本申请为国际申请日2001年10月5日、国际申请号PCT/GB2001/004434于2003年5月27日进入中国国家阶段、申请号01819566.0、发明名称“乙醇生产”的分案申请。
许多细菌有天然的代谢能力,通过糖酵解途径将单糖转变为酸性和中性发酵产物的混合物。糖酵解是一系列酶促反应,六碳葡萄糖在此过程中降解,经多种中间产物变成两分子三碳化合物丙酮酸。许多细菌的糖酵解途径都产生丙酮酸作为普遍的中间产物,接着,丙酮酸代谢生成净产物NADH和ATP以及通常被认为是发酵产物的副产物。在有氧条件下,大约95%由糖酵解产生的丙酮酸在许多短的代谢途径中被消耗,同时NAD+经氧化反应再生。在此过程中,NADH被氧化,经一系列的步骤供给氢同剂量的氧以生成水,维持糖酵解的持续进行和ATP的产生。
大多数兼性厌氧菌无论在有氧还是无氧条件下都不能大量产生乙醇。它们在丙酮酸脱氢酶(PDH)的作用下经三羧酸循环(TCA)无氧代谢丙酮酸。在无氧条件下,丙酮酸主要的能量代谢途径是通过丙酮酸甲酸裂解酶(PFL)途径提供甲酸和乙酰CoA,然后乙酰CoA经磷酸转乙酰酶(PTA)和乙酸激酶(AK)作用转化成乙酸并同时产生ATP,或者,经乙醛脱氢酶(AcDH)和醇脱氢酶(ADH)催化还原为乙醇。为维持反应的计量平衡,糖酵解产生的过量NADH在丙酮酸还原成乳酸时被乳酸脱氢酶(LDH)重新氧化成NAD+。NADH也可以在乙酰CoA还原为乙醇时被AcDH和ADH重新氧化,但当细胞中有功能性LDH存在时,后者就是次要反应。为了促使ATP再生,大部分乙酰CoA转变成了乙酸,而且糖酵解产生的过量NADH也被LDH氧化,因此,乙醇的理论产量就很低。
产乙醇的微生物,例如Zymomonasmobilis和酵母,有第二种无氧发酵的方式,通常被称为乙醇发酵。在这种途径中丙酮酸被丙酮酸脱羧酶(PDC)代谢成乙醛和CO2。然后,乙醛被ADH还原为乙醇同时NAD+再生。乙醇发酵的结果是1分子葡萄糖转变为2分子乙醇和2分子的CO2。编码Z.mobilis菌体内这两种酶的DNA已经被分离、克隆,并且可以在通过以上的合成途径大量产生乙醇的宿主菌中表达。例如专利US5,000,000和Ingram等人(1997)BiotechnologyandBioengineering58,Nos.2和3已经报道了编码来源于Z.mobilis的PDC(pdc)和ADH(adh)基因可以掺入到“pet”操纵子,它可用来转化EscherichiaColi菌株形成共同表达Z.mobilispdc和adh两种基因的E.coli重组子。这样的结果是无论在有氧还是无氧条件下,在E.coli的生长过程中产生了从丙酮酸到乙醇为中心代谢的合成途径。专利US5554520也有类似报道,来源于Z.mobilis的pdc和adh均可以使用“pet”操纵子整合产生革兰氏阴性重组宿主菌,包括Erwina.Klebsiella和Xanthomonas品系。这些品系表达Z.mobilis外源基因,通过由丙酮酸到乙醇的合成途径大量产生乙醇。
专利US5482846报道了用编码PDC和ADH酶的异源基因同时转化中温(mesophilic)革兰氏阳性菌Bacillussp,这样,转化菌就可以产生乙醇作为它的初级发酵产物,并无报道提及采用pdc基因单独转化的细菌能产生乙醇。
Biwas等人(JBacteriol.,175,3628-3635,1993)介绍了一种同源重组的方法,通过这一方法,LactococcusLactis的一个染色体基因被携带修饰拷贝的质粒所取代。这个方法使用了温敏性的质粒,所以不能用来转化嗜热菌。
生物催化剂催化生产乙醇时,采用耐热的微生物并在高温下操作取得了重大进步,糖类转化为乙醇的速率大大加快。例如:在嗜热Bacillus中的生产乙醇的能力比用传统的酵母30℃下发酵工艺的能力大10倍。所以,生产能力在一定体积时所需的工厂规模可以减少,工厂的构建费用也相应降低。在高温下,发酵罐中其它微生物对产物产生污染的可能性减小了,后续操作的时间相应减少,生产能力提高,对原料灭菌时所消耗的能量也降低了。而且,不再要求发酵冷却,进一步降低了操作费用。发酵产生的热量有助于乙醇的蒸发,降低了高度的乙醇对菌体的生长抑制,而这是存在于细菌发酵生产的普遍问题。发酵罐上部的乙醇蒸汽也有利于产物回收。
本发明人所用的菌株来自于一种野生型的分离菌,具有天然堆肥能力,比传统的中温微生物更适合将农业原料中的糖类转化为乙醇。这种基础菌株拥有全部转化遗传机器,可把己糖、戊糖以及纤维二糖转化为乙醇;本发明人只是简单地阻断了LDH途径来提高乙醇产量。这个过程被称为自我克隆并不涉及外源基因的表达,故符合遗传修饰有机体(GMO)的安全使用准则。
相反,传统的生物催化剂要么是不能利用戊糖的良好的乙醇生产催化剂,要么是可以利用戊糖的较差的乙醇生产催化剂。这些有机体已经用复杂的遗传学技术在遗传上进行了修饰,这样它们可以将已糖和戊糖都转化为乙醇。然而,这些重组有机体的稳定性以及安全性受到怀疑,因为这些有机体达不到GMO的安全准则。此外,重组中温菌对营养的要求较高,对高盐浓度和原种抑制物也较敏感。
由代谢反应引起的乳酸的形成(LDH途径)已经被体内化学诱变所阻断,因此得到的TN菌株是不产乳酸的,但能大量产生乙醇。然而,这种突变的菌株并不稳定,且会自发回复为产乳酸的野生型。在低pH和高糖环境下,回复突变体比突变体生长快,在连续培养时快速“取代”突变体。随着“取代”的进行,主要的发酵产物由乙醇变成了乳酸。
本发明人首创了一种分子生物学的方法来解决稳定性问题,并且了解了与乙醇生成有关的遗传体系。本发明人首选开发了遗传学技术对有机体进行特异性的操作,连续培养后得到了芽胞形成缺陷型突变体。接着,本发明人对几个关键的代谢基因:乳酸脱氢酶(ldh),乳糖酶通透酶(lp),醇脱氢酶(adh)和一个新的定位于ldh基因内的插入序列进行测序。来源于化学突变菌株的ldh基因的DNA序列分析结果显示在编码区插入一个自发的插入序列元件(IE,也被称为IS元件)之后使基因失活。转座入/出ldh基因以及接下来的基因失活本身都是不稳定的,其结果就是产生回复。
通过质粒和ldh基因插入序列两者间的特异性同源重组,ldh基因突变的稳定性得到了提高。得到的菌株为芽胞形成缺陷型、兼性厌氧和革兰氏阳性的Bacillus菌株,在连续培养过程中显示其生产乙醇能力的提高。以上结果表明新的突变型相当稳定,而且,较之于由体内化学诱变形成的第一代突变型有着更为优越的生长特性和乙醇生产能力。
Southern杂交分析显示了有3个拷贝的转位插入序列元件(IE)存在于BacillusLLD-R菌株的染色体上。该插入序列长3.2Kb并包含三个可以被翻译成蛋白质的开放性的阅读框序列(ORF’s)。ORF1与国家生物技术信息中心(NCBI)数据库(www.ncbi.nlm.nih.gov)公布的任何蛋白都无同源性,而istA和istB与一个已知的转座酶家族有高度的同源性。由LLD-R经体内化学诱变得到BacillusTN菌株(图9A和9B),在结构基因ldh中发现一个拷贝的插入序列,正是这个插入序列使得ldh基因失活,从而得到乳酸阴性的表型,主要的发酵代谢产物也因此由乳酸变成了乙醇。在图1中列出了来源于BacillusTN菌株的ldh基因和IE序列(划线)的DNA序列,在图11中列出了L-LDH的氨基酸序列。然而,这种插入并不稳定,TN突变株会自发回复突变成带有功能性ldh基因的TN-R菌株,主要的发酵代谢产物由乙醇变成了乳酸,如图2所示。这也表明BacillusTN突变株在遗传上的不稳定性。
经PCR扩增,IE序列从TN染色体DNA上获得,PCR引物选自位于插入序列两侧的ldh基因序列。上游引物加入一个HindIII限制性酶切位点。下游引物加入一个XbaI限制性酶切位点,加入的限制性酶切位点便于下一步克隆到pUBUC质粒中,一个长3.2kb的含有插入序列的PCR片段经HindIII和XbaI限制性内切酶切割后克隆到pUBUC质粒中,得到了pUBUC-IE质粒(图5)。
质粒DNA经转化后在携带pMETH质粒的E.coliTOP10细胞中扩增并纯化,转化Bacillussp之后对其进行体内甲基化以防止限制性酶切。甲基化的pUBUC-IE用于转化BacillusTN菌株。转化子首先在52℃下用TGP琼脂平板(卡那霉素)上分离,再用PCR扩增ldh基因进行筛选。用LDH引物未得到目的PCR产物(在设定的PCR条件下大于10kb)说明至少一个拷贝的质粒已经被整合到染色体中。
新菌株TN-T9在pH稳定且不含卡那霉素选择的条件下进行连续培养以检查此菌株的稳定性。采用发酵培养基中含有残余糖类的亚理想的发酵培养条件和偏向于回复突变的条件,证实了TN-T9菌株的稳定性。750小时的连续发酵除了培养基中残余的糖类、丙酮酸分泌物和许多与设定条件有所偏离的情况外,没有任何迹象表明有乳酸产生。在整个发酵过程中,乙醇大量地产生(图4),表明在规定的实验条件下连续培养,TN-T9菌株的ldh基因突变是稳定的。
总而言之,本发明人发明了一个双重体系,在此体系中使用pdc/adh操纵子选择性地表达pdc和adh基因,提高了ldh突变株的稳定性。本发明人分离并测序了一个新的ldh基因和插入序列元件,以及新的乳酸通透酶和乙醇脱氢酶基因。此外,本发明人开发了新的技术,用于质粒DNA与染色体的整合以及筛选重组的Bacillussp,同时,建立了一系列细菌发酵生产乙醇的最佳化条件。
本发明同时详细介绍了一种重组的嗜热革兰氏阳性细菌,它经过质粒和细菌染色体DNA间的同源重组使ldh突变的稳定性得到了提高,形成了一种以产生乙醇作为细菌发酵产物的新菌株。
更可取地,革兰氏阳性菌已经被携带如图1所示的IE序列或其功能性部分或变体的质粒转化,并且如图1所示的IE序列或其功能性部分或变体通过同源重组被稳定地整合到重组菌的染色体中。
本发明的第二个方面是提供一种革兰氏阳性菌,它的天然ldh基因因同源重组而失活,一个或更多的可表达基因被插入到菌体染色体DNA中。此外,质粒可以单拷贝或多拷贝的方式从多个插入位点整合到插入序列中,由此基因的拷贝数增加,基因表达也得到提高。一个或多个可表达的基因可能被插入到菌体染色体DNA中的一个或多个IE序列中,例如在TN-T9和TN-TK两种菌株的染色体上有三个IE序列。
表达的基因对Bacillus而言可能是天然存在的,例如乙醇脱氢酶,或者是外源的(即异源的),例如来源于Z.mobilis的丙酮酸脱羧酶和来源于B.stearothemophilus的α-淀粉酶,这些基因在相同的转录控制下被重排到一个操纵子中,基因表达可以通过增加基因的拷贝数和使用不同的转录启动子序列调控。
本发明的第四个方面是提供一个穿梭载体,它能够在高达54℃的温度下在E.coli和Bacillussp菌株中复制,它有氨苄青霉素和卡那霉素抗性并携带来源于BacillusTN菌株的如图1所示的IE序列或其一部分。
本发明的第五个方面是提供一种在高温下筛选Bacillussp重组子的方法,包括质粒DNA经同源重组稳定地整合到重组子细菌的ldh基因中,以及用PCR筛选不含天然ldh基因和IE序列的重组子。
功能性的部分或变体至少保留如图13,图14或图15所示多肽的转座酶功能部分,且至少含20个氨基酸,更好至少含50个。此外,比较好地其变体与如图13,图14或图15所示的多肽至少有80%的同源性,90%的同源性更好,95%的同源性最好。同源性是优先用BLAST程序检测。
在本说明书中,功能性变体含有序列增加、删除或替代得到的DNA序列,或含有遗传密码子简并性、杂交和/或编码一个有乳酸脱氢酶、乳酸通透酶或乙醇脱氢酶活性多肽的DNA序列。更可取地,这些变体与如图所示的序列至少有80%的同源性,90%的同源性更好,95%的同源性最好。同源性是优先用BLAST程序检测。
本发明最后的第十一个方面,是提供用革兰氏阳性菌发酵生产乙醇的工艺方法,包括细胞生长和乙醇生产的pH值、温度、氧还值和特异稀释率等发酵条件的最佳化选择。发酵条件优先选用5.5-5.7的pH范围,40-75℃的温度范围,-360--400mV的氧还值以及0.3-0.8h-1的稀释率。
实施例2
卡那霉素敏感的TN-TK菌株的筛选
BacillusTN-TK是一种卡那霉素敏感的TN-T9衍生菌株。它对ldh突变相当稳定,对以卡那霉素作为选择标记的质粒表达是一个理想的宿主。
TN-T9首先于68℃在5ml补加卡那霉素(10μg/ml)的TGP培养基中生长24小时,大约100ml培养物铺于两块TGP(km)琼脂板上,68℃孵育过夜。得到几百个菌落,将其中100个菌落用无菌牙签转移到新鲜的TGP(km)平板,68℃过夜生长之后,菌落(通过影印铺板)转移到新鲜的TGP平板和TGP(km)平板,68℃过夜培养。
在TGP平板而非相应的TGP(Km)平板上得到两个卡那霉素敏感的菌落。来自于这两个菌落的ldh区域经PCR扩增,其大小与来源于TN-T9(亲本菌株)的被打断的ldh基因相当。用PCR证明菌株已经失去了卡那霉素抗性的基因。筛选出来一个称为TN-TK的衍生菌株用于进一步生长实验。以上的实验证实了卡那霉素敏感性和ldh突变都是相当稳定的。
序列表
<110>埃尔斯沃思生物技术有限公司
穆罕默德·贾维德
法伊奥纳·库斯丁
保罗·迈尔纳
爱德华·格林
<120>乙醇生产
<130>SPI093746-61
<140>PCT/GB01/04434
<141>2001-10-05
<150>US60/247,017
<151>2000-11-13
<150>UK0024554.8
<151>2000-10-06
<160>16
<170>FastSEQforWindowsVersion4.0
<210>1
<211>4913
<212>DNA
<213>BacillusTN菌株
<220>
<221>misc_feature
<222>(1)...(4913)
<223>n=A,T,CorG
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gaattcaccgaagtcgctcgaagagctaaacgagcgattttggaagtggttggaggaaga2400
ttaccatcgaaaaccgcatgcctcgttgaacgggaagacgccacatgaagtgtttcaatc2460
gcaagtccatttggtgtcgttcgtcgaggattcggattggctcgactcgatctttttgaa2520
acgcgaataccgtaaagtgaaggccgatggtacggtcacgttgaacaagcagctgtatga2580
agttccgccccggttcatcggacaatcgatcgaactccgttatgatgaacaaggcgtgta2640
tgtgtacgaagacggtcaacgggtcgccgaagcggtccttgttcgcttcgaggacaatgc2700
ctatgtgaaacgccatcggtcaccgtttgcggcggttccggtagagggaggcgaaaacga2760
tgtataaaacgttttattccctttcccgagagccgttttcgaaggagacgaatccaccag2820
aggcttatcaaggggcctcgtatcaagaggccctcgccgctttggactacgtgaaacgaa2880
caagagggatcgggctattgatcggtgaaccaggggccggcaagacattcgcccttcggg2940
cgtttaaggaatccctgaatccgtcactgtatcacgtcgtttattttccattgtcaacgg3000
gaagcgtgatggacttttatcgcggccttgccttcgggctcggggaagagccgaaatacc3060
gcaaggtcgacttgttttatcaaatccaacaagggatcgagcgcttgtatcatgaacaac3120
gggtaacgtcagtgttcatcctcgatgaaatgcatttagcgaaggatgcctttctgcagg3180
atatcgcgatcctgttcaactttcacatggactcaacaaatccgtttgtcttgattttgg3240
cggggctgccccatttacaggcaaaactacggttgaaatcaacaccgtccgcttcaccaa3300
cgaatcatcatgcgataccagatggggcctcttgataaggaagaagtggtaggatatatc3360
gaacaccgctgaaacaggcgggggcgaaacacccgatttttaccccagctgccttagaag3420
cgatcgccctgcagtcgcaggggtggccgcggatcatcaacaacctcgccaccacttgcc3480
tgttatacggcgctcaattaaaaaaacatatgattgacgaagacattgtgcgtatggcag3540
ccgaagaaatggggtactgacacagcaggggctgatcggcccctgttatgtttcatcccg3600
atccatcctcattctagttaatcatccgaaataatgtgcaaatgttcggaaataatctgc3660
aaaacctggaataattcgcaaagattttgcacattatttccgaatccgtccgaaataatt3720
tgaaaaagggattctgaaataatgtgctaatttacatttcttgtggcaacgaacccagtg3780
gatattttaacgtatgctacttggaaatttagcgggttaccgaaagagcgggtaatcggc3840
tcaggaacgattcttgatacagcaagattccgcttcttgctaagtgaatattttcaagtg3900
gctccgaccaatgtacatgcgtatattattggcgagcatggggatacagagctgcctgtt3960
tggagccatgcggaaattggaagcattccagttgagcaaatattgatgcaaaacgataac4020
tatagaaaagaggatttagacaatatctttgttaatgttcgtgatgcggcatatcaaatc4080
attgagaaaaaaggggcaacgtattacggcattgcaatgggattagtccgtatcactcgt4140
gctattttgcacaatgaaaatgccatcttaaccgtttctgctcatttggacggccaatat4200
ggcgaacgaaatgtttatattggcgtgcctgccattatcaaccgaaacggtattcgtgaa4260
gtgatggaattgacgctaaatgaaacagaacaacaacaattccatcatagtgtaactgta4320
ttaaaagacattctttcccgttattttgatgatgtaaaataatactgactttgaatacaa4380
caaggtgaacatcgtgtggatacaacattacaatcccttgcataacacatatctttcggc4440
atttattgcggcgtttccgatcgttttatttctattatgcttaactgtgtttaggatgag4500
gggagtaaaagccgcttttctcattctttgttttggtttagtaacagctgttttgttttt4560
ccatatgccgatttcaaaggcgattgctgcgtccgtctatggaatcgcaaacggtttatg4620
gccgattggctatattgtgattatggccgtctggctgtataaaattgctgtgaaaacggg4680
gaaatttgatattatccgcagcagtattgcaaacatttcggaggatcagcggcttcaact4740
gctgctcatcggctttagctttaatgcttttttagaaggagctgcaggattcggtgttcc4800
gattgccatttcggctgctttgctttcagaactaggatttcatccattaaaagcggccgc4860
gctttgcttaattgccaatgctgcctctggcgcgtttggagcgataggaattc4913
<210>2
<211>1255
<212>DNA
<213>BacillusLN
<400>2
agggcaatctgaaaggaagggaaaattcctttcggattgtccttttagttatttttatgg60
ggagtgaatattatataggcattacggaaatgataatggcagagttttttcatttattag120
actgcttgatgtaattggatgtgatgatacaaaaataatgttgtgtaaacaaaatgttaa180
caaaaaagacaaatttcattcatagttgatacttgataaagattgtgaaataatgcacaa240
tatatcaatgtatgagcagtttcacaaattcattttttggaaaggatgacagacagcgat300
gaaacaacaaggcatgaatcgagtagcacttataggaacggggttcgttggggccagcta360
tgcatttgcccttatgaaccaaggaatagcagatgagttagtattgattgatgtaaataa420
gaataaggcagagggcgatgtgatggatttaaatcacggaaaagtattcgcgccgaagcc480
gatgaatatttggtttggagattatcaagattgccaagacgccgatttggtggtgatttg540
tgcaggggctaaccaaaagccgggagaaacaagactggatcttgttgacaaaaatattaa600
tatcttcaaaacgattgtcgattctgtgatgaaatccggatttgatggcgtttttcttgt660
ggcaacgaacccagtggatattttaacgtatgctacttggaaatttagcgggttaccgaa720
agagcgggtaatcggctcaggaacgattcttgatacagcaagattccgcttcttgctaag780
tgaatattttcaagtggctccgaccaatgtacatgcgtatattattggcgagcatgggga840
tacagagctgcctgtttggagccatgcggaaattggaagcattccagttgagcaaatatt900
gatgcaaaacgataactatagaaaagaggatttagacaatatctttgttaatgttcgtga960
tgcggcatatcaaatcattgagaaaaaaggggcaacgtattacggcattgcaatgggatt1020
agtccgtatcactcgtgctattttgcacaatgaaaatgccatcttaaccgtttctgctca1080
tttggacggccaatatggcgaacgaaatgtttatattggcgtgcctgccattatcaaccg1140
aaacggtattcgtgaagtgatggaattgacgctaaatgaaacagaacaacaacaattcca1200
tcatagtgtaactgtattaaaagacattctttcccgttattttgatgatgtaaaa1255
<210>3
<211>319
<212>PRT
<213>BacillusLN
<400>3
MetLysGlnGlnGlyMetAsnArgValAlaLeuIleGlyThrGlyPhe
151015
ValGlyAlaSerTyrAlaPheAlaLeuMetAsnGlnGlyIleAlaAsp
202530
GluLeuValLeuIleAspValAsnLysAsnLysAlaGluGlyAspVal
354045
MetAspLeuAsnHisGlyLysValPheAlaProLysProMetAsnIle
505560
TrpPheGlyAspTyrGlnAspCysGlnAspAlaAspLeuValValIle
65707580
CysAlaGlyAlaAsnGlnLysProGlyGluThrArgLeuAspLeuVal
859095
AspLysAsnIleAsnIlePheLysThrIleValAspSerValMetLys
100105110
SerGlyPheAspGlyValPheLeuValAlaThrAsnProValAspIle
115120125
LeuThrTyrAlaThrTrpLysPheSerGlyLeuProLysGluArgVal
130135140
IleGlySerGlyThrIleLeuAspThrAlaArgPheArgPheLeuLeu
145150155160
SerGluTyrPheGlnValAlaProThrAsnValHisAlaTyrIleIle
165170175
GlyGluHisGlyAspThrGluLeuProValTrpSerHisAlaGluIle
180185190
GlySerIleProValGluGlnIleLeuMetGlnAsnAspAsnTyrArg
195200205
LysGluAspLeuAspAsnIlePheValAsnValArgAspAlaAlaTyr
210215220
GlnIleIleGluLysLysGlyAlaThrTyrTyrGlyIleAlaMetGly
225230235240
LeuValArgIleThrArgAlaIleLeuHisAsnGluAsnAlaIleLeu
245250255
ThrValSerAlaHisLeuAspGlyGlnTyrGlyGluArgAsnValTyr
260265270
IleGlyValProAlaIleIleAsnArgAsnGlyIleArgGluValMet
275280285
GluLeuThrLeuAsnGluThrGluGlnGlnGlnPheHisHisSerVal
290295300
ThrValLeuLysAspIleLeuSerArgTyrPheAspAspValLys
305310315
<210>4
<211>528
<212>DNA
<213>BacillusLN
<400>4
gtgaacatcgtgtggatacaacattacaatcccttgcataacacatatctttcggcattt60
attgcggcgtttccgatcgttttatttctattatgcttaactgtgtttaggatgagggga120
gtaaaagccgcttttctcattctttgttttggtttagtaacagctgttttgtttttccat180
atgccgatttcaaaggcgattgctgcgtccgtctatggaatcgcaaacggtttatggccg240
attggctatattgtgattatggccgtctggctgtataaaattgctgtgaaaacggggaaa300
tttgatattatccgcagcagtattgcaaacatttcggaggatcagcggcttcaactgctg360
ctcatcggctttagctttaatgcttttttagaaggagctgcaggattcggtgttccgatt420
gccatttcggctgctttgctttcagaactaggatttcatccattaaaagcggccgcgctt480
tgcttaattgccaatgctgcctctggcgcgtttggagcgataggaatt528
<210>5
<211>176
<212>PRT
<213>BacillusLN
<400>5
ValAsnIleValTrpIleGlnHisTyrAsnProLeuHisAsnThrTyr
151015
LeuSerAlaPheIleAlaAlaPheProIleValLeuPheLeuLeuCys
202530
LeuThrValPheArgMetArgGlyValLysAlaAlaPheLeuIleLeu
354045
CysPheGlyLeuValThrAlaValLeuPhePheHisMetProIleSer
505560
LysAlaIleAlaAlaSerValTyrGlyIleAlaAsnGlyLeuTrpPro
65707580
IleGlyTyrIleValIleMetAlaValTrpLeuTyrLysIleAlaVal
859095
LysThrGlyLysPheAspIleIleArgSerSerIleAlaAsnIleSer
100105110
GluAspGlnArgLeuGlnLeuLeuLeuIleGlyPheSerPheAsnAla
115120125
PheLeuGluGlyAlaAlaGlyPheGlyValProIleAlaIleSerAla
130135140
AlaLeuLeuSerGluLeuGlyPheHisProLeuLysAlaAlaAlaLeu
145150155160
CysLeuIleAlaAsnAlaAlaSerGlyAlaPheGlyAlaIleGlyIle
165170175
<210>6
<211>1704
<212>DNA
<213>BacillusLN
<400>6
gtgaacatcgtgtggatacaacattacaatcccttgcataacacatatctttcggcattt60
attgcggcgtttccgatcgttttatttctattatgcttaactgtgtttaggatgagggga120
gtaaaagccgcttttctcattctttgttttggtttagtaacagctgttttgtttttccat180
atgccgatttcaaaggcgattgctgcgtccgtctatggaatcgcaaacggtttatggccg240
attggctatattgtgattatggccgtctggctgtataaaattgctgtgaaaacggggaaa300
tttgatattatccgcagcagtattgcaaacatttcggaggatcagcggcttcaactgctg360
ctcatcggctttagctttaatgcttttttagaaggagctgcaggattcggtgttccgatt420
gccatttcggctgctttgctttcagaactaggatttcatccattaaaagcggccgcgctt480
tgcttaattgccaatgctgcctctggcgcgtttggagcgataggaattccggttattgtc540
ggagcgcaaatgggggatttaacgccgattgagttatcccgtacgcttgcttggattttg600
ccgtttatctcgtttctcattccattcttattagtgtttgtcttggataagtggaagggt660
attaaagaaacattgcctgctcttttcgtggtaagcggaagttacaccattgtccaaaca720
ttgacgatcattgtgcttggtcctgagctggccaacattttagcggcgcttgtcagcatg780
ggggcactggctcttttcttgagaaaatggcagccttcaaatatatatcgagtgaatccg840
gatgaaaaaggcggagaaaaatgcaaatacagcttgaaagacatcatcagtgcgtggtcc900
ccgttttatattttaacggtgctcgttattatatggagtctgcctggttttaaagctctg960
tttgccgagggaggcgcactgcagcgaacgacgcttttgtttaaggtgccatttttgcat1020
ggcgaagtcgcgaaaattcctccggtggcgccggcccagacggcattagatgccatattt1080
aagctagaccttgtatcggcaacaggcacggcgattttactggcggtgctgttcacaggg1140
atgtttagcaaaaacattacgttcgcggaaggaatacaaagtttaaaagaaacatgtaaa1200
gagctattcattcctgtattaacgatttgttttatcatgggatttgccaacttagccgac1260
tatgcaggtttatccgctgcgattggtttggcattggcggagacaggcgatgcatttcca1320
tttgtttccccgttattagggtggcttggtgtgtttattacaggatctgtcgtgagcaat1380
aatgctttattcggccatttacaagctgttacaggagcgcaaatagggacaagctcttcg1440
ttattgcttgcggctaacaccagcgggggagtgatggggaaacttatttccccgcagtcc1500
atcgccatagcaactgctgcagtgaaagagacaggcaaagaatctcacctgtttaaaatg1560
acgatttattatagcttgatcctgctattgtttgtgggagtatggacgtactttctttcg1620
accgcaggaatgtagatcattatggtttgttattcagttaggcaccgtgtataaatgaaa1680
aaagatgtattttgcggccttttc1704
<210>7
<211>566
<212>PRT
<213>BacillusLN
<400>7
ValAsnIleValTrpIleGlnHisTyrAsnProLeuHisAsnThrTyr
151015
LeuSerAlaPheIleAlaAlaPheProIleValLeuPheLeuLeuCys
202530
LeuThrValPheArgMetArgGlyValLysAlaAlaPheLeuIleLeu
354045
CysPheGlyLeuValThrAlaValLeuPhePheHisMetProIleSer
505560
LysAlaIleAlaAlaSerValTyrGlyIleAlaAsnGlyLeuTrpPro
65707580
IleGlyTyrIleValIleMetAlaValTrpLeuTyrLysIleAlaVal
859095
LysThrGlyLysPheAspIleIleArgSerSerIleAlaAsnIleSer
100105110
GluAspGlnArgLeuGlnLeuLeuLeuIleGlyPheSerPheAsnAla
115120125
PheLeuGluGlyAlaAlaGlyPheGlyValProIleAlaIleSerAla
130135140
AlaLeuLeuSerGluLeuGlyPheHisProLeuLysAlaAlaAlaLeu
145150155160
CysLeuIleAlaAsnAlaAlaSerGlyAlaPheGlyAlaIleGlyIle
165170175
ProValIleValGlyAlaGlnMetGlyAspLeuThrProIleGluLeu
180185190
SerArgThrLeuAlaTrpIleLeuProPheIleSerPheLeuIlePro
195200205
PheLeuLeuValPheValLeuAspLysTrpLysGlyIleLysGluThr
210215220
LeuProAlaLeuPheValValSerGlySerTyrThrIleValGlnThr
225230235240
LeuThrIleIleValLeuGlyProGluLeuAlaAsnIleLeuAlaAla
245250255
LeuValSerMetGlyAlaLeuAlaLeuPheLeuArgLysTrpGlnPro
260265270
SerAsnIleTyrArgValAsnProAspGluLysGlyGlyGluLysCys
275280285
LysTyrSerLeuLysAspIleIleSerAlaTrpSerProPheTyrIle
290295300
LeuThrValLeuValIleIleTrpSerLeuProGlyPheLysAlaLeu
305310315320
PheAlaGluGlyGlyAlaLeuGlnArgThrThrLeuLeuPheLysVal
325330335
ProPheLeuHisGlyGluValAlaLysIleProProValAlaProAla
340345350
GlnThrAlaLeuAspAlaIlePheLysLeuAspLeuValSerAlaThr
355360365
GlyThrAlaIleLeuLeuAlaValLeuPheThrGlyMetPheSerLys
370375380
AsnIleThrPheAlaGluGlyIleGlnSerLeuLysGluThrCysLys
385390395400
GluLeuPheIleProValLeuThrIleCysPheIleMetGlyPheAla
405410415
AsnLeuAlaAspTyrAlaGlyLeuSerAlaAlaIleGlyLeuAlaLeu
420425430
AlaGluThrGlyAspAlaPheProPheValSerProLeuLeuGlyTrp
435440445
LeuGlyValPheIleThrGlySerValValSerAsnAsnAlaLeuPhe
450455460
GlyHisLeuGlnAlaValThrGlyAlaGlnIleGlyThrSerSerSer
465470475480
LeuLeuLeuAlaAlaAsnThrSerGlyGlyValMetGlyLysLeuIle
485490495
SerProGlnSerIleAlaIleAlaThrAlaAlaValLysGluThrGly
500505510
LysGluSerHisLeuPheLysMetThrIleTyrTyrSerLeuIleLeu
515520525
LeuLeuPheValGlyValTrpThrTyrPheLeuSerThrAlaGlyMet
530535540
IleIleMetValCysTyrSerValArgHisArgValMetLysLysAsp
545550555560
ValPheCysGlyLeuPhe
565
<210>8
<211>1573
<212>DNA
<213>BacillusLN
<400>8
gtggctccaagctatgtatttttagcaagcgaagaggcatcctatattacggggcaaatg60
atacatgtgaatggcggaaagattgtcaatggatagaaagcggcggaaaacggaacgttc120
ttcatgactgcgcaaattttatgtaatattttctgacatagttgttgtgcggtctgtata180
tcaccgttatgataaaaatacatgctatttcttatccatgttcctcaaccttttgtatgg240
attgcaaaaggcatcctctcttccttcacttgcaacaatttattgcaagtttttgtgtat300
ttaggaatattatatgccgcatcaacggacaggcaatgtgaacaaagctgctccaaacat360
aacatattattgatatttcttgaaatattactaatattttgattaaatgtttttgtttgc420
actgcagcttttacaaaagtagaatattttatggtgtttatccgaagaatatcatcatga480
taccctatgggagggaattgtgatgaaagctgcagtagtagagcaatttaaggaaccatt540
aaaaattaaagaagtggaaaagccatccatttcatatggcgaagtattagtccgcattaa600
agcatgcggtgtatgccatacggacttgcatgccgctcacggcgattggccagtaaaacc660
aaaacttcctttaatccctggccatgaaggagtcggaattgttgaagaagtcggtccggg720
ggtaacccatttaaaagtgggagaccgcgttggaattccttggttatattctgcttgcgg780
ccattgcgaatattgtttaagcggacaagagacattatgtgaacatcaagaaaacgccgg840
ctactcagtcgacggggggtatgcagaatattgcagagctgcggcagactatgtggtgaa900
aattcctgacaacttgtcgtttgaagaagctgctcctattttctgcgccggagttactac960
ttataaagcgttaaaagtcacaggtacaaaaccgggagaatgggtagcgatctatggcat1020
cggtggccttggacatgttgccgtccagtatgcgaaagcgatggggcttcatgttgttgc1080
agtggatatcggcgatgagaaactggaacttgcaaaagagcttggcgccgatcttgttgt1140
aaatcctgcaaaagaaaatgcggcacaatttatgaaagagaaagtcggcggagtacacgc1200
ggctgttgtgacagctgtatctaaacctgcttttcaatctgcgtacaattctgtccgcag1260
aggcggcacgtgcgtgcttgtcggattaccgccggaagaaatgcctattccaatctttga1320
tacggtattaaacggaattaaaattatcggttccattgtcggcacgcggaaagacttgca1380
agaagcgcttcagttcgctgcagaaggtaaagtaaaaaccattattgaagtgcaacctct1440
tgaaaaaattaacgaagtatttgacagaatgctaaaaggagaaattaacggacgggttgt1500
tttaacgttagaaaataataattaacgtcaacaacacaatgttgacaacccggcattcta1560
gggctgtctcatc1573
<210>9
<211>319
<212>PRT
<213>BacillusTN菌株
<400>9
MetLysGlnGlnGlyMetAsnArgValAlaLeuIleGlyThrGlyPhe
151015
ValGlyAlaSerTyrAlaPheAlaLeuMetAsnGlnGlyIleAlaAsp
202530
GluLeuValLeuIleAspValAsnLysAsnLysAlaGluGlyAspVal
354045
MetAspLeuAsnHisGlyLysValPheAlaProLysProMetAsnIle
505560
TrpPheGlyAspTyrGlnAspCysGlnAspAlaAspLeuValValIle
65707580
CysAlaGlyAlaAsnGlnLysProGlyGluThrArgLeuAspLeuVal
859095
AspLysAsnIleAsnIlePheLysThrIleValAspSerValMetLys
100105110
SerGlyPheAspGlyValPheLeuValAlaThrAsnProValAspIle
115120125
LeuThrTyrAlaThrTrpLysPheSerGlyLeuProLysGluArgVal
130135140
IleGlySerGlyThrIleLeuAspThrAlaArgPheArgPheLeuLeu
145150155160
SerGluTyrPheGlnValAlaProThrAsnValHisAlaTyrIleIle
165170175
GlyGluHisGlyAspThrGluLeuProValTrpSerHisAlaGluIle
180185190
GlySerIleProValGluGlnIleLeuMetGlnAsnAspAsnTyrArg
195200205
LysGluAspLeuAspAsnIlePheValAsnValArgAspAlaAlaTyr
210215220
GlnIleIleGluLysLysGlyAlaThrTyrTyrGlyIleAlaMetGly
225230235240
LeuValArgIleThrArgAlaIleLeuHisAsnGluAsnAlaIleLeu
245250255
ThrValSerAlaHisLeuAspGlyGlnTyrGlyGluArgAsnValTyr
260265270
IleGlyValProAlaIleIleAsnArgAsnGlyIleArgGluValMet
275280285
GluLeuThrLeuAsnGluThrGluGlnGlnGlnPheHisHisSerVal
290295300
ThrValLeuLysAspIleLeuSerArgTyrPheAspAspValLys
305310315
<210>10
<211>340
<212>PRT
<213>BacillusTN菌株
<400>10
MetLysAlaAlaValValGluGlnPheLysGluProLeuLysIleLys
151015
GluValGluLysProSerIleSerTyrGlyGluValLeuValArgIle
202530
LysAlaCysGlyValCysHisThrAspLeuHisAlaAlaHisGlyAsp
354045
TrpProValLysProLysLeuProLeuIleProGlyHisGluGlyVal
505560
GlyIleValGluGluValGlyProGlyValThrHisLeuLysValGly
65707580
AspArgValGlyIleProTrpLeuTyrSerAlaCysGlyHisCysGlu
859095
TyrCysLeuSerGlyGlnGluThrLeuCysGluHisGlnGluAsnAla
100105110
GlyTyrSerValAspGlyGlyTyrAlaGluTyrCysArgAlaAlaAla
115120125
AspTyrValValLysIleProAspAsnLeuSerPheGluGluAlaAla
130135140
ProIlePheCysAlaGlyValThrThrTyrLysAlaLeuLysValThr
145150155160
GlyThrLysProGlyGluTrpValAlaIleTyrGlyIleGlyGlyLeu
165170175
GlyHisValAlaValGlnTyrAlaLysAlaMetGlyLeuHisValVal
180185190
AlaValAspIleGlyAspGluLysLeuGluLeuAlaLysGluLeuGly
195200205
AlaAspLeuValValAsnProAlaLysGluAsnAlaAlaGlnPheMet
210215220
LysGluLysValGlyGlyValHisAlaAlaValValThrAlaValSer
225230235240
LysProAlaPheGlnSerAlaTyrAsnSerValArgArgGlyGlyThr
245250255
CysValLeuValGlyLeuProProGluGluMetProIleProIlePhe
260265270
AspThrValLeuAsnGlyIleLysIleIleGlySerIleValGlyThr
275280285
ArgLysAspLeuGlnGluAlaLeuGlnPheAlaAlaGluGlyLysVal
290295300
LysThrIleIleGluValGlnProLeuGluLysIleAsnGluValPhe
305310315320
AspArgMetLeuLysGlyGluIleAsnGlyArgValValLeuThrLeu
325330335
GluAsnAsnAsn
340
<210>11
<211>226
<212>PRT
<213>Bacillus
<400>11
MetGlyAspPheGlnArgGlyAspGluAspValIleArgPheHisAsp
151015
PheGlnValAspValGlnThrTyrAlaGlnArgGlyLysGlnAsnAsp
202530
PheProLeuLeuLysArgCysProHisCysGlnAlaLysArgProLeu
354045
TyrArgHisGlyTyrTyrGluArgAsnAlaValThrSerHisGlnSer
505560
TyrArgIleTrpIleAlaArgTyrArgCysProGluCysArgArgThr
65707580
ValAlaValLeuProSerPheLeuLeuProTyrPheGlnTyrThrLeu
859095
ProThrIleTrpArgValValLysGluArgLeuGlyLeuThrProLys
100105110
ArgGlyMetGluGluAlaProLeuLeuProThrAspGluGlyValLeu
115120125
PheTyrValProThrPheIleProLysPheGluProProSerLeuVal
130135140
PheCysGlyAlaLeuGluLysAsnTrpSerCysHisArgProSerArg
145150155160
GluAsnGluProTyrGlyGlySerArgArgTrpArgArgSerAlaSer
165170175
PheSerSerSerLysArgTyrGlySerThrAspArgArgIlePheLeu
180185190
HisValHisSerValProAspLeuLeuIleSerProTyrMetGluSer
195200205
PheIleAspSerGlnThrPheProLeuAspGlyArgGlyAsnAspPro
210215220
IleGly
225
<210>12
<211>415
<212>PRT
<213>Bacillus
<400>12
MetAsnGluSerMetArgGlnGluIleAlaLeuPheArgTyrGlyLeu
151015
IleAlaProLeuValAsnGlyGlnValAspProLysThrTyrLeuLys
202530
GluValAlaGluArgIleHisGlnValProHisHisGlyGluLysArg
354045
IleAlaAlaLysThrIleLeuAspTrpCysThrGlnTyrLysLysGly
505560
GlyPheGluAlaLeuLysProLysArgArgSerAspArgGlyHisSer
65707580
ArgArgLeuSerProGluGluGluAspHisIleLeuAlaLeuArgLys
859095
LysHisProHisMetProValThrValPheTyrGlnHisLeuIleGlu
100105110
GlnGlyGluIleGlnSerIleSerTyrPheThrIleTyrArgLeuLeu
115120125
LysLysTyrAsnLeuValGlyLysGluIIeLeuProIleProGluArg
130135140
LysArgPheAlaTyrAspGlnValAsnGluLeuTrpGlnGlyAspLeu
145150155160
SerHisGlyProLeuIleArgValAsnGlyLysThrGlnLysThrPhe
165170175
LeuIleAlaTyrIleAspAspCysSerArgValValProTyrAlaGln
180185190
PhePheSerSerGluLysPheAspGlyLeuArgIleValThrLysGlu
195200205
AlaLeuLeuArgTyrGlyLysProLysArgIleTyrSerAspAsnGly
210215220
LysIleTyrArgAlaGluProLeuGlnTyrAlaCysAlaGluLeuGly
225230235240
IleThrLeuIleHisThrGlnProTyrAspProGlnSerLysGlyLys
245250255
IleGluArgPhePheArgThrValGlnThrArgPheTyrProLeuLeu
260265270
GluMetAsnSerProLysSerLeuGluGluLeuAsnGluArgPheTrp
275280285
LysTrpLeuGluGluAspTyrHisArgLysProHisAlaSerLeuAsn
290295300
GlyLysThrProHisGluValPheGlnSerGlnValHisLeuValSer
305310315320
PheValGluAspSerAspTrpLeuAspSerIlePheLeuLysArgGlu
325330335
TyrArgLysValLysAlaAspGlyThrValThrLeuAsnLysGlnLeu
340345350
TyrGluValProProArgPheIleGlyGlnSerIleGluLeuArgTyr
355360365
AspGluGlnGlyValTyrValTyrGluAspGlyGlnArgValAlaGlu
370375380
AlaValLeuValArgPheGluAspAsnAlaTyrValLysArgHisArg
385390395400
SerProPheAlaAlaValProValGluGlyGlyGluAsnAspVal
405410415
<210>13
<211>266
<212>PRT
<213>Bacillus
<400>13
MetTyrLysThrPheTyrSerLeuSerArgGluProPheSerLysGlu
151015
ThrAsnProProGluAlaTyrGlnGlyAlaSerTyrGlnGluAlaLeu
202530
AlaAlaLeuAspTyrValLysArgThrArgGlyIleGlyLeuLeuIle
354045
GlyGluProGlyAlaGlyLysThrPheAlaLeuArgAlaPheLysGlu
505560
SerLeuAsnProSerLeuTyrHisValValTyrPheProLeuSerThr
65707580
GlySerValMetAspPheTyrArgGlyLeuAlaPheGlyLeuGlyGlu
859095
GluProLysTyrArgLysValAspLeuPheTyrGlnIleGlnGlnGly
100105110
IleGluArgLeuTyrHisGluGlnArgValThrSerValPheIleLeu
115120125
AspGluMetHisLeuAlaLysAspAlaPheLeuGlnAspIleAlaIle
130135140
LeuPheAsnPheHisMetAspSerThrAsnProPheValLeuIleLeu
145150155160
AlaGlyLeuProHisLeuGlnAlaLysLeuArgLeuAsnGlnHisArg
165170175
ProLeuHisGlnArgIleIleMetArgTyrGlnMetGlyProLeuAsp
180185190
LysGluGluValValGlyTyrIleGluHisArgLeuLysGlnAlaGly
195200205
AlaLysHisProIlePheThrProAlaAlaLeuGluAlaIleAlaLeu
210215220
GlnSerGlnGlyTrpProArgIleIleAsnAsnLeuAlaThrThrCys
225230235240
LeuLeuTyrGlyAlaGlnLeuLysLysHisMetIleAspGluAspIle
245250255
ValArgMetAlaAlaGluGluMetGlyTyr
260265
<210>14
<211>27
<212>DNA
<213>Bacilllus
<400>14
aagcttgatgaaatccggatttgatgg27
<210>15
<211>24
<212>DNA
<213>Bacillus
<400>15
tctagagctaaatttccaagtagc24
<210>16
<211>3149
<212>DNA
<213>BacillusTN菌株
<220>
<221>misc_feature
<222>(1)...(3149)
<223>n=A,T,CorG
<400>16
angtcttgttgtaaattatcactttatttccgcacaaaaaagactcttttttgcacattc60
cttcggaatatccctctccccctttccgaaagaatgtgctaaattttttgtgaattattt120
cggaatgggacatgggtgatttccagaggggggacgaggacgtgattcggtttcatgact180
ttcaggtcgatgtccagacatatgcccagcggggaaaacaaaacgactttccccttctta240
agcggtgccctcattgccaggcaaaacgccctctttatcgccatgggtactacgaacgaa300
atgccgtgacgtcgcatcagtcttatcgcatttggatcgctcggtatcgctgcccggagt360
gcaggaggacggtggccgtgttgccttcatttcttctcccttattttcagtatacgttgc420
ccaccatatggagagtggtgaaagaacggttgggcctgactccgaaacgggggatggagg480
aggctccactccttcctacggatgaaggggttttattttatgtcccgacgtttattccga540
aatttgaaccaccttcattggttttttgcggagcgctgggagaaaaattggtcctgccat600
cgcccaagccgagagaacgagccctatggtggatccagacgatggaggagatcggcctct660
ttttcgtcatccaagagatatgggagcaccgatcgacgcatctttttgcacgtacattca720
gttcctgatttacttatatccccttatatggaatcatttatagattcccaaacctttcct780
ctcgacggtcgggggaatgatccgataggatagagacaggatggaccgataaggtcctag840
aatgggatgaacgaaggaggagatcgaaatgaatgagtcgatgagacaggagatcgcttt900
atttcggtatggattgatcgctccattggtgaatggacaagtcgatccaaaaacgtactt960
gaaggaagtagcggaacggatccatcaagttccccaccatggagagaaacgcatcgccgc1020
caaaacgatcctcgactggtgcacgcagtacaaaaaagggggctttgaggcgctgaagcc1080
gaaacgacggtcggaccgtggccattcccggaggctgtcacctgaagaagaggatcacat1140
tttagccctgagaaaaaaacacccccacatgcccgtgacggtgttttaccaacaccttat1200
cgagcagggggaaatccaatccatctcttatttcactatataccgacttttaaaaaaata1260
caacctcgtggggaaagaaattttaccgattcctgaacgaaaacgattcgcgtacgatca1320
ggtcaatgagctctggcaaggtgatttgtcccatggcccgttgattcgcgtgaatggcaa1380
aacgcaaaaaacgtttttgattgcctatatcgatgactgctcgcgggtcgtgccgtacgc1440
tcagtttttctcttccgagaaatttgacgggttgcggatcgtaaccaaggaagcgctgct1500
tcgatacggaaagccgaagcgaatttactcggataacggcaagatttatcgggcggaacc1560
cttgcagtacgcctgcgcggagttagggatcaccttgatccatacccagccgtacgatcc1620
gcaaagcaaagggaaaatcgaacgatttttccgcaccgtacagacgcggttttacccgtt1680
gctcgaaatgaattcaccgaagtcgctcgaagagctaaacgagcgattttggaagtggtt1740
ggaggaagattaccatcgaaaaccgcatgcctcgttgaacgggaagacgccacatgaagt1800
gtttcaatcgcaagtccatttggtgtcgttcgtcgaggattcggattggctcgactcgat1860
ctttttgaaacgcgaataccgtaaagtgaaggccgatggtacggtcacgttgaacaagca1920
gctgtatgaagttccgccccggttcatcggacaatcgatcgaactccgttatgatgaaca1980
aggcgtgtatgtgtacgaagacggtcaacgggtcgccgaagcggtccttgttcgcttcga2040
ggacaatgcctatgtgaaacgccatcggtcaccgtttgcggcggttccggtagagggagg2100
cgaaaacgatgtataaaacgttttattccctttcccgagagccgttttcgaaggagacga2160
atccaccagaggcttatcaaggggcctcgtatcaagaggccctcgccgctttggactacg2220
tgaaacgaacaagagggatcgggctattgatcggtgaaccaggggccggcaagacattcg2280
cccttcgggcgtttaaggaatccctgaatccgtcactgtatcacgtcgtttattttccat2340
tgtcaacgggaagcgtgatggacttttatcgcggccttgccttcgggctcggggaagagc2400
cgaaataccgcaaggtcgacttgttttatcaaatccaacaagggatcgagcgcttgtatc2460
atgaacaacgggtaacgtcagtgttcatcctcgatgaaatgcatttagcgaaggatgcct2520
ttctgcaggatatcgcgatcctgttcaactttcacatggactcaacaaatccgtttgtct2580
tgattttggcggggctgccccatttacaggcaaaactacggttgaaatcaacaccgtccg2640
cttcaccaacgaatcatcatgcgataccagatggggcctcttgataaggaagaagtggta2700
ggatatatcgaacaccgctgaaacaggcgggggcgaaacacccgatttttaccccagctg2760
ccttagaagcgatcgccctgcagtcgcaggggtggccgcggatcatcaacaacctcgcca2820
ccacttgcctgttatacggcgctcaattaaaaaaacatatgattgacgaagacattgtgc2880
gtatggcagccgaagaaatggggtactgacacagcaggggctgatcggcccctgttatgt2940
ttcatcccgatccatcctcattctagttaatcatccgaaataatgtgcaaatgttcggaa3000
ataatctgcaaaacctggaataattcgcaaagattttgcacattatttccgaatccgtcc3060
gaaataatttgaaaaagggattctgaaataatgtgctaatttacatttcttgtggcaacg3120
aacccagtggatattttaacgtatgctac3149