly Ile Phe Thr Arg Lys Leu Lys Val Thr Glu Ala Phe His
755 760 765
tca agc cac atg cga cca atg gca gat gcc ttt ggg gcg tca ctg aga 2352
Ser Ser His Met Arg Pro Met Ala Asp Ala Phe Gly Ala Ser Leu Arg
770 775 780
gat ctg ttc aac tcg gat aac aac aac gac aat ccc aat gct gac acc 2400
Asp Leu Phe Asn Ser Asp Asn Asn Asn Asp Asn Pro Asn Ala Asp Thr
785 790 795 800
tca aag ggt gta tta tat tca tca cct aag act ggt agt cgc atg acc 2448
Ser Lys Gly Val Leu Tyr Ser Ser Pro Lys Thr Gly Ser Arg Met Thr
805 810 815
gat ctt aaa ttg cta ttg gat ccc aca cac tgg atg gat agt atg cta 2496
Asp Leu Lys Leu Leu Leu Asp Pro Thr His Trp Met Asp Ser Met Leu
820 825 830
cag ccg gta gag ttc gag tcc tca ctc cgc gag atg tgc ttt gat ccc 2544
Gln Pro Val Glu Phe Glu Ser Ser Leu Arg Glu Met Cys Phe Asp Pro
835 840 845
aac acc aaa gag aaa gcc gtc gat gtg att att gaa ata ggg cct cac 2592
Asn Thr Lys Glu Lys Ala Val Asp Val Ile Ile Glu Ile Gly Pro His
850 855 860
gga gcg ctt ggt ggt cca atc aac caa gtc atg cag gat ctg ggt ctg 2640
Gly Ala Leu Gly Gly Pro Ile Asn Gln Val Met Gln Asp Leu Gly Leu
865 870 875 880
aaa gga aca gat ata aac tat ctc agt tgc ctt tct cgc ggc aga agc 2688
Lys Gly Thr Asp Ile Asn Tyr Leu Ser Cys Leu Ser Arg Gly Arg Ser
885 890 895
tcg ttg gag aca atg tat cgt gct gct acg gag ttg ata agc aag ggt 2736
Ser Leu Glu Thr Met Tyr Arg Ala Ala Thr Glu Leu Ile Ser Lys Gly
900 905 910
tat ggg ctc aaa atg gac gct ata aac ttt cct cat gga aga aaa gag 2784
Tyr Gly Leu Lys Met Asp Ala Ile Asn Phe Pro His Gly Arg Lys Glu
915 920 925
ccc aga gtg aag gta ctg agc gat ttg ccg gcg tac ccg tgg aat cac 2832
Pro Arg Val Lys Val Leu Ser Asp Leu Pro Ala Tyr Pro Trp Asn His
930 935 940
caa acc cgt tat tgg aga gag cct cgc ggc agt cgt gag tcc aaa cag 2880
Gln Thr Arg Tyr Trp Arg Glu Pro Arg Gly Ser Arg Glu Ser Lys Gln
945 950 955 960
aga acc cat ccg cct cac act ttg ata ggc tca cgg gaa tct ctc tct 2928
Arg Thr His Pro Pro His Thr Leu Ile Gly Ser Arg Glu Ser Leu Ser
965 970 975
cct cat ttc gcg cct aaa tgg aaa cat gtt ctc cgt ctg tca gat att 2976
Pro His Phe Ala Pro Lys Trp Lys His Val Leu Arg Leu Ser Asp Ile
980 985 990
cca tgg ata cga gat cac gtc gtt ggt tcg agc atc atc ttt ccg gga 3024
Pro Trp Ile Arg Asp His Val Val Gly Ser Ser Ile Ile Phe Pro Gly
995 1000 1005
gct ggc ttc atc agc atg gcc atc gag ggg ttt tca caa gtc tgc 3069
Ala Gly Phe Ile Ser Met Ala Ile Glu Gly Phe Ser Gln Val Cys
1010 1015 1020
cca cca gtt gcg ggg gct agc atc aac tac aac ttg cgt gac gtt 3114
Pro Pro Val Ala Gly Ala Ser Ile Asn Tyr Asn Leu Arg Asp Val
1025 1030 1035
gaa ctc gcg cag gct ctc ata ata ccc gct gat gca gaa gca gag 3159
Glu Leu Ala Gln Ala Leu Ile Ile Pro Ala Asp Ala Glu Ala Glu
1040 1045 1050
gtt gac ctg cgc cta acg atc cgt tca tgt gag gaa agg tcc ctc 3204
Val Asp Leu Arg Leu Thr Ile Arg Ser Cys Glu Glu Arg Ser Leu
1055 1060 1065
ggc aca aag aac tgg cat caa ttt tct gtg cac tca att tcg ggc 3249
Gly Thr Lys Asn Trp His Gln Phe Ser Val His Ser Ile Ser Gly
1070 1075 1080
gaa aat aat acc tgg aca gaa cac tgc acc gga tta ata cgt tcg 3294
Glu Asn Asn Thr Trp Thr Glu His Cys Thr Gly Leu Ile Arg Ser
1085 1090 1095
gag agc gaa aga agc cac ctt gac tgt tca act gtg gaa gcc tca 3339
Glu Ser Glu Arg Ser His Leu Asp Cys Ser Thr Val Glu Ala Ser
1100 1105 1110
cgc agg ttg aat cta ggc tca gat aac cgg agc att gat ccc aac 3384
Arg Arg Leu Asn Leu Gly Ser Asp Asn Arg Ser Ile Asp Pro Asn
1115 1120 1125
gat ctc tgg gag tcc tta cac gcg aat ggg ata tgc cac gga ccc 3429
Asp Leu Trp Glu Ser Leu His Ala Asn Gly Ile Cys His Gly Pro
1130 1135 1140
att ttt cag aac att cag cga att caa aac aat gga cag ggc tcg 3474
Ile Phe Gln Asn Ile Gln Arg Ile Gln Asn Asn Gly Gln Gly Ser
1145 1150 1155
ttt tgc aga ttt tcc att gct gac act gcc tcg gct atg cct cac 3519
Phe Cys Arg Phe Ser Ile Ala Asp Thr Ala Ser Ala Met Pro His
1160 1165 1170
tcg tac gag aat cga cac atc gtc cat cct act act ctg gac tcg 3564
Ser Tyr Glu Asn Arg His Ile Val His Pro Thr Thr Leu Asp Ser
1175 1180 1185
gtg atc cag gcg gca tac acg gtg tta ccc tac gcg gga aca cgt 3609
Val Ile Gln Ala Ala Tyr Thr Val Leu Pro Tyr Ala Gly Thr Arg
1190 1195 1200
atg aaa acg gcc atg gta cca agg agg cta aga aat gtc aaa ata 3654
Met Lys Thr Ala Met Val Pro Arg Arg Leu Arg Asn Val Lys Ile
1205 1210 1215
tcc tct agc ctg gct gac ttg gag gct ggt gat gct ctg gac gca 3699
Ser Ser Ser Leu Ala Asp Leu Glu Ala Gly Asp Ala Leu Asp Ala
1220 1225 1230
cag gcc agc atc aag gat cgc aac tct caa tcc ttc tct acc gac 3744
Gln Ala Ser Ile Lys Asp Arg Asn Ser Gln Ser Phe Ser Thr Asp
1235 1240 1245
ttg gca gtg ttt gat gac tat gat agc ggt tct tct ccc tcg gac 3789
Leu Ala Val Phe Asp Asp Tyr Asp Ser Gly Ser Ser Pro Ser Asp
1250 1255 1260
gga atc cca gtc ata gag att gaa ggc ctt gtt ttc cag tcg gtt 3834
Gly Ile Pro Val Ile Glu Ile Glu Gly Leu Val Phe Gln Ser Val
1265 1270 1275
gga agc agc ttc tct gac caa aag tca gac tcc aac gac aca gaa 3879
Gly Ser Ser Phe Ser Asp Gln Lys Ser Asp Ser Asn Asp Thr Glu
1280 1285 1290
aat gcc tgc agc tcc tgg gtt tgg gcc cct gac atc agc ttg ggt 3924
Asn Ala Cys Ser Ser Trp Val Trp Ala Pro Asp Ile Ser Leu Gly
1295 1300 1305
gac tcc act tgg ctc aaa gaa aag ttg agc act gag gct gag acg 3969
Asp Ser Thr Trp Leu Lys Glu Lys Leu Ser Thr Glu Ala Glu Thr
1310 1315 1320
aaa gaa acg gaa ctc atg atg gac ctc cga aga tgc acg atc aac 4014
Lys Glu Thr Glu Leu Met Met Asp Leu Arg Arg Cys Thr Ile Asn
1325 1330 1335
ttt ata cag gag gct gtc act gat ttg aca aat tct gat atc caa 4059
Phe Ile Gln Glu Ala Val Thr Asp Leu Thr Asn Ser Asp Ile Gln
1340 1345 1350
cat ctg gat ggc cac ctt cag aag tat ttc gat tgg atg aat gtc 4104
His Leu Asp Gly His Leu Gln Lys Tyr Phe Asp Trp Met Asn Val
1355 1360 1365
caa ttg gac ctt gcg aga caa aac aag ctc agc cca gcc agt tgc 4149
Gln Leu Asp Leu Ala Arg Gln Asn Lys Leu Ser Pro Ala Ser Cys
1370 1375 1380
gac tgg cta agt gac gat gct gag cag aag aaa tgc cta cag gcc 4194
Asp Trp Leu Ser Asp Asp Ala Glu Gln Lys Lys Cys Leu Gln Ala
1385 1390 1395
aga gtc gct gga gaa agc gtc aat ggc gag atg att tct cgt cta 4239
Arg Val Ala Gly Glu Ser Val Asn Gly Glu Met Ile Ser Arg Leu
1400 1405 1410
gga cct cag tta ata gca atg cta cgc cgc gaa aca gag cca ctt 4284
Gly Pro Gln Leu Ile Ala Met Leu Arg Arg Glu Thr Glu Pro Leu
1415 1420 1425
gag ttg atg atg caa gat cag ctg cta agc aga tac tac gtc aac 4329
Glu Leu Met Met Gln Asp Gln Leu Leu Ser Arg Tyr Tyr Val Asn
1430 1435 1440
gca atc aaa tgg agc cga tca aac gca caa gcc agc gag ctg atc 4374
Ala Ile Lys Trp Ser Arg Ser Asn Ala Gln Ala Ser Glu Leu Ile
1445 1450 1455
cga ctt tgc gcc cac aag aac ccg cgt tct cgc att ttg gag att 4419
Arg Leu Cys Ala His Lys Asn Pro Arg Ser Arg Ile Leu Glu Ile
1460 1465 1470
ggc gga ggc acg ggc ggc tgc aca aag ctt att gtc aat gca ttg 4464
Gly Gly Gly Thr Gly Gly Cys Thr Lys Leu Ile Val Asn Ala Leu
1475 1480 1485
gga aac acc aag ccg atc gat cgt tat gac ttc acc gat gtg tct 4509
Gly Asn Thr Lys Pro Ile Asp Arg Tyr Asp Phe Thr Asp Val Ser
1490 1495 1500
gcc ggg ttt ttc gag tcg gcg cgt gag caa ttt gcg gat tgg caa 4554
Ala Gly Phe Phe Glu Ser Ala Arg Glu Gln Phe Ala Asp Trp Gln
1505 1510 1515
gac gtg atg act ttc aaa aaa ttg gat att gaa agc gat ccc gag 4599
Asp Val Met Thr Phe Lys Lys Leu Asp Ile Glu Ser Asp Pro Glu
1520 1525 1530
caa caa ggg ttt gaa tgt gcc acc tac gat gtg gtc gtg gct tgc 4644
Gln Gln Gly Phe Glu Cys Ala Thr Tyr Asp Val Val Val Ala Cys
1535 1540 1545
cag gtc ctg cat gca act cga tgc atg aaa cga aca ctg agt aac 4689
Gln Val Leu His Ala Thr Arg Cys Met Lys Arg Thr Leu Ser Asn
1550 1555 1560
gtt cga aaa ttg ctc aag cct ggg ggc aac ttg att ttg gtt gag 4734
Val Arg Lys Leu Leu Lys Pro Gly Gly Asn Leu Ile Leu Val Glu
1565 1570 1575
act acc agg gat cag ctc gat ttg ttc ttt acc ttc gga ctg ttg 4779
Thr Thr Arg Asp Gln Leu Asp Leu Phe Phe Thr Phe Gly Leu Leu
1580 1585 1590
cca ggt tgg tgg ctc agt gag gag cct gag cgg aag tcg acg cca 4824
Pro Gly Trp Trp Leu Ser Glu Glu Pro Glu Arg Lys Ser Thr Pro
1595 1600 1605
tcg ctc act acc gat ctt tgg aac acc atg ttg gac acg agc ggt 4869
Ser Leu Thr Thr Asp Leu Trp Asn Thr Met Leu Asp Thr Ser Gly
1610 1615 1620
ttc aac ggt gtg gaa ttg gag gtt cgt gat tgt gaa gac gat gag 4914
Phe Asn Gly Val Glu Leu Glu Val Arg Asp Cys Glu Asp Asp Glu
1625 1630 1635
ttt tac atg atc agc aca atg cta tcg acg gct aga aaa gag aat 4959
Phe Tyr Met Ile Ser Thr Met Leu Ser Thr Ala Arg Lys Glu Asn
1640 1645 1650
aca acc ccg gat aca gtg gca gaa tcg gag gtg ctt ttg ctg cac 5004
Thr Thr Pro Asp Thr Val Ala Glu Ser Glu Val Leu Leu Leu His
1655 1660 1665
gga gcg ctc cga cct cct tca tct tgg ctg gaa agt ctc cag gca 5049
Gly Ala Leu Arg Pro Pro Ser Ser Trp Leu Glu Ser Leu Gln Ala
1670 1675 1680
gca att tgt gaa aag acc agt tct agc cca tcg atc aac gct ctg 5094
Ala Ile Cys Glu Lys Thr Ser Ser Ser Pro Ser Ile Asn Ala Leu
1685 1690 1695
ggc gag gta gat acc act gga agg aca tgc att ttt ctt ggg gaa 5139
Gly Glu Val Asp Thr Thr Gly Arg Thr Cys Ile Phe Leu Gly Glu
1700 1705 1710
atg gag tcc tcg ctc ctt gga gag gtg gga agc gag acc ttc aaa 5184
Met Glu Ser Ser Leu Leu Gly Glu Val Gly Ser Glu Thr Phe Lys
1715 1720 1725
tcc atc acc gcg atg ctg aat aac tgc aac gca ctt ctc tgg gtg 5229
Ser Ile Thr Ala Met Leu Asn Asn Cys Asn Ala Leu Leu Trp Val
1730 1735 1740
tct aga gga gca gcc atg agc tcc gag gat cca tgg aaa gct cta 5274
Ser Arg Gly Ala Ala Met Ser Ser Glu Asp Pro Trp Lys Ala Leu
1745 1750 1755
cat att ggt ctg ctg cgt acc atc cgc aac gaa aat aac ggg aag 5319
His Ile Gly Leu Leu Arg Thr Ile Arg Asn Glu Asn Asn Gly Lys
1760 1765 1770
gaa tat gta tcg ttg gat ctc gat cct tct cga aac gca tac acc 5364
Glu Tyr Val Ser Leu Asp Leu Asp Pro Ser Arg Asn Ala Tyr Thr
1775 1780 1785
cac gag tcc ctg tat gct atc tgc aat atc ttc aat ggc cgc ctc 5409
His Glu Ser Leu Tyr Ala Ile Cys Asn Ile Phe Asn Gly Arg Leu
1790 1795 1800
ggc gac ctt tcc gaa gac aag gag ttt gaa ttt gca gag aga aac 5454
Gly Asp Leu Ser Glu Asp Lys Glu Phe Glu Phe Ala Glu Arg Ash
1805 1810 1815
ggc gtc atc cac gta ccg cga ctt ttc aat gac ccg cac tgg aag 5499
Gly Val Ile His Val Pro Arg Leu Phe Asn Asp Pro His Trp Lys
1820 1825 1830
gac caa gaa gcg gtt gag gtc aca ctg cag ccg ttc gag caa ccc 5544
Asp Gln Glu Ala Val Glu Val Thr Leu Gln Pro Phe Glu Gln Pro
1835 1840 1845
ggg cgt cgt ctg cgg atg gag gtt gag acg cca ggg ctc tta gac 5589
Gly Arg Arg Leu Arg Met Glu Val Glu Thr Pro Gly Leu Leu Asp
1850 1855 1860
tcc ctg caa ttt cga gac gac gaa gga cgt gaa ggc aag gat ctt 5634
Ser Leu Gln Phe Arg Asp Asp Glu Gly Arg Glu Gly Lys Asp Leu
1865 1870 1875
ccg gat gat tgg gta gaa atc gaa ccc aaa gct ttc ggt ctc aat 5679
Pro Asp Asp Trp Val Glu Ile Glu Pro Lys Ala Phe Gly Leu Asn
1880 1885 1890
ttt cgg gat gtc atg gtt gcc atg ggt caa ttg gag gcc aac cgt 5724
Phe Arg Asp Val Met ValAla Met Gly Gln Leu Glu Ala Asn Arg
1895 1900 1905
gtg atg ggc ttc gaa tgc gcc gga gtg atc aca aag ctc ggt gga 5769
Val Met Gly Phe Glu Cys Ala Gly Val Ile Thr Lys Leu Gly Gly
1910 1915 1920
gct gct gcc gct agc caa ggc ctc aga tta ggg gac cgc gta tgt 5814
Ala Ala Ala Ala Ser Gln Gly Leu Arg Leu Gly Asp Arg Val Cys
1925 1930 1935
gca cta ctg aaa ggc cat tgg gcg acc aga aca cag acg ccg tac 5859
Ala Leu Leu Lys Gly His Trp Ala Thr Arg Thr Gln Thr Pro Tyr
1940 1945 1950
act aat gtc gtc cgt att ccg gac gaa atg ggc ttc cca gaa gcc 5904
Thr Asn Val Val Arg Ile Pro Asp Glu Met Gly Phe Pro Glu Ala
1955 1960 1965
gct tcg gtc ccc ctg gct ttc act acc gca tat att gcg ctt tat 5949
Ala Ser Val Pro Leu Ala Phe Thr Thr Ala Tyr Ile Ala Leu Tyr
1970 1975 1980
acc acg gca aag cta cga cga ggc gaa aga gtc ttg atc cac agt 5994
Thr Thr Ala Lys Leu Arg Arg Gly Glu Arg Val Leu Ile His Ser
1985 1990 1995
gga gct gga ggc gtc ggt caa gca gcg atc att ttg tcc cag ctt 6039
Gly Ala Gly Gly Val Gly Gln Ala Ala Ile Ile Leu Ser Gln Leu
2000 2005 2010
gcg ggt gcc gag gtc ttc gtc aca gcg gga act caa gcc aag cgt 6084
Ala Gly Ala Glu Val Phe Val Thr Ala Gly Thr Gln Ala Lys Arg
2015 2020 2025
gac ttt gtc ggc gat aaa ttc ggc atc aat ccg gat cat atc ttc 6129
Asp Phe Val Gly Asp Lys Phe Gly Ile Asn Pro Asp His Ile Phe
2030 2035 2040
tcg agc agg aat gac tta ttc gtc gac ggc atc aaa gcc tac acg 6174
Ser Ser Arg Asn Asp Leu Phe Val Asp Gly Ile Lys Ala Tyr Thr
2045 2050 2055
ggc gga ctt ggc gtt cat gtc gtt cta aac tca ttg gca ggt caa 6219
Gly Gly Leu Gly Val His Val Val Leu Asn Ser Leu Ala Gly Gln
2060 2065 2070
ctc ctc caa gca agc ttt gac tgc atg gcc gaa ttc ggc aga ttt 6264
Leu Leu Gln Ala Ser Phe Asp Cys Met Ala Glu Phe Gly Arg Phe
2075 2080 2085
gtt gag att gga aaa aag gac ctg gag caa aac agc aga ctt gac 6309
Val Glu Ile Gly Lys Lys Asp Leu Glu Gln Asn Ser Arg Leu Asp
2090 2095 2100
atg ctg cca ttc acc cgg gac gtc tct ttc aca tca att gat ctt 6354
Met Leu Pro Phe Thr Arg Asp Val Ser Phe Thr Ser Ile Asp Leu
2105 2110 2115
ctc tcg tgg caa aga gcc aaa agt gaa gaa gta tcc gaa gcg ttg 6399
Leu Ser Trp Gln Arg Ala Lys Ser Glu Glu Val Ser Glu Ala Leu
2120 2125 2130
aac cat gtc aca aaa ctc ctc gag aca aaa gcg att ggc ttg att 6444
Asn His Val Thr Lys Leu Leu Glu Thr Lys Ala Ile Gly Leu Ile
2135 2140 2145
ggt cca atc cag cag cac tcc ttg tca aac atc gag aag gcc ttc 6489
Gly Pro Ile Gln Gln His Ser Leu Ser Asn Ile Glu Lys Ala Phe
2150 2155 2160
cgt acg atg cag agt ggt cag cat gtt ggc aaa gtt gtg gtc aat 6534
Arg Thr Met Gln Ser Gly Gln His Val Gly Lys Val Val Val Asn
2165 2170 2175
gta tct ggg gac gaa ctg gtc cca gtc ggc gat gga ggg ttc tcg 6579
Val Ser Gly Asp Glu Leu Val Pro Val Gly Asp Gly Gly Phe Ser
2180 2185 2190
ctg aag ctg aag cct gac agt tct tac cta gtt gct ggt ggg ctg 6624
Leu Lys Leu Lys Pro Asp Ser Ser Tyr Leu Val Ala Gly Gly Leu
2195 2200 2205
ggg gga att gga aag cag atc tgt cag tgg ctt gtt gat cat ggc 6669
Gly Gly Ile Gly Lys Gln Ile Cys Gln Trp Leu Val Asp His Gly
2210 2215 2220
gcg aag cac ttg att atc cta tcg aga agt gca aag gcc agt cca 6714
Ala Lys His Leu Ile Ile Leu Ser Arg Ser Ala Lys Ala Ser Pro
2225 2230 2235
ttc ata acc agc ttg caa aat caa cag tgc gct gtc tat cta cac 6759
Phe Ile Thr Ser Leu Gln Asn Gln Gln Cys Ala Val Tyr Leu His
2240 2245 2250
gca tgt gac atc tca gat caa gat cag gtc acc aag gtg ctc cgg 6804
Ala Cys Asp Ile Ser Asp Gln Asp Gln Val Thr Lys Val Leu Arg
2255 2260 2265
ttg tgc gaa gaa gca cat gca ccg cca att cga ggt atc ata caa 6849
Leu Cys Glu Glu Ala His Ala Pro Pro Ile Arg Gly Ile Ile Gln
2270 2275 2280
ggt gcc atg gtt ctc aag gac gcg ctt cta tcg cga atg aca ttg 6894
Gly Ala Met Val Leu Lys Asp Ala Leu Leu Ser Arg Met Thr Leu
2285 2290 2295
gat gaa ttt aat gca gca aca cgc cca aaa gta cag ggt agt tgg 6939
Asp Glu Phe Asn Ala Ala Thr Arg Pro Lys Val Gln Gly Ser Trp
2300 2305 2310
tat ctt cac aag atc gca cag gat gtt gac ttc ttc gtg atg ctc 6984
Tyr Leu His Lys Ile Ala Gln Asp Val Asp Phe Phe Val Met Leu
2315 2320 2325
tca tcc ctt gtt ggg gtc atg ggt ggg gca ggc cag gcc aat tac 7029
Ser Ser Leu Val Gly Val Met Gly Gly Ala Gly Gln Ala Asn Tyr
2330 2335 2340
gca gct gct ggt gca ttc cag gac gca ctt gcg cac cac cgg aga 7074
Ala Ala Ala Gly Ala Phe Gln Asp Ala Leu Ala His His Arg Arg
2345 2350 2355
gcc cat ggc atg ccg gct gtc acc att gac ttg ggc atg gtc aag 7119
Ala His Gly Met Pro Ala Val Thr Ile Asp Leu Gly Met Val Lys
2360 2365 2370
tct gtt gga tac gtg gct gaa act ggc cgt ggt gtg gcc gac cgg 7164
Ser Val Gly Tyr Val Ala Glu Thr Gly Arg Gly Val Ala Asp Arg
2375 2380 2385
ctc gct aga ata ggt tac aag cct atg cat gaa aag gac gtc atg 7209
Leu Ala Arg Ile Gly Tyr Lys Pro Met His Glu Lys Asp Val Met
2390 2395 2400
gat gtg ttg gag aag gca atc ctg tgt tct tcc cct caa ttt cca 7254
Asp Val Leu Glu Lys Ala Ile Leu Cys Ser Ser Pro Gln Phe Pro
2405 2410 2415
tca cct ccc gca gct gtg gtt aca gga atc aac aca tcc ccg ggt 7299
Ser Pro Pro Ala Ala Val Val Thr Gly Ile Asn Thr Ser Pro Gly
2420 2425 2430
gct cac tgg acc gag gca aac tgg ata cag gaa cag cgg ttt gtg 7344
Ala His Trp Thr Glu Ala Asn Trp Ile Gln Glu Gln Arg Phe Val
2435 2440 2445
gga ctt aaa tac cgc caa gtc ctt cat gca gac caa tcc ttt gtc 7389
Gly Leu Lys Tyr Arg Gln Val Leu His Ala Asp Gln Ser Phe Val
2450 2455 2460
tct tcg cat aaa aaa gga cca gat ggc gtg cgg gcc caa cta agc 7434
Ser Ser His Lys Lys Gly Pro Asp Gly Val Arg Ala Gln Leu Ser
2465 2470 2475
agg gtc acc tct cac gac gag gcc att tct atc gtc ctc aaa gca 7479
Arg Val Thr Ser His Asp Glu Ala Ile Ser Ile Val Leu Lys Ala
2480 2485 2490
atg acg gaa aag ctg atg cga atg ttt ggt ctg gca gaa gac gac 7524
Met Thr Glu Lys Leu Met Arg Met Phe Gly Leu Ala Glu Asp Asp
2495 2500 2505
atg tcc tcg tcc aaa aac ctg gca ggt gtc ggc gta gac tca ctc 7569
Met Ser Ser Ser Lys Asn Leu Ala Gly Val Gly Val Asp Ser Leu
2510 2515 2520
gtc gcc att gaa ctt cga aac tgg atc aca tct gaa atc cat gtt 7614
Val Ala Ile Glu Leu Arg Asn Trp Ile Thr Ser Glu Ile His Val
2525 2530 2535
gat gtg tcg atc ttt gag ctc atg aat ggt aac acc atc gcc ggc 7659
Asp Val Ser Ile Phe Glu Leu Met Asn Gly Asn Thr Ile Ala Gly
2540 2545 2550
ctc gtc gag tta gtt gtg gcg aaa tgc agt taa 7692
Leu Val Glu Leu Val Val Ala Lys Cys Ser
2555 2560
<210>46
<211>2563
<212>PRT
<213>柑桔青霉
<400>46
Met Asn Asn Thr Pro Ala Val Thr Ala Thr Ala Thr Ala Thr Ala Thr
1 5 10 15
Ala Thr Ala Met Ala Gly Ser Ala Cys Ser Asn Thr Ser Thr Pro Ile
20 25 30
Ala Ile Val Gly Met Gly Cys Arg Phe Ala Gly Asp Ala Thr Ser Pro
35 40 45
Gln Lys Leu Trp Glu Met Val Glu Arg Gly Gly Ser Ala Trp Ser Lys
50 55 60
Val Pro Ser Ser Arg Phe Asn Val Arg Gly Val Tyr His Pro Asn Gly
65 70 75 80
Glu Arg Val Gly Ser Thr His Val Lys Gly Gly His Phe Ile Asp Glu
85 90 95
Asp Pro Ala Leu Phe Asp Ala Ala Phe Phe Asn Met Thr Thr Glu Val
100 105 110
Ala Ser Cys Met Asp Pro Gln Tyr Arg Leu Met Leu Glu Val Val Tyr
115 120 125
Glu Ser Leu Glu Ser Ala Gly Ile Thr Ile Asp Gly Met Ala Gly Ser
130 135 140
Asn Thr Ser Val Phe Gly Gly Val Met Tyr His Asp Tyr Gln Asp Ser
145 150 155 160
Leu Asn Arg Asp Pro Glu Thr Val Pro Arg Tyr Phe Ile Thr Gly Asn
165 170 175
Ser Gly Thr Met Leu Ser Asn Arg Ile Ser His Phe Tyr Asp Leu Arg
180 185 190
Gly Pro Ser Val Thr Val Asp Thr Ala Cys Ser Thr Thr Leu Thr Ala
195 200 205
Leu His Leu Ala Cys Gln Ser Leu Arg Thr Gly Glu Ser Asp Thr Ala
210 215 220
Ile Val Ile Gly Ala Asn Leu Leu Leu Asn Pro Asp Val Phe Val Thr
225 230 235 240
Met Ser Asn Leu Gly Phe Leu Ser Pro Asp Gly Ile Ser Tyr Ser Phe
245 250 255
Asp Pro Arg Ala Asn Gly Tyr Gly Arg Gly Glu Gly Ile Ala Ala Leu
260 265 270
Val Ile Lys Ala Leu Pro Asn Ala Leu Arg Asp Gln Asp Pro Ile Arg
275 280 285
Ala Val Ile Arg Glu Thr Ala Leu Asn Gln Asp Gly Lys Thr Pro Ala
290 295 300
Ile Thr Ala Pro Ser Asp Val Ala Gln Lys Ser Leu Ile Gln Glu Cys
305 310 315 320
Tyr Asp Lys Ala Gly Leu Asp Met Ser Leu Thr Ser Tyr Val Glu Ala
325 330 335
His Gly Thr Gly Thr Pro Thr Gly Asp Pro Leu Glu Ile Ser Ala Ile
340 345 350
Ser Ala Ala Phe Lys Gly His Pro Leu His Leu Gly Ser Val Lys Ala
355 360 365
Asn Ile Gly His Thr Glu Ala Ala Ser Gly Leu Ala Ser Ile Ile Lys
370 375 380
Val Ala Leu Ala Leu Glu Lys Gly Leu Ile Pro Pro ASn Ala Arg Phe
385 390 395 400
Leu Gln Lys Asn Ser Lys Leu Met Leu Asp Gln Lys Asn Ile Lys Ile
405 410 415
Pro Met Ser Ala Gln Asp Trp Pro Val Lys Asp Gly Thr Arg Arg Ala
420 425 430
Ser Val Asn Asn Phe Gly Phe Gly Gly Ser Asn Ala His Val Ile Leu
435 440 445
Glu Ser Tyr Asp Arg Ala Ser Leu Ala Leu Pro Glu Asp Gln Val His
450 455 460
Val Asn Gly Asn Ser Glu His Gly Arg Val Glu Asp Gly Ser Lys Gln
465 470 475 480
Ser Arg Ile Tyr Val Val Arg Ala Lys Asp Glu Gln Ala Cys Arg Arg
485 490 495
Thr Ile Ala Ser Leu Arg Asp Tyr Ile Lys Ser Val Ala Asp Ile Asp
500 505 510
Gly Glu Pro Phe Leu Ala Ser Leu Ala Tyr Thr Leu Gly Ser Arg Arg
515 520 525
Ser Ile Leu Pro Trp Thr Ser Val Tyr Val Ala Asp Ser Leu Gly Gly
530 535 540
Leu Val Ser Ala Leu Ser Asp Glu Ser Asn Gln Pro Lys Arg Ala Asn
545 550 555 560
Glu Lys Val Arg Leu Gly Phe Val Phe Thr Gly Gln Gly Ala Gln Trp
565 570 575
His Ala Met Gly Arg Glu Leu Val Asn Thr Phe Pro Val Phe Lys Gln
580 585 590
Ala Ile Leu Glu Cys Asp Gly Tyr Ile Lys Gln Leu Gly Ala Ser Trp
595 600 605
Asn Phe Met Glu Glu Leu His Arg Asp Glu Leu Thr Thr Arg Val Asn
610 615 620
Asp Ala Glu Tyr Ser Leu Pro Leu Ser Thr Ala Ile Gln Ile Ala Leu
625 630 635 640
Val Arg Leu Leu Trp Ser Trp Gly Ile Arg Pro Thr Gly Ile Thr Ser
645 650 655
His Ser Ser Gly Glu Ala Ala Ala Ala Tyr Ala Ala Gly Ala Leu Ser
660 665 670
Ala Arg Ser Ala Ile Gly Ile Thr Tyr Ile Arg Gly Val Leu Thr Thr
675 680 685
Lys Pro Lys Pro Ala Leu Ala Ala Lys Gly Gly Met Met Ala Val Gly
690 695 700
Leu Gly Arg Ser Glu Thr Asn Val Tyr Ile Ser Arg Leu Asn Gln Glu
705 710 715 720
Asp Gly Cys Val Val Val Gly Cys Ile Asn Ser Gln Cys Ser Val Thr
725 730 735
Val Ser Gly Asp Leu Gly Ala Ile Glu Lys Leu Glu Lys Leu Leu His
740 745 750
Ala Asp Gly Ile Phe Thr Arg Lys Leu Lys Val Thr Glu Ala Phe His
755 760 765
Ser Ser His Met Arg Pro Met Ala Asp Ala Phe Gly Ala Ser Leu Arg
770 775 780
Asp Leu Phe Asn Ser Asp Asn Asn Asn Asp Asn Pro Asn Ala Asp Thr
785 790 795 800
Ser Lys Gly Val Leu Tyr Ser Ser Pro Lys Thr Gly Ser Arg Met Thr
805 810 815
Asp Leu Lys Leu Leu Leu Asp Pro Thr His Trp Met Asp Ser Met Leu
820 825 830
Gln Pro Val Glu Phe Glu Ser Ser Leu Arg Glu Met Cys Phe Asp Pro
835 840 845
Asn Thr Lys Glu Lys Ala Val Asp Val Ile Ile Glu Ile Gly Pro His
850 855 860
Gly Ala Leu Gly Gly Pro Ile Asn Gln Val Met Gln Asp Leu Gly Leu
865 870 875 880
Lys Gly Thr Asp Ile Asn Tyr Leu Ser Cys Leu Ser Arg Gly Arg Ser
885 890 895
Ser Leu Glu Thr Met Tyr Arg Ala Ala Thr Glu Leu Ile Ser Lys Gly
900 905 910
Tyr Gly Leu Lys Met Asp Ala Ile Asn Phe Pro His Gly Arg Lys Glu
915 920 925
Pro Arg Val Lys Val Leu Ser Asp Leu Pro Ala Tyr Pro Trp Asn His
930 935 940
Gln Thr Arg Tyr Trp Arg Glu Pro Arg Gly Ser Arg Glu Ser Lys Gln
945 950 955 960
Arg Thr His Pro Pro His Thr Leu Ile Gly Ser Arg Glu Ser Leu Ser
965 970 975
Pro His Phe Ala Pro Lys Trp Lys His Val Leu Arg Leu Ser Asp Ile
980 985 990
Pro Trp Ile Arg Asp His Val Val Gly Ser Ser Ile Ile Phe Pro Gly
995 1000 1005
Ala Gly Phe Ile Ser Met Ala Ile Glu Gly Phe Ser Gln Val Cys
1010 1015 1020
Pro Pro Val Ala Gly Ala Ser Ile Asn Tyr Asn Leu Arg Asp Val
1025 1030 1035
Glu Leu Ala Gln Ala Leu Ile Ile Pro Ala Asp Ala Glu Ala Glu
1040 1045 1050
Val Asp Leu Arg Leu Thr Ile Arg Ser Cys Glu Glu Arg Ser Leu
1055 1060 1065
Gly Thr Lys Asn Trp His Gln Phe Ser Val His Ser Ile Ser Gly
1070 1075 1080
Glu Asn Asn Thr Trp Thr Glu His Cys Thr Gly Leu Ile Arg Ser
1085 1090 1095
Glu Ser Glu Arg Ser His Leu Asp Cys Ser Thr Val Glu Ala Ser
1100 1105 1110
Arg Arg Leu Asn Leu Gly Ser Asp Asn Arg Ser Ile Asp Pro Asn
1115 1120 1125
Asp Leu Trp Glu Ser Leu His Ala Asn Gly Ile Cys His Gly Pro
1130 1135 1140
Ile Phe Gln Asn Ile Gln Arg Ile Gln Asn Asn Gly Gln Gly Ser
1145 1150 1155
Phe Cys Arg Phe Ser Ile Ala Asp Thr Ala Ser Ala Met Pro His
1160 1165 1170
Ser Tyr Glu Asn Arg His Ile Val His Pro Thr Thr Leu Asp Ser
1175 1180 1185
Val Ile Gln Ala Ala Tyr Thr Val Leu Pro Tyr Ala Gly Thr Arg
1190 1195 1200
Met Lys Thr Ala Met Val Pro Arg Arg Leu Arg Asn Val Lys Ile
1205 1210 1215
Ser Ser Ser Leu Ala Asp Leu Glu Ala Gly Asp Ala Leu Asp Ala
1220 1225 1230
Gln Ala Ser Ile Lys Asp Arg Asn Ser Gln Ser Phe Ser Thr Asp
1235 1240 1245
Leu Ala Val Phe Asp Asp Tyr Asp Ser Gly Ser Ser Pro Ser Asp
1250 1255 1260
Gly Ile Pro Val Ile Glu Ile Glu Gly Leu Val Phe Gln Ser Val
1265 1270 1275
Gly Ser Ser Phe Ser Asp Gln Lys Ser Asp Ser Asn Asp Thr Glu
1280 1285 1290
Asn Ala Cys Ser Ser Trp Val Trp Ala Pro Asp Ile Ser Leu Gly
1295 1300 1305
Asp Ser Thr Trp Leu Lys Glu Lys Leu Ser Thr Glu Ala Glu Thr
1310 1315 1320
Lys Glu Thr Glu Leu Met Met Asp Leu Arg Arg Cys Thr Ile Asn
1325 1330 1335
Phe Ile Gln Glu Ala Val Thr Asp Leu Thr Asn Ser Asp Ile Gln
1340 1345 1350
His Leu Asp Gly His Leu Gln Lys Tyr Phe Asp Trp Met Asn Val
1355 1360 1365
Gln Leu Asp Leu Ala Arg Gln Asn Lys Leu Ser Pro Ala Ser Cys
1370 1375 1380
Asp Trp Leu Ser Asp Asp Ala Glu Gln Lys Lys Cys Leu Gln Ala
1385 1390 1395
Arg Val Ala Gly Glu Ser Val Asn Gly Glu Met Ile Ser Arg Leu
1400 1405 1410
Gly Pro Gln Leu Ile Ala Met Leu Arg Arg Glu Thr Glu Pro Leu
1415 1420 1425
Glu Leu Met Met Gln Asp Gln Leu Leu Ser Arg Tyr Tyr Val Asn
1430 1435 1440
Ala Ile Lys Trp Ser Arg Ser Asn Ala Gln Ala Ser Glu Leu Ile
1445 1450 1455
Arg Leu Cys Ala His Lys Asn Pro Arg Ser Arg Ile Leu Glu Ile
1460 1465 1470
Gly Gly Gly Thr Gly Gly Cys Thr Lys Leu Ile Val Asn Ala Leu
1475 1480 1485
Gly Asn Thr Lys Pro Ile Asp Arg Tyr Asp Phe Thr Asp Val Ser
1490 1495 1500
Ala Gly Phe Phe Glu Ser Ala Arg Glu Gln Phe Ala Asp Trp Gln
1505 1510 1515
Asp Val Met Thr Phe Lys Lys Leu Asp Ile Glu Ser Asp Pro Glu
1520 1525 1530
Gln Gln Gly Phe Glu Cys Ala Thr Tyr Asp Val Val Val Ala Cys
1535 1540 1545
Gln Val Leu His Ala Thr Arg Cys Met Lys Arg Thr Leu Ser Asn
1550 1555 1560
Val Arg Lys Leu Leu Lys Pro Gly Gly Asn Leu Ile Leu Val Glu
1565 1570 1575
Thr Thr Arg Asp Gln Leu Asp Leu Phe Phe Thr Phe Gly Leu Leu
1580 1585 1590
Pro Gly Trp Trp Leu Ser Glu Glu Pro Glu Arg Lys Ser Thr Pro
1595 1600 1605
Ser Leu Thr Thr Asp Leu Trp Asn Thr Met Leu Asp Thr Ser Gly
1610 1615 1620
Phe Asn Gly Val Glu Leu Glu Val Arg Asp Cys Glu Asp Asp Glu
1625 1630 1635
Phe Tyr Met Ile Ser Thr Met Leu Ser Thr Ala Arg Lys Glu Asn
1640 1645 1650
Thr Thr Pro Asp Thr Val Ala Glu Ser Glu Val Leu Leu Leu His
1655 1660 1665
Gly Ala Leu Arg Pro Pro Ser Ser Trp Leu Glu Ser Leu Gln Ala
1670 1675 1680
Ala Ile Cys Glu Lys Thr Ser Ser Ser Pro Ser Ile Asn Ala Leu
1685 1690 1695
Gly Glu Val Asp Thr Thr Gly Arg Thr Cys Ile Phe Leu Gly Glu
1700 1705 1710
Met Glu Ser Ser Leu Leu Gly Glu Val Gly Ser Glu Thr Phe Lys
1715 1720 1725
Ser Ile Thr Ala Met Leu Asn Asn Cys Asn Ala Leu Leu Trp Val
1730 1735 1740
Ser Arg Gly Ala Ala Met Ser Ser Glu Asp Pro Trp Lys Ala Leu
1745 1750 1755
His Ile Gly Leu Leu Arg Thr Ile Arg Asn Glu Asn Asn Gly Lys
1760 1765 1770
Glu Tyr Val Ser Leu Asp Leu Asp Pro Ser Arg Asn Ala Tyr Thr
1775 1780 1785
His Glu Ser Leu Tyr Ala Ile Cys Asn Ile Phe Asn Gly Arg Leu
1790 1795 1800
Gly Asp Leu Ser Glu Asp Lys Glu Phe Glu Phe Ala Glu Arg Asn
1805 1810 1815
Gly Val Ile His Val Pro Arg Leu Phe Asn Asp Pro His Trp Lys
1820 1825 1830
Asp Gln Glu Ala Val Glu Val Thr Leu Gln Pro Phe Glu Gln Pro
1835 1840 1845
Gly Arg Arg Leu Arg Met Glu Val Glu Thr Pro Gly Leu Leu Asp
1850 1855 1860
Ser Leu Gln Phe Arg Asp Asp Glu Gly Arg Glu Gly Lys Asp Leu
1865 1870 1875
Pro Asp Asp Trp Val Glu Ile Glu Pro Lys Ala Phe Gly Leu Asn
1880 1885 1890
Phe Arg Asp Val Met Val Ala Met Gly Gln Leu Glu Ala Asn Arg
1895 1900 1905
Val Met Gly Phe Glu Cys Ala Gly Val Ile Thr Lys Leu Gly Gly
1910 1915 1920
Ala Ala Ala Ala Ser Gln Gly Leu Arg Leu Gly Asp Arg Val Cys
1925 1930 1935
Ala Leu Leu Lys Gly His Trp Ala Thr Arg Thr Gln Thr Pro Tyr
1940 1945 1950
Thr Asn Val Val Arg Ile Pro Asp Glu Met Gly Phe Pro Glu Ala
1955 1960 1965
Ala Ser Val Pro Leu Ala Phe Thr Thr Ala Tyr Ile Ala Leu Tyr
1970 1975 1980
Thr Thr Ala Lys Leu Arg Arg Gly Glu Arg Val Leu Ile His Ser
1985 1990 1995
Gly Ala Gly Gly Val Gly Gln Ala Ala Ile Ile Leu Ser Gln Leu
2000 2005 2010
Ala Gly Ala Glu Val Phe Val Thr Ala Gly Thr Gln Ala Lys Arg
2015 2020 2025
Asp Phe Val Gly Asp Lys Phe Gly Ile Asn Pro Asp His Ile Phe
2030 2035 2040
Ser Ser Arg Asn Asp Leu Phe Val Asp Gly Ile Lys Ala Tyr Thr
2045 2050 2055
Gly Gly Leu Gly Val His Val Val Leu Asn Ser Leu Ala Gly Gln
2060 2065 2070
Leu Leu Gln Ala Ser Phe Asp Cys Met Ala Glu Phe Gly Arg Phe
2075 2080 2085
Val Glu Ile Gly Lys Lys Asp Leu Glu Gln Asn Ser Arg Leu Asp
2090 2095 2100
Met Leu Pro Phe Thr Arg Asp Val Ser Phe Thr Ser Ile Asp Leu
2105 2110 2115
Leu Ser Trp Gln Arg Ala Lys Ser Glu Glu Val Ser Glu Ala Leu
2120 2125 2130
Asn His Val Thr Lys Leu Leu Glu Thr Lys Ala Ile Gly Leu Ile
2135 2140 2145
Gly Pro Ile Gln Gln His Ser Leu Ser Asn Ile Glu Lys Ala Phe
2150 2155 2160
Arg Thr Met Gln Ser Gly Gln His Val Gly Lys Val Val Val Asn
2165 2170 2175
Val Ser Gly Asp Glu Leu Val Pro Val Gly Asp Gly Gly Phe Ser
2180 2185 2190
Leu Lys Leu Lys Pro Asp Ser Ser Tyr Leu Val Ala Gly Gly Leu
2195 2200 2205
Gly Gly Ile Gly Lys Gln Ile Cys Gln Trp Leu Val Asp His Gly
2210 2215 2220
Ala Lys His Leu Ile Ile Leu Ser Arg Ser Ala Lys Ala Ser Pro
2225 2230 2235
Phe Ile Thr Ser Leu Gln Asn Gln Gln Cys Ala Val Tyr Leu His
2240 2245 2250
Ala Cys Asp Ile Ser Asp Gln Asp Gln Val Thr Lys Val Leu Arg
2255 2260 2265
Leu Cys Glu Glu Ala His Ala Pro Pro Ile Arg Gly Ile Ile Gln
2270 2275 2280
Gly Ala Met Val Leu Lys Asp Ala Leu Leu Ser Arg Met Thr Leu
2285 2290 2295
Asp Glu Phe Asn Ala Ala Thr Arg Pro Lys Val Gln Gly Ser Trp
2300 2305 2310
Tyr Leu His Lys Ile Ala Gln Asp Val Asp Phe Phe Val Met Leu
2315 2320 2325
Ser Ser Leu Val Gly Val Met Gly Gly Ala Gly Gln Ala Asn Tyr
2330 2335 2340
Ala Ala Ala Gly Ala Phe Gln Asp Ala Leu Ala His His Arg Arg
2345 2350 2355
Ala His Gly Met Pro Ala Val Thr Ile Asp Leu Gly Met Val Lys
2360 2365 2370
Ser Val Gly Tyr Val Ala Glu Thr Gly Arg Gly Val Ala Asp Arg
2375 2380 2385
Leu Ala Arg Ile Gly Tyr Lys Pro Met His Glu Lys Asp Val Met
2390 2395 2400
Asp Val Leu Glu Lys Ala Ile Leu Cys Ser Ser Pro Gln Phe Pro
2405 2410 2415
Ser Pro Pro Ala Ala Val Val Thr Gly Ile Asn Thr Ser Pro Gly
2420 2425 2430
Ala His Trp Thr Glu Ala Asn Trp Ile Gln Glu Gln Arg Phe Val
2435 2440 2445
Gly Leu Lys Tyr Arg Gln Val Leu His Ala Asp Gln Ser Phe Val
2450 2455 2460
Ser Ser His Lys Lys Gly Pro Asp Gly Val Arg Ala Gln Leu Ser
2465 2470 2475
Arg Val Thr Ser His Asp Glu Ala Ile Ser Ile Val Leu Lys Ala
2480 2485 2490
Met Thr Glu Lys Leu Met Arg Met Phe Gly Leu Ala Glu Asp Asp
2495 2500 2505
Met Ser Ser Ser Lys Asn Leu Ala Gly Val Gly Val Asp Ser Leu
2510 2515 2520
Val Ala Ile Glu Leu Arg Asn Trp Ile Thr Ser Glu Ile His Val
2525 2530 2535
Asp Val Ser Ile Phe Glu Leu Met Asn Gly Asn Thr Ile Ala Gly
2540 2545 2550
Leu Val Glu Leu Val Val Ala Lys Cys Ser
2555 2560
<210>47
<211>1557
<212>DNA
<213>柑桔青霉
<220>
<221>CDS
<222>(1)..(1557)
<400>47
atg ctc ggc cag gtt ctt ctg acc gtc gaa tcg tac caa tgg gta tcg 48
Met Leu Gly Gln Val Leu Leu Thr Val Glu Ser Tyr Gln Trp Val Ser
1 5 10 15
acc cct caa gcc ctt gtg gcg gtc gca gtg ctt ctt agt ctc atc gcc 96
Thr Pro Gln Ala Leu Val Ala Val Ala Val Leu Leu Ser Leu Ile Ala
20 25 30
tac cgt ttg cgg ggg cgc cag tcc gaa ctg caa gtc tat aat ccc aaa 144
Tyr Arg Leu Arg Gly Arg Gln Ser Glu Leu Gln Val Tyr Asn Pro Lys
35 40 45
aaa tgg tgg gag ttg acg acc atg agg gct agg cag gac ttc gat acg 192
Lys Trp Trp Glu Leu Thr Thr Met Arg Ala Arg Gln Asp Phe Asp Thr
50 55 60
tat ggt ccg agc tgg atc gaa gct tgg ttc tcg aaa aac gac aag ccc 240
Tyr Gly Pro Ser Trp Ile Glu Ala Trp Phe Ser Lys Asn Asp Lys Pro
65 70 75 80
ctg cgc ttc att gtt gat tcc ggc tat tgc acc atc ctc cca tcg tcc 288
Leu Arg Phe Ile Val Asp Ser Gly Tyr Cys Thr Ile Leu Pro Ser Ser
85 90 95
atg gcc gac gag ttt cgg aaa atc aaa gat atg tgc atg tac aag ttt 336
Met Ala Asp Glu Phe Arg Lys Ile Lys Asp Met Cys Met Tyr Lys Phe
100 105 110
ttg gcg gat gac ttt cac tct cat ctc cct gga ttc gac ggg ttc aag 384
Leu Ala Asp Asp Phe His Ser His Leu Pro Gly Phe Asp Gly Phe Lys
115 120 125
gaa atc tgc cag gat gca cat ctt gtc aac aaa gtt gtt ttg aac cag 432
Glu Ile Cys Gln Asp Ala His Leu Val Asn Lys Val Val Leu Asn Gln
130 135 140
tta caa acc caa gcc ccc aag tac aca aag cca ttg gct acc ttg gcc 480
Leu Gln Thr Gln Ala Pro Lys Tyr Thr Lys Pro Leu Ala Thr Leu Ala
145 150 155 160
gac gct act att gcc aag ttg ttc ggt aaa agc gag gag tgg caa acc 528
Asp Ala Thr Ile Ala Lys Leu Phe Gly Lys Ser Glu Glu Trp Gln Thr
165 170 175
gca cct gtc tat tcc aat gga ttg gac ctt gtc aca cga aca gtc aca 576
Ala Pro Val Tyr Ser Asn Gly Leu Asp Leu Val Thr Arg Thr Val Thr
180 185 190
ctc att atg gtc ggc gac aaa atc tgc cac aat gag gag tgg ctg gat 624
Leu Ile Met Val Gly Asp Lys Ile Cys His Asn Glu Glu Trp Leu Asp
195 200 205
att gca aag aac cat gcc gtg agt gtg gcg gta caa gct cgc caa ctt 672
Ile Ala Lys Asn His Ala Val Ser Val Ala Val Gln Ala Arg Gln Leu
210 215 220
cgc gta tgg ccc atg cta ctg cga ccg ctc gct cac tgg ttt caa ccg 720
Arg Val Trp Pro Met Leu Leu Arg Pro Leu Ala His Trp Phe Gln Pro
225 230 235 240
caa gga cgc aaa ttg cgt gac caa gtg cgc cgc gca cga aag atc att 768
Gln Gly Arg Lys Leu Arg Asp Gln Val Arg Arg Ala Arg Lys Ile Ile
245 250 255
gat cct gag att cag cga cga cgt gct gaa aag gcc gca tgt gta gcg 816
Asp Pro Glu Ile Gln Arg Arg Arg Ala Glu Lys Ala Ala Cys Val Ala
260 265 270
aag ggc gtg cag ccg ccc cag tac gtc gat acc atg caa tgg ttt gaa 864
Lys Gly Val Gln Pro Pro Gln Tyr Val Asp Thr Met Gln Trp Phe Glu
275 280 285
gac acc gcc gac ggc cgc tgg tac gat gtg gcg ggt gct cag ctc gct 912
Asp Thr Ala Asp Gly Arg Trp Tyr Asp Val Ala Gly Ala Gln Leu Ala
290 295 300
atg gat ttc gcc ggc atc tac gcc tcg acg gat ctt ttc gtc ggt gcc 960
Met Asp Phe Ala Gly Ile Tyr Ala Ser Thr Asp Leu Phe Val Gly Ala
305 310 315 320
ctt gtg gac att gcc agg cac cca gac ctt att cag cct ctc cgc caa 1008
Leu Val Asp Ile Ala Arg His Pro Asp Leu Ile Gln Pro Leu Arg Gln
325 330 335
gag atc cgc act gta atc gga gaa ggg ggc tgg acg cct gcc tct ctg 1056
Glu Ile Arg Thr Val Ile Gly Glu Gly Gly Trp Thr Pro Ala Ser Leu
340 345 350
ttc aag ctg aag ctc ctc gac agc tgc atg aaa gag acg cag cga atc 1104
Phe Lys Leu Lys Leu Leu Asp Ser Cys Met Lys Glu Thr Gln Arg Ile
355 360 365
aag ccg gtc gag tgc gcc act atg cgc agt acc gct ctc aga gac atc 1152
Lys Pro Val Glu Cys Ala Thr Met Arg Ser Thr Ala Leu Arg Asp Ile
370 375 380
act cta tcc aat ggc ctc ttc att ccc aag ggc gag ttg gcc gct gtg 1200
Thr Leu Ser Asn Gly Leu Phe Ile Pro Lys Gly Glu Leu Ala Ala Val
385 390 395 400
gct gca gac cgc atg aac aac cct gat gtg tgg gaa aac ccc gaa aat 1248
Ala Ala Asp Arg Met Asn Asn Pro Asp Val Trp Glu Asn Pro Glu Asn
405 410 415
tat gat ccc tac cga ttt atg cgc atg cgc gag gat cca gac aag gcc 1296
Tyr Asp Pro Tyr Arg Phe Met Arg Met Arg Glu Asp Pro Asp Lys Ala
420 425 430
ttc acc gct caa ttg gag aat acc aac ggt gat cac atc ggc ttc ggc 1344
Phe Thr Ala Gln Leu Glu Asn Thr Asn Gly Asp His Ile Gly Phe Gly
435 440 445
tgg aac cca cgc gct tgt ccc ggg cgg ttc ttc gcc tcg aag gaa atc 1392
Trp Asn Pro Arg Ala Cys Pro Gly Arg Phe Phe Ala Ser Lys Glu Ile
450 455 460
aag att ctc ctc gct cat ata ctg att cag tat gat gtg aag cct gta 1440
Lys Ile Leu Leu Ala His Ile Leu Ile Gln Tyr Asp Val Lys Pro Val
465 470 475 480
cca gga gac gat gac aaa tac tac cgt cac gct ttt agc gtt cgt atg 1488
Pro Gly Asp Asp Asp Lys Tyr Tyr Arg His Ala Phe Ser Val Arg Met
485 490 495
cat cca acc aca aag ctc atg gta cgc cgg cgc aac gag gac atc ccg 1536
His Pro Thr Thr Lys Leu Met Val Arg Arg Arg Asn Glu Asp Ile Pro
500 505 510
ctc cct cat gac cgg tgc taa 1557
Leu Pro His Asp Arg Cys
515
<210>48
<211>518
<212>PRT
<213>柑桔青霉
<400>48
Met Leu Gly Gln Val Leu Leu Thr Val Glu Ser Tyr Gln Trp Val Ser
1 5 10 15
Thr Pro Gln Ala Leu Val Ala Val Ala Val Leu Leu Ser Leu Ile Ala
20 25 30
Tyr Arg Leu Arg Gly Arg Gln Ser Glu Leu Gln Val Tyr Asn Pro Lys
35 40 45
Lys Trp Trp Glu Leu Thr Thr Met Arg Ala Arg Gln Asp Phe Asp Thr
50 55 60
Tyr Gly Pro Ser Trp Ile Glu Ala Trp Phe Ser Lys Asn Asp Lys Pro
65 70 75 80
Leu Arg Phe Ile Val Asp Ser Gly Tyr Cys Thr Ile Leu Pro Ser Ser
85 90 95
Met Ala Asp Glu Phe Arg Lys Ile Lys Asp Met Cys Met Tyr Lys Phe
100 105 1l0
Leu Ala Asp Asp Phe His Ser His Leu Pro Gly Phe Asp Gly Phe Lys
115 120 125
Glu Ile Cys Gln Asp Ala His Leu Val Asn Lys Val Val Leu Asn Gln
130 135 140
Leu Gln Thr Gln Ala Pro Lys Tyr Thr Lys Pro Leu Ala Thr Leu Ala
145 150 155 160
Asp Ala Thr Ile Ala Lys Leu Phe Gly Lys Ser Glu Glu Trp Gln Thr
165 170 175
Ala Pro Val Tyr Ser Asn Gly Leu Asp Leu Val Thr Arg Thr Val Thr
180 185 190
Leu Ile Met Val Gly Asp Lys Ile Cys His Asn Glu Glu Trp Leu Asp
195 200 205
Ile Ala Lys Asn His Ala Val Ser Val Ala Val Gln Ala Arg Gln Leu
210 215 220
Arg Val Trp Pro Met Leu Leu Arg Pro Leu Ala His Trp Phe Gln Pro
225 230 235 240
Gln Gly Arg Lys Leu Arg Asp Gln Val Arg Arg Ala Arg Lys Ile Ile
245 250 255
Asp Pro Glu Ile Gln Arg Arg Arg Ala Glu Lys Ala Ala Cys Val Ala
260 265 270
Lys Gly Val Gln Pro Pro Gln Tyr Val Asp Thr Met Gln Trp Phe Glu
275 280 285
Asp Thr Ala Asp Gly Arg Trp Tyr Asp Val Ala Gly Ala Gln Leu Ala
290 295 300
Met Asp Phe Ala Gly Ile Tyr Ala Ser Thr Asp Leu Phe Val Gly Ala
305 310 315 320
Leu Val Asp Ile Ala Arg His Pro Asp Leu Ile Gln Pro Leu Arg Gln
325 330 335
Glu Ile Arg Thr Val Ile Gly Glu Gly Gly Trp Thr Pro Ala Ser Leu
340 345 350
Phe Lys Leu Lys Leu Leu Asp Ser Cys Met Lys Glu Thr Gln Arg Ile
355 360 365
Lys Pro Val Glu Cys Ala Thr Met Arg Ser Thr Ala Leu Arg Asp Ile
370 375 380
Thr Leu Ser Asn Gly Leu Phe Ile Pro Lys Gly Glu Leu Ala Ala Val
385 390 395 400
Ala Ala Asp Arg Met Asn Asn Pro Asp Val Trp Glu Asn Pro Glu Asn
405 410 415
Tyr Asp Pro Tyr Arg Phe Met Arg Met Arg Glu Asp Pro Asp Lys Ala
420 425 430
Phe Thr Ala Gln Leu Glu Asn Thr Asn Gly Asp His Ile Gly Phe Gly
435 440 445
Trp Asn Pro Arg Ala Cys Pro Gly Arg Phe Phe Ala Ser Lys Glu Ile
450 455 460
Lys Ile Leu Leu Ala His Ile Leu Ile Gln Tyr Asp Val Lys Pro Val
465 470 475 480
Pro Gly Asp Asp Asp Lys Tyr Tyr Arg His Ala Phe Ser Val Arg Met
485 490 495
His Pro Thr Thr Lys Leu Met Val Arg Arg Arg Asn Glu Asp Ile Pro
500 505 510
Leu Pro His Asp Arg Cys
515
<210>49
<211>3522
<212>DNA
<213>柑桔青霉
<220>
<221>CDS
<222>(1)..(3522)
<400>49
atg gtc gct tcg ttg cta ccc tct cgc ttt cgc ggt agg gaa tca atg 48
Met Val Ala Ser Leu Leu Pro Ser Arg Phe Arg Gly Arg Glu Ser Met
1 5 10 15
aat cag cag cac cct cta cgc tcg gga aat cgg gca ttg acc tcc aca 96
Asn Gln Gln His Pro Leu Arg Ser Gly Asn Arg Ala Leu Thr Ser Thr
20 25 30
ctc caa ttt cta tcc aaa acg gcg tgt cta cac ccg atc cat acc gtt 144
Leu Gln Phe Leu Ser Lys Thr Ala Cys Leu His Pro Ile His Thr Val
35 40 45
tgc acc ata gct att cta gct agt acc aca tac gtt gga cta ctc aaa 192
Cys Thr Ile Ala Ile Leu Ala Ser Thr Thr Tyr Val Gly Leu Leu Lys
50 55 60
gac agc ttc ttc cat ggc ccc gca aac gtt gat aaa gca gaa tgg ggc 240
Asp Ser Phe Phe His Gly Pro Ala Asn Val Asp Lys Ala Glu Trp Gly
65 70 75 80
tct ttg gtc gaa gga agt cga agc ttg atc acc ggc cca cag aat ggc 288
Ser Leu Val Glu Gly Ser Arg Ser Leu Ile Thr Gly Pro Gln Asn Gly
85 90 95
tgg aag tgg cag agc ttc gac ggg gat gca gat gtt ctc gga gat ttc 336
Trp Lys Trp Gln Ser Phe Asp Gly Asp Ala Asp Val Leu Gly Asp Phe
100 105 110
aac cat caa gca cta atg acc ttg gta ttc ccg ggg tca tat ggg gtt 384
Asn His Gln Ala Leu Met Thr Leu Val Phe Pro Gly Ser Tyr Gly Val
115 120 125
gca tct caa gca gcc tca cca ttc ctt gct ccc ctc cct gtg aac cta 432
Ala Ser Gln Ala Ala Ser Pro Phe Leu Ala Pro Leu Pro Val Asn Leu
130 135 140
tct gtg att gac ctt ccc tca acg tcg agc cct tta acc gcc tat tcg 480
Ser Val Ile Asp Leu Pro Ser Thr Ser Ser Pro Leu Thr Ala Tyr Ser
145 150 155 160
aaa gat aaa gtt ttc gcc ttc tct gtg gaa tac agc agc gcg ccg gaa 528
Lys Asp Lys Val Phe Ala Phe Ser Val Glu Tyr Ser Ser Ala Pro Glu
165 170 175
ctc gtg gct gct gtt caa gaa atc ccc aac aac agt gcc gac ctg aaa 576
Leu Val Ala Ala Val Gln Glu Ile Pro Asn Asn Ser Ala Asp Leu Lys
180 185 190
ttg cag gag acg caa ttg atc gag atg gaa cgc cag atg tgg atc atg 624
Leu Gln Glu Thr Gln Leu Ile Glu Met Glu Arg Gln Met Trp Ile Met
195 200 205
aag gct gcc agg gct cac aca aaa cgc agc ctt gct caa tgg gtg cac 672
Lys Ala Ala Arg Ala His Thr Lys Arg Ser Leu Ala Gln Trp Val His
210 215 220
gat acc tgg aca gag tct ctt gat ctt atc aag agc gct caa acg ctc 720
Asp Thr Trp Thr Glu Ser Leu Asp Leu Ile Lys Ser Ala Gln Thr Leu
225 230 235 240
gac gtg gtt gtc atg gtg cta ggt tat ata tca atg cac ttg act ttc 768
Asp Val Val Val Met Val Leu Gly Tyr Ile Ser Met His Leu Thr Phe
245 250 255
gtc tca ctc ttc ctc agc atg aaa aaa ttg gga tcg aag gtt tgg ctg 816
Val Ser Leu Phe Leu Ser Met Lys Lys Leu Gly Ser Lys Val Trp Leu
260 265 270
gct aca agc gtc ctt ttg tcg tca aca ttt gcc ttt ctc ctc ggt ctc 864
Ala Thr Ser Val Leu Leu Ser Ser Thr Phe Ala Phe Leu Leu Gly Leu
275 280 285
gac gtg gcc ata aga cta ggg gtt ccg atg agc atg agg ttg cta tcc 912
Asp Val Ala Ile Arg Leu Gly Val Pro Met Ser Met Arg Leu Leu Ser
290 295 300
gaa ggc ctc ccc ttc ttg gtg gtg atc gtt ggc ttt gag aag agc atc 960
Glu Gly Leu Pro Phe Leu Val Val Ile Val Gly Phe Glu Lys Ser Ile
305 310 315 320
act ctg acc agg gct gtt ttg tcc tat gct gtg cag cac cga aag ccc 1008
Thr Leu Thr Arg Ala Val Leu Ser Tyr Ala Val Gln His Arg Lys Pro
325 330 335
cag aag ata cag tct gac cag ggt agc gtg aca gcc att gct gaa agt 1056
Gln Lys Ile Gln Ser Asp Gln Gly Ser Val Thr Ala Ile Ala Glu Ser
340 345 350
acc atc aat tac gcc gta cga agc gcc att cgg gag aag ggt tac aat 1104
Thr Ile Asn Tyr Ala Val Arg Ser Ala Ile Arg Glu Lys Gly Tyr Asn
355 360 365
atc gtg tgc cac tac gtg gtc gag atc ctg ctc cta gtt atc ggt gct 1152
Ile Val Cys His Tyr Val Val Glu Ile Leu Leu Leu Val Ile Gly Ala
370 375 380
gtc tta ggc atc caa ggt ggg cta cag cac ttc tgt gtt cta gct gca 1200
Val Leu Gly Ile Gln Gly Gly Leu Gln His Phe Cys Val Leu Ala Ala
385 390 395 400
ttg atc ctg ttc ttt gac tgt ctg ctg ctg ttt aca ttc tac act gcg 1248
Leu Ile Leu Phe Phe Asp Cys Leu Leu Leu Phe Thr Phe Tyr Thr Ala
405 410 415
att ctg tct atc aag ctc gag gta aac cgc ctc aaa cgt cat atc aac 1296
Ile Leu Ser Ile Lys Leu Glu Val Asn Arg Leu Lys Arg His Ile Asn
420 425 430
atg cgg tac gcg ttg gaa gat gag ggt ctc agt cag cgg acg gcg gag 1344
Met Arg Tyr Ala Leu Glu Asp Glu Gly Leu Ser Gln Arg Thr Ala Glu
435 440 445
agt gtc gcg acc agc aat gat gcc caa gac agt gca cgt aca tat ctg 1392
Ser Val Ala Thr Ser Asn Asp Ala Gln Asp Ser Ala Arg Thr Tyr Leu
450 455 460
ttt ggc aat gat atg aaa ggc agc agt gtt ccg aag ttc aaa ttc tgg 1440
Phe Gly Asn Asp Met Lys Gly Ser Ser Val Pro Lys Phe Lys Phe Trp
465 470 475 480
atg gtc gtt ggt ttc ctt atc gtc aac ctc gtc aac atc ggc tcc acc 1488
Met Val Val Gly Phe Leu Ile Val Asn Leu Val Asn Ile Gly Ser Thr
485 490 495
ctt ttc caa gcc tct tct agt gga tcg ttg tcc agt ata tca tct tgg 1536
Leu Phe Gln Ala Ser Ser Ser Gly Ser Leu Ser Ser Ile Ser Ser Trp
500 505 510
acc gaa agt ctg agc gga tcg gcc att aaa ccc ccg ctt gag ccc ttc 1584
Thr Glu Ser Leu Ser Gly Ser Ala Ile Lys Pro Pro Leu Glu Pro Phe
515 520 525
aag gta gct gga agt gga cta gat gaa cta ctt ttc cag gca aga ggg 1632
Lys Val Ala Gly Ser Gly Leu Asp Glu Leu Leu Phe Gln Ala Arg Gly
530 535 540
cgc ggt caa tcg act atg gtc act gtc ctc gcc ccc atc aag tac gaa 1680
Arg Gly Gln Ser Thr Met Val Thr Val Leu Ala Pro Ile Lys Tyr Glu
545 550 555 560
cta gag tat cct tcc att cac cgt ggt acc tcg cag cta cac gag tat 1728
Leu Glu Tyr Pro Ser Ile His Arg Gly Thr Ser Gln Leu His Glu Tyr
565 570 575
gga gtt ggt gga aaa atg gtc ggt agc ctg ctc acc agc ctg gaa gat 1776
Gly Val Gly Gly Lys Met Val Gly Ser Leu Leu Thr Ser Leu Glu Asp
580 585 590
ccc gtc ctc tcc aaa tgg gtg ttt gtg gca ctt gcc cta agt gtc gct 1824
Pro Val Leu Ser Lys Trp Val Phe Val Ala Leu Ala Leu Ser Val Ala
595 600 605
ctg aac agc tat ctg ttc aag gcc gcc aga ctg gga atc aaa gat cct 1872
Leu Asn Ser Tyr Leu Phe Lys Ala Ala Arg Leu Gly Ile Lys Asp Pro
610 615 620
aat ctc ccg agt cac cca gtt gat cca gtt gag ctt gac cag gcc gaa 1920
Asn Leu Pro Ser His Pro Val Asp Pro Val Glu Leu Asp Gln Ala Glu
625 630 635 640
agc ttc aac gct gcc cag aac cag acc cct cag att caa tca agt ctc 1968
Ser Phe Asn Ala Ala Gln Asn Gln Thr Pro Gln Ile Gln Ser Ser Leu
645 650 655
caa gct cct cag acc aga gtg ttc act cct acc acc acc gac agt gac 2016
Gln Ala Pro Gln Thr Arg Val Phe Thr Pro Thr Thr Thr Asp Ser Asp
660 665 670
agt gat gcc tca tta gtc tta att aaa gca tct cta aag gtc act aag 2064
Ser Asp Ala Ser Leu Val Leu Ile Lys Ala Ser Leu Lys Val Thr Lys
675 680 685
cga gca gaa gga aag aca gcc act agt gaa ctt ccc gtg tct cgc aca 2112
Arg Ala Glu Gly Lys Thr Ala Thr Ser Glu Leu Pro Val Ser Arg Thr
690 695 700
caa atc gaa ctg gac aat ttg ctg aag cag aac aca atc agc gag ttg 2160
Gln Ile Glu Leu Asp Asn Leu Leu Lys Gln Asn Thr Ile Ser Glu Leu
705 710 715 720
aac gat gag gat gtc gtt gcc ttg tct ttg cgg gga aag gtt ccc ggg 2208
Asn Asp Glu Asp Val Val Ala Leu Ser Leu Arg Gly Lys Val Pro Gly
725 730 735
tat gcc cta gag aag agt ctc aaa gac tgc act cgt gcc gtc aag gtt 2256
Tyr Ala Leu Glu Lys Ser Leu Lys Asp Cys Thr Arg Ala Val Lys Val
740 745 750
cgc cgc tct atc att tcg agg aca ccg gct acc gca gag ctt aca agt 2304
Arg Arg Ser Ile Ile Ser Arg Thr Pro Ala Thr Ala Glu Leu Thr Ser
755 760 765
atg ctg gag cac tcg aag ctg ccg tac gaa aac tac gcc tgg gaa cgc 2352
Met Leu Glu His Ser Lys Leu Pro Tyr Glu Asn Tyr Ala Trp Glu Arg
770 775 780
gtg ctc ggt gca tgt tgc gag aac gtt att ggc tat atg cca gtc cct 2400
Val Leu Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr Met Pro Val Pro
785 790 795 800
gtt ggc gtc gcc ggt cct att gtt atc gac ggc aag agt tat ttc att 2448
Val Gly Val Ala Gly Pro Ile Val Ile Asp Gly Lys Ser Tyr Phe Ile
805 810 815
cct atg gca acc acc gag ggc gtc ctc gtc gct agt gct agc cgt ggc 2496
Pro Met Ala Thr Thr Glu Gly Val Leu Val Ala Ser Ala Ser Arg Gly
820 825 830
agt aag gca atc aac ctc ggt ggc ggt gcc gtg aca gtc ctg act ggc 2544
Ser Lys Ala Ile Asn Leu Gly Gly Gly Ala Val Thr Val Leu Thr Gly
835 840 845
gac ggt atg aca cga ggc ccg tgt gtg aag ttt gat gtc ctt gaa cga 2592
Asp Gly Met Thr Arg Gly Pro Cys Val Lys Phe Asp Val Leu Glu Arg
850 855 860
gct ggt gct gct aag atc tgg ctc gat tcg gac gtc ggc cag acc gta 2640
Ala Gly Ala Ala Lys Ile Trp Leu Asp Ser Asp Val Gly Gln Thr Val
865 870 875 880
atg aaa gaa gcc ttc aat tca acc agc aga ttt gcg cgc tta caa agt 2688
Met Lys Glu Ala Phe Asn Ser Thr Ser Arg Phe Ala Arg Leu Gln Ser
885 890 895
atg cgg aca act atc gcc ggt act cac tta tat att cga ttt aag act 2736
Met Arg Thr Thr Ile Ala Gly Thr His Leu Tyr Ile Arg Phe Lys Thr
900 905 910
act act ggc gac gct atg gga atg aat atg att tct aag ggc gtg gag 2784
Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser Lys Gly Val Glu
915 920 925
cat gca ctg aat gtt atg gcg aca gag gca ggt ttc agc gat atg aat 2832
His Ala Leu Asn Val Met Ala Thr Glu Ala Gly Phe Ser Asp Met Asn
930 935 940
att att acc cta tca gga aat tac tgt acg gat aag aaa cct tca gct 2880
Ile Ile Thr Leu Ser Gly Asn Tyr Cys Thr Asp Lys Lys Pro Ser Ala
945 950 955 960
ttg aat tgg atc gat gga cgg ggc aag ggc att gtg gcc gaa gcc atc 2928
Leu Asn Trp Ile Asp Gly Arg Gly Lys Gly Ile Val Ala Glu Ala Ile
965 970 975
ata ccg gcg aac gtt gtc agg gat gtc tta aag agc gat gtg gat agc 2976
Ile Pro Ala Asn Val Val Arg Asp Val Leu Lys Ser Asp Val Asp Ser
980 985 990
atg gtt cag ctc aac ata tcg aaa aat ctg att ggg tcc gct atg gct 3024
Met Val Gln Leu Asn Ile Ser Lys Asn Leu Ile Gly Ser Ala Met Ala
995 1000 1005
ggc tca gtt ggc ggc ttc aac gcc caa gct gcc aat ctt gcg gca 3069
Gly Ser Val Gly Gly Phe Asn Ala Gln Ala Ala Asn Leu Ala Ala
1010 1015 1020
gcc att ttc att gcc aca ggt cag gat ccg gcg caa gtt gtg gag 3114
Ala Ile Phe Ile Ala Thr Gly Gln Asp Pro Ala Gln Val Val Glu
1025 1030 1035
agc gct aac tgc atc act ctc atg aac aat ctt cgc gga tcg ctt 3159
Ser Ala Asn Cys Ile Thr Leu Met Asn Asn Leu Arg Gly Ser Leu
1040 1045 1050
caa atc tct gtc tcc atg ccg tct att gag gtt gga acg ttg ggc 3204
Gln Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Leu Gly
1055 1060 1065
ggt ggt acg att ctg gag ccc cag ggc gca atg ctt gac atg ctt 3249
Gly Gly Thr Ile Leu Glu Pro Gln Gly Ala Met Leu Asp Met Leu
1070 1075 1080
ggt gtc cgc gga tca cac ccg acc act ccc ggt gag aat gca cgt 3294
Gly Val Arg Gly Ser His Pro Thr Thr Pro Gly Glu Asn Ala Arg
1085 1090 1095
caa ctt gcg cgc atc atc gga agc gct gtt ttg gct ggg gag ctc 3339
Gln Leu Ala Arg Ile Ile Gly Ser Ala Val Leu Ala Gly Glu Leu
1100 1105 1110
tcg cta tgt gct gcc cta gcc gcc ggt cac ctg gtc aag gcg cac 3384
Ser Leu Cys Ala Ala Leu Ala Ala Gly His Leu Val Lys Ala His
1115 1120 1125
atg gcg cac aac cgt tct gcc ccg gca tct tca gcc cct tct cga 3429
Met Ala His Asn Arg Ser Ala Pro Ala Ser Ser Ala Pro Ser Arg
1130 1135 1140
agt gtc tcc ccg tca ggc gga acc agg aca gtc cct gtt cct aac 3474
Ser Val Ser Pro Ser Gly Gly Thr Arg Thr Val Pro Val Pro Asn
1145 1150 1155
aat gca ctg agg ccg agt gct gca gct act gat cgg gct cga cgc 3519
Asn Ala Leu Arg Pro Ser Ala Ala Ala Thr Asp Arg Ala Arg Arg
1160 1165 1170
tga 3522
<210>50
<211>1173
<212>PRT
<213>柑桔青霉
<400>50
Met Val Ala Ser Leu Leu Pro Ser Arg Phe Arg Gly Arg Glu Ser Met
1 5 10 15
Asn Gln Gln His Pro Leu Arg Ser Gly Asn Arg Ala Leu Thr Ser Thr
20 25 30
Leu Gln Phe Leu Ser Lys Thr Ala Cys Leu His Pro Ile His Thr Val
35 40 45
Cys Thr Ile Ala Ile Leu Ala Ser Thr Thr Tyr Val Gly Leu Leu Lys
50 55 60
Asp Ser Phe Phe His Gly Pro Ala Asn Val Asp Lys Ala Glu Trp Gly
65 70 75 80
Ser Leu Val Glu Gly Ser Arg Ser Leu Ile Thr Gly Pro Gln Asn Gly
85 90 95
Trp Lys Trp Gln Ser Phe Asp Gly Asp Ala Asp Val Leu Gly Asp Phe
100 105 110
Asn His Gln Ala Leu Met Thr Leu Val Phe Pro Gly Ser Tyr Gly Val
115 120 125
Ala Ser Gln Ala Ala Ser Pro Phe Leu Ala Pro Leu Pro Val Asn Leu
130 135 140
Ser Val Ile Asp Leu Pro Ser Thr Ser Ser Pro Leu Thr Ala Tyr Ser
145 150 155 160
Lys Asp Lys Val Phe Ala Phe Ser Val Glu Tyr Ser Ser Ala Pro Glu
165 170 175
Leu Val Ala Ala Val Gln Glu Ile Pro Asn Asn Ser Ala Asp Leu Lys
180 185 190
Leu Gln Glu Thr Gln Leu Ile Glu Met Glu Arg Gln Met Trp Ile Met
195 200 205
Lys Ala Ala Arg Ala His Thr Lys Arg Ser Leu Ala Gln Trp Val His
210 215 220
Asp Thr Trp Thr Glu Ser Leu Asp Leu Ile Lys Ser Ala Gln Thr Leu
225 230 235 240
Asp Val Val Val Met Val Leu Gly Tyr Ile Ser Met His Leu Thr Phe
245 250 255
Val Ser Leu Phe Leu Ser Met Lys Lys Leu Gly Ser Lys Val Trp Leu
260 265 270
Ala Thr Ser Val Leu Leu Ser Ser Thr Phe Ala Phe Leu Leu Gly Leu
275 280 285
Asp Val Ala Ile Arg Leu Gly Val Pro Met Ser Met Arg Leu Leu Ser
290 295 300
Glu Gly Leu Pro Phe Leu Val Val Ile Val Gly Phe Glu Lys Ser Ile
305 310 315 320
Thr Leu Thr Arg Ala Val Leu Ser Tyr Ala Val Gln His Arg Lys Pro
325 330 335
Gln Lys Ile Gln Ser Asp Gln Gly Ser Val Thr Ala Ile Ala Glu Ser
340 345 350
Thr Ile Asn Tyr Ala Val Arg Ser Ala Ile Arg Glu Lys Gly Tyr Asn
355 360 365
Ile Val Cys His Tyr Val Val Glu Ile Leu Leu Leu Val Ile Gly Ala
370 375 380
Val Leu Gly Ile Gln Gly Gly Leu Gln His Phe Cys Val Leu Ala Ala
385 390 395 400
Leu Ile Leu Phe Phe Asp Cys Leu Leu Leu Phe Thr Phe Tyr Thr Ala
405 410 415
Ile Leu Ser Ile Lys Leu Glu Val Asn Arg Leu Lys Arg His Ile Asn
420 425 430
Met Arg Tyr Ala Leu Glu Asp Glu Gly Leu Ser Gln Arg Thr Ala Glu
435 440 445
Ser Val Ala Thr Ser Asn Asp Ala Gln Asp Ser Ala Arg Thr Tyr Leu
450 455 460
Phe Gly Asn Asp Met Lys Gly Ser Ser Val Pro Lys Phe Lys Phe Trp
465 470 475 480
Met Val Val Gly Phe Leu Ile Val Asn Leu Val Asn Ile Gly Ser Thr
485 490 495
Leu Phe Gln Ala Ser Ser Ser Gly Ser Leu Ser Ser Ile Ser Ser Trp
500 505 510
Thr Glu Ser Leu Ser Gly Ser Ala Ile Lys Pro Pro Leu Glu Pro Phe
515 520 525
Lys Val Ala Gly Ser Gly Leu Asp Glu Leu Leu Phe Gln Ala Arg Gly
530 535 540
Arg Gly Gln Ser Thr Met Val Thr Val Leu Ala Pro Ile Lys Tyr Glu
545 550 555 560
Leu Glu Tyr Pro Ser Ile His Arg Gly Thr Ser Gln Leu His Glu Tyr
565 570 575
Gly Val Gly Gly Lys Met Val Gly Ser Leu Leu Thr Ser Leu Glu Asp
580 585 590
Pro Val Leu Ser Lys Trp Val Phe Val Ala Leu Ala Leu Ser Val Ala
595 600 605
Leu Asn Ser Tyr Leu Phe Lys Ala Ala Arg Leu Gly Ile Lys Asp Pro
610 615 620
Asn Leu Pro Ser His Pro Val Asp Pro Val Glu Leu Asp Gln Ala Glu
625 630 635 640
Ser Phe Asn Ala Ala Gln Asn Gln Thr Pro Gln Ile Gln Ser Ser Leu
645 650 655
Gln Ala Pro Gln Thr Arg Val Phe Thr Pro Thr Thr Thr Asp Ser Asp
660 665 670
Ser Asp Ala Ser Leu Val Leu Ile Lys Ala Ser Leu Lys Val Thr Lys
675 680 685
Arg Ala Glu Gly Lys Thr Ala Thr Ser Glu Leu Pro Val Ser Arg Thr
690 695 700
Gln Ile Glu Leu Asp Asn Leu Leu Lys Gln Asn Thr Ile Ser Glu Leu
705 710 715 720
Asn Asp Glu Asp Val Val Ala Leu Ser Leu Arg Gly Lys Val Pro Gly
725 730 735
Tyr Ala Leu Glu Lys Ser Leu Lys Asp Cys Thr Arg Ala Val Lys Val
740 745 750
Arg Arg Ser Ile Ile Ser Arg Thr Pro Ala Thr Ala Glu Leu Thr Ser
755 760 765
Met Leu Glu His Ser Lys Leu Pro Tyr Glu Asn Tyr Ala Trp Glu Arg
770 775 780
Val Leu Gly Ala Cys Cys Glu Asn Val Ile Gly Tyr Met Pro Val Pro
785 790 795 800
Val Gly Val Ala Gly Pro Ile Val Ile Asp Gly Lys Ser Tyr Phe Ile
805 810 815
Pro Met Ala Thr Thr Glu Gly Val Leu Val Ala Ser Ala Ser Arg Gly
820 825 830
Ser Lys Ala Ile Asn Leu Gly Gly Gly Ala Val Thr Val Leu Thr Gly
835 840 845
Asp Gly Met Thr Arg Gly Pro Cys Val Lys Phe Asp Val Leu Glu Arg
850 855 860
Ala Gly Ala Ala Lys Ile Trp Leu Asp Ser Asp ValGly Gln Thr Val
865 870 875 880
Met Lys Glu Ala Phe Asn Ser Thr Ser Arg Phe Ala Arg Leu Gln Ser
885 890 895
Met Arg Thr Thr Ile Ala Gly Thr His Leu Tyr Ile Arg Phe Lys Thr
900 905 910
Thr Thr Gly Asp Ala Met Gly Met Asn Met Ile Ser Lys Gly Val Glu
915 920 925
His Ala Leu Asn Val Met Ala Thr Glu Ala Gly Phe Ser Asp Met Asn
930 935 940
Ile Ile Thr Leu Ser Gly Asn Tyr Cys Thr Asp Lys Lys Pro Ser Ala
945 950 955 960
Leu Asn Trp Ile Asp Gly Arg Gly Lys Gly Ile Val Ala Glu Ala Ile
965 970 975
Ile Pro Ala Asn Val Val Arg Asp Val Leu Lys Ser Asp Val Asp Ser
980 985 990
Met Val Gln Leu Asn Ile Ser Lys Asn Leu Ile Gly Ser Ala Met Ala
995 1000 1005
Gly Ser Val Gly Gly Phe Asn Ala Gln Ala Ala Asn Leu Ala Ala
1010 1015 1020
Ala Ile Phe Ile Ala Thr Gly Gln Asp Pro Ala Gln Val Val Glu
1025 1030 1035
Ser Ala Asn Cys Ile Thr Leu Met Asn Asn Leu Arg Gly Ser Leu
1040 1045 1050
Gln Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Leu Gly
1055 1060 1065
Gly Gly Thr Ile Leu Glu Pro Gln Gly Ala Met Leu Asp Met Leu
1070 1075 1080
Gly Val Arg Gly Ser His Pro Thr Thr Pro Gly Glu Asn Ala Arg
1085 1090 1095
Gln Leu Ala Arg Ile Ile Gly Ser Ala Val Leu Ala Gly Glu Leu
1100 1105 1110
Ser Leu Cys Ala Ala Leu Ala Ala Gly His Leu Val Lys Ala His
1115 1120 1125
Met Ala His Asn Arg Ser Ala Pro Ala Ser Ser Ala Pro Ser Arg
1130 1135 1140
Ser Val Ser Pro Ser Gly Gly Thr Arg Thr Val Pro Val Pro Asn
1145 1150 1155
Asn Ala Leu Arg Pro Ser Ala Ala Ala Thr Asp Arg Ala Arg Arg
1160 1165 1170
<210>51
<211>20
<212>DNA
<213>柑桔青霉
<400>51
gcaagctctg ctaccagcac 20
<210>52
<211>20
<212>DNA
<213>柑桔青霉
<400>52
ctaggccaac ttcagagccg 20
<210>53
<211>20
<212>DNA
<213>柑桔青霉
<400>53
agtcatgcag gatctgggtc 20
<210>54
<211>20
<212>DNA
<213>柑桔青霉
<400>54
gcagacacat cggtgaagtc 20
<210>55
<211>20
<212>DNA
<213>柑桔青霉
<400>55
aaaccgcacc tgtctattcc 20
<210>56
<211>20
<212>DNA
<213>柑桔青霉
<400>56
ctttgtggtt ggatgcatac 20
<210>57
<211>20
<212>DNA
<213>柑桔青霉
<400>57
cgctctatca tttcgaggac 20
<210>58
<211>20
<212>DNA
<213>柑桔青霉
<400>58
tcaatagacg gcatggagac 20
<210>59
<211>20
<212>DNA
<213>柑桔青霉
<400>59
atgtcagaac ctctaccccc 20
<210>60
<211>20
<212>DNA
<213>柑桔青霉
<400>60
tcaagcatca gtctcaggca 20
<210>61
<211>20
<212>DNA
<213>柑桔青霉
<400>61
atgtccctgc cgcatgcaac 20
<210>62
<211>20
<212>DNA
<213>柑桔青霉
<400>62
ctaagcaata ttgtgtttct 20
Claims (28)
1.编码由氨基酸序列SEQ ID NO:42组成的蛋白质的多核苷酸。
2.权利要求1的多核苷酸,所述多核苷酸由SEQ ID NO:41的核苷酸序列组成。
3.与权利要求1或2的多核苷酸互补的多核苷酸,其特征在于当导入产ML-236B的微生物中时加速该产ML-236B的微生物中的ML-236B的生物合成。
4.与权利要求3的多核苷酸互补的mRNA。
5.如权利要求1的多核苷酸,由可获自转化的大肠杆菌pSAKexpR SANK72599的DNA组成,所述pSAKexpR SANK 72599于2000年1月25日以保藏号FERM BP-7006保藏在工业科技署的生命科学和技术研究院。
6.如权利要求1或2的多核苷酸,它是DNA。
7.如权利要求1或2的多核苷酸,它是基因组DNA。
8.如权利要求1或2的多核苷酸,它是cDNA。
9.含有如前面任一项权利要求的多核苷酸的载体。
10.如权利要求9的载体,其中该载体不含SEQ ID NOS:37、43、45、47或49的多核苷酸序列。
11.如权利要求9的载体,其中该载体不含SEQ ID NOS:43、45、47或49的多核苷酸序列。
12.如权利要求9的载体,能由大肠杆菌pSAKexpR SANK 72599获得,所述pSAKexpR SANK 72599于2000年1月25日以保藏号FERM BP-7006保藏在工业科技署的生命科学和技术研究院。
13.如权利要求9的载体,它是表达载体。
14.由权利要求9-13任一项的载体转化的宿主细胞。
15.如权利要求14的宿主细胞,其特征在于其为产ML-236B的微生物。
16.如权利要求15的宿主细胞,其特征在于其为青霉属物种。
17.如权利要求15或16的宿主细胞,其特征在于其为柑桔青霉。
18.如权利要求14的宿主细胞,其特征在于其为大肠杆菌。
19.如权利要求18的宿主细胞,特征在于其为大肠杆菌pSAKexpR SANK72599,所述pSAKexpR SANK 72599于2000年1月25日以保藏号FERMBP-7006保藏在工业科技署的生命科学和技术研究院。
20.由权利要求1-2或4-8的任一项的多核苷酸编码的多肽。
21.由SEQ ID NO:42的序列组成的多肽。
22.一种生产ML-236B的方法,包括将权利要求14-17任一项的宿主细胞培养,然后从该培养物中回收ML-236B。
23.如权利要求22的方法,其中所述宿主细胞用含有SEQ ID NO:41的核苷酸序列的载体转化。
24.如权利要求23的方法,其中所述载体不含SEQ ID NO:37的多核苷酸序列。
25.如权利要求23或24的方法,其中该载体不含至少一个编码SEQ ID NO:44、46、48和/或50的氨基酸序列的多核苷酸。
26.如权利要求22的方法,其中在没有具有相应于SEQ ID NO:38、44、46、48和/或50的氨基酸序列的一种或多种重组多肽的情况下进行生产。
27.如权利要求22的方法,其中在没有相应于SEQ ID NO:37、43、45、47和/或49的一种或多种cDNA的情况下进行生产。
28.一种生产帕伐他丁的方法,包括进行权利要求22-27任一项的方法,并将ML-236B转变成帕伐他丁。
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JP2000117458 | 2000-04-19 |
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EP (1) | EP1149919B1 (zh) |
KR (1) | KR100632174B1 (zh) |
CN (1) | CN1325959B (zh) |
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WO2010034686A1 (en) * | 2008-09-24 | 2010-04-01 | Dsm Ip Assets B.V. | Improved statin production |
WO2010069914A1 (en) * | 2008-12-19 | 2010-06-24 | Dsm Ip Assets B.V. | Statin transcription regulators |
WO2015161856A1 (en) * | 2014-04-23 | 2015-10-29 | Danmarks Tekniske Universitet | Statin resistance and export |
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WO1995012661A1 (en) * | 1993-11-02 | 1995-05-11 | Merck & Co., Inc. | Dna encoding triol polyketide synthase |
CN1225687A (zh) * | 1996-08-09 | 1999-08-11 | 永进药品工业株式会社 | 生产帕瓦它丁前体ml-236b的新方法 |
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JPS572240A (en) | 1980-06-06 | 1982-01-07 | Sankyo Co Ltd | Ml-236b derivative |
US5179013A (en) * | 1987-02-02 | 1993-01-12 | Sankyo Company, Limited | Cytochrome P-450 enzymes |
US6391583B1 (en) | 1998-12-18 | 2002-05-21 | Wisconsin Alumni Research Foundation | Method of producing antihypercholesterolemic agents |
AU6475800A (en) | 1999-08-11 | 2001-03-13 | Sankyo Company Limited | Ml-236b biosynthesis-associated dna |
FR2801648B1 (fr) * | 1999-11-30 | 2002-06-21 | Commissariat Energie Atomique | Injecteur a vapeur haute pression comportant un drain axial |
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