CN114908027B - 泛酸生产相关菌株及其构建方法与应用 - Google Patents
泛酸生产相关菌株及其构建方法与应用 Download PDFInfo
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Abstract
本发明提供了泛酸生产菌及其构建方法与应用，菌株从谷氨酸棒杆菌ATCC 13032 rpsLK43R出发，在D‑泛酸生产领域具有广泛的应用前景。通过整合第二拷贝ptsG基因，在基因组水平引入需钠弧菌中ED途径的edd和eda基因，获得缬氨酸生产菌株解除反馈抑制的乙酰羟酸合酶编码基因ilvB和ilvN突变体，与乙酰羟酸异构还原酶基因ilvC一起，整合到基因组中，敲除avtA、ilvE来减弱支链氨基酸缬氨酸、亮氨酸、异亮氨酸的合成，过表达枯草芽孢杆菌panBCD基因，获得了D‑泛酸生产菌株；强化pyc、aspB基因的表达和引入大肠杆菌aspA基因，增加前体物β‑丙氨酸的供应；敲除ilvA以阻断α‑酮基丁酸合成，避免与底物3‑甲基‑2‑氧代丁酸竞争panB基因编码的羟甲基转移酶，且避免α‑酮基丁酸对ilvBN编码的乙酰羟酸合酶的抑制。
Description
技术领域
本发明涉及基因工程技术领域，尤其是泛酸生产相关菌株及其构建方法与应用。
背景技术
泛酸(VB5)是辅酶A(CoA)和酰基载体蛋白(ACP)生物合成的重要前体物质，维持生物体正常生理功能所必须的维生素。参与生物体内碳水化合物、脂肪酸、蛋白质和能量代谢。在人体中还参与类固醇、褪黑激素、抗体和亚铁血红素的合成。泛酸在食品、药物、饲料添加剂等方面具有重要作用。
传统的生产方法采用化学方法，手性拆分剂成本高，分离困难，并有环境污染和毒性问题。随着人们对绿色环保的追求，微生物法生产D-泛酸产品越来越受到人们的重视。微生物发酵法条件温和、环境友好、产品质量稳定，具有发展前途。谷氨酸棒杆菌(Corynebacterium glutamicum)为革兰氏阳性菌，由于其安全性和发酵稳定性被广泛作为氨基酸等的工业生产菌，目前利用谷氨酸棒杆菌生产D-泛酸的报道较少，即使在添加β-丙氨酸的条件下也产率极低。
发明内容
本发明所要解决的技术问题在于提供一种用于制备泛酸生产菌的菌株。
本发明所要解决的技术问题在于提供一种泛酸生产菌。
本发明所要解决的技术问题在于提供上述泛酸生产菌的构建方法。
本发明所要解决的技术问题在于提供上述泛酸生产菌的应用。
为解决上述技术问题，本发明的技术方案是：
一种用于制备泛酸生产菌的菌株，为VB5-1，是由下述方法构建而成的：在链霉素抗性谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC 13032 rpsLK43R(Wang et al.,Anupdate of the suicide plasmid-mediated genome editing system inCorynebacterium glutamicum，Microbial Biotechnology，2019，12(5),907–919)基因组的cg1890假基因位点整合由Ptrc启动子启动的ptsG基因，得到C.glutamicum ATCC13032rpsLK43R(cg1890::Ptrc-ptsG)菌株。
一种用于制备泛酸生产菌的菌株，为VB5-2，是由下述方法构建而成的：在VB5-1基因组cg1895假基因位点整合由Ptuf启动子启动的来源于需钠弧菌的eddeda基因，得到VB5-1(cg1895::Ptuf-eddeda)菌株。
一种用于制备泛酸生产菌的菌株，为VB5-3，是由下述方法构建而成的：在VB5-2基因组cg1995假基因位点整合由Ptuf启动子启动的ilvB*N*C基因，ilvB*N*C来源高产L-缬氨酸诱变菌C.glutamicum XV的ilvB* (ilvBK30Q,G128S,A138V,Y252H,T362S)和ilvN*(ilvNH47L)，得到VB5-2(cg1995:: Ptuf-ilvB*N*C*)菌株。
一种用于制备泛酸生产菌的菌株，为VB5-4，是由下述方法构建而成的：将Vb5-3中avtA基因敲除，得到VB5-3(△avtA)菌株。
一种用于制备泛酸生产菌的菌株，为VB5-5，是由下述方法构建而成的：将Vb5-4中ilvE基因敲除，得到VB5-4(△ilvE)菌株。
一种用于制备泛酸生产菌的菌株，为VB5-6，是由下述方法构建而成的：将VB5-5中pyc基因编码的酶第458位氨基酸由脯氨酸突变为丝氨酸，得到 VB5-5(pyc*)菌株。
一种泛酸生产菌，为VB5-7，是由下述方法构建而成的：在Vb5-6基因组cg1960假基因位点整合由Ptuf启动子启动的aspB基因，得到VB5-6 (cg1960::Ptuf-aspB)菌株。
一种泛酸生产菌，为VB5-8，是由下述方法构建而成的：在VB5-7基因组Ncgl2850a假基因位点整合由Ptuf启动子启动的来源于大肠杆菌的 aspAEco基因，得到VB5-7(Ncgl2850a::Ptuf-aspAEco)菌株。
一种泛酸生产菌，为VB5-9，是由下述方法构建而成的：将VB5-8中ilvA 基因敲除，得到VB5-8(△ilvA)菌株。
一种泛酸生产菌，为VB5-10，是由下述方法构建而成的：将pXtuf质粒 (pXMJ19衍生质粒，将Ptac启动子替换为Ptuf；Wang et al.,An update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum，MicrobialBiotechnology，2019，12(5),907–919)与片段来源于枯草芽胞杆菌的panBCDBsu进行连接，构建pXtuf-panBCDBsu质粒；将质粒转化到VB5-9中，得到VB5-9/pXtuf-panBCDBsu(D-泛酸生产菌)。
上述泛酸生产菌的构建方法，具体步骤如下：
(1)以菌株Corynebacterium glutamicum ATCC 13032 rpsLK43R为出发菌株，运用自杀质粒pK18mobrpsL介导的谷氨酸棒杆菌基因编辑技术(Wang et al.,An update ofthe suicide plasmid-mediated genome editing system in Corynebacteriumglutamicum，Microbial Biotechnology，2019， 12(5),907–919)，将cg1890假基因位点整合由Ptrc启动子启动的ptsG 基因，得到C.glutamicum ATCC 13032rpsLK43R(cg1890::Ptrc-ptsG)菌株，记为VB5-1；
(2)运用自杀质粒pK18mobrpsL介导的谷氨酸棒杆菌基因编辑技术，在VB5-1基因组cg1895假基因位点整合由Ptuf启动子启动的来源于需纳弧菌的eddeda基因，得到VB5-1(cg1895::Ptuf-eddeda)菌株，记为VB5-2；
(3)运用CRISPR-Cpf1基因编辑技术，在VB5-2基因组cg1995假基因位点整合由Ptuf启动子启动的ilvB*N*C基因，ilvB*N*C来源高产L-缬氨酸诱变菌C.glutamicum XV的ilvB*(ilvBK30Q,G128S,A138V,Y252H,T362S)和 ilvN*(ilvNH47L)，得到VB5-2(cg1995::Ptuf-ilvB*N*C*)菌株，记为VB5-3；
(4)运用CRISPR-Cpf1基因编辑技术，将VB5-3中avtA基因敲除，得到VB5-3(△avtA)菌株，记为VB5-4；
(5)运用CRISPR-Cpf1基因编辑技术，将VB5-4中ilvE基因敲除，得到VB5-4(△ilvE)菌株，记为VB5-5；
(6)运用自杀质粒pK18mobrpsL介导的谷氨酸棒杆菌基因编辑技术，将VB5-5中pyc基因编码的酶第458位氨基酸由脯氨酸突变为丝氨酸，得到 VB5-5(pyc*)菌株，记为VB5-6；
(7)运用CRISPR-Cpf1基因编辑技术，在VB5-6基因组cg1960假基因位点整合由Ptuf启动子启动的aspB基因，得到VB5-6(cg1960::Ptuf-aspB) 菌株，记为VB5-7；
(8)运用CRISPR-Cpf1基因编辑技术，在VB5-7基因组Ncgl2850a假基因位点整合由Ptuf启动子启动的来源于大肠杆菌的aspAEco基因，得到 VB5-7(Ncgl2850a::Ptuf-aspAEco)菌株，记为VB5-8；
(9)运用CRISPR-Cpf1基因编辑技术，将VB5-8中ilvA基因敲除，得到VB5-8(△ilvA)菌株，记为VB5-9；
(10)将pXtuf质粒与片段panBCDBsu进行连接，构建pXtuf-panBCDbsu质粒；将质粒转化到VB5-9中，得到VB5-9/pXtuf-panBCDbsu，记为VB5-10 (D-泛酸生产菌)。
上述泛酸生产菌在发酵生产D-泛酸方面的应用。
优选的，上述泛酸生产菌的应用，具体发酵生产方法如下：
(1)菌体活化：用接种环从保菌管中取一环划线接种于斜面试管活化， 32℃恒温静置培养20-24h，即可得到一代活化斜面；然后从该斜面上用接种环将菌体接种于斜面茄形瓶中，32℃恒温静置培养12-24h，即可得到二代活化斜面；
(2)种子罐培养：取上一步活化较好的二代斜面茄子瓶，取适量无菌水于茄形瓶中，将菌悬液接入种子培养基中并最终定容至3L，通过流加氨水使pH稳定在7.0-7.2，温度恒定在32℃，控制溶氧在20-30％，培养菌体生长至OD600达到15-30；
(3)发酵罐培养：按照5％-10％接种量接入新鲜的发酵培养基开始发酵，发酵过程中通过流加氨水控制pH稳定在7.0-7.2，温度维持在32℃，溶氧在20％以上；当培养基中的葡萄糖消耗完之后，流加80％(m/v)的葡萄糖溶液，维持发酵培养基中的葡萄糖浓度在0.1-5g/L；发酵周期一般在40-48h。
优选的，上述泛酸生产菌的应用，所述种子培养基的组成如下：葡萄糖 80g/L，(NH4)2SO4 5g/L，KH2PO4 2.5g/L，MgSO4·7H2O 1.6g/L，FeSO4·7H2O 20mg/L， MnSO4·H2O20mg/L，B族维生素VB1、VB3、VB12各0.3mg/L，蛋白胨2g/L，酵母粉3g/L，亮氨酸1g/L，异亮氨酸1g/L，缬氨酸1g/L，蛋氨酸1g/L，谷氨酸5g/L，丝肽粉5g/L，柠檬酸2g/L，玉米浆5g/L，用自来水配置培养基。
优选的，上述泛酸生产菌的应用，所述发酵培养基的组成如下：葡萄糖 80g/L，(NH4)2SO4 5g/L，KH2PO4 2.5g/L，MgSO4·7H2O 1.6g/L，FeSO4·7H2O 20mg/L ，MnSO4·H2O 20mg/L，VB1、VB3、VB12各0.3mg/L，蛋白胨2g/L，酵母粉3g/L ，亮氨酸1g/L，异亮氨酸1g/L，缬氨酸1g/L，蛋氨酸1g/L，谷氨酸5g/L，丝肽粉5g/L，柠檬酸2g/L，玉米浆20g/L，豆浓10ml/L，其余为水。
有益效果：
上述泛酸生产菌，无需添加β-丙氨酸即可高产泛酸，在发酵过程中实现发酵液中D-泛酸的有效积累，其构建方法从谷氨酸棒杆菌ATCC 13032 rpsLK43R出发，通过(1)整合第二拷贝ptsG基因，加快葡萄糖的运输与利用。 (2)在基因组水平引入需钠弧菌中ED途径的edd和eda基因，使葡萄糖能更快的转化为丙酮酸。(3)获得缬氨酸生产菌株解除反馈抑制的乙酰羟酸合酶编码基因ilvB和ilvN突变体，与乙酰羟酸异构还原酶基因ilvC一起，整合到基因组中，既能解除反馈抑制又能强化代谢流。(4)通过减弱竞争途径来减少副产物生产，敲除avtA、ilvE来减弱支链氨基酸缬氨酸、亮氨酸、异亮氨酸的合成，该菌株能够有效积累D-泛酸前体物3-甲基-2-氧代丁酸。(5)过表达枯草芽孢杆菌panBCD基因，获得了D-泛酸生产菌株。(6) 强化pyc、aspB基因的表达和引入大肠杆菌aspA基因，增加前体物β-丙氨酸的供应，进一步提升了D-泛酸的产量。(7)敲除ilvA以阻断α-酮基丁酸的合成，避免与底物3-甲基-2-氧代丁酸竞争panB基因编码的羟甲基转移酶，且避免α-酮基丁酸对ilvBN编码的乙酰羟酸合酶的抑制。最终5L发酵罐在无需外源添加β-丙氨酸的情况下D-泛酸产量达到18.62g/L，为目前报道的以葡萄糖从头合成D-泛酸的最高产量。所述构建方法简单易操作，获得的泛酸生产菌在D-泛酸生产领域具有广泛的应用前景。
附图说明
图1为基因工程改造谷氨酸棒状杆菌生产D-泛酸代谢路径示意图，包括泛酸生物合成途径及代谢工程改造策略；
图中，首先整合ptsG基因，加快葡萄糖的运输与利用。随后引入需钠弧菌中的edd和eda基因整合到基因组上，使葡萄糖能更快的转化为丙酮酸。其次解除合成缬氨酸的关键酶乙酰羟酸合酶的反馈抑制，对ilvB和ilvN基因进行突变，来解除反馈抑制的乙酰羟酸合酶。然后将突变的乙酰羟酸异构还原酶整合到同一位点，构成完整的操纵子，既能解除反馈抑制又能强化代谢流。此外通过阻断竞争途径来减少副产物生成，敲除avtA、ilvE来减弱支链氨基酸缬氨酸、亮氨酸、异亮氨酸的合成。该菌株能够有效积累D-泛酸前体物3-甲基-2-氧代丁酸。通过强化pyc和aspB、引入大肠杆菌aspA基因，促进了了D-泛酸另一前体物β-丙氨酸的合成。选择敲除ilvA以减少 2-氧代丁酸的积累来减弱与乙酰羟酸形成竞争性底物。最后质粒过表达来源于枯草芽孢杆菌的panBCD基因，最终获得D-泛酸的高效合成。
图2为质粒pXtuf-panBCDBsu(B.subtilis)图谱。
图3为VB5-10菌株发酵过程OD600和D-泛酸效价变化，其中，三角代表泛酸产量，方框代表菌体OD值。
具体实施方式
下面结合具体实施例对本发明所述技术方案作进一步的说明。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。
以下实施例中，所述卡那霉素的使用浓度为:在大肠杆菌中的工作浓度为50mg/L，在谷氨酸棒杆菌中的工作浓度为10mg/L；所述氯霉素的使用浓度为:在大肠杆菌中的工作浓度为30mg/L，在谷氨酸棒杆菌中的工作浓度为 12mg/L；所述异丙基硫代半乳糖苷在谷氨酸棒杆菌中的工作浓度为 0.1mmol/L；所述链霉素在谷氨酸棒杆菌中的工作浓度为0.5g/L；所述硫酸壮观霉素在谷氨酸棒杆菌中的工作浓度为0.2g/L。
实施例1：VB5-1菌株的构建
(1)pK18mobrpsL-cg1890::Ptrc-ptsG质粒的构建
以C.glutamicum ATCC 13032基因组为模板，引物cg1890-up-1和 cg1890-up-2来扩增上游同源臂，引物cg1890-down-1和cg1890-down-2来扩增下游同源臂，引物ptsG-1和ptsG-2来扩增ptsG基因(SEQ ID NO.3 所示序列)。以pEC-XK99E质粒为模板，引物cg1890-Ptrc-1和cg1890-Ptrc-2 来扩增Ptrc启动子(SEQ ID NO.1所示序列)。具体引物设计见表1。然后通过重叠PCR，利用引物cg1890-up-1和cg1890-down-2扩增得到重叠片段 cg1890::Ptrc-ptsG，经琼脂糖凝胶电泳验证后进行纯化回收。载体 pK18mobrpsl选取XbaⅠ和KpnⅠ两个酶切位点进行双酶切回收。将重叠片段与线性化载体pK18mobrpsl进行同源重组，再转化到E.coliDH5α中。卡那霉素平板筛选，通过菌落PCR验证正确的转化子，即为 pK18mobrpsL-cg1890::Ptrc-ptsG质粒。
(2)pK18mobrpsL-cg1890::Ptrc-ptsG质粒电击转化
将构建好的质粒pK18mobrpsL-cg1890::Ptrc-ptsG提取出来电转化到底盘菌Corynebacterium glutamicum ATCC 13032rpsLK43R感受态中，电转化后涂布于含有卡那霉素抗性(10mg/L)的平板上，将在卡那霉素抗性平板上长出的转化子，挑取单菌落通过设计的两对特异性鉴定引物(验证采用两对引物，前一对为上游同源臂前和下游同源臂下游引物，后一对为上游同源臂上游引物和下游同源臂后)，菌落PCR验证发生一轮交换菌株时所使用的引物为cg1890-jd-1，cg1890-down-2，只有鉴定出条带大小为3514bp则为正确；利用菌落PCR扩增进行验证，经琼脂糖凝胶电泳检测，得到的片段的大小与理论值一致，证明穿梭质粒已成功整合到菌株的基因组上，完成了第一次同源交换，即为单交换菌株；将在对应平板长起来的阳性转化子接入含有卡那霉素抗性(10mg/L)的BHI摇管中32℃培养12h，将培养好的菌液保菌，储存于-80℃，利用剩余的菌液通过BHI复苏液稀释500倍涂布到含有链霉素抗性(0.5g/L)的平板中；将平板上长出的单菌落涂布链霉素抗性平板(0.5g/L)，菌落PCR验证发生二轮交换菌株时所使用的引物为 cg1890-jd-1，cg1890-jd-2，若鉴定出条带大小为3686bp则为正确的菌株，若鉴定出条带大小为2250bp则是回复为原菌。
(3)正确菌株测序验证
将菌落验证正确的菌株提基因组，扩增cg1890::Ptrc-ptsG部分，测序正确得到菌株VB5-1。
实施例2：VB5-2菌株的构建
(1)pK18mobrpsL-cg1895::Ptuf-eddeda质粒的构建
以C.glutamicum ATCC 13032基因组为模板引物cg1895-up-1和 cg1895-up-2来扩增上游同源臂，引物cg1895-down-1和cg1895-down-2来扩增下游同源臂。以pXtuf质粒为模板，引物cg1895-Ptuf-1和cg1895-Ptuf-2来扩增Ptuf启动子(SEQ ID NO.2所示序列)。以V. natriegens ATCC 14048基因组为模板，引物ed-1和ed-2来扩增eddedaVna基因(SEQ IDNO.4所示序列)，具体引物设计见表1。然后通过重叠PCR，利用引物cg1895-up-1和cg1895-down-2扩增得到重叠片段 cg1895::Ptuf-eddeda，经琼脂糖凝胶电泳验证后进行纯化回收。载体 pK18mobrpsl选取XbaⅠ和KpnⅠ两个酶切位点进行双酶切回收。将重叠片段与线性化载体pK18mobrpsl进行同源重组，再转化到E.coli DH5α中。卡那霉素平板筛选，通过菌落PCR验证正确的转化子，即为pK18mobrpsL-cg1895::Ptuf-eddeda质粒。
(2)pK18mobrpsL-cg1895::Ptuf-eddeda质粒电击转化
将构建好的质粒pK18mobrpsL-cg1895::Ptuf-eddeda提取出来电转化到 Vb5-1感受态中，经过一轮筛选和二轮筛选以及菌落PCR验证，得到菌株 Vb5-2。其中，菌落PCR验证发生一轮交换菌株时所使用的引物为 cg1895-jd-1，cg1895-down-2，只有鉴定出条带大小为4048bp则为正确；菌落PCR验证发生二轮交换菌株时所使用的引物为cg1895-jd-1， cg1895-jd-2，若鉴定出条带大小为4289bp则为正确的菌株，若鉴定出条带大小为1808bp则是回复为原菌。
(3)正确菌株测序验证
将菌落验证正确的菌株提基因组，扩增cg1895::Ptuf-eddeda部分，测序正确得到菌株VB5-2。
实施例3：VB5-3菌株的构建
(1)pXMJ19sacB-cg1995crRNA-cg1995::Ptuf-ilvB*N*C*质粒的构建
以C.glutamicum ATCC 13032基因组为模板,引物cg1995-up-1和 cg1995-up-2来扩增上游同源臂，引物cg1995-down-1和cg1995-down-2来扩增下游同源臂。以pXtuf质粒为模板，引物cg1995-Ptuf-1和cg1995-Ptuf-2来扩增Ptuf启动子。以实验室高产L-缬氨酸诱变菌C. glutamicum XV基因组基因组为模板，引物ilvBNC-1和ilvBNC-2来扩增 XVilvB*N*C*基因(SEQ ID NO.5所示序列)，具体引物设计见表1。然后通过重叠PCR，利用引物cg1995-up-1和cg1995-down-2扩增得到重叠片段cg1995::Ptuf-ilvB*N*C*，经琼脂糖凝胶电泳验证后进行纯化回收。载体 pXMJ19sacB-crRNA(SEQ ID NO.10所示序列)选取XbaⅠ和EcoRⅠ两个酶切位点进行双酶切回收。将重叠片段与线性化载体pXMJ19sacB-crRNA进行同源重组，再转化到E.coliDH5α中。氯霉素平板筛选，通过菌落PCR验证正确的转化子，即为pXMJ19sacB-crRNA-cg1995::Ptuf-ilvB*N*C*质粒。
(2)pEC-XK99E△perI-Cpf1-RecET质粒电击转化
将pEC-XK99E△perI-Cpf1-RecET质粒(SEQ ID NO.11所示序列)电击转化入VB5-2菌株中。硫酸壮观霉素平板筛选，通过菌落PCR验证正确的转化子，即为VB5-2/pEC-XK99E△perI-Cpf1-RecET菌株。
(3)pXMJ19sacB-crRNA-cg1995::Ptuf-ilvB*N*C*质粒电击转化
将构建好的pXMJ19sacB-crRNA-cg1995::Ptuf-ilvB*N*C*质粒电击转化入VB5-2/pEC-XK99E△perI-Cpf1-RecET感受态中，感受态制备过程中 OD600＝0.8-1.0时，需添加终浓度1mM的茶碱，最后在硫酸壮观霉素、氯霉素、异丙基硫代半乳糖苷混合平板筛选，通过菌落PCR验证，若鉴定出条带大小为6082bp则为正确的菌株，若鉴定出条带大小为2400bp则为原菌。
(4)正确菌株测序验证
将菌落验证正确的菌株提基因组，扩增cg1995::Ptuf-ilvB*N*C*部分，测序正确得到菌株Vb5-3。
(5)pXMJ19sacB-crRNA-cg1995::Ptuf-ilvB*N*C*质粒消除
挑取单菌落接种到含2.2mM蔗糖的BHI试管，32℃培养过夜，次日菌液涂于硫酸壮观霉素平板，32℃培养24h，挑取单菌落对点于氯霉素、硫酸壮观霉素平板，在硫酸壮观霉素平板生长，不能在氯霉素平板生长的单菌落，说明pXMJ19sacB-crRNA-cg1995::Ptuf-ilvB*N*C*质粒消除成功。消除质粒的菌株制备感受态用于下一轮基因编辑。
(6)pEC-XK99E△perI-Cpf1-RecET质粒消除
将pXMJ19sacB-crRNA-cg1995::Ptuf-ilvB*N*C*质粒消除成功的单菌落接入无抗的BHI试管，32℃培养过夜，次日菌液涂于无抗平板，32℃培养24h，挑取单菌落对点于硫酸壮观霉素、无抗平板。在无抗平板生长，不能在硫酸壮观霉素平板生长的单菌落，说明pEC-XK99E△perI-Cpf1-RecET质粒消除成功。最终得到无质粒的VB5-3菌株。
实施例4：VB5-4菌株的构建
(1)pXMJ19sacB-avtAcrRNA质粒的构建和敲除模板△avtA的获得
以C.glutamicum ATCC 13032基因组为模板,引物avtA-up-1和 avtA-up-2来扩增上游同源臂，引物avtA-down-1和avtA-down-2来扩增下游同源臂。具体引物设计见表1。然后通过重叠PCR，利用引物avtA-up-1 和avtA-down-2扩增得到片段△avtA，经琼脂糖凝胶电泳验证后进行纯化回收备用。用反向互补引物crRNA-avtA-1和crRNA-avtA-2扩增crRNA-avtA，与线性化载体pXMJ19sacB-crRNA进行同源重组，再转化到E.coli DH5α中。氯霉素平板筛选，通过菌落PCR验证正确的转化子，即为 pXMJ19sacB-avtAcrRNA质粒。
(2)△avtA片段和pXMJ19sacB-avtAcrRNA质粒电击转化
将构建好的△avtA片段和pXMJ19sacB-avtAcrRNA质粒电击转化入 VB5-3/pEC-XK99E△perI-Cpf1-RecET感受态中，感受态制备过程中 OD600＝0.8-1.0时，需添加终浓度1mM的茶碱，最后在硫酸壮观霉素、氯霉素、异丙基硫代半乳糖苷混合平板筛选，通过菌落PCR验证，若鉴定出条带大小为1422bp则为正确的菌株，若鉴定出条带大小为2184bp则为原菌。
(3)pXMJ19sacB-avtAcrRNA质粒消除
同实施例3。消除质粒后用于下一轮基因编辑。
(4)pEC-XK99E△perI-Cpf1-RecET质粒消除
同实施例3。消除质粒后得到VB5-4菌株。
实施例5：VB5-5菌株的构建
(1)pXMJ19sacB-ilvEcrRNA质粒的构建和敲除模板△ilvE的获得
以C.glutamicum ATCC 13032基因组为模板,引物ilvE-up-1和 ilvE-up-2来扩增上游同源臂，引物ilvE-down-1和ilvE-down-2来扩增下游同源臂。具体引物设计见表1。然后通过重叠PCR，利用引物ilvE-up-1 和ilvE-down-2扩增得到重叠片段△ilvE，经琼脂糖凝胶电泳验证后进行纯化回收备用。用反向互补引物crRNA-ilvE-1和crRNA-ilvE-2扩增crRNA-ilvE，与线性化载体pXMJ19sacB-crRNA进行同源重组，再转化到 E.coli DH5α中。氯霉素平板筛选，通过菌落PCR验证正确的转化子，即为pXMJ19sacB-ilvEcrRNA质粒。
(2)△ilvE片段和pXMJ19sacB-ilvEcrRNA质粒电击转化
将构建好的△ilvE片段和pXMJ19sacB-ilvEcrRNA质粒电击转化入 VB5-4/pEC-XK99E△perI-Cpf1-RecET感受态中，感受态制备过程中 OD600＝0.8-1.0时，需添加终浓度1mM的茶碱，最后在硫酸壮观霉素、氯霉素、异丙基硫代半乳糖苷混合平板筛选，通过菌落PCR验证，若鉴定出条带大小为1460bp则为正确的菌株，若鉴定出条带大小为1963bp则为原菌。
(3)pXMJ19sacB-ilvEcrRNA质粒消除
同实施例3。
(4)pEC-XK99E△perI-Cpf1-RecET质粒消除
同实施例3，得到VB5-5菌株。
实施例6：VB5-6菌株的构建
(1)pK18mobrpsl-pyc*质粒的构建
以C.glutamicum ATCC 13032基因组为模板进行PCR扩增，设计引物 pyc-up-1和pyc-up-2来扩增上游同源臂，引物pyc-down-1和pyc-down-2 来扩增下游同源臂。具体引物设计见表1。然后通过重叠PCR，利用引物 pyc-up-1和pyc-down-2扩增得到重叠片段pyc*(SEQ ID NO.6所示序列)，经琼脂糖凝胶电泳验证后进行纯化回收。载体pK18mobrpsl选取XbaⅠ和Kpn Ⅰ两个酶切位点进行双酶切回收。将重叠片段与线性化载体pK18mobrpsl进行同源重组，再转化到E.coliDH5α中。卡那霉素平板筛选，通过菌落PCR 验证正确的转化子，即为pK18mobrpsL-pyc*质粒。
(2)质粒测序验证
将菌落验证正确的菌株提质粒，测序得到正确突变的pK18mobrpsL-pyc* 质粒。
(3)pK18mobrpsL-pyc*质粒电击转化
将测序正确的质粒pK18mobrpsL-pyc*提取出来电转化到Vb5-5感受态中，经过一轮筛选和二轮筛选以及菌落PCR验证，得到菌株Vb5-6。其中，菌落PCR验证发生一轮交换菌株时所使用的引物为pyc-jd-1，pyc-down-2，只有鉴定出条带大小为582bp则为正确；菌落PCR验证发生二轮交换菌株时所使用的引物为pyc-jd-1，pyc-jd-2，若鉴定出条带大小为622bp则为正确的菌株。
实施例7：VB5-7菌株的构建
(1)pXMJ19sacB-cg1960crRNA-cg1960::Ptuf-aspB质粒的构建
以C.glutamicum ATCC 13032基因组为模板,引物cg1960-up-1和 cg1960-up-2来扩增上游同源臂，引物cg1960-down-1和cg1960-down-2来扩增下游同源臂，引物aspB-1和aspB-2来扩增aspB基因。以pXtuf质粒为模板，引物cg1960-Ptuf-1和cg1960-Ptuf-2来扩增Ptuf启动子。引物aspB-1和aspB-2来扩增aspB基因(SEQ ID NO.7所示序列)，具体引物设计见表1。然后通过重叠PCR，利用引物cg1960-up-1和cg1960-down-2扩增得到重叠片段cg1960::Ptuf-aspB，经琼脂糖凝胶电泳验证后进行纯化回收。载体pXMJ19sacB-crRNA选取XbaⅠ和EcoRⅠ两个酶切位点进行双酶切回收。将重叠片段与线性化载体pXMJ19sacB-crRNA进行同源重组，再转化到E.coli DH5α中。氯霉素平板筛选，通过菌落PCR验证正确的转化子，即为pXMJ19sacB-cg1960crRNA-cg1960::Ptuf-aspB质粒。
(2)pXMJ19sacB-cg1960crRNA-cg1960::Ptuf-aspB质粒电击转化
将pEC-XK99E△perI-Cpf1-RecET质粒电击转化入VB5-6菌株中。硫酸壮观霉素平板筛选，通过菌落PCR验证正确的转化子，即为VB5-6/pEC-XK99E △perI-Cpf1-RecET菌株。
(3)pXMJ19sacB-cg1960crRNA-cg1960::Ptuf-aspB质粒电击转化
将构建好的pXMJ19sacB-cg1960crRNA-cg1960::Ptuf-aspB质粒电击转化入VB5-6/pEC-XK99E△perI-Cpf1-RecET感受态中，感受态制备过程中 OD600＝0.8-1.0时，需添加终浓度1mM的茶碱，最后在硫酸壮观霉素、氯霉素、异丙基硫代半乳糖苷混合平板筛选，通过菌落PCR验证，若鉴定出条带大小为2971bp则为正确的菌株，若鉴定出条带大小为2126bp则为原菌。
(4)正确菌株测序验证
将菌落验证正确的菌株提基因组，扩增cg1960::Ptuf-aspB部分，测序正确得到菌株VB5-7。
(5)VB5-6/pEC-XK99E△perI-Cpf1-RecET质粒消除
同实施例3。消除质粒后用于下一轮基因编辑。
(6)pEC-XK99E△perI-Cpf1-RecET质粒消除
同实施例3。消除质粒后得到VB5-7菌株。
实施例8：VB5-8菌株的构建
(1)pXMJ19sacB-Ncgl2850acrRNA-Ncgl2850a::Ptuf-aspAEco质粒的构建
以C.glutamicum ATCC 13032基因组为模板,引物Ncgl2850a-up-1和 Ncgl2850a-up-2来扩增上游同源臂，引物Ncgl2850a-down-1和 Ncgl2850a-down-2来扩增下游同源臂。以pXtuf质粒为模板，引物Ncgl2850a-Ptuf-1和Ncgl2850a-Ptuf-2来扩增Ptuf启动子。以Escherichia coli W3110基因组为模板,引物aspA-1和aspA-2来扩增aspAEco基因(SEQ IDNO.8所示序列)，具体引物设计见表1。然后通过重叠PCR，利用引物Ncgl2850a-up-1和Ncgl2850a-down-2扩增得到重叠片段Ncgl2850a:: Ptuf-aspAEco，经琼脂糖凝胶电泳验证后进行纯化回收。载体 pXMJ19sacB-crRNA选取XbaⅠ和EcoRⅠ两个酶切位点进行双酶切回收。将重叠片段与线性化载体pXMJ19sacB-crRNA进行同源重组，再转化到E.coli DH5α中。氯霉素平板筛选，通过菌落PCR验证正确的转化子，即为 pXMJ19sacB-Ncgl2850acrRNA-Ncgl2850a::Ptuf-aspAEco质粒。
(2)pXMJ19sacB-Ncgl2850acrRNA-Ncgl2850a::Ptuf-aspAEco质粒电击转化
将构建好的pXMJ19sacB-Ncgl2850acrRNA-Ncgl2850a::Ptuf-aspAEco质粒电击转化入VB5-7/pEC-XK99E-Cpf1-RecET感受态中，感受态制备过程中 OD600＝0.8-1.0时，需添加终浓度1mM的茶碱，最后在硫酸壮观霉素、氯霉素、异丙基硫代半乳糖苷混合平板筛选，通过菌落PCR验证，若鉴定出条带大小为3697bp则为正确的菌株，若鉴定出条带大小为2468bp则为原菌。
(3)正确菌株测序验证
将菌落验证正确的菌株提基因组，扩增Ncgl2850a::Ptuf-aspAEco部分，测序正确得到菌株VB5-8。
(4)pXMJ19sacB-Ncgl2850acrRNA-Ncgl2850a::Ptuf-aspAEco质粒消除
同实施例3。消除质粒后用于下一轮基因编辑。
(5)pEC-XK99E-Cpf1-RecET质粒消除
同实施例3，得到VB5-8菌株。
实施例9：VB5-9菌株的构建
(1)pXMJ19sacB-ilvAcrRNA质粒的构建和敲除模板△ilvA的获得
以C.glutamicum ATCC 13032基因组为模板,引物ilvA-up-1和 ilvA-up-2来扩增上游同源臂，引物ilvA-down-1和ilvA-down-2来扩增下游同源臂。具体引物设计见表1。然后通过重叠PCR，利用引物ilvA-up-1 和ilvA-down-2扩增得到重叠片段△ilvA，经琼脂糖凝胶电泳验证后进行纯化回收备用。用反向互补引物crRNA-ilvA-1和crRNA-ilvA-2扩增crRNA-ilvA，与线性化载体pXMJ19sacB-crRNA进行同源重组，再转化到 E.coli DH5α中。氯霉素平板筛选，通过菌落PCR验证正确的转化子，即为pXMJ19sacB-ilvAcrRNA质粒。
(2)△ilvA片段和pXMJ19sacB-ilvAcrRNA质粒电击转化
将构建好的△ilvA片段和pXMJ19sacB-ilvAcrRNA质粒电击转化入 VB5-8/pEC-XK99E-Cpf1-RecET感受态中，感受态制备过程中OD600＝0.8-1.0 时，需添加终浓度1mM的茶碱，最后在硫酸壮观霉素、氯霉素、异丙基硫代半乳糖苷混合平板筛选，通过菌落PCR验证，若鉴定出条带大小为1277bp 则为正确的菌株，若鉴定出条带大小为1962bp则为原菌。
(3)pXMJ19sacB-ilvAcrRNA质粒消除
同实施例3。
(4)pEC-XK99E-Cpf1-RecET质粒消除
同实施例3，得到VB5-9菌株。
实施例10：VB5-9/pXtuf-panBCDBsu菌株的构建
(1)pXtuf-panBCDBsu质粒的构建
以Bacillus subtilis 168基因组为模板，引物panBCD-1和panBCD-2 来扩增panBCD基因，具体引物设计见表1。经琼脂糖凝胶电泳验证后进行纯化回收。载体pXtuf，选用酶切位点Hind III和BamH I进行双酶切回收。将重叠片段与线性化载体pXtuf进行同源重组，再转化到E.coli DH5α中。氯霉素平板筛选，通过菌落PCR验证正确的转化子，即为所构建的质粒 pXtuf-panBCDBsu(SEQ ID NO.9所示序列)。
(2)质粒测序验证
将菌落验证正确的菌株提质粒，测序得到正确pXtuf-panBCDBsu质粒。
(3)pXtuf-panBCDBsu质粒电击转化
将测序正确的质粒pXtuf-panBCDBsu提取出来电转化到VB5-9感受态中，得到菌株VB5-9/pXtuf-panBCDBsu，记为VB5-10(D-泛酸生产菌)。菌落PCR 验证鉴定出条带大小为2331bp则为正确。
实施例11：菌株VB5-10的5-L发酵罐发酵
(1)菌体活化：用接种环从保菌管中取一环划线接种于斜面试管活化， 32℃恒温静置培养20-24h，即可得到一代活化斜面。然后从该斜面上用接种环将菌体接种于斜面茄形瓶中，32℃恒温静置培养12-24h，即可得到二代活化斜面；
(2)种子罐培养：取上一步活化较好的二代斜面茄子瓶，取适量无菌水于茄形瓶中，将菌悬液接入种子培养基中并最终定容至3L，通过流加氨水使pH稳定在7.0-7.2左右，温度恒定在32℃，控制溶氧在20-30％之间，培养菌体生长至OD600达到18左右；所述种子培养基组成如下：葡萄糖80 g/L，(NH4)2SO4 5g/L，KH2PO4 2.5g/L，MgSO4·7H2O 1.6g/L，FeSO4·7H2O 20 mg/L，MnSO4·H2O 20mg/L，VB1、VB3、VB12 0.3mg/L，蛋白胨2g/L，酵母粉3g/L，亮氨酸1g/L，异亮氨酸1g/L，L-缬氨酸1g/L，蛋氨酸1g/L，谷氨酸5g/L，丝肽粉5g/L，柠檬酸2g/L，玉米浆5g/L；
(3)发酵罐培养：按照10％的接种量接入新鲜的发酵培养基开始发酵，发酵过程中通过流加氨水控制pH稳定在7.0-7.2左右，温度维持在32℃，溶氧在20％之间；当培养基中的葡萄糖消耗完之后，流加80％(m/v)的葡萄糖溶液，维持发酵培养基中的葡萄糖浓度在0.1-5g/L；发酵周期一般在 40-48h；所述发酵培养基组成如下：葡萄糖80g/L，(NH4)2SO45g/L，KH2PO4 2.5g/L，MgSO4·7H2O 1.6g/L，FeSO4·7H2O 20mg/L，MnSO4·H2O 20mg/L， VB1、VB3、VB12 0.3mg/L，蛋白胨2g/L，酵母粉3g/L，亮氨酸1g/L，异亮氨酸1g/L，L-缬氨酸1g/L，蛋氨酸1g/L，谷氨酸5g/L，丝肽粉 5g/L，柠檬酸2g/L，玉米浆20g/L，豆浓10ml/L。
实施例12：D-泛酸含量的HPLC测定
色谱柱：C18柱(250×4.6mm，5um)
检测波长：200nm
柱温：30℃
流动相：水：乙腈：磷酸＝947：50：3混匀后，过滤，超声30min。
流速：0.9ml/L
出峰时间：10.4min
通过基因工程操作后，基因工程菌VB5-10通过5-L发酵罐发酵D-泛酸产量达到18.62g/L，产量稳定。本发明所构建的D-泛酸基因工程菌在发酵过程中能实现发酵液中D-泛酸的有效积累，降低了生产成本，具有较大的应用潜能。
表1实施例所用引物序列
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以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 天津科技大学
<120> 泛酸生产相关菌株及其构建方法与应用
<130> 2022
<160> 102
<170> SIPOSequenceListing 1.0
<210> 1
<211> 246
<212> DNA
<213> promoter
<220>
<221> promoter
<222> (1)..(246)
<400> 1
cgactgcacg gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc 60
tgtgcaggtc gtaaatcact gcataattcg tgtcgctcaa ggcgcactcc cgttctggat 120
aatgtttttt gcgccgacat cataacggtt ctggcaaata ttctgaaatg agctgttgac 180
aattaatcat ccggctcgta taatgtgtgg aattgtgagc ggataacaat ttcacacagg 240
aaacag 246
<210> 2
<211> 368
<212> DNA
<213> promoter
<220>
<221> promoter
<222> (1)..(368)
<400> 2
gttaacagat cgtttagatc cgaaggaaaa cgtcgaaaag caatttgctt ttcgacgccc 60
caccccgcgc gttttagcgt gtcagtaggc gcgtagggta agtggggtag cggcttgtta 120
gatatcttga aatcggcttt caacagcatt gatttcgatg tatttagctg gccgttaccc 180
tgcgaatgtc cacagggtag ctggtagttt gaaaatcaac gccgttgccc ttaggattca 240
gtaactggca cattttgtaa tgcgctagat ctgtgtgctc agtcttccag gctgcttatc 300
acagtgaaag caaaaccaat tcgtggctgc gaaagtcgta gccaccacga agtccaggag 360
gaaagctt 368
<210> 3
<211> 2052
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(2052)
<400> 3
atggcgtcca aactgacgac gacatcgcaa catattctgg aaaaccttgg tggaccagac 60
aatattactt cgatgactca ctgtgcgact cgccttcgct tccaagtgaa ggatcaatcc 120
attgttgatc aacaagaaat tgactccgac ccatcagttc ttggcgtagt accccaagga 180
tccaccggta tgcaggtggt gatgggtgga tctgttgcaa actattacca agaaatcctc 240
aaacttgatg gaatgaagca cttcgccgac ggtgaagcta cagagagttc atccaagaag 300
gaatacggcg gagtccgtgg caagtactcg tggattgact acgccttcga gttcttgtct 360
gatactttcc gaccaatcct gtgggccctg cttggtgcct cactgattat taccttgttg 420
gttcttgcgg atactttcgg tttgcaagac ttccgcgctc caatggatga gcagcctgat 480
acttatgtat tcctgcactc catgtggcgc tcggtcttct acttcctgcc aattatggtt 540
ggtgccaccg cagctcgaaa gctcggcgca aacgagtgga ttggtgcagc tattccagcc 600
gcacttctta ctccagaatt cttggcactg ggttctgccg gcgataccgt cacagtcttt 660
ggcctgccaa tggttctgaa tgactactcc ggacaggtat tcccaccgct gattgcagca 720
attggtctgt actgggtgga aaagggactg aagaagatca tccctgaagc agtccaaatg 780
gtgttcgtcc cattcttctc cctgctgatt atgatcccag cgaccgcatt cctgcttgga 840
cctttcggca tcggtgttgg taacggaatt tccaacctgc ttgaagcgat taacaacttc 900
agcccattta ttctttccat cgttatccca ttgctctacc cattcttggt tccacttgga 960
ttgcactggc cactaaacgc catcatgatc cagaacatca acaccctggg ttacgacttc 1020
attcagggac caatgggtgc ctggaacttc gcctgcttcg gcctggtcac cggcgtgttc 1080
ttgctctcca ttaaggaacg aaacaaggcc atgcgtcagg tttccctggg tggcatgttg 1140
gctggtttgc tcggcggcat ttccgagcct tccctctacg gtgttctgct ccgattcaag 1200
aagacctact tccgcctcct gccgggttgt ttggcaggcg gtatcgtgat gggcatcttc 1260
gacatcaagg cgtacgcttt cgtgttcacc tccttgctta ccatcccagc aatggaccca 1320
tggttgggct acaccattgg tatcgcagtt gcattcttcg tttccatgtt ccttgttctc 1380
gcactggact accgttccaa cgaagagcgc gatgaggcac gtgcaaaggt tgctgctgac 1440
aagcaggcag aagaagatct gaaggcagaa gctaatgcaa ctcctgcagc tccagtagct 1500
gctgcaggtg cgggagccgg tgcaggtgca ggagccgctg ctggcgctgc aaccgccgtg 1560
gcagctaagc cgaagctggc cgctggggaa gtagtggaca ttgtttcccc actcgaaggc 1620
aaggcaattc cactttctga agtacctgac ccaatctttg cagcaggcaa gcttggacca 1680
ggcattgcaa tccaaccaac tggaaacacc gttgttgctc cagcagacgc tactgtcatc 1740
cttgtccaga aatctggaca cgcagtggca ttgcgcttag atagcggagt tgaaatcctt 1800
gtccacgttg gattggacac cgtgcaattg ggcggcgaag gcttcaccgt tcacgttgag 1860
cgcaggcagc aagtcaaggc gggggatcca ctgatcactt ttgacgctga cttcattcga 1920
tccaaggatc tacctttgat caccccagtt gtggtgtcta acgccgcgaa attcggtgaa 1980
attgaaggta ttcctgcaga tcaggcaaat tcttccacga ctgtgatcaa ggtcaacggc 2040
aagaacgagt aa 2052
<210> 4
<211> 2421
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(2421)
<400> 4
atgacccact ccgttattct caatgtaacc gaacgtctta tcgaacgaag ccgtgaagct 60
cgcgccgaat ttttagcaca tactcgtatt cagtcggaag cgggtaaagg tcgtaccggt 120
ttatcctgtg gaaacctagc gcatgcggtg gctgcgtcat gttcttccga gaaagaaaat 180
attctcaact ttacacagtc taatatcgcg ctcatcagtg cctacaatga catgttgagt 240
gcccaccagc cttaccaaga atatccagcg caaattaaac aagtactggc gcaatatggg 300
cacaccgctc aagtagcagg ctgcgtaccc gccatgtgcg acggcgtgac tcaaggtcag 360
cctggcatgg atatgtcgct gttttctcgt gacttaattg ctcaatcgac ggcgttatct 420
ttaagccata atgtctttga cgcgaccctg ctgcttggta tctgcgacaa aatcgcacca 480
ggacaactga tgggggcgct ctcttacgct cacctgccaa cggcctttgt acctgctggt 540
ttaatggcaa ccggcatcag caacgaagaa aaagtcgatg tacgccaaaa atacgcggct 600
ggcgaagtag gcaaagaagc actgctggac atggagtgcc gtgcttatca ctctcctggc 660
acctgcactt tctacggcac cgcaaatacc aaccaactgg tctttgaagc catgggccta 720
atgctaccgg gctctgcgtt tatccacccg catagtgaac tgcgtaaagc actaacagac 780
catgcggcac tgaagatagc gtccatgact gcaggctcca gcaatttccg tcctctggct 840
gaagttgtaa cggaaaagag tttaatcaac ggtatcattg cgctgctggc atctggcggc 900
agtactaacc acaccattca tatggtcgcc gtcgctcgcg cagcaggcat cttgctcacc 960
tggcaagaca tcagtgactt gtccgaagtg gtgcctctgc tggcgcgtgt ttatccaaac 1020
ggtccggcgg atatgaatgc atttcaggcc gctggcggcg ttccagcctt gttgcatcga 1080
ctgaatgaat ccgagctact gcaccgcgat gtaaaaccgg ttttcggaga gttctctgat 1140
cagatgacga ttccatcttt gaacaatggc cagttggtat ggacagcctg tcagcgaagc 1200
ctagatggcg atgtcatcgc tgccccggaa gcagtattcc aacacactgg tggcacacgt 1260
gttttagatg gcaatttagg taaagcggtc gtgaaagtct cggcagtaaa agaagaacag 1320
cgcattatcg aagcacctgc ggtcgttttc cagtgccagc atgaagtgga agcggcatac 1380
aaacgaagtg agctcaacaa agactgcatt gtcgttgtaa cgcacaacgg tccagctgcg 1440
aacggcatgc cagagctgca taagttgatg ccaatattag gtaatgtgca aaaaatgggc 1500
tttaaagttg cgttggtaac cgatggccgt ttgtcaggcg catccggcaa aattccatcc 1560
gcgatccatg tatcacctga agcgattcgc ggtggtgcaa taggtttagt ccgtgatggt 1620
gatgttattc gcgtggattg ccaaacgggt gagctgaaca acttaaccga caccagtgga 1680
cgtgaagtca ttcagcttga tactgaatca acccagaaaa catggggacg tggatttttc 1740
gaagtgatcc gccacaacgt ttccagtgca gatcagggcg cgagctttat tgtttagagg 1800
ttacccatga caactcttga acaacgatta aaagaaatca aaatcgtccc agttatcgct 1860
attaacgatg tcgctcacgc actgccactg gcgaaagtgt tagtagaaaa cggcctgcca 1920
tgtgccgaag tgactttccg taccgaagcg gcggcagaat caattcgtat tatgcgcgaa 1980
gcgtatccag acctgctaat tggtgcaggc acagtactga cgaccgagca ggttgatctt 2040
gctattgatg cgggtgcgga ctttatcgtt agcccgggct ttaacccaac gacagtgaaa 2100
tactgtcagc agcgtaatat cgcgattgta ccgggtgtga acaacccaag cttggttgag 2160
caagcgatgg aaatgggcct gcgtacactg aagtttttcc cggcagaacc atcgggtggc 2220
gtgaacatgc taaaagcact gactgcggtt tacccggtaa actttatgcc aacgggtggc 2280
gttagccctg ctaacgtaga agattacctt gcacttaagt cggttatcgc atgtggcggt 2340
acctggatgg taccaacgaa actgatggat gatggcgact gggaaggctt ggctgaattg 2400
gttcgcgcgg ttaattcata a 2421
<210> 5
<211> 3610
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(3610)
<400> 5
atgaatgtgg cagcttctca acagcccact cccgccacgg ttgcaagccg tggtcgatcc 60
gccgcccctg agcggatgac aggtgcacag gcaattgttc gatcgctcga ggagcttaac 120
gccgacatcg tgttcggtat tcctggtggt gcggtgctac cggtgtatga cccgctctat 180
tcctccacaa aggtgcgcca cgtcctagtg cgccacgagc agggcgcagg ccacgcagca 240
accggctacg cgcaggttac tggacgcgtt ggcgtctgca ttgcaacctc tggcccaggc 300
gcaaccaact tggttacccc aatcgctgat gcaaacttgg actccgttcc catggttgcc 360
atcaccggcc aggtcggaag tagcctgctg ggtaccgatg ctttccagga agtcgatatc 420
cgcggcatca ccatgccagt gaccaagcac aacttcatgg tcaccaaccc caacgacatt 480
ccacaggcat tggctgaggc attccacctc gcgattactg gtcgccctgg tcctgttcta 540
gtggatatcc ccaaggatgt tcagaacgct gaattggatt tcgtctggcc accaaagatc 600
gacctgccag gctaccgccc agtttcaaca ccgcatgctc gacagattga gcaggctgtc 660
aaactgatcg gtgagtctaa gaagcctgtc ctttacgttg gcggcggcgt tatcaaggct 720
gatgcccacg aagagcttcg tgcgttcgct gagcacaccg gcattccagt tgtcaccaca 780
ttgatggcgc tgggaacctt cccagagtcc cacgagctgc acatgggtat gccaggcatg 840
catggcactg tgtccgctgt tggtgcactg cagcgcagcg acctgctgat tgctatcggc 900
tcccgctttg atgaccgcgt caccggtgac gttgacactt tcgcacctga tgccaagatc 960
attcacgccg acattgatcc tgccgaaatc ggcaagatca agcaggttga ggttccaatc 1020
gtgggcgatg cccgcgaggt tcttgctcgt ctgctcgaaa ccaccaaggc aagcaaggca 1080
gagtctgagg acatctccga gtgggttgac tacctcaagg gcctcaaggc acgtttccca 1140
cgtggctacg acgagcagcc aggcgatctg ctggcaccac agtttgtcat tgaaaccctg 1200
tccaaggaag ttggccccga cgcaatttac tgcgccggcg ttggccagca ccagatgtgg 1260
gcagctcagt tcgttgactt cgaaaagcca cgcacctggc tcaactccgg tggactgggc 1320
accatgggct acgcagttcc tgcggctctt ggagcaaagg ctggcgcacc tgacaaggaa 1380
gtctgggcta tcgacggcga cggctgtttc cagatgacca accaggaact caccaccgcc 1440
gcagttgaag gtttccccat taagatcgca ctaatcaaca acggaaacct gggtatggtt 1500
cgccaatggc agaccctatt ctatgaagga cggtactcaa atactaaact tcgtaaccag 1560
ggcgagtaca tgcccgactt tgttaccctt tctgagggac ttggctgtgt tgccatccgc 1620
gtcaccaaag cggaggaagt actgccagcc atccaaaagg ctcgagagat caacgaccgc 1680
ccagtagtca tcgacttcat cgtcggtgaa gacgcacagg tatggccaat ggtgtctgct 1740
ggatcatcca actccgatat ccagtacgca ctcggattgc gcccattctt tgatggtgat 1800
gaatctgcag cagaagatcc tgccgacatt cacgaagccg tcagcgacat tgatgccgcc 1860
gttgaatcga ccgaggcata aggagagacc caagatggct aattctgacg tcacccgcca 1920
catcctgtcc gtactcgttc aggacgtaga cggaatcatt tcccgcgtat caggtatgtt 1980
cacccgacgc gcattcaacc tcgtgtccct cgtgtctgca aagaccgaaa cactcggcat 2040
caaccgcatc acggttgttg tcgacgccga cgagctcaac attgagcaga tcaccaagca 2100
gctcaacaag ctgatccccg tgctcaaagt cgtgcgactt gatgaagaga ccactatcgc 2160
ccgcgcaatc atgctggtta aggtttctgc ggacagcacc aaccgtccgc agatcgtcga 2220
cgccgcgaac atcttccgcg cccgagtcgt cgacgtggct ccagactctg tggttattga 2280
atccacaggc accccaggca agctccgcgc actgcttgac gtgatggaac cattcggaat 2340
ccgcgaactg atccaatccg gacagattgc actcaaccgc ggtccgaaga ccatggctcc 2400
ggccaagatc taaacagcaa ttaatctgat tgcacctgct gcataaatgt gactagtcaa 2460
acaccgtcta attacatgtg tgtggtagaa caataatgta gttgtctgcc caagcgagtt 2520
aaactcccac gatttacagt ggggggcaga catcttttca ccaaaatttt tacgaaaggc 2580
gagattttct cccatggcta ttgaactgct ttatgatgct gacgctgacc tctccttgat 2640
ccagggccgt aaggttgcca tcgttggcta cggctcccag ggccacgcac actcccagaa 2700
cctccgcgat tctggcgttg aggttgtcat tggtctgcgc gagggctcca agtccgcaga 2760
gaaggcaaag gaagcaggct tcgaggtcaa gaccaccgct gaggctgcag cttgggctga 2820
cgtcatcatg ctcctggctc cagacacctc ccaggcagaa atcttcacca acgacatcga 2880
gccaaacctg aacgcaggcg acgcactgct gttcggccac ggcctgaaca ttcacttcga 2940
cctgatcaag ccagctgacg acatcatcgt tggcatggtt gcgccaaagg gcccaggcca 3000
cttggttcgc cgtcagttcg ttgatggcaa gggtgttcct tgcctcatcg cagtcgacca 3060
ggacccaacc ggaaccgcac aggctctgac cctgtcctac gcagcagcaa tcggtggcgc 3120
acgcgcaggc gttatcccaa ccaccttcga agctgagacc gtcaccgacc tcttcggcga 3180
gcaggctgtt ctctgcggtg gcaccgagga actggtcaag gttggcttcg aggttctcac 3240
cgaagctggc tacgagccag agatggcata cttcgaggtt cttcacgagc tcaagctcat 3300
cgttgacctc atgttcgaag gtggcatcag caacatgaac tactctgttt ctgacaccgc 3360
tgagttcggt ggctacctct ccggcccacg cgtcatcgat gcagacacca agtcccgcat 3420
gaaggacatc ctgaccgata tccaggacgg caccttcacc aagcgcctca tcgcaaacgt 3480
tgagaacggc aacaccgagc ttgagggcct tcgtgcttcc tacaacaacc acccaatcga 3540
ggagaccggc gctaagctcc gcgacctcat gagctgggtc aaggttgacg ctcgcgcaga 3600
aaccgcttaa 3610
<210> 6
<211> 3423
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(3423)
<400> 6
gtgtcgactc acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 60
ggcgaaatcg cggtccgtgc tttccgtgca gcactcgaaa ccggtgcagc cacggtagct 120
atttaccccc gtgaagatcg gggatcattc caccgctctt ttgcttctga agctgtccgc 180
attggtaccg aaggctcacc agtcaaggcg tacctggaca tcgatgaaat tatcggtgca 240
gctaaaaaag ttaaagcaga tgccatttac ccgggatacg gcttcctgtc tgaaaatgcc 300
cagcttgccc gcgagtgtgc ggaaaacggc attactttta ttggcccaac cccagaggtt 360
cttgatctca ccggtgataa gtctcgcgcg gtaaccgccg cgaagaaggc tggtctgcca 420
gttttggcgg aatccacccc gagcaaaaac atcgatgaga tcgttaaaag cgctgaaggc 480
cagacttacc ccatctttgt gaaggcagtt gccggtggtg gcggacgcgg tatgcgtttt 540
gttgcttcac ctgatgagct tcgcaaatta gcaacagaag catctcgtga agctgaagcg 600
gctttcggcg atggcgcggt atatgtcgaa cgtgctgtga ttaaccctca gcatattgaa 660
gtgcagatcc ttggcgatca cactggagaa gttgtacacc tttatgaacg tgactgctca 720
ctgcagcgtc gtcaccaaaa agttgtcgaa attgcgccag cacagcattt ggatccagaa 780
ctgcgtgatc gcatttgtgc ggatgcagta aagttctgcc gctccattgg ttaccagggc 840
gcgggaaccg tggaattctt ggtcgatgaa aagggcaacc acgtcttcat cgaaatgaac 900
ccacgtatcc aggttgagca caccgtgact gaagaagtca ccgaggtgga cctggtgaag 960
gcgcagatgc gcttggctgc tggtgcaacc ttgaaggaat tgggtctgac ccaagataag 1020
atcaagaccc acggtgcagc actgcagtgc cgcatcacca cggaagatcc aaacaacggc 1080
ttccgcccag ataccggaac tatcaccgcg taccgctcac caggcggagc tggcgttcgt 1140
cttgacggtg cagctcagct cggtggcgaa atcaccgcac actttgactc catgctggtg 1200
aaaatgacct gccgtggttc cgactttgaa actgctgttg ctcgtgcaca gcgcgcgttg 1260
gctgagttca ccgtgtctgg tgttgcaacc aacattggtt tcttgcgtgc gttgctgcgg 1320
gaagaggact tcacttccaa gcgcatcgcc accggattca ttgccgatca ctcgcacctc 1380
cttcaggctc cacctgctga tgatgagcag ggacgcatcc tggattactt ggcagatgtc 1440
accgtgaaca agcctcatgg tgtgcgtcca aaggatgttg cagctcctat cgataagctg 1500
cctaacatca aggatctgcc actgccacgc ggttcccgtg accgcctgaa gcagcttggc 1560
ccagccgcgt ttgctcgtga tctccgtgag caggacgcac tggcagttac tgataccacc 1620
ttccgcgatg cacaccagtc tttgcttgcg acccgagtcc gctcattcgc actgaagcct 1680
gcggcagagg ccgtcgcaaa gctgactcct gagcttttgt ccgtggaggc ctggggcggc 1740
gcgacctacg atgtggcgat gcgtttcctc tttgaggatc cgtgggacag gctcgacgag 1800
ctgcgcgagg cgatgccgaa tgtaaacatt cagatgctgc ttcgcggccg caacaccgtg 1860
ggatacaccc cgtacccaga ctccgtctgc cgcgcgtttg ttaaggaagc tgccagctcc 1920
ggcgtggaca tcttccgcat cttcgacgcg cttaacgacg tctcccagat gcgtccagca 1980
atcgacgcag tcctggagac caacaccgcg gtagccgagg tggctatggc ttattctggt 2040
gatctctctg atccaaatga aaagctctac accctggatt actacctaaa gatggcagag 2100
gagatcgtca agtctggcgc tcacatcttg gccattaagg atatggctgg tctgcttcgc 2160
ccagctgcgg taaccaagct ggtcaccgca ctgcgccgtg aattcgatct gccagtgcac 2220
gtgcacaccc acgacactgc gggtggccag ctggcaacct actttgctgc agctcaagct 2280
ggtgcagatg ctgttgacgg tgcttccgca ccactgtctg gcaccacctc ccagccatcc 2340
ctgtctgcca ttgttgctgc attcgcgcac acccgtcgcg ataccggttt gagcctcgag 2400
gctgtttctg acctcgagcc gtactgggaa gcagtgcgcg gactgtacct gccatttgag 2460
tctggaaccc caggcccaac cggtcgcgtc taccgccacg aaatcccagg cggacagttg 2520
tccaacctgc gtgcacaggc caccgcactg ggccttgcgg atcgtttcga actcatcgaa 2580
gacaactacg cagccgttaa tgagatgctg ggacgcccaa ccaaggtcac cccatcctcc 2640
aaggttgttg gcgacctcgc actccacctc gttggtgcgg gtgtggatcc agcagacttt 2700
gctgccgatc cacaaaagta cgacatccca gactctgtca tcgcgttcct gcgcggcgag 2760
cttggtaacc ctccaggtgg ctggccagag ccactgcgca cccgcgcact ggaaggccgc 2820
tccgaaggca aggcacctct gacggaagtt cctgaggaag agcaggcgca cctcgacgct 2880
gatgattcca aggaacgtcg caatagcctc aaccgcctgc tgttcccgaa gccaaccgaa 2940
gagttcctcg agcaccgtcg ccgcttcggc aacacctctg cgctggatga tcgtgaattc 3000
ttctacggcc tggtcgaagg ccgcgagact ttgatccgcc tgccagatgt gcgcacccca 3060
ctgcttgttc gcctggatgc gatctctgag ccagacgata agggtatgcg caatgttgtg 3120
gccaacgtca acggccagat ccgcccaatg cgtgtgcgtg accgctccgt tgagtctgtc 3180
accgcaaccg cagaaaaggc agattcctcc aacaagggcc atgttgctgc accattcgct 3240
ggtgttgtca ccgtgactgt tgctgaaggt gatgaggtca aggctggaga tgcagtcgca 3300
atcatcgagg ctatgaagat ggaagcaaca atcactgctt ctgttgacgg caaaatcgat 3360
cgcgttgtgg ttcctgctgc aacgaaggtg gaaggtggcg acttgatcgt cgtcgtttcc 3420
taa 3423
<210> 7
<211> 804
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(804)
<400> 7
atgctgaaca ttgtgatgat cggatgtgga gcaattggtg cgggagttct ggagcttctt 60
gaaaacgacc ctcagcttcg tgttgatgca gtaatcgtcc cgcgcgactc tgaaacacag 120
gtacgccatc gtttggcatc cctgcgtcgt ccacctcgcg tattatcggc cttgcctgcg 180
ggtgagcgcc cggacctgtt agtggagtgt gcaggacatc gcgctattga gcaacacgtc 240
ctgcctgcat tggctcaggg catcccctgc ctggtggtgt ctgtgggtgc gttatcggaa 300
ccgggattgg tagaacgttt agaagctgcg gcccaagctg gaggcagccg cattgaatta 360
ctgcccggtg caatcggagc aattgatgca ctgagtgccg cacgtgttgg gggattggaa 420
tccgtgcgtt acactggtcg caaacccgct tcggcatggc ttggcacgcc tggggaaacg 480
gtgtgcgact tacagcgttt ggaaaaagca cgcgttatct ttgacggcag cgcacgcgaa 540
gccgcccgct tatatcctaa aaatgccaac gtggcggcaa ccctttcttt agccggactt 600
gggcttgatc gcacacaagt acgcttaatt gcggaccccg agtcctgtga gaacgtacac 660
caggtggagg cttcaggcgc ttttggcggg ttcgagctta ccttgcgtgg caaaccatta 720
gcagcgaacc cgaaaacgtc ggctttaaca gtgtactcag tggtccgtgc tttaggaaat 780
catgcccatg ccatttcaat ctaa 804
<210> 8
<211> 1437
<212> DNA
<213> gene
<220>
<221> gene
<222> (1)..(1437)
<400> 8
atgtcaaaca acattcgtat cgaagaagat ctgttgggta ccagggaagt tccagctgat 60
gcctactatg gtgttcacac tctgagagcg attgaaaact tctatatcag caacaacaaa 120
atcagtgata ttcctgaatt tgttcgcggt atggtaatgg ttaaaaaagc cgcagctatg 180
gcaaacaaag agctgcaaac cattcctaaa agtgtagcga atgccatcat tgccgcatgt 240
gatgaagtcc tgaacaacgg aaaatgcatg gatcagttcc cggtagacgt ctaccagggc 300
ggcgcaggta cttccgtaaa catgaacacc aacgaagtgc tggccaatat cggtctggaa 360
ctgatgggtc accaaaaagg tgaatatcag tacctgaacc cgaacgacca tgttaacaaa 420
tgtcagtcca ctaacgacgc ctacccgacc ggtttccgta tcgcagttta ctcttccctg 480
attaagctgg tagatgcgat taaccaactg cgtgaaggct ttgaacgtaa agctgtcgaa 540
ttccaggaca tcctgaaaat gggtcgtacc cagctgcagg acgcagtacc gatgaccctc 600
ggtcaggaat tccgcgcttt cagcatcctg ctgaaagaag aagtgaaaaa catccaacgt 660
accgctgaac tgctgctgga agttaacctt ggtgcaacag caatcggtac tggtctgaac 720
acgccgaaag agtactctcc gctggcagtg aaaaaactgg ctgaagttac tggcttccca 780
tgcgtaccgg ctgaagacct gatcgaagcg acctctgact gcggcgctta tgttatggtt 840
cacggcgcgc tgaaacgcct ggctgtgaag atgtccaaaa tctgtaacga cctgcgcttg 900
ctctcttcag gcccacgtgc cggcctgaac gagatcaacc tgccggaact gcaggcgggc 960
tcttccatca tgccagctaa agtaaacccg gttgttccgg aagtggttaa ccaggtatgc 1020
ttcaaagtca tcggtaacga caccactgtt accatggcag cagaagcagg tcagctgcag 1080
ttgaacgtta tggagccggt cattggccag gccatgttcg aatccgttca cattctgacc 1140
aacgcttgct acaacctgct ggaaaaatgc attaacggca tcactgctaa caaagaagtg 1200
tgcgaaggtt acgtttacaa ctctatcggt atcgttactt acctgaaccc gttcatcggt 1260
caccacaacg gtgacatcgt gggtaaaatc tgtgccgaaa ccggtaagag tgtacgtgaa 1320
gtcgttctgg aacgcggtct gttgactgaa gcggaacttg acgatatttt ctccgtacag 1380
aatctgatgc acccggctta caaagcaaaa cgctatactg atgaaagcga acagtaa 1437
<210> 9
<211> 7775
<212> DNA
<213> plasmid
<220>
<221> misc_feature
<222> (1)..(7775)
<400> 9
gttaacagat cgtttagatc cgaaggaaaa cgtcgaaaag caatttgctt ttcgacgccc 60
caccccgcgc gttttagcgt gtcagtaggc gcgtagggta agtggggtag cggcttgtta 120
gatatcttga aatcggcttt caacagcatt gatttcgatg tatttagctg gccgttaccc 180
tgcgaatgtc cacagggtag ctggtagttt gaaaatcaac gccgttgccc ttaggattca 240
gtaactggca cattttgtaa tgcgctagat ctgtgtgctc agtcttccag gctgcttatc 300
acagtgaaag caaaaccaat tcgtggctgc gaaagtcgta gccaccacga agtccaggag 360
gaaagcttat gaaaacaaaa ctggattttc taaaaatgaa ggagtctgaa gaaccgattg 420
tcatgctgac cgcttatgat tatccggcag ctaaacttgc tgaacaagcg ggagttgaca 480
tgattttagt cggtgattca cttggaatgg tcgtcctcgg ccttgattca actgtcggtg 540
tgacagttgc ggacatgatc catcatacaa aagccgttaa aaggggtgcg ccgaatacct 600
ttattgtgac agatatgccg tttatgtctt atcacctgtc taaggaagat acgctgaaaa 660
atgcagcggc tatcgttcag gaaagcggag ctgacgcact gaagcttgag ggcggagaag 720
gcgtgtttga atccattcgc gcattgacgc ttggaggcat tccagtagtc agtcacttag 780
gtttgacacc gcagtcagtc ggcgtactgg gcggctataa agtacagggc aaagacgaac 840
aaagcgccaa aaaattaata gaagacagta taaaatgcga agaagcagga gctatgatgc 900
ttgtgctgga atgtgtgccg gcagaactca cagccaaaat tgccgagacg ctaagcatac 960
cggtcattgg aatcggggct ggtgtgaaag cggacggaca agttctcgtt tatcatgata 1020
ttatcggcca cggtgttgag agaacaccta aatttgtaaa gcaatatacg cgcattgatg 1080
aaaccatcga aacagcaatc agcggatatg ttcaggatgt aagacatcgt gctttccctg 1140
aacaaaagca ttcctttcaa atgaaccaga cagtgcttga cggcttgtac gggggaaaat 1200
aagatgagac agattactga tatttcacag ctgaaagaag ccataaaaca ataccattca 1260
gagggcaagt caatcggatt tgttccgacg atggggtttc tgcatgaggg gcatttaacc 1320
ttagcagaca aagcaagaca agaaaacgac gccgttatta tgagtatttt tgtgaatcct 1380
gcacaattcg gccctaatga agattttgaa gcatatccgc gcgatattga gcgggatgca 1440
gctcttgcag aaaacgccgg agtcgatatt ctttttacgc cagatgctca tgatatgtat 1500
cccggtgaaa agaatgtcac gattcatgta gaaagacgca cagacgtgtt atgcgggcgc 1560
tcaagagaag gacattttga cggggtcgcg atcgtactga cgaagctttt caatctagtc 1620
aagccgactc gtgcctattt cggtttaaaa gatgcgcagc aggtagctgt tgttgatggg 1680
ttaatcagcg acttcttcat ggatattgaa ttggttcctg tcgatacggt cagagaggaa 1740
gacggcttag ccaaaagctc tcgcaatgta tacttaacag ctgaggaaag aaaagaagcg 1800
cctaagctgt atcgggccct tcaaacaagt gcggaacttg tccaagccgg tgaaagagat 1860
cctgaagcgg tgataaaagc tgcaaaagat atcattgaaa cgactagcgg aaccatagac 1920
tatgtagagc tttattccta tccggaactc gagcctgtga atgaaattgc tggaaagatg 1980
attctcgctg ttgcagttgc tttttcaaaa gcgcgtttaa tagataatat cattattgat 2040
attcgagaaa tggagagaat ataatatgta tcgaacaatg atgagcggca aacttcacag 2100
ggcaactgtt acggaagcaa acctgaacta tgtgggaagc attacaattg atgaagatct 2160
cattgatgct gtgggaatgc ttcctaatga aaaagtacaa attgtgaata ataataatgg 2220
agcacgtctt gaaacgtata ttattcctgg taaacgggga agcggcgtca tatgcttaaa 2280
cggtgcagcc gcacgccttg tgcaggaagg agataaggtc attattattt cctacaaaat 2340
gatgtctgat caagaagcgg caagccatga gccgaaagtg gctgttctga atgatcaaaa 2400
caaaattgaa caaatgctgg ggaacgaacc agcccgtaca attttgtagg gatccccggg 2460
taccgagctc gaattcagct tggctgtttt ggcggatgag agaagatttt cagcctgata 2520
cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc 2580
gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt 2640
agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc 2700
tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag 2760
taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg 2820
ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga 2880
tggccttttt gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt 2940
atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 3000
tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 3060
tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 3120
gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 3180
aagaacgttt tccaatgatg agcacttttg cttcctcgct cactgactcg ctgcgctcgg 3240
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 3300
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 3360
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 3420
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 3480
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 3540
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 3600
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 3660
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 3720
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 3780
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 3840
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 3900
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 3960
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 4020
aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 4080
ttttggggtg ggcgaagaac tccagcatga gatccccgcg ctggaggatc atccagccat 4140
tcggggtcgt tcactggttc ccctttctga tttctggcat agaagaaccc ccgtgaactg 4200
tgtggttccg ggggttgctg atttttgcga gacttctcgc gcaattccct agcttaggtg 4260
aaaacaccat gaaacactag ggaaacaccc atgaaacacc cattagggca gtagggcggc 4320
ttcttcgtct agggcttgca tttgggcggt gatctggtct ttagcgtgtg aaagtgtgtc 4380
gtaggtggcg tgctcaatgc actcgaacgt cacgtcattt accgggtcac ggtgggcaaa 4440
gagaactagt gggttagaca ttgttttcct cgttgtcggt ggtggtgagc ttttctagcc 4500
gctcggtaaa cgcggcgatc atgaactctt ggaggttttc accgttctgc atgcctgcgc 4560
gcttcatgtc ctcacgtagt gccaaaggaa cgcgtgcggt gaccacgacg ggcttagcct 4620
ttgcctgcgc ttctagtgct tcgatggtgg cttgtgcctg cgcttgctgc gcctgtagtg 4680
cctgttgagc ttcttgtagt tgctgttcta gctgtgcctt ggttgccatg ctttaagact 4740
ctagtagctt tcctgcgata tgtcatgcgc atgcgtagca aacattgtcc tgcaactcat 4800
tcattatgtg cagtgctcct gttactagtc gtacatactc atatttacct agtctgcatg 4860
cagtgcatgc acatgcagtc atgtcgtgct aatgtgtaaa acatgtacat gcagattgct 4920
gggggtgcag ggggcggagc caccctgtcc atgcggggtg tggggcttgc cccgccggta 4980
cagacagtga gcaccggggc acctagtcgc ggataccccc cctaggtatc ggacacgtaa 5040
ccctcccatg tcgatgcaaa tctttaacat tgagtacggg taagctggca cgcatagcca 5100
agctaggcgg ccaccaaaca ccactaaaaa ttaatagtcc ctagacaaga caaacccccg 5160
tgcgagctac caactcatat gcacgggggc cacataaccc gaaggggttt caattgacaa 5220
ccatagcact agctaagaca acgggcacaa cacccgcaca aactcgcact gcgcaacccc 5280
gcacaacatc gggtctaggt aacactgagt aacactgaaa tagaagtgaa cacctctaag 5340
gaaccgcagg tcaatgaggg ttctaaggtc actcgcgcta gggcgtggcg taggcaaaac 5400
gtcatgtaca agatcaccaa tagtaaggct ctggcggggt gccataggtg gcgcagggac 5460
gaagctgttg cggtgtcctg gtcgtctaac ggtgcttcgc agtttgaggg tctgcaaaac 5520
tctcactctc gctgggggtc acctctggct gaattggaag tcatgggcga acgccgcatt 5580
gagctggcta ttgctactaa gaatcacttg gcggcgggtg gcgcgctcat gatgtttgtg 5640
ggcactgttc gacacaaccg ctcacagtca tttgcgcagg ttgaagcggg tattaagact 5700
gcgtactctt cgatggtgaa aacatctcag tggaagaaag aacgtgcacg gtacggggtg 5760
gagcacacct atagtgacta tgaggtcaca gactcttggg cgaacggttg gcacttgcac 5820
cgcaacatgc tgttgttctt ggatcgtcca ctgtctgacg atgaactcaa ggcgtttgag 5880
gattccatgt tttcccgctg gtctgctggt gtggttaagg ccggtatgga cgcgccactg 5940
cgtgagcacg gggtcaaact tgatcaggtg tctacctggg gtggagacgc tgcgaaaatg 6000
gcaacctacc tcgctaaggg catgtctcag gaactgactg gctccgctac taaaaccgcg 6060
tctaaggggt cgtacacgcc gtttcagatg ttggatatgt tggccgatca aagcgacgcc 6120
ggcgaggata tggacgctgt tttggtggct cggtggcgtg agtatgaggt tggttctaaa 6180
aacctgcgtt cgtcctggtc acgtggggct aagcgtgctt tgggcattga ttacatagac 6240
gctgatgtac gtcgtgaaat ggaagaagaa ctgtacaagc tcgccggtct ggaagcaccg 6300
gaacgggtcg aatcaacccg cgttgctgtt gctttggtga agcccgatga ttggaaactg 6360
attcagtctg atttcgcggt taggcagtac gttctcgatt gcgtggataa ggctaaggac 6420
gtggccgctg cgcaacgtgt cgctaatgag gtgctggcaa gtctgggtgt ggattccacc 6480
ccgtgcatga tcgttatgga tgatgtggac ttggacgcgg ttctgcctac tcatggggac 6540
gctactaagc gtgatctgaa tgcggcggtg ttcgcgggta atgagcagac tattcttcgc 6600
acccactaaa agcggcataa accccgttcg atattttgtg cgatgaattt atggtcaatg 6660
tcgcgggggc aaactatgat gggtcttgtt gttggcgtcc cggaaaacga ttccgaagcc 6720
caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc 6780
ttggtcggtc atttcgaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc 6840
cactcatcgc agtactgttg taattcatta agcattctgc cgacatggaa gccatcacag 6900
acggcatgat gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat 6960
ttgcccatgg tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa 7020
ctggtgaaac tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta 7080
gggaaatagg ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac 7140
tgccggaaat cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg 7200
aaaacggtgt aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc 7260
atacggaact ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa 7320
aacttgtgct tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc 7380
tggttatagg tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat 7440
tgggatatat caacggtggt atatccagtg atttttttct ccattttagc ttccttagct 7500
cctgaaaatc tcgtcgaagc tcggcggatt tgtcctactc aagctgatcc gacaaaatcc 7560
acacattatc ccaggtgtcc ggatcggtca aatacgctgc cagctcatag accgtatcca 7620
aagcatccgg ggctgatccc cggcgccagg gtggtttttc ttttcaccag tgagacgggc 7680
aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgtgg 7740
tttgccccag caggcgaaaa tcctgtttga tggtg 7775
<210> 10
<211> 7395
<212> DNA
<213> plasmid
<220>
<221> misc_feature
<222> (1)..(7395)
<400> 10
aacgtaaatg ccgcttcgcc ttcgcgcgcg aattgcaagc tgatccgggc ttatcgactg 60
cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta tggctgtgca 120
ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt 180
ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgg aattccttat 240
cggtaccttg acagctagct cagtcctagg tataatcccg gggaatttct actgttgtag 300
attctagagg ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag 360
aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac 420
ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc 480
cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac 540
tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg 600
ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc aggacgcccg 660
ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg cctttttgcg 720
tttctacaaa ctcttttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 780
acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 840
tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 900
agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat 960
cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 1020
aatgatgagc acttttgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 1080
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 1140
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 1200
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 1260
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 1320
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 1380
cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta 1440
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 1500
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 1560
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 1620
gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 1680
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 1740
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 1800
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 1860
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt tggggtgggc 1920
gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccattcg gggtcgttca 1980
ctggttcccc tttctgattt ctggcataga agaacccccg tgaactgtgt ggttccgggg 2040
gttgctgatt tttgcgagac ttctcgcgca attccctagc ttaggtgaaa acaccatgaa 2100
acactaggga aacacccatg aaacacccat tagggcagta gggcggcttc ttcgtctagg 2160
gcttgcattt gggcggtgat ctggtcttta gcgtgtgaaa gtgtgtcgta ggtggcgtgc 2220
tcaatgcact cgaacgtcac gtcatttacc gggtcacggt gggcaaagag aactagtggg 2280
ttagacattg ttttcctcgt tgtcggtggt ggtgagcttt tctagccgct cggtaaacgc 2340
ggcgatcatg aactcttgga ggttttcacc gttctgcatg cctgcgcgct tcatgtcctc 2400
acgtagtgcc aaaggaacgc gtgcggtgac cacgacgggc ttagcctttg cctgcgcttc 2460
tagtgcttcg atggtggctt gtgcctgcgc ttgctgcgcc tgtagtgcct gttgagcttc 2520
ttgtagttgc tgttctagct gtgccttggt tgccatgctt taagactcta gtagctttcc 2580
tgcgatatgt catgcgcatg cgtagcaaac attgtcctgc aactcattca ttatgtgcag 2640
tgctcctgtt actagtcgta catactcata tttacctagt ctgcatgcag tgcatgcaca 2700
tgcagtcatg tcgtgctaat gtgtaaaaca tgtacatgca gattgctggg ggtgcagggg 2760
gcggagccac cctgtccatg cggggtgtgg ggcttgcccc gccggtacag acagtgagca 2820
ccggggcacc tagtcgcgga taccccccct aggtatcgga cacgtaaccc tcccatgtcg 2880
atgcaaatct ttaacattga gtacgggtaa gctggcacgc atagccaagc taggcggcca 2940
ccaaacacca ctaaaaatta atagtcccta gacaagacaa acccccgtgc gagctaccaa 3000
ctcatatgca cgggggccac ataacccgaa ggggtttcaa ttgacaacca tagcactagc 3060
taagacaacg ggcacaacac ccgcacaaac tcgcactgcg caaccccgca caacatcggg 3120
tctaggtaac actgagtaac actgaaatag aagtgaacac ctctaaggaa ccgcaggtca 3180
atgagggttc taaggtcact cgcgctaggg cgtggcgtag gcaaaacgtc atgtacaaga 3240
tcaccaatag taaggctctg gcggggtgcc ataggtggcg cagggacgaa gctgttgcgg 3300
tgtcctggtc gtctaacggt gcttcgcagt ttgagggtct gcaaaactct cactctcgct 3360
gggggtcacc tctggctgaa ttggaagtca tgggcgaacg ccgcattgag ctggctattg 3420
ctactaagaa tcacttggcg gcgggtggcg cgctcatgat gtttgtgggc actgttcgac 3480
acaaccgctc acagtcattt gcgcaggttg aagcgggtat taagactgcg tactcttcga 3540
tggtgaaaac atctcagtgg aagaaagaac gtgcacggta cggggtggag cacacctata 3600
gtgactatga ggtcacagac tcttgggcga acggttggca cttgcaccgc aacatgctgt 3660
tgttcttgga tcgtccactg tctgacgatg aactcaaggc gtttgaggat tccatgtttt 3720
cccgctggtc tgctggtgtg gttaaggccg gtatggacgc gccactgcgt gagcacgggg 3780
tcaaacttga tcaggtgtct acctggggtg gagacgctgc gaaaatggca acctacctcg 3840
ctaagggcat gtctcaggaa ctgactggct ccgctactaa aaccgcgtct aaggggtcgt 3900
acacgccgtt tcagatgttg gatatgttgg ccgatcaaag cgacgccggc gaggatatgg 3960
acgctgtttt ggtggctcgg tggcgtgagt atgaggttgg ttctaaaaac ctgcgttcgt 4020
cctggtcacg tggggctaag cgtgctttgg gcattgatta catagacgct gatgtacgtc 4080
gtgaaatgga agaagaactg tacaagctcg ccggtctgga agcaccggaa cgggtcgaat 4140
caacccgcgt tgctgttgct ttggtgaagc ccgatgattg gaaactgatt cagtctgatt 4200
tcgcggttag gcagtacgtt ctcgattgcg tggataaggc taaggacgtg gccgctgcgc 4260
aacgtgtcgc taatgaggtg ctggcaagtc tgggtgtgga ttccaccccg tgcatgatcg 4320
ttatggatga tgtggacttg gacgcggttc tgcctactca tggggacgct actaagcgtg 4380
atctgaatgc ggcggtgttc gcgggtaatg agcagactat tcttcgcacc cactaaaagc 4440
ggcataaacc ccgttcgata ttttgtgcga tgaatttatg gtcaatgtcg cgggggcaaa 4500
ctatgatggg tcttgttgtt ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag 4560
aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt 4620
tcgaagggca ccaataactg ccttaaaaaa attacgcccc gccctgccac tcatcgcagt 4680
actgttgtaa ttcattaagc attctgccga catggaagcc atcacagacg gcatgatgaa 4740
cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga 4800
aaacgggggc gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca 4860
cccagggatt ggctgagacg aaaaacatat tctcaataaa ccctttaggg aaataggcca 4920
ggttttcacc gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt 4980
cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac 5040
aagggtgaac actatcccat atcaccagct caccgtcttt cattgccata cggaactccg 5100
gatgagcatt catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat 5160
ttttctttac ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac 5220
attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa 5280
cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg 5340
tcgaagctcg gcggatttgt cctactcaag ctgatccgac aaaatccaca cattatccca 5400
ggtgtccgga tcggtcaaat acgctgccag ctcatagacc gtatccaaag catccggggc 5460
tgatccccgg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 5520
cctttatttg ttaactgtta attgtccttg ttcaaggatg ctgtctttga caacagatgt 5580
tttcttgcct ttgatgttca gcaggaagct cggcgcaaac gttgattgtt tgtctgcgta 5640
gaatcctctg tttgtcatat agcttgtaat cacgacattg tttcctttcg cttgaggtac 5700
agcgaagtgt gagtaagtaa aggttacatc gttaggatca agatccattt ttaacacaag 5760
gccagttttg ttcagcggct tgtatgggcc agttaaagaa ttagaaacat aaccaagcat 5820
gtaaatatcg ttagacgtaa tgccgtcaat cgtcattttt gatccgcggg agtcagtgaa 5880
caggtaccat ttgccgttca ttttaaagac gttcgcgcgt tcaatttcat ctgttactgt 5940
gttagatgca atcagcggtt tcatcacttt tttcagtgtg taatcatcgt ttagctcaat 6000
cataccgaga gcgccgtttg ctaactcagc cgtgcgtttt ttatcgcttt gcagaagttt 6060
ttgactttct tgacggaaga atgatgtgct tttgccatag tatgctttgt taaataaaga 6120
ttcttcgcct tggtagccat cttcagttcc agtgtttgct tcaaatacta agtatttgsa 6180
cbtggccttt atcttctacg tagtgaggat ctctcagcgt atggttgtcg cctgagctgt 6240
agttgccttc atcgatgaac tgctgtacat tttgatacgt ttttccgtca ccgtcaaaga 6300
ttgatttata atcctctaca ccgttgatgt tcaaagagct gtctgatgct gatacgttaa 6360
cttgtgcagt tgtcagtgtt tgtttgccgt aatgtttacc ggagaaatca gtgtagaata 6420
aacggatttt tccgtcagat gtaaatgtgg ctgaacctga ccattcttgt gtttggtctt 6480
ttaggataga atcatttgca tcgaatttgt cgctgtcttt aaagacgcgg ccagcgtttt 6540
tccagctgtc aatagaagtt tcgccgactt tttgatagaa catgtaaatc gatgtgtcat 6600
ccgcattttt aggatctccg gctaatgcaa agacgatgtg gtagccgtga tagtttgcga 6660
cagtgccgtc agcgttttgt aatggccagc tgtcccaaac gtccaggcct tttgcagaag 6720
agatattttt aattgtggac gaatcaaatt cagaaacttg atatttttca tttttttgct 6780
gttcagggat ttgcagcata tcatggcgtg taatatggga aatgccgtat gtttccttat 6840
atggcttttg gttcgtttct ttcgcaaacg cttgagttgc gcctcctgcc agcagtgcgg 6900
tagtaaaggt taatactgtt gcttgttttg caaacttttt gatgttcatc gttcatgtct 6960
ccttttttat gtactgtgtt agcggtctgc ttcttccagc cctcctgttt gaagatggca 7020
agttagttac gcacaataaa aaaagaccta aaatatgtaa ggggtgacgc caaagtatac 7080
actttgccct ttacacattt taggtcttgc ctgctttatc agtaacaaac ccgcgcgatt 7140
tacttttcga cctcattcta ttagactctc gtttggattg caactggtct attttcctct 7200
tttgtttgat agaaaatcat aaaaggattt gcagactacg ggcctaaaga actaaaaaat 7260
ctatctgttt cttttcattc tctgtatttt ttatagtttc tgttgcatgg gcataaagtt 7320
gcctttttaa tcacaattca gaaaatatca taatatctca tttcactaaa taatagtgaa 7380
cggcaggtat atgtg 7395
<210> 11
<211> 14725
<212> DNA
<213> plasmid
<220>
<221> misc_feature
<222> (1)..(14725)
<400> 11
gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt 60
tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg 120
gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag 180
tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc 240
gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa 300
cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt 360
gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc 420
actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt 480
ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag 540
caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc 600
tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg 660
agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact 720
gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc 780
gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca 840
tgttatatcc cgccgtcaac caccatcaaa caggattttc gcctgctggg gcaaaccagc 900
gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc 960
gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc 1020
gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 1080
tgagcgcaac gcaattaatg tgagttagcg cgaattgatc tggtttgaca gcttatcatc 1140
gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct 1200
gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata 1260
atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca 1320
attaatcatc cggctcgtat aatgtgtgga attgtgagcg gataacaatt tcacacagga 1380
aacagaccat gagcggataa caatttcaca caggaaacag accatggaat tcatgtccat 1440
ctaccaagag tttgtgaata aatactccct gtccaagacc ctccgttttg agctgatccc 1500
ccaaggcaag accctcgaaa acatcaaggc acgcggcctc atcctggatg acgaaaagcg 1560
cgctaaggat tacaagaagg caaagcagat catcgacaag taccaccagt tcttcatcga 1620
agagatcctg tcctccgtgt gcatctccga ggacctgctc cagaactact ccgatgtcta 1680
cttcaagctc aagaagtccg atgacgataa cctgcagaag gacttcaagt ccgctaagga 1740
taccatcaag aagcagatct ccgaatacat caaggattcc gagaagttca agaacctctt 1800
caaccagaac ctgatcgacg caaagaaggg ccaggaatcc gatctcatcc tgtggctcaa 1860
gcagtccaag gataacggca tcgagctctt caaggccaac tccgacatca ccgacatcga 1920
tgaagctctg gagatcatca agtccttcaa gggctggacc acctacttca agggcttcca 1980
cgaaaaccgc aagaacgtgt actcctccaa cgatatccca acctctatca tctaccgcat 2040
cgtcgacgat aacctgccaa agttcctcga aaacaaggca aagtacgagt ccctgaagga 2100
taaggcccca gaagctatca actacgagca gatcaagaag gacctggccg aagagctcac 2160
cttcgacatc gattacaaga cctctgaagt gaaccagcgc gtcttctccc tcgatgaagt 2220
gttcgagatc gccaacttca acaactacct gaaccagtcc ggcatcacca agttcaacac 2280
catcatcggc ggcaagttcg tcaacggcga aaacaccaag cgcaagggca tcaacgagta 2340
catcaacctc tactcccagc agatcaacga taagaccctg aagaagtaca agatgtccgt 2400
gctcttcaag cagatcctgt ccgacaccga atccaagtcc ttcgtcatcg acaagctgga 2460
ggacgattcc gatgtggtca ccaccatgca gtccttctac gaacagatcg cagccttcaa 2520
gaccgtggaa gagaagtcca tcaaggagac cctctccctg ctcttcgacg atctgaaggc 2580
tcagaagctg gatctctcca agatctactt caagaacgac aagtccctga ccgatctctc 2640
ccagcaggtc ttcgacgatt actccgtgat cggcaccgca gtcctggaat acatcaccca 2700
gcagatcgcc ccaaagaacc tcgataaccc atccaagaag gaacaggagc tgatcgccaa 2760
gaagaccgaa aaggctaagt acctgtccct cgagaccatc aagctggctc tcgaagagtt 2820
caacaagcac cgcgacatcg ataagcagtg ccgcttcgaa gagatcctcg caaacttcgc 2880
tgcaatccca atgatcttcg acgaaatcgc acagaacaag gataacctgg cccagatctc 2940
catcaagtac cagaaccagg gcaagaagga tctgctccag gcctccgctg aggacgatgt 3000
gaaggcaatc aaggacctgc tcgatcagac caacaacctg ctccacaagc tgaagatctt 3060
ccacatctcc cagtccgaag acaaggccaa catcctcgac aaggatgagc acttctacct 3120
ggtgttcgaa gagtgctact tcgaactcgc taacatcgtc ccactgtaca acaagatccg 3180
caactacatc acccagaagc catactccga tgaaaagttc aagctcaact tcgagaactc 3240
caccctggca aacggctggg acaagaacaa ggaaccagat aacaccgcca tcctcttcat 3300
caaggacgat aagtactacc tgggcgtgat gaacaagaag aacaacaaga tcttcgacga 3360
taaggccatc aaggaaaaca agggcgaggg ctacaagaag atcgtgtaca agctgctccc 3420
aggcgctaac aagatgctcc caaaggtctt cttctccgca aagtccatca agttctacaa 3480
cccatccgaa gatatcctgc gcatccgcaa ccactccacc cacaccaaga acggctcccc 3540
acagaagggc tacgaaaagt tcgagttcaa catcgaagac tgccgcaagt tcatcgattt 3600
ctacaagcag tccatctcca agcacccaga gtggaaggac ttcggcttcc gcttctccga 3660
tacccagcgc tacaactcca tcgatgaatt ctaccgcgaa gtggagaacc agggctacaa 3720
gctgaccttc gaaaacatct ccgagtccta catcgattcc gtggtcaacc agggcaagct 3780
gtacctcttc cagatctaca acaaggactt ctccgcttac tccaagggcc gcccaaacct 3840
gcacaccctc tactggaagg cactcttcga cgaacgcaac ctgcaggatg tggtctacaa 3900
gctcaacggc gaagcagagc tgttctaccg caagcagtcc atcccaaaga agatcaccca 3960
cccagccaag gaagcaatcg ccaacaagaa caaggataac ccaaagaagg aatccgtgtt 4020
cgagtacgac ctgatcaagg ataagcgctt caccgaggac aagttcttct tccactgccc 4080
aatcaccatc aacttcaagt cctccggcgc caacaagttc aacgatgaaa tcaacctgct 4140
cctgaaggag aaggctaacg acgtgcacat cctgtccatc gatcgcggcg aacgccacct 4200
cgcctactac accctggtcg acggcaaggg caacatcatc aagcaggaca ccttcaacat 4260
catcggcaac gatcgcatga agaccaacta ccacgacaag ctggccgcta tcgagaagga 4320
ccgcgattcc gctcgcaagg attggaagaa gatcaacaac atcaaggaaa tgaaggaagg 4380
ctacctctcc caggtggtcc acgaaatcgc taagctggtg atcgagtaca acgcaatcgt 4440
ggtcttcgaa gacctgaact tcggcttcaa gcgcggccgc ttcaaggtgg agaagcaggt 4500
ctaccagaag ctggaaaaga tgctcatcga gaagctgaac tacctcgtgt tcaaggacaa 4560
cgaattcgat aagaccggcg gcgtcctccg tgcataccag ctgaccgccc cattcgagac 4620
cttcaagaag atgggcaagc agaccggcat catctactac gtgccagctg gcttcacctc 4680
taagatctgc ccagtgaccg gcttcgtcaa ccagctctac ccaaagtacg aatccgtctc 4740
caagtcccag gagttcttct ccaagttcga caagatctgc tacaacctgg ataagggcta 4800
cttcgaattc tccttcgact acaagaactt cggcgataag gcagccaagg gcaagtggac 4860
catcgcatcc ttcggctccc gcctcatcaa cttccgcaac tccgacaaga accacaactg 4920
ggatacccgc gaagtgtacc caaccaagga actggagaag ctcctgaagg attactccat 4980
cgaatacggc cacggcgagt gcatcaaggc tgcaatctgc ggcgaatccg acaagaagtt 5040
cttcgcaaag ctgacctctg tgctcaacac catcctgcag atgcgcaact ccaagaccgg 5100
caccgagctg gattacctca tctccccagt ggccgacgtc aacggcaact tcttcgattc 5160
ccgccaggct ccaaagaaca tgccacagga cgctgatgca aacggcgcct accacatcgg 5220
tctgaagggt ctcatgctcc tgggtcgcat caagaacaac caggaaggca agaagctgaa 5280
tctcgtcatt aagaacgaag aatactttga atttgtccag aaccgcaata actaaggtac 5340
ctgtaaggcc tgcaccaaca atgattgagc gaagctccaa aatgtcctcc ccgggttgat 5400
attagatttc ataaatatac taaaaatctt gagagttttt ccgttgaaaa ctaaaaagct 5460
gggaaggtga atcgaatttc ggggctttaa agcaaaaatg aacagcttgg tctatagtgg 5520
ctaggtaccc tttttgtttt ggacacatgt agggtggccg aaacaaagta ataggacaac 5580
aacgctcgac cgcgattatt tttggagaat catgagcaca aaaccactct tcctgttacg 5640
gaaagcgaaa aaatcatccg gtgaacctga cgtcgtcctg tgggcaagca acgattttga 5700
atcgacctgt gccactctgg actacctgat cgttaagtca ggtaaaaaac tgagcagcta 5760
ttttaaagct gttgccacga attttcctgt cgttaatgac ctgcccgctg aaggtgagat 5820
cgattttacc tggagtgaac gctatcaact cagcaaagac tccatgacat gggaactaaa 5880
accgggagca gcaccagaca acgctcacta tcaaggcaat accaacgtca acggcgaaga 5940
catgactgag attgaggaga atatgctact cccaatttct ggccaggaac tgcccattcg 6000
ttggcttgct caacacggca gcgaaaaacc ggtaacgcac gtttcacgcg acggactcca 6060
ggcattacac attgctcggg ctgaagaact accggctgtt actgccctgg ctgtttccca 6120
caaaaccagc ctgctcgacc cgctggaaat tcgcgaactc cacaaactgg ttcgtgacac 6180
tgacaaagtt ttccctaatc ctggtaattc aaacctggga ctgataactg cttttttcga 6240
agcatacctg aacgctgact acaccgatcg aggactgctg acaaaagagt ggatgaaggg 6300
taatcgtgtt tcacacatca ctcgcacggc ttccggtgct aatgctggcg gcggaaacct 6360
caccgatcgc ggcgaaggtt tcgtacacga tctgacgtca ctggcgcgcg acgtagccac 6420
tggcgtactg gcccgttcaa tggatctgga catctataac cttcatccgg cacacgctaa 6480
acgcattgag gaaattatcg ctgaaaataa accgcccttt tctgttttcc gcgacaaatt 6540
catcaccatg cctggcgggc tggattattc ccgcgccatc gtggttgcgt ccgtaaaaga 6600
agcaccaatt gggatcgagg tcatccccgc gcacgtcact gaatatctga acaaagtact 6660
gactgaaacc gatcatgcca accctgatcc ggaaatcgtg gatattgcct gcggtcgctc 6720
ctctgccccg atgccgcagc gagtaacaga agaaggaaaa caggatgatg aagaaaaacc 6780
gcaaccatct ggaacaacgg cagttgaaca gggagaggct gaaacaatgg aaccggacgc 6840
aactgaacat catcaggaca cgcagccgct ggatgctcag tcacaggtaa attctgttga 6900
tgcgaaatat caggaactgc gggcagaact ccatgaagcc cggaaaaaca ttccatcaaa 6960
aaatcctgtc gatgacgata aattgcttgc tgcatcacgt ggtgaatttg ttgacggaat 7020
tagcgacccg aacgatccga aatgggtaaa ggggatccag actcgcgatt gtgtgtacca 7080
gaaccagcca gaaacggaaa aaaccagccc agatatgaat caacctgagc cagtagtgca 7140
acaggaaccg gaaatagcct gcaatgcctg cggccagact ggcggggata actgccctga 7200
ctgtggtgcg gtgatgggcg acgcaacata ccaggaaaca ttcgatgaag agagtcaggt 7260
tgaagctaag gaaaatgatc cggaggaaat ggaaggcgct gaacatccgc acaatgagaa 7320
tgctggcagc gatccgcatc gcgattgcag tgatgaaact ggcgaagtcg cagatcccgt 7380
aatcgtagaa gacatagagc caggtattta ttacggaatt tcgaatgaga attaccacgc 7440
gggtcccggt atcagtaagt ctcagctcga tgacattgct gatactccgg cactatattt 7500
gtggcgtaaa aatgcccccg tggacaccac aaagacaaaa acgctcgatt taggaactgc 7560
tttccactgc cgggtacttg aaccggaaga attcagtaac cgctttatcg tagcacctga 7620
atttaaccgc cgtacaaacg ccggaaaaga agaagagaaa gcgtttctga tggaatgcgc 7680
aagcacagga aaaacggtta tcactgcgga agaaggccgg aaaattgaac tcatgtatca 7740
aagcgttatg gctttgccgc tggggcaatg gcttgttgaa agcgccggac acgctgaatc 7800
atcaatttac tgggaagatc ctgaaacagg aattttgtgt cggtgccgtc cggacaaaat 7860
tatccctgaa tttcactgga tcatggacgt gaaaactacg gcggatattc aacgattcaa 7920
aaccgcttat tacgactacc gctatcacgt tcaggatgca ttctacagtg acggttatga 7980
agcacagttt ggagtgcagc caactttcgt ttttctggtt gccagcacaa ctattgaatg 8040
cggacgttat ccggttgaaa ttttcatgat gggcgaagaa gcaaaactgg caggtcaaca 8100
ggaatatcac cgcaatctgc gaaccctgtc tgactgcctg aataccgatg aatggccagc 8160
tattaagaca ttatcactgc cccgctgggc taaggaatat gcaaatgact aagcaaccac 8220
caatcgcaaa agccgatctg caaaaaactc agggaaaccg tgcaccagca gcagttaaaa 8280
atagcgacgt gattagtttt attaaccagc catcaatgaa agagcaactg gcagcagctc 8340
ttccacgcca tatgacggct gaacgtatga tccgtatcgc caccacagaa attcgtaaag 8400
ttccggcgtt aggaaactgt gacactatga gttttgtcag tgcgatcgta cagtgttcac 8460
agctcggact tgagccaggt agcgccctcg gtcatgcata tttactgcct tttggtaata 8520
aaaacgaaaa gagcggtaaa aagaacgttc agctaatcat tggctatcgc ggcatgattg 8580
atctggctcg ccgttctggt caaatcgcca gcctgtcagc ccgtgttgtc cgtgaaggtg 8640
acgagtttag cttcgaattt ggccttgatg aaaagttaat acaccgcccg ggagaaaacg 8700
aagatgcccc ggttacccac gtctatgctg tcgcaagact gaaagacgga ggtactcagt 8760
ttgaagttat gacgcgcaaa cagattgagc tggtgcgcag cctgagtaaa gctggtaata 8820
acgggccgtg ggtaactcac tgggaagaaa tggcaaagaa aacggctatt cgtcgcctgt 8880
tcaaatattt gcccgtatca attgagatcc agcgtgcagt atcaatggat gaaaaggaac 8940
cactgacaat cgatcctgca gattcctctg tattaaccgg ggaatacagt gtaatcgata 9000
attcagagga ataatctaga gtcgacctgc aggcatgcaa gcttggctgt tttggcggat 9060
gagagaagat tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac 9120
agaatttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag 9180
tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc 9240
aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt 9300
ttgtcggtga acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg 9360
aagcaacggc ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt 9420
aagcagaagg ccatcctgac ggatggcctt tttgcgtttc tacaaactct ttttgtttat 9480
ttttctaaat acattcaaat atgtatccgc tcatgaatta attccgctag atgacgtgcg 9540
gcttcgacct cctgggcgtg gcgcttgttg gcgcgctcgc ggctggctgc ggcacgacac 9600
gcgtctgagc agtattttgc gcgccgtcct cgtgggtcag gccggggtgg gatcaggcca 9660
ccgcagtagg cgcagctgat gcgatcctcc accgctgatg cttcaggcca gttttggtac 9720
ttcgtcgtga aggtcatgac accattataa cgaacgttcg ttaaaaattc tagccccaat 9780
tctgataatt tcttccggca ctcctgcgaa aacctgcgag acttcttgcc cagaaaaaac 9840
gccaagcgca gcggttaccg cacttttttt ccaggtgatt tcaccctgac cagcgaagcg 9900
gcactttagt gcatgaggtg tgcccctggt ttcccctctt tggagggttc aacccaaaaa 9960
agcacacaag caaaaatgaa aatcatcatg agcaagttgg tgcgaagcag caacgcgcta 10020
gctccaaaaa ggtctccagg atctcgagga gatttttgag ggggagggag tcgaggaaga 10080
gccagagcag aaggcgggga accgttctct gccgacagcg tgagcccccc ttaaaaatca 10140
ggccggggag gaaccgggga gggatcagag ctaggagcga gacaccctaa agggggggaa 10200
ccgttttctg ctgacggtgt ttcgtttatt agttttcagc ccgtggatag cggagggtga 10260
gggcaagtga gagccagagc aaggacggga cccctaaagg ggggaaccgt tttctgctga 10320
cggtgtttcg tttattagtt ttcagcccgt ggacggccgc gtttagcttc cattccaagt 10380
gcctttctga cttgttggat gcgcctttca ctgacaccta gttcgcctgc aagctcacga 10440
gtcgagggat cagcaaccga ttgagaacgg gcatccagga tcgcagtttt gacgcgaagt 10500
tcgagcaact cgcctgtcat ttctcggcgt ttgtttgctt ccgctaatcg ctgtcgcgtc 10560
tcctgcgcat acttactttc tgggtcagcc catctgcgtg cattcgatgt agctgcgccc 10620
cgtcgcccca tcgtcgctag agctttccgc cctcggctgc tctgcgtttc cacccgacga 10680
gcagggacga ctggctggcc tttagccacg tagccgcgca cacgacgcgc catcgtcagg 10740
cgatcacgca tggcgggaag atccggctcc cggccgtctg caccgaccgc ctgggcaacg 10800
ttgtacgcca cttcatacgc gtcgatgatc ttggcatctt ttaggcgctc accagcagct 10860
ttgagctggt atcccacggt caacgcgtgg cgaaacgcgg tctcgtcgcg cgctcgctct 10920
ggatttgtcc agagcactcg cacgccgtcg atcaggtcgc cggacgcgtc cagggcgctc 10980
ggcaggctcg cgtccaaaat cgctagcgcc ttggcttctg cggtggcgcg ttgtgccgct 11040
tcaatgcggg cgcgtccgct ggaaaagtcc tgctcaatgt actttttcgg cttctgtgat 11100
ccggtcatcg ttcgagcaat ctccattagg tcggccagcc gatccacacg atcatgctgg 11160
cagtgccatt tataggctgt cggatcgtct gagacgtgca gcggccaccg gctcagccta 11220
tgcgaaaaag cctggtcagc gccgaaaaca cgagtcattt cttccgtcgt tgcagccagc 11280
aggcgcatat ttgggctggt tttacctgct gcggcataca ccgggtcaat gagccagatg 11340
agctggcatt tcccgctcag cggattcacg ccgatccaag ccggcgcttt ttctaggcgt 11400
gcccatttct ctaaaatcgc gtagacctgc gggtttacgt gctcaatctt cccgccggcc 11460
tggtggctgg gcacatcgat gtcaagcacg atcaccgcgg catgttgcgc gtgcgtcagc 11520
gcaacgtact ggcaccgcgt cagcgctttt gagccagccc ggtagagctt tggttgggtt 11580
tcgccggtat ccgggttttt aatccaggcg ctcgcgaaat ctcttgtctt gctgccctgg 11640
aagctttcgc gtcccaggtg agcgagcagt tcgcggcgat cttctgccgt ccagccgcgt 11700
gagccgcagc gcatagcttc ggggtgggtg tcgaacagat cggcggacaa tttccacgcg 11760
ctagctgtga ctgtgtcctg cggatcggct agagtcatgt cttgagtgct ttctcccagc 11820
tgatgactgg gggttagccg acgccctgtg agttcccgct cacggggcgt tcaacttttt 11880
caggtatttg tgcagcttat cgtgttttct tcgtaaatga acgcttaact accttgttaa 11940
acgtggcaaa taggcaggat tgatggggat ctagcttcac gctgccgcaa gcactcaggg 12000
cgcaagggct gctaaaggaa gcggaacacg tagaaagcca gtccgcagaa acggtgctga 12060
ccccggatga atgtcgagcc gttccataca gaagctgggc gaacaaacga tgctcgcctt 12120
ccagaaaacc gaggatgcga accacttcat ccggggtcag caccaccggc aagcgccgcg 12180
acggccgagg tcttccgatc tcctgaagcc agggcagatc cgtgcacagc accttgccgt 12240
agaagaacag caaggccgcc aatgcctgac gatgcgtgga gaccgaaacc ttgcgctcgt 12300
tcgccagcca ggacagaaat gcctcgactt cgctgctgcc caaggttgcc gggtgacgca 12360
caccgtggaa acggatgaag gcacgaaccc agtggacata agcctgttcg gttcgtaagc 12420
tgtaatgcaa gtagcgtatg cgctcacgca actggtccag aaccttgacc gaacgcagcg 12480
gtggtaacgg cgcagtggcg gttttcatgg cttgttatga ctgttttttt ggggtacagt 12540
ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat 12600
ggagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta aacatcatga 12660
gggaagcggt gatcgccgaa gtatcgactc aactatcaga ggtagttggc gtcatcgagc 12720
gccatctcga accgacgttg ctggccgtac atttgtacgg ctccgcagtg gatggcggcc 12780
tgaagccaca cagtgatatt gatttgctgg ttacggtgac cgtaaggctt gatgaaacaa 12840
cgcggcgagc tttgatcaac gaccttttgg aaacttcggc ttcccctgga gagagcgaga 12900
ttctccgcgc tgtagaagtc accattgttg tgcacgacga catcattccg tggcgttatc 12960
cagctaagcg cgaactgcaa tttggagaat ggcagcgcaa tgacattctt gcaggtatct 13020
tcgagccagc cacgatcgac attgatctgg ctatcttgct gacaaaagca agagaacata 13080
gcgttgcctt ggtaggtcca gcggcggagg aactctttga tccggttcct gaacaggatc 13140
tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg 13200
atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa 13260
tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc 13320
ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc 13380
gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg 13440
gcaaataagc gggactctgg ggttcgcgga atcatgacca aaatccctta acgtgagttt 13500
tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 13560
tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 13620
ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 13680
ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 13740
gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 13800
aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 13860
ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 13920
agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 13980
aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 14040
aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 14100
ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 14160
cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 14220
tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 14280
accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc 14340
cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 14400
gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg 14460
cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 14520
ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 14580
catcaccgaa acgcgcgagg cagcagatca attcgcgcgc gaaggcgaag cggcatgcat 14640
ttacgttgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga 14700
agagagtcaa ttcagggtgg tgaat 14725
<210> 12
<211> 24
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(24)
<400> 12
cgccagggtt ttcccagtca cgac 24
<210> 13
<211> 24
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(24)
<400> 13
agcggataac aatttcacac agga 24
<210> 14
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 14
cgacacggaa atgttgaata ctcat 25
<210> 15
<211> 39
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(39)
<400> 15
tgcctgcagg tcgacgtgca cctgatcagg taaatgagt 39
<210> 16
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 16
tgcaccgtgc agtcgggtac cttggtatca atgtggttga agaaat 46
<210> 17
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 17
cggcaagaac gagtaatcta gagctcttac cgaagtagcc tttgtc 46
<210> 18
<211> 40
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(40)
<400> 18
tacgaattcg agctcgaggt tttctcgcac attattcata 40
<210> 19
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 19
tcttcaacca cattgatacc aaggtacccg actgcacggt gcacca 46
<210> 20
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 20
gtcgtcagtt tggacgccat ctgtttcctg tgtgaaattg ttatcc 46
<210> 21
<211> 45
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(45)
<400> 21
gataacaatt tcacacagga aacagatggc gtccaaactg acgac 45
<210> 22
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 22
ggctacttcg gtaagagctc tagattactc gttcttgccg ttgacc 46
<210> 23
<211> 23
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(23)
<400> 23
gggtagtgat ctttttgctc agc 23
<210> 24
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 24
caatacatct actctggggg ctaga 25
<210> 25
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 25
tgcctgcagg tcgactctag aggtttttta gttttctggg gacatg 46
<210> 26
<211> 45
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(45)
<400> 26
gatctaaacg atctgttaac ctgcttcagt gagatcaata ccctg 45
<210> 27
<211> 45
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(45)
<400> 27
gttcgcgcgg ttaattcata aaatacttac cgcgacgatg aaggt 45
<210> 28
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 28
tacgaattcg agctcggtac cagcgtcacc cttatcaccc ttatta 46
<210> 29
<211> 45
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(45)
<400> 29
cagggtattg atctcactga agcaggttaa cagatcgttt agatc 45
<210> 30
<211> 44
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(44)
<400> 30
ttgagaataa cggagtgggt cataagcttt cctcctggac ttcg 44
<210> 31
<211> 44
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(44)
<400> 31
cgaagtccag gaggaaagct tatgacccac tccgttattc tcaa 44
<210> 32
<211> 45
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(45)
<400> 32
accttcatcg tcgcggtaag tattttatga attaaccgcg cgaac 45
<210> 33
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 33
gcttggatca tctgaacaga gtgaa 25
<210> 34
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 34
cagcagtaca ggtgttgttc tcacc 25
<210> 35
<211> 70
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(70)
<400> 35
ggaatttcta ctgttgtaga tgtgcgattg ctcgcgaggg tgaggcttct tgctagttgt 60
cagtggtcaa 70
<210> 36
<211> 47
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(47)
<400> 36
ccttcggatc taaacgatct gttaacttgc gagatgctca tcaacaa 47
<210> 37
<211> 47
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(47)
<400> 37
ttgttgatga gcatctcgca agttaacaga tcgtttagat ccgaagg 47
<210> 38
<211> 44
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(44)
<400> 38
tgttgagaag ctgccacatt cacaagcttt cctcctggac ttcg 44
<210> 39
<211> 44
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(44)
<400> 39
cgaagtccag gaggaaagct tgtgaatgtg gcagcttctc aaca 44
<210> 40
<211> 43
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(43)
<400> 40
caacactctc ttcggacagt gatagttaag cggtttctgc gcg 43
<210> 41
<211> 43
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(43)
<400> 41
cgcgcagaaa ccgcttaact atcactgtcc gaagagagtg ttg 43
<210> 42
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 42
tccgccaaaa cagcctctag agagtaaggg cgatattgtt accaag 46
<210> 43
<211> 24
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(24)
<400> 43
gtgcgattgc tcgcgagggt gagg 24
<210> 44
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 44
gttttatctt gtgcgtgttt accgt 25
<210> 45
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 45
aatagtcctg ctttatcgcc atgag 25
<210> 46
<211> 18
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(18)
<400> 46
cggcgagttc gatggaat 18
<210> 47
<211> 44
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(44)
<400> 47
ccatttcttc gatgtacttg tgctgcagga tccataggaa tcgg 44
<210> 48
<211> 44
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(44)
<400> 48
ccgattccta tggatcctgc agcacaagta catcgaagaa atgg 44
<210> 49
<211> 22
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(22)
<400> 49
cttccacccc ctacgtctca ta 22
<210> 50
<211> 23
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(23)
<400> 50
cgtcctgatg aaaacgccga gcg 23
<210> 51
<211> 65
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(65)
<400> 51
ggaatttcta ctgttgtaga tcgtcctgat gaaaacgccg agcgtctaga ggctgttttg 60
gcgga 65
<210> 52
<211> 65
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(65)
<400> 52
tccgccaaaa cagcctctag acgctcggcg ttttcatcag gacgatctac aacagtagaa 60
attcc 65
<210> 53
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 53
ggtactgaag atgaaatcga ggatt 25
<210> 54
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 54
tacatccagg tccaatcatc aatgt 25
<210> 55
<211> 66
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(66)
<400> 55
ggaatttcta ctgttgtaga tctgggctgg tgacaataac agccttctag aggctgtttt 60
ggcgga 66
<210> 56
<211> 66
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(66)
<400> 56
tccgccaaaa cagcctctag aaggctgtta ttgtcaccag cccagatcta caacagtaga 60
aattcc 66
<210> 57
<211> 24
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(24)
<400> 57
ctgggctggt gacaataaca gcct 24
<210> 58
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 58
cagccatgac caggctttgg tactg 25
<210> 59
<211> 48
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(48)
<400> 59
gtttatcgcc accttggatc acaaggaaga caccattgaa ggtgtgcg 48
<210> 60
<211> 48
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(48)
<400> 60
cgcacacctt caatggtgtc ttccttgtga tccaaggtgg cgataaac 48
<210> 61
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 61
cagataacgt tcttcacgct ggtac 25
<210> 62
<211> 20
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(20)
<400> 62
cgaagtgggt agcggttgag 20
<210> 63
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 63
aaacccaggc tgtactggca ctact 25
<210> 64
<211> 35
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(35)
<400> 64
tgcctgcagg tcgacagatc caaacaacgg cttcc 35
<210> 65
<211> 30
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(30)
<400> 65
ctgaaggagg tgcgagtgat cggcaatgaa 30
<210> 66
<211> 30
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(30)
<400> 66
ttcattgccg atcactcgca cctccttcag 30
<210> 67
<211> 34
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(34)
<400> 67
tacgaattcg agctccggag atcacgagca aacg 34
<210> 68
<211> 24
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(24)
<400> 68
ctgacccaag ataagatcaa gacc 24
<210> 69
<211> 21
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(21)
<400> 69
cgcggaaggt ggtatcagta a 21
<210> 70
<211> 67
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(67)
<400> 70
ggaatttcta ctgttgtaga tgatctgcgg gcaggtctac tgagccgcag atgtagccct 60
ccacaat 67
<210> 71
<211> 51
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(51)
<400> 71
ccttcggatc taaacgatct gttaacgtct gagacgttgt aggcaatgag a 51
<210> 72
<211> 51
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(51)
<400> 72
tctcattgcc tacaacgtct cagacgttaa cagatcgttt agatccgaag g 51
<210> 73
<211> 44
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(44)
<400> 73
ccgatcatca caatgttcag cataagcttt cctcctggac ttcg 44
<210> 74
<211> 44
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(44)
<400> 74
cgaagtccag gaggaaagct tatgctgaac attgtgatga tcgg 44
<210> 75
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 75
ggtcggtgat gttgtcatag aagaattaga ttgaaatggc atgggc 46
<210> 76
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 76
gcccatgcca tttcaatcta attcttctat gacaacatca ccgacc 46
<210> 77
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 77
tccgccaaaa cagcctctag agccactgtg tgtagatctt gatcat 46
<210> 78
<211> 24
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(24)
<400> 78
gatctgcggg caggtctact gagc 24
<210> 79
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 79
tattgatccg atcaccatgg atgac 25
<210> 80
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 80
cttgagcgac aaactcccac tcata 25
<210> 81
<211> 24
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(24)
<400> 81
gaaagctatc tcgcgacggt gggg 24
<210> 82
<211> 70
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(70)
<400> 82
ggaatttcta ctgttgtaga tgaaagctat ctcgcgacgg tggggaacac ctacacggac 60
aaggacatct 70
<210> 83
<211> 50
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(50)
<400> 83
cagtgcagac gaaaaggtat tgccccttct tagggttact ttcgactgct 50
<210> 84
<211> 50
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(50)
<400> 84
taagaagcga accagctgta tggcccttca acagcaagct gatcactatg 50
<210> 85
<211> 46
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(46)
<400> 85
tccgccaaaa cagcctctag agagcggatt agccattatc agtcac 46
<210> 86
<211> 47
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(47)
<400> 86
gggcaatacc ttttcgtctg cactgagtgg ggtagcggct tgttaga 47
<210> 87
<211> 48
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(48)
<400> 87
cgcttttgaa gcagaagcct acctttgtat gtcctcctgg acttcgtg 48
<210> 88
<211> 50
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(50)
<400> 88
aaggtaggct tctgcttcaa aagcgatgtc aaacaacatt cgtatcgaag 50
<210> 89
<211> 52
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(52)
<400> 89
ggccatacag ctggttcgct tcttattact gttcgctttc atcagtatag cg 52
<210> 90
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 90
gttgttctgg atcgtgctca gctat 25
<210> 91
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 91
ctgcattctc atcgatcttg ttgtc 25
<210> 92
<211> 66
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(66)
<400> 92
ggaatttcta ctgttgtaga ttctccttgg tggtcactgg caatatctag aggctgtttt 60
ggcgga 66
<210> 93
<211> 66
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(66)
<400> 93
tccgccaaaa cagcctctag atattgccag tgaccaccaa ggagaatcta caacagtaga 60
aattcc 66
<210> 94
<211> 24
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(24)
<400> 94
tctccttggt ggtcactggc aata 24
<210> 95
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 95
tggcaataaa tatgcggatt tacta 25
<210> 96
<211> 50
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(50)
<400> 96
aagttcacca agaagtagtg cttcatggag tctgcacagg aacatagatg 50
<210> 97
<211> 50
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(50)
<400> 97
catctatgtt cctgtgcaga ctccatgaag cactacttct tggtgaactt 50
<210> 98
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 98
gcttacaagc agctgttgca cctgc 25
<210> 99
<211> 48
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(48)
<400> 99
cgaagtccag gaggaaagct tatgaaaaca aaactggatt ttctaaaa 48
<210> 100
<211> 44
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(44)
<400> 100
gagctcggta cccggggatc cctacaaaat tgtacgggct ggtt 44
<210> 101
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 101
gcttatcaca gtgaaagcaa aacca 25
<210> 102
<211> 25
<212> DNA
<213> primer
<220>
<221> primer_bind
<222> (1)..(25)
<400> 102
gatttgtcct actcaggaga gcgtt 25
Claims (6)
1.一种泛酸生产菌，其特征在于：为VB5-10，是由下述方法构建而成的：
在链霉素抗性谷氨酸棒杆菌ATCC 13032 rpsL K43R基因组的cg1890假基因位点整合由Ptrc启动子启动的ptsG基因，得到菌株VB5-1,所述ptsG基因的序列如SEQ ID NO.3所示；
在VB5-1基因组cg1895假基因位点整合由Ptuf启动子启动的来源于需钠弧菌的eddeda基因，得到菌株VB5-2，所述eddeda基因的序列如SEQ ID NO.4所示；
在VB5-2基因组cg1995假基因位点整合由Ptuf启动子启动的ilvB*N*C*基因，得到菌株VB5-3，所述ilvB*N*C*基因的序列如SEQ ID NO.5所示；
将Vb5-3中avtA基因敲除，得到菌株VB5-4；
将VB5-4中ilvE基因敲除，得到菌株VB5-5；
将VB5-5中pyc基因编码的酶第458位氨基酸由脯氨酸突变为丝氨酸，得到菌株VB5-6，所述pyc基因的序列如SEQ ID NO.6所示；
在VB5-6基因组cg1960假基因位点整合由Ptuf启动子启动的aspB基因，得到菌株VB5-7，所述aspB基因的序列如SEQ ID NO.7所示；
在VB5-7基因组Ncgl2850a假基因位点整合由Ptuf启动子启动的来源于大肠杆菌的aspA Eco 基因，得到菌株VB5-8，所述aspA Eco 基因的序列如SEQ ID NO.8所示；
将VB5-8中ilvA基因敲除，得到菌株VB5-9；
将质粒pXtuf-panBCD Bsu 转化到VB5-9中，得到VB5-10，所述质粒pXtuf-panBCD Bsu 的序列如SEQ ID NO.9所示。
2.权利要求1所述泛酸生产菌的构建方法，其特征在于：具体步骤如下：
（1）以菌株Corynebacterium glutamicum ATCC 13032 rpsL K43R为出发菌株，运用自杀质粒pK18mobrpsL介导的谷氨酸棒杆菌基因编辑技术，将cg1890假基因位点整合由Ptrc启动子启动的ptsG基因，得到菌株VB5-1；
（2）运用自杀质粒pK18mobrpsL介导的谷氨酸棒杆菌基因编辑技术，在VB5-1基因组cg1895假基因位点整合由Ptuf启动子启动的来源于需纳弧菌的eddeda基因，得到菌株VB5-2；
（3）运用CRISPR-Cpf1基因编辑技术，在VB5-2基因组cg1995假基因位点整合由Ptuf启动子启动的ilvB*N*C*基因，得到菌株VB5-3；
（4）运用CRISPR-Cpf1基因编辑技术，将VB5-3中avtA基因敲除，得到菌株VB5-4；
（5）运用CRISPR-Cpf1基因编辑技术，将VB5-4中ilvE基因敲除，得到菌株VB5-5；
（6）运用自杀质粒pK18mobrpsL介导的谷氨酸棒杆菌基因编辑技术，将VB5-5中pyc基因编码的酶第458位氨基酸由脯氨酸突变为丝氨酸，得到菌株VB5-6；
（7）运用CRISPR-Cpf1基因编辑技术，在VB5-6基因组cg1960假基因位点整合由Ptuf启动子启动的aspB基因，得到菌株VB5-7；
（8）运用CRISPR-Cpf1基因编辑技术，在VB5-7基因组Ncgl2850a假基因位点整合由Ptuf启动子启动的来源于大肠杆菌的aspA Eco 基因，得到菌株VB5-8；
（9）运用CRISPR-Cpf1基因编辑技术，将VB5-8中ilvA基因敲除，得到菌株VB5-9；
（10）将pXtuf质粒与片段panBCD Bsu 进行连接，构建pXtuf-panBCD bsu质粒；将质粒转化到VB5-9中，得到VB5-9/pXtuf-panBCD bsu，记为VB5-10。
3.权利要求1所述泛酸生产菌在发酵生产D-泛酸方面的应用。
4.根据权利要求3所述泛酸生产菌的应用，其特征在于：具体发酵生产方法如下：
（1）菌体活化：用接种环从保菌管中取一环划线接种于斜面试管活化，32℃恒温静置培养20-24 h，即可得到一代活化斜面；然后从该斜面上用接种环将菌体接种于斜面茄形瓶中，32℃恒温静置培养12-24 h，即可得到二代活化斜面；
（2）种子罐培养：取上一步活化的二代斜面茄子瓶，取适量无菌水于茄形瓶中，将菌悬液接入种子培养基中并最终定容至3 L，通过流加氨水使pH稳定在7.0-7.2，温度恒定在32℃，控制溶氧在20-30%，培养菌体生长至OD600达到15-30；
（3）发酵罐培养：按照5%-10%接种量接入新鲜的发酵培养基开始发酵，发酵过程中通过流加氨水控制pH稳定在7.0-7.2，温度维持在32℃，溶氧在20%以上；当培养基中的葡萄糖消耗完之后，流加80% (m/v)的葡萄糖溶液，维持发酵培养基中的葡萄糖浓度在0.1-5 g/L；发酵周期在40-48 h。
5.根据权利要求4所述泛酸生产菌的应用，其特征在于：所述种子培养基的组成如下：葡萄糖80 g/L，(NH4)2SO4 5 g/L，KH2PO4 2.5 g/L，MgSO4 •7H2O 1.6g/L，FeSO4•7H2O 20 mg/L，MnSO4·H2O 20 mg/L，B族维生素VB1、VB3、VB12各0.3 mg/L，蛋白胨2 g/L，酵母粉3 g/L，亮氨酸1 g/L，异亮氨酸1 g/L，缬氨酸1 g/L，蛋氨酸1 g/L ，谷氨酸5 g/L ，丝肽粉5 g/L ，柠檬酸2 g/L，玉米浆5 g/L，用自来水配置培养基。
6.根据权利要求4所述泛酸生产菌的应用，其特征在于：所述发酵培养基的组成如下：葡萄糖80g/L，(NH4)2SO4 5g/L，KH2PO4 2.5g/L，MgSO4•7H2O 1.6g/L，FeSO4 •7H2O 20mg/L，MnSO4 •H2O 20mg/L，VB1、VB3、VB12 各 0.3 mg/L，蛋白胨2g/L，酵母粉3g/L，亮氨酸1g/L，异亮氨酸1g/L，缬氨酸1g/L，蛋氨酸1g/L ，谷氨酸5g/L，丝肽粉5g/L，柠檬酸2g/L，玉米浆20g/L，豆浓10ml/L，其余为水。
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