CN108048472A - 一株高效异源表达Disorazole Z的工程菌株和构建该菌株的基因簇及其应用 - Google Patents
一株高效异源表达Disorazole Z的工程菌株和构建该菌株的基因簇及其应用 Download PDFInfo
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Abstract
本发明公开了一种Disorazole Z的生物合成基因簇dis427,其核苷酸序列如SEQ ID No.1所示。本发明还公开了利用所述dis427基因簇构建的高效异源表达Disorazole Z的工程菌株DK1622::Km‑Ptet‑dis427,是利用黄色粘球菌Myxococcus xanthus DK1622为出发菌株,通过转座的方法在其基因组上整合了Disorazole Z的生物合成基因簇dis427获得。本发明还公开了工程菌株DK1622::Km‑Ptet‑dis427在制备Disorazole Z中的应用。本发明所提供的Disorazole Z生物合成途径及其在异源宿主中的高效表达方法为开发新的抗肿瘤或抗感染药物、降低发酵生产成本具有重要的研究和应用价值。
Description
技术领域
本发明属于微生物基因资源和生物合成技术领域,具体涉及一种Disorazole Z生物合成基因簇和利用该基因簇构建的一株高效异源表达Disorazole Z的工程菌株及其应用。
背景技术
Disorazoles是最早由Jansen等人于1994年从纤维堆囊菌Sorangium cellulosumSo ce 12的发酵液中分离得到的结构新颖的大环双内酯类化合物。至今,在Sorangiumcellulosum So ce12中已经发现29个Disorazoles衍生物,分别为Disorazole A1至Disorazole I。
研究表明,Disorazoles类化合物能够抑制微管蛋白聚合,促进微管蛋白解聚,从而干扰细胞分裂,诱导细胞凋亡,对于多种肿瘤细胞系,包括多药耐药肿瘤细胞系均具有很强的生物活性,是一类新型的细胞微管抗稳定剂。Disorazole Al和Disorazole C1是目前研究较多的组分,对于多种人类肿瘤细胞系,包括多药耐药肿瘤细胞系,其半抑制浓度IC50在pM至nM水平。近期研究发现,Disorazoles类化合物还对A群链球菌的入侵细胞感染途径具有抑制作用。虽然活性显著,但是来源于Sorangium cellulosum So ce 12的Disorazoles类化合物在生物体内的半衰期非常短,是其成药的瓶颈。
Disorazole Z是来源于纤维堆囊菌Sorangium cellulosum So ce 427的Disorazoles家族化合物,与来源于Sorangium cellulosum So ce 12的Disorazoles类化合物相比也具有显著的抗肿瘤活性,同时具有较小的环状骨架,结构更为稳定,在生物体内具有更长的半衰期。已有报道将该化合物与促黄体激素释放激素偶联用于三阴性乳腺癌的靶向治疗已进入二期临床研究。因此,Disorazole Z是一种优良的潜在抗肿瘤或者抗感染新药物。
尽管Disorazole Z作为一种极具开发前景的抗肿瘤药物或者抗感染药物有望在不远的将来作为商品药物推广,但是如何得到大剂量的纯品物质是当今最大限制之一。一方面,由于野生菌株Sorangium cellulosum So ce 427生长非常缓慢、不易培养因而不适合大规模发酵,另一方面,人工全合成方法制备Disorazole Z非常困难,至今尚未有成功合成的报道。鉴于此,如何高效地生产并提纯Disorazole Z是目前亟待解决的问题。因此,获取其生物合成途径基因簇并将该基因簇转移至生长快速且易于培养的宿主菌中进行异源生物合成显得十分必要,对于开发新的抗肿瘤或抗感染药物、降低发酵生产成本具有重要的应用价值。经检索,Disorazole Z的生物合成基因簇(dis427)以及利用该基因簇在异源宿主菌黄色粘球菌Myxococcus xanthus DK1622中实现高效表达Disorazole Z的文献或专利还未见报道。
发明内容
针对目前产Disorazole Z的野生菌株Sorangium cellulosum So ce 427生长非常缓慢、不易培养因而不适合大规模发酵的不足,本发明要解决的问题是基因组挖掘原始产生菌So ce427来提供一种Disorazole Z生物合成途径基因簇(dis427)以及利用该基因簇构建一株高效异源表达Disorazole Z的工程菌株用于Disorazole Z的高效异源生物合成。
本发明所述Disorazole Z的生物合成基因簇,其特征在于:该基因簇命名为dis427,其包含Disorazole Z生物合成所必需的编码聚酮合成酶及非核糖体多肽合成酶的四个核心基因disA,disB,disC和disD,一个假设蛋白基因orf4和一个后修饰基因orf6;该基因簇来源于纤维堆囊菌Sorangium cellulosum So ce 427,其核苷酸序列如SEQ IDNo.1所示。所述基因簇对应的Disorazole Z生物合成途径如图1所示。
本发明所述的高效异源表达Disorazole Z的工程菌株,其特征在于:该菌株命名为工程菌株DK1622::Km-Ptet-dis427,其基因型为:Myxococcus xanthus DK1622,kanamycin resistance,tetracycline inducible Ptet promoter,disA,disB,disC,orf4,disD and orf6,是利用黄色粘球菌Myxococcus xanthus DK1622为出发菌株,通过转座的方法在其基因组上整合了Disorazole Z的生物合成基因簇dis427获得。
本发明所述高效异源表达Disorazole Z的工程菌株DK1622::Km-Ptet-dis427的构建方法,步骤是:
(1)利用Red/ET DNA重组技术将Disorazole Z的生物合成基因簇dis427直接克隆至p15A-cm-tetR-tetO-hyg-ccdB载体上,构建得到质粒p15A-cm-dis427;
(2)在步骤(1)构建的质粒p15A-cm-dis427上插入反向筛选标记amp-ccdB,构建得到质粒p15A-cm-amp-ccdB-dis427;
(3)步骤(2)构建的质粒p15A-cm-amp-ccdB-dis427通过限制性内切酶PacI和PmeI酶切后与tetR-tetO PCR片段进行线线重组,构建得到质粒p15A-cm-tetR-tetO-dis427;
(4)在步骤(3)构建的质粒p15A-cm-tetR-tetO-dis427上插入转座元件,构建得到表达质粒p15A-tnpA-kan-tetR-tetO-dis427;
(5)将步骤(4)构建的表达质粒p15A-tnpA-kan-tetR-tetO-dis427电转至Myxococcus xanthus DK1622中,表达质粒在Myxococcus xanthus DK1622中表达转座酶将Disorazole Z的生物合成基因簇dis427整合到Myxococcus xanthus DK1622的基因组上,得到能高效异源表达Disorazole Z的工程菌株,命名为工程菌株DK1622::Km-Ptet-dis427。
本发明还公开了所述高效异源表达Disorazole Z的工程菌株DK1622::Km-Ptet-dis427在制备Disorazole Z中的应用。
本发明所涉及的工程菌株DK1622::Km-Ptet-dis427在文献中未见报道,是首次对Disorazole Z的生物合成基因簇(dis427)在异源宿主菌Myxococcus xanthus DK1622中实现高效表达。实验证实:本发明提供的工程菌株DK1622::Km-Ptet-dis427与原始产生菌Sorangium cellulosum So ce 427相比,Disorazole Z的产量提高了1倍,而且缩短了发酵生产周期,这对于降低发酵生产成本,开发新的抗肿瘤或抗感染药物具有重要的研究和应用价值。
附图说明
图1:Disorazole Z生物合成基因簇(dis427)及其合成途径。
其中:模块1至模块6编码聚酮合成酶,模块8编码非核糖体多肽合成酶,各模块中KS为酮基合成酶结构域,KR为酮基还原酶结构域,DH为脱水酶结构域,ACP为酰基载体蛋白结构域,MT为甲基转移酶结构域,HC为杂环化结构域,A为腺苷酰化结构域,AT为酰基转移酶结构域。
图2:Disorazole Z生物合成基因簇(dis427)的直接克隆过程。
图3:表达质粒p15A-tnpA-kan-tetR-tetO-dis427的构建过程。
图4:Disorazole Z生物合成基因簇(dis427)直接克隆重组子质粒p15A-cm-dis427(1),构建的质粒p15A-cm-amp-ccdB-dis427(2)、p15A-cm-tetR-tetO-dis427(3)及表达质粒p15A-tnpA-kan-tetR-tetO-dis427(4)的酶切分析。
用SphI和EcoRV对质粒进行双酶切分析。左图为理论酶切图,右图为实际酶切图。
图5:菌落PCR检测构建的工程菌株DK1622::Km-Ptet-dis427。
A为利用引物Colony PCR chk01-F和Colony PCR chk01-R进行检测的结果;B为利用引物Colony PCR chk02-F和Colony PCR chk02-R进行检测的结果;C为利用引物ColonyPCR chk03-F和Colony PCR chk03-R进行检测的结果;M为TaKaRa DL1000DNAMarker;N为原始异源宿主Myxococcus xanthus DK1622,作为阴性对照;P为重组载体p15A-tnpA-Kan-tetR-tetO-dis427,作为阳性对照;数字1-6代表不同的单克隆。
图6:工程菌株DK1622::Km-Ptet-dis427表达Disorazole Z的高效液相色谱-质谱检测。
其中,So ce 427_WT为Disorazole Z原始产生菌Sorangium cellulosum So ce427发酵液粗提物,为阳性对照组;DK1622_WT为野生型异源宿主菌Myxococcus xanthusDK1622发酵液粗提物,为阴性对照组;DK1622::Km-Ptet-dis427为四环素诱导启动子调控下的Disorazole Z生物合成基因簇在异源宿主中进行表达的发酵液粗提物。
具体实施方式
以下结合附图及具体实例详细描述本发明,以便更好地理解本发明,但所述内容并不限制本发明的保护内容。
一般性说明:如下实施例所涉及的大肠杆菌GB05、GB05-dir和GBred-gyrA462,重组酶表达质粒pSC101-BAD-ETgA-tet以及质粒p15A-cm-tetR-tetO-hyg-ccdB、pR6K-amp-cddB和pR6K-oriT-tnpA-kan均购于德国GeneBridges公司;T4DNA聚合酶和限制性内切酶购于NEB公司,用于PCR扩增的DNA聚合酶购于TaKaRa公司;质粒提取试剂和DNA琼脂糖凝胶回收试剂盒购于天根公司;野生型黄色粘球菌Myxococcus xanthus DK1622和纤维堆囊菌Sorangium cellulosum So ce427为山东大学-亥姆霍兹生物技术研究所保藏;DisorazoleZ生物合成基因簇(dis427)核苷酸序列见序列表SEQ ID No.1;基因测序由华大基因公司完成;寡核苷酸合成由上海生工生物公司完成;其他涉及的试剂和耗材均为国产,实施例中的实验方法及试剂如无特殊说明,均为本领域常规方法与市售试剂。
实施例1:Disorazole Z生物合成基因簇(dis427)的挖掘
将纤维堆囊菌Sorangium cellulosum So ce 427接种至VY/2固体培养基(5g/L安琪酵母、1.36g/L二水合氯化钙、0.5mg/L维生素B12、15g/L琼脂粉,调节pH值为7.2)中,置于30℃培养至扩散生长状态。刮取边缘菌膜转接到M26液体培养基(8g/L马铃薯淀粉、2g/L大豆蛋白胨、2g/L酵母提取物、1g/L七水合硫酸镁、1g/L二水合氯化钙、1mL/L微量元素溶液,调节pH值为7.2)中,置于30℃摇床培养至足够的菌体量以用于制备基因组DNA。
离心收集菌体后,将其重悬于10mM Tris-HCl缓冲液中(pH值为8.0)。向菌悬液中加入终浓度为1mg/ml的蛋白酶K及终浓度为1%的SDS,置于50℃水浴处理至少2h。向处理后的裂解液中加入等体积的DNA提取液(苯酚:氯仿:异戊醇=25:24:1),充分混匀后离心得到上清液。向上清液中加入1/10体积的3M醋酸钠(pH值为8.0),混匀后再加入3倍体积的无水乙醇,充分混匀后可见絮状基因组DNA沉淀。将絮状沉淀挑取至75%乙醇中,离心后弃上清得到基因组DNA,自然晾干后溶解于10mM Tris-HCl缓冲液中(pH值为8.0)置于4℃备用。
上述方法制备的Sorangium cellulosum So ce 427基因组DNA经过RNA酶消化处理之后送至华大基因公司进行全基因组测序。将获得的基因组DNA序列信息提交至antiSMASH(https://antismash.secondarymetabolites.org)进行次生代谢产物生物合成基因簇预测,分析得到Disorazole Z的生物合成基因簇。将得到的基因簇结构域构成与Disorazole Z化学结构进行比较分析,最终确定了Disorazole Z的生物合成途径,如图1所示。
实施例2:Disorazole Z生物合成基因簇(dis427)的直接克隆
Disorazole Z生物合成基因簇(dis427)直接克隆过程见图2。
2.1 Disorazole Z生物合成基因簇(dis427)直接克隆载体的制备
具体步骤为:限制性内切酶AvaI酶切质粒p15A-cm-tetR-tetO-hyg-ccdB得到片段p15A-cm-tetR-tetO(酶切回收大片段,胶跑到底部再切胶,胶回收具体做法参照天根试剂盒说明书)。然后以p15A-cm-tetR-tetO作为PCR模板,用引物p15A-Cm BstBI and AflIIfor dis427-F和p15A-Cm BstBI and AflII for dis427-R进行PCR扩增,得到的PCR产物p15A-cm vector for dis427末端带有Disorazole Z生物合成基因簇(dis427)两端序列的同源臂。
PCR引物序列如下(序列中大写字母为同源臂,小写字母为引物):
p15A-cm BstBI and AflII for dis427-F:AAGCCGTCACGGGCGCTCTGGTCTCCCTTAGTAGCAGGACACGGGCCAGGGCTCGGCCTGACAGATTTCCCGCGTTTACCagttacggatcttaaggatctc
p15A-cm BstBI and AflII for dis427-R:CGATTGCTCGGGGGCGCCGGAGACCGCCGGCAGGGGCTTCGATTTCCGCGGGTATCTGGCGCGCATGGCCGCCACGGAGActtattcggccttgaattgatc
用引物p15A-Cm BstBI and AflII for dis427-F和p15A-Cm BstBI and AflIIfor dis427-RPCR扩增片段p15A-cm vector for dis427的具体做法如下:
PCR扩增体系:
PCR程序:95℃预变性3min;98℃变性15s;58℃(根据引物Tm值设定)退火15s;72℃延伸2min(延伸时间根据所扩增的长度确定,1kb/1min);循环30次;最后72℃,10min。实验过程中所用的引物是p15A-Cm BstBI and AflII for dis427-F和p15A-Cm BstBI andAflII for dis427-R。模板是p15A-cm-tetR-tetO-hyg-ccdB用限制性内切酶AvaI线性化的产物。
2.2基因组DNA的限制性内切酶处理
将制备的Sorangium cellulosum So ce 427基因组DNA用限制性内切酶BstBI和AflII进行酶切处理以释放出待克隆的目的基因片段,酶切体系如下表所示:
将酶切反应液置于37℃处理4h,取10μl进行琼脂糖凝胶电泳检测,剩余的反应液利用苯酚:氯仿:异戊醇(25:24:1)抽提,然后用醋酸钠-乙醇沉淀。酶切后的基因组DNA最终溶解于适量无菌去离子水,利用Nanodrop 2000测浓度,大约2μg/μl,置于4℃备用。
2.3 Disorazole Z生物合成基因簇(dis427)克隆子的获得
克隆载体片段和酶切后的基因组DNA首先利用T4DNA聚合酶进行处理,然后电击转化表达重组酶的大肠杆菌来进一步在体内完成最终的重组反应。
体外T4DNA聚合酶处理的反应体系如下表所示:
体外T4DNA聚合酶处理的反应条件如下表所示:
电转化步骤为:将含有温敏复制子的重组酶表达质粒pSC101-BAD-ETgA-tet的菌株GB05-dir在加有4μg/ml四环素的LB培养基(low salt,1%Triptone,0.5%yeastextract,0.1%NaCl)中30℃培养过夜(OD600=3~4)。将40μl过夜培养物(OD600=3~4)转接到加有4μg/ml四环素的1.3ml LB中,置于Eppendorf thermomixer上30℃,950rpm培养2h(OD600=0.35~0.4)。向培养物中加35μl 10%L-阿拉伯糖,置于Eppendorf thermomixer上37℃,950rpm培养40min。9400g离心30sec收集细胞。弃上清,沉淀用1ml H2O悬浮。重复离心、重悬、再离心、弃上清,用20μl H2O悬浮细胞。加入T4聚合酶处理并脱盐的DNA,将细胞和DNA的混合液转入1mm电击杯中,用Eppendorf electroporator 2510进行电击,电压1350V,电容10Μf,电阻600Ω。加1ml LB至电击杯中,洗涤细胞并将其转移至扎孔的1.5ml管中,置于Eppendorf thermomixer上37℃,950rpm培养1h。最后将所有菌液涂布到加有15μg/ml氯霉素的LB平板上,37℃过夜培养。
挑取单菌落在加有10μg/ml氯霉素的LB培养基中置于37℃培养过夜,利用碱裂解和异丙醇沉淀法提取质粒DNA,经限制性内切酶SphI和EcoRV消化后进行电泳检测,筛选得到正确的重组质粒p15A-cm-dis427(酶切电泳分析见图4)。
实施例3:Disorazole Z生物合成基因簇(dis427)表达质粒p15A-tnpA-kan-tetR-tetO-dis427的构建
3.1质粒p15A-cm-tetR-tetO-disZ427的构建
质粒p15A-cm-tetR-tetO-disZ427的构建过程见图3。
已有报道Disorazoles类化合物生物合成基因簇的组成型表达可能影响异源宿主生长及正常代谢过程,因此本发明构建了一种对dis427基因簇进行启动子改造以严谨调控其表达的质粒。
具体步骤为:首先用引物Amp-ccdB PCR-F和Amp-ccdB PCR-R通过PCR扩增含有amp-ccdB的DNA片段,PCR反应体系及扩增条件参照实施例2.1。胶回收之后用无菌去离子水洗脱,利用Nanodrop 2000测浓度,大约200ng/μl,将该DNA片段与重组表达载体在低温条件下共同转化阿拉伯糖诱导的大肠杆菌GBred-gyrA462,37℃复苏1h后涂布至加有15μg/ml氯霉素和100μg/ml氨苄霉素双抗的LB平板,37℃过夜培养至长出单菌落。
然后挑取单菌落制备质粒DNA,用限制性内切酶SphI和EcoRV酶切,筛选得到正确的重组质粒p15A-cm-amp-ccdB-dis427(酶切电泳分析见图4),并对酶切正确的质粒用引物Promoter substitution seq-01和Promoter substitution seq-02进行测序。
PCR引物序列如下(序列中大写字母为同源臂,小写字母为引物,下划线字母为限制性内切酶PacI和PmeI的酶切位点):
Amp-ccdB PCR-F:CCGCATATGATCAATTCAAGGCCGAATAAGTTAATTAAGTTTAAACtttgttcaaaaaaaagcc
Amp-ccdB PCR-R:CGTCCTGCTCTACGTGATTCCCGCTGCTCATTTAATTAAGTTTAAACtttgtttatttttctaaatac
测序引物序列如下:
Promoter substitution seq-01:CAACGGTGGTATATCCAGTG
Promoter substitution seq-02:CGAAATCAGGGGAATAATAGG
3.2质粒p15A-cm-tetR-tetO-dis427的构建
质粒p15A-cm-tetR-tetO-dis427的构建过程见图3。
用限制性内切酶PacI和PmeI对质粒p15A-cm-amp-ccdB-disZ427进行双酶切,酶切反应产物经醋酸钠-乙醇沉淀后溶解于适量无菌去离子水中得到线性片段。用引物tetR-tetO PCR-F和tetR-tetO PCR-R通过PCR扩增含有四环素诱导启动子的DNA片段得到tetR-tetO PCR for dis427,PCR反应体系及扩增条件参照实施例2.1。参照实施例2.3中的T4DNA聚合酶作用条件将酶切后的线性DNA片段和PCR扩增的启动子片段tetR-tetO PCR fordis427进行体外连接,脱盐处理后电击转化大肠杆菌GB05,涂布至加有15μg/ml氯霉素的LB平板,37℃过夜培养至长出单菌落。
挑取单菌落制备质粒DNA,用限制性内切酶SphI和EcoRV酶切,筛选正确的重组质粒p15A-cm-tetR-tetO-dis427(酶切电泳分析见图4)。并对酶切正确的质粒用测序引物Promoter substitution seq-03和Promoter substitution seq-04进行测序。
PCR引物序列如下(序列中大写字母为同源臂,小写字母为引物):
tetR-tetO PCR-F:CCGCATATGATCAATTC
tetR-tetO PCR-R:CGTCCTGCTCTACGTGATTCCCGCTGCTCAtagatcctttctcctctttagatc
测序引物序列如下:
Promoter substitution seq-03:GTGAGTATGGTGCCTATCTA
Promoter substitution seq-04:GAAGGGGAAAGCTGGCAAGA
3.3表达质粒p15A-tnpA-kan-tetR-tetO-dis427的构建
表达质粒p15A-tnpA-kan-tetR-tetO-dis427的构建过程见图5。
具体步骤为:限制性内切酶AseI酶切质粒pR6K-oriT-tnpA-kan得到片段oriT-tnpA-kan(酶切回收大片段,胶跑到底部再切胶,胶回收具体做法参照天根试剂盒说明书)。片段oriT-tnpA-kan两端带有质粒p15A-cm-tetR-tetO-dis427中氯霉素基因两端的同源臂。然后将200ng DNA片段oriT-tnpA-kan和200ng质粒p15A-cm-tetR-tetO-dis427共电转化到35μl 10%L-阿拉伯糖诱导表达了Redα/β/γ重组酶的菌株GBred-gyrA462中进行线环重组。在重组酶的作用下,质粒p15A-cm-tetR-tetO-dis427上的氯霉素基因被oriT-tnpA-kan替换,从而得到重组质粒p15A-tnpA-kan-tetR-tetO-dis427。复苏之后的菌液涂布到加有15μg/ml卡那霉素的LB平板上,37℃培养过夜。然后挑取单菌落制备质粒DNA,用限制性内切酶SphI和EcoRV酶切,筛选正确的重组质粒p15A-tnpA-kan-tetR-tetO-dis427(酶切电泳分析见图4)。
实施例4:本发明所述表达Disorazole Z的工程菌株DK1622::Km-Ptet-dis427的构建
将质粒p15A-tnpA-kan-tetR-tetO-dis427常温脱盐处理之后电转化至黄色粘球菌Myxococcus xanthus DK1622,电转化步骤为:将Myxococcus xanthus DK1622接种于CTT液体培养基(Casitone 10g/L,MgSO4-7H2O 1.97g/L,1mol/L Tris HCI(pH=7.6)10mL,0.1mol/L KPO4buffer(pH=7.6)10mL,pH=7.6)中,置于30℃摇床培养过夜,取100μL过夜培养物转接到新的1.7mL CTT液体培养基中继续培养约24h至OD600为0.6,低温9400g离心1min收集菌体,将菌体重悬于1mL预冷的无菌去离子水中,重复一次,菌体最终重悬于50μL无菌去离子水中以用作电转感受态细胞。取3μg除盐处理之后的质粒DNA加入到制备的感受态细胞中混匀,将混匀液转入1mm电转杯中并置于1250V电压下进行电击转化,电转化之后将菌体重悬于1mL CTT液体培养基中,置于30℃摇床复苏培养4-6h。向复苏培养液中加入1mLCTT液体培养基和1mL融化并冷却至42℃的CTT固体培养基(含1.5%Agar)混匀以形成软琼脂菌悬液,倾倒含50μg/mL卡那霉素的CTT平板(含1.5%Agar),待软琼脂凝固之后将平板倒置于30℃培养箱中培养5-7d至长出单菌落。
挑取单菌落接种到加有50μg/mL卡那霉素的1.5mL CTT液体培养基中置于30℃摇床培养过夜以用于菌落PCR鉴定。分别用3对引物(Colony PCR chk01-F和Colony PCRchk01-R、Colony PCR chk02-F和Colony PCR chk02-R、Colony PCR chk03-F和Colony PCRchk03-R)对其进行菌落PCR鉴定,鉴定结果见图5。
上述菌落PCR引物的序列为:
Colony PCR chk01-F:CAGAAGAACTCGTCAAGAAG
Colony PCR chk01-R:GAACAAGATGGATTGCACGC
Colony PCR chk02-F:GGATCGTGAGTACCTGGAGAAG
Colony PCR chk02-R:GAGCGTCCGGGAGGTCGTGGGC
Colony PCR chk03-F:GCAGAAGTACGTGGGCCTCAGC
Colony PCR chk03-R:CGACGAGCAGGGTGGCGTATCC
菌落PCR扩增体系:
PCR程序:94℃预变性1min;98℃变性10s;55℃(根据引物Tm值设定)退火15s;68℃延伸1min(延伸时间根据所扩增的长度确定,1kb/1min);循环30次;后延伸68℃,10min;最后4℃保温。
实施例5:本发明所述工程菌株DK1622::Km-Ptet-dis427在制备Disorazole Z中的应用
将工程菌株DK1622::Km-Ptet-dis427接种至含有卡那霉素(50μg/mL)的CTT液体培养基中,30℃摇床培养过夜。按1%的接种量,将过夜培养物接种到含有50ml新鲜CTT液体培养基的摇瓶中。30℃,200rpm培养2d之后加入终浓度为0.5μg/ml的无水四环素。继续培养1d之后加入2%的XAD-16大孔吸附树脂,然后继续培养1d至发酵结束。8000rpm离心10min收集细胞和大孔吸附树脂,然后用甲醇提取。甲醇提取液用滤纸过滤,将滤液在40℃下减压旋转蒸干得到粗提物,并将得到的粗提物溶解于1ml色谱甲醇中。
利用0.22μm滤膜过滤之后取5μl用于HPLC-MS分析。高效液相色谱仪型号为UltiMateTM3000RSLC。色谱条件为:AcclaimTM RSLC 120C18,5μm,4.6×250mm;溶剂A为超纯水(0.1%甲酸)和B乙腈(0.1%甲酸);溶剂梯度为,0–5min,5%B,5–25min,5%–95%B,25–30min,95%B;柱流速是0.75ml/min。高分辨质谱仪的型号为Bruker microOTOF-Q II,ESI-Q-TOF MS(电喷雾四级杆飞行时间质谱仪)。质谱条件为:Auto MS2,Mass range(50-1500),precursor ion 2。
采用Data Analysis软件对采集到的液质数据进行分析,以Disorazole Z原始产生菌Sorangium cellulosum So ce 427的粗提物为阳性对照,以野生型异源宿主菌Myxococcus xanthus DK1622的粗提物为阴性对照,提取[M+H]+的峰进行比较和分析,结果显示,Disorazole Z生物合成基因簇(dis427)在Myxococcus xanthus DK1622中能够成功表达,结果见图6。
实施例6:构建的工程菌株DK1622::Km-Ptet-dis427与原始产生菌Sorangiumcellulosum So ce 427产Disorazole Z的量的比较
本发明构建的工程菌株DK1622::Km-Ptet-dis427与野生菌株Sorangiumcellulosum So ce427产Disorazole Z的量的比较主要是采用峰面积比较法,具体如下:首先,对Disorazole Z提取离子流(EIC)的[M+H]+峰(EIC 747.3121±0.05+All MS)进行积分,得到峰面积;然后对峰面积进行比值,比值接近2:1。实验证明,本发明构建的表达Disorazole Z的工程菌株DK1622::Km-Ptet-dis427与野生菌株Sorangium cellulosum Soce 427相比,Disorazole Z的产量提高了1倍。
序列表
<110>山东大学
<120>一株高效异源表达Disorazole Z的工程菌株和构建该菌株的基因簇及其应用
<141>2017-12-3
<160>3
<210>1
<211>48309
<212>DNA
<213>纤维堆囊菌Sorangium cellulosum So ce 427
<220>
<221> Disorazole Z生物合成基因簇dis427核苷酸序列
<222>(1)…(48309)
<400>1
aattttgcgc ggactctttg tattctcgcg caccgcgttg acaccgcgat tttgtggtct 60
ataaaacgag ggcatagcct gactccgtcg agagcatggc ggcgccgctg accgacccgc 120
tctcgatgac gggctgaatg gacatcgtga gaaagtatac ggcacgtggg tagggtcccg 180
cgtgactcgt ggcgttctgc gttctcggcg cgggccgtga tgcgcgaaaa agagaaggag 240
ccatgcggaa aggctgaagg attgctcacc atgcaggcat tcagcctggg gtaagacacg 300
cgctcgttcc tcgaacggcc atcgctttga cctggctcgc gccgctcctc gccgcgcaat 360
cgcgcggcgc agctggccgc gctttggcca atgcgcatgc ctcggcaacg aaggagacac 420
tggttgagca gcgggaatca cgtagagcag gacggcattg ccatcatcgg catggcctgc 480
cggtttcctg ggtctccgga ctacagagga tactggcagc tcctcgagcg ggaagagcac 540
gcgatccggg agatcccatc gagcaggtgg gacccaggga cctattattc ccctgatttc 600
gacgaaccca acaagagcat cagcaaatgg tgcgggctcg tcgacgacat cgccggcttc 660
gacaaccgct tgttcaatat ctccgagcgc gaagcgaaga gcatggaccc gcagcagcgc 720
ctgctcctgg aggagacgtg gcgctgcatc gaggacgccg gcgtgcccct gaggcagctc 780
cgcgccgggg cgacctcggt gtacgtgggc ttcatggcca gcgattacca ccaggaatcc 840
gcggccctga atcgatcgat cgacagctat gccgccctgg ggagctacag ctcgatcctc 900
gccaaccgga tctcctatac cctggggctg cgcggcgcga gcgtggccct ggacgccgca 960
tgcgcgtcct ccctggtcgc gctccacgag gcccggcgct ccctgcagcg aggcgagagc 1020
gacttcgcga tcgccgcggg cgtgagcctc aacttccacc cctggaagta catctccttc 1080
tccaggtcgc gcatgctcag cccggacggg ctgtgcaaga cgttcgacag ggacgcgaac 1140
ggctatgtcc ccggagacgg ggtgggcgtc ctcctcctgc ggccgctctc cagggccatc 1200
gcggcaggag accatatcca cggcgtcatc tcgggctccg cggtcaatca caccggcgcc 1260
tcgcgttcca tcaccgcgcc tcgggtggcc tcccagcggg atgtcatcct cgaggcgtac 1320
gaggacgcgg gctggagccc cgagacggtg acctacgtgg aggcgcacgg caccggcacc 1380
tccctcggcg acccgatcga gctggaggcg ctcacccagg cattccgccg ccacacacag 1440
aagcgccagt actgcgggat cgggtcggtc aaatcgaaca taggccacct cgaggccgcc 1500
gcgggcgtgg ccggggtcat caaggtgctc atgatgttga agcaccggac tatcccccgg 1560
acgctgcacg tcaagacgct caaccccctc atcgccttcg acgagacgcc cttcgtcgtc 1620
gcgacccgca gcagcgaatg gcgatcggcc gatgacctgc cgctgcgggc aggggtgagc 1680
tcgttcggct tcggcggcgc gaacgctcac gtcctcctgt ccgcgtacga gcgcaggtcc 1740
gcggagcgcg gccccctcgg ccccgctgag gagcgcgaag gcaccctctt catcgcctcc 1800
gcccagtccg ctccttgcct gacgaggacc atgcaacgct ggtcgaccct cgccgacgag 1860
ctcctcgaga aggagagccg ggagatctcg ctccgcgacg tgggcgcgac gatggccacc 1920
gggcgggaga gcttcgcgta tcgtcacggc ttccacgcgc gcgacgagca ggagttccgc 1980
cgcctcatca aggaggcgcc cggccgcctg gaaaagagca ggccgcctcg ctggataacg 2040
cgcttcggcg ctcctgccct caagccaggc gagcccgtct cgacgctgct cggcgcgcga 2100
cacctgatcg gccgccacat cgaggccatc cggatctccc tccaggagct cgatacaggg 2160
cgccaggtgg cgcggatcta cgaaggcgac agcgcgcccg agcaccacga gccgctgcat 2220
gcgttcctct tcgcgcacgc gtacatgtcg gcgctggccg atctgaatct gaggccgtgg 2280
gcgaccaccg gtgatggtca cggcatctgg ttggcgctcg cccagagcgg gatcctgccg 2340
ctgagcgcga tcgtggcggg cctccagggc ggcgaggagt ggcgacgcgt cccgcctcgc 2400
cgccccgcgc tgcccttctt cgatcccgtc cgatcgacct acctgatgcc gtatctcctg 2460
gacgccgagt acctgtcttc cctcgtggag gggctgccgg tgcacacggc gacggccgag 2520
ggcgtgctcg cgcgagccag ggcgctgctg cgcgctcagt tcaccttcaa gaagttcctg 2580
gacgagtggt cgccggcgct gcgagccctg gacacgacgc ccgagcgcct gctccaggag 2640
gagctccgcg ccccggacgc gcgcctgtcg ctcgcggcca tcgtcgcgca gagcgccatg 2700
cgcaagctga accgtcgatg gcagctgtcg gaggcgggct cctccggcga cgcgcgggtg 2760
aacgagctcg tggacctcgt cgtcgacggg ctcattcctc acgaggcggc ggtgcagctc 2820
gtcctcgacc ctcgaccgga cctccacggc atcgccgagc tcctgcgcca gcgccaggag 2880
atgctcgatc tcgatcagcc ctacgccgtg ctccggaggc acagcgagcg cctcgacgag 2940
cgggagatcg gcgacttccc ggggtggatc cagcgcatcg tcgagctcga gccagcgagc 3000
cttcccctcg acgacggcgt cgcgttcctg gagctcgggc agctggcgcg gccctctccc 3060
cgggtatcgg ggccggggct ggccatcccc gtgctggatc agcccctgca gctcacggcg 3120
ctgcgcctgt ggctgcaagg gaccgacatc cggtgggagg agctctttcc ggacggccag 3180
ttctcgaaga tcccgctgcc gggctacgcc ttcgacagga ggcacttctg gttgccggag 3240
ggcgaaggcg tcccctcgcc ggtcagggct gccgggcaca tgagcggccg cccggaggag 3300
gcggccgccg ctccgccgct cccggccgcc cagggcaccg acggcgccct cgtctccacg 3360
tgggccggcg cgcgccccgc ggcgagcgcc gagccgcgcg cggacgctgc gggcgcgacc 3420
ccggcgcgac catcgccctt cacgtccgag gagaggccag cccaggcgga gcgagcgctc 3480
acctcgacgg accgcctggt ggccgatcac gtcatctcgg ggcgctccat cgtgcccggc 3540
gccctcctga tcgagatggc cctggaggcg tcgcagcggc gtcacgctcg cccggcgacc 3600
ttcctgaagg acgtggtctt ccagcgcgcg gtcccggtgg gctcctccgt ggatctcacg 3660
ttcgagatcg agcctgaacg cgggcggttc agcgggaaac acgccggtca cagcgtctgc 3720
cgtggagctt acgggcacga gcccccgccc ccgctggagg ccctcgacgc ggcggcgcgc 3780
gggtgcgaac gccgggcaga ccccgagctc tacagcgacc tggcgcgcgt cggttatcgc 3840
tatggcgaga gcttgcaggt gatcgccgcg gtcgggcggg ccggcacgcg tcacatcgtc 3900
gagctccgcc cggcggcggc cccctgcgag cgtctcgccg gcttcgaccc cgcgctcttc 3960
gacggcctcc tgcaggcggc gctcgtcgtg gggcggggcc tcgggctgtt cagcgggagc 4020
gacgcgctct acgtgccgca ggccatcggg ctgctcgagc agctcgcccc gctgagcggc 4080
ggctgcctcg tctgcatcga tgagcgcgac gtcgcgatcg aggaccacgg catggtcgcc 4140
gacctgcgcg tccacgatct ctcgggagcc ggcctgctcc gggcgaatgg cgtcttcttc 4200
cgcagggtgc cccgaggctt cctgggcagc tcgcctgaag cgcccgccga gcgcgccccg 4260
gaggtgcggc ggcgccacga cgaggacgac ccgtccaggc tcaccgcggc ttgctatcta 4320
cccgtctggg agcgacagcc gccctccgat cgcggcggta cagccctgag ccgccgcgcg 4380
gtggcgatcc tccgctcgga ggcgcagtcc gcggcctggc tcgagccgct gcgagagcgc 4440
tatgcgcacc tcaccgtcgc gcggctcagc agctccccgg cgcaagcggg cgacgacggt 4500
cggctcgtcc tgcgcgacga ccaggaagag gacttctcgg cgctgctgcg ccgggtagag 4560
cgagaggcgg ccggcgaggc cgcggacatc tactttctgg cagcgctcac gcccgcggac 4620
gatctcccgc ccccggcgcc tgggccgctc gagccggcgc tcgccccgga ggacgaggcc 4680
gtcgcgcgcg gcatgttcct gctggccaag gccctcgtga agagcggggt gccccatcat 4740
ctgatcgtcg gcgcgcggcg ctgccaggtg gtgctgcacg acgaccgggg agaagggttc 4800
cgccatgagg tgcttggcgg catcgccagg accctggccc aggagaaccc gcagctccgc 4860
gtccacctcg tggatctcga cacagccgat ccgcgctcgt gcgcgagcca cctcatcgag 4920
gagcgcggcg tgctcgacca ggtagactgg gtagcttacc gcggcggcgc ccgtcacgta 4980
cgcgcgttcg cgcagctcga ggaccccggc gcggcgccct cgccgttcca ggacggtcgg 5040
gtctatctgc tgctcggcgg cgccggaggg atcggcctcc gcctcgccga gcacatcgcc 5100
tctcgggtcc atgctcggct cgtcctggtc ggccgctcgg agctccgcga cgaggcgaag 5160
cgccgcctcg ccgcgctgag cggcgagggc agcgaggtcc ttcacctgat cgcggatatc 5220
ggcgatccac ggcagtgcca ggaggtcgtg gcggcggcgc gccagcgctt cggcgccatc 5280
cacggcgtgg tgcagctggc cggcgtcgtg gaggacaggc tgctcgccgg caagccctgg 5340
gactcggtgc ggcgagagat ggcgccgaag gtgcagggca catggtcctt gcaccggctc 5400
acccagggcg agccgctcga tttcttcgtc accttctcct ctgtggtctc cctcctcggc 5460
aaccgcggcc aggtgggcta cgcggccgcc aacagcttcc tcgacgggtt catccaccac 5520
cgagcccggg ccggcgcgcc aggcaggagc ctcggcgtga actggaccct gtgggaggac 5580
ggcgggatgg gcgcgaaccc cgagatcgcg cgtcgcttct cggcgcgcgg gctcccgccc 5640
atcggcgagc gcgcagcgtt ccacgcgctc gaccggctga tgacccggtg cccgtcgcct 5700
caaggggtcg tcctcgctcg agctgcagag cacctcctgg cgagaccgtc gacccgacct 5760
gccgcacacg cggtccatca cgagccggcg cgtgatggcc tggctcgaaa ccgagataac 5820
gaacaagggc tggcaaacgc gagcatggca catatgtcgc aatcatcgag ttctcgtgag 5880
aaggtcctcg ctgcggcggg agacgacggg caccgggcgg cgcgcatcga gggcgatctc 5940
cgccggctcg tcgccgccaa ggtccaggcg gactcgagcg atatcgacgc ggaggagtcg 6000
ttcttctccc tgggggtcga ctccgtggct ctccaggaga tcacggagca gctcgagcac 6060
gtccatgggt cgttgccgcc cacgctgctc ttcgagagcc cgaacatccg caggctggcc 6120
cgctacctcg cggagcgcgc ctcctcggcg gtcgccgcgc ccggggagga ggaccggggt 6180
ccggcgccgg cgcccccggg cgcggccgcg cccgcgccgc ccgccgcgcc ccctgtcgtc 6240
ccctcccccg ccccggcagc tcccccggac gccgcagccc acgccgcggg ggcagagccg 6300
gtcgtgagca ggcaggagcg cgatgcgccg ggtatgccgt ccgccccgct catcaggcgc 6360
ccgcggccat cctccgcgat cgcgatcgtc ggcatgagcg cccgcttccc gaagtccccc 6420
gatgtggacg ccttctggga gaacctccgc tcgggccgcg attgcatcga ggagatcccc 6480
gccgagcgct gggaccaccg gcgctatttc gcggagaccc cgcagcccga caagacctac 6540
gggaagtggg gcggcttcat cgaggacgtg gcctgcttcg acccgctgtt cttcaacatc 6600
tcccctcgtg aggcggagct gatggatccg cagcagcgcg tcttcctgga gtgcgcctgg 6660
gcgaccatgg agcacgcggg ctacggcgat ccgcgcgcgt acaaggacga cgccgtgggc 6720
ctgttcgtcg gggtgatgtg gaatgaatac agccgcatcg gcggccggct cacccaccag 6780
accgggcgct acgccggacc gggctcgctc tactgggcga tcgccaaccg ggtctcctac 6840
tggatgaact tcaccggtcc gagcctcgcc atcgacacgg cctgctcctc gtcgctcgtc 6900
gccgtccacc aggcctgcgc gagcatccag aacggagagt gcgacatggc ggtggccggc 6960
gggatcaacc tgtcgatcga tcccgacaag tatctctatc tggcgcagtc caagttcctg 7020
tccctcgacg ggcgctgccg cagctttggc gagggcggca ccggctacgt gcccagcgag 7080
ggtgtcggcg ccgtcctcct caagccgctg gaccgcgccc tgagcgacgg cgatcacgtg 7140
tacggcatca tccgcggctc ggcgctgaac cacggcggca gggcgaccgg gttcaccgtg 7200
ccggatccgg aagcccaggc gaggctcgtg ttcgacgcgc tgcaacgcgc gcgcgtgtcg 7260
cccgatcagc tgggctatat cgagtgccac ggcacgggga cggcgctggg cgatcccatc 7320
gagatcgccg gcctcagcaa ggcgttccgc aaggccggcg ccacgcgccg gagcttcccg 7380
atcggctcgg tcaaatccaa cctcggccac ctggaggccg ccgccgggat cgcggcgttg 7440
atcaaggtcc tcctgtccat gcggcaccag gcgatcccca ggagccttca tagcgagacc 7500
aggaacccca acatcgattt caacgacgtc ccgttcgagc ccgtgaacga gcttcgccca 7560
tggcaggcgg acggcggggg ctcccgcttc gccggcatca gctccttcgg cgcgggcggc 7620
tccaacgccc atgccatcgt cgaggcctac gagccgcatg tgcgccgcgg cgcgggcgag 7680
gacgccgcgg gcgaggaggc cctgatcctg ctctcggcga ggaaccgcga gcggctcaac 7740
gccgcgacgg agcggctgcg ggattttctg cgcgagcagc cagccgggtc cccctccctg 7800
ggcgacatgg cctatacgct gcagctgggg cgccaggcca tggatcagcg gctggcgatc 7860
atcgcctcca gccgggaaga gctgctcgcc aagctggacg ccgtgctctc cggtcgcggc 7920
gacgtgcccg gcgtgtttca aggtcaggtc cagggccaca agaccgcttc gttctcgatg 7980
gatggggacg acgaggatcg tgagtacctg gagaagctcg tccgcaacca caagctgccc 8040
aagctcgccg gcctgtggat gcaggggctc tcgatcccct gggagcacct tcaccagggt 8100
cgcggccgca agcggaccgc tctgcccacg tatcctttcg cgcgcgagca ttactggttg 8160
cccagcgtgg agggctcatc ctccgcgcac gccgcgcccg cgcccgtgag ctccgccccc 8220
gcgctcggag ggcccgccgc gcgcgtggaa gcgcccgcgc cccgcgcggc agcaggctct 8280
ctcgagggct tcttcttcca ccagcaatgg tcgctggctc cgctggaccc ggcgacggcg 8340
gcgggcggcg cagccgtcca gaccgcgctc gtgatccata cgccggaggg cgcgcgcctc 8400
gcggacgccc tggccgcgaa ccatcccggt gcccgtatcg cccgtgtcct cctcggcgcg 8460
cagcgggaga ccgccgccca cgacctcccg gacgctcggg gcagctcggc cgccagcgcc 8520
gtacggccat ccctcgcggc ttcccgagcg gtggaggttc aagccgagga tcccggcgcc 8580
ctggagcggg cgctccggga cctggccgcc gcgggcctcg accgtctcga cgccgtgtat 8640
ttcctcggcg ggctgtccgc gcaggagccc gctgccggcg atctggacgc cctggagcgc 8700
tgccagcagc gagggttgct gtccctgttc cgcctggtga aggccctgga cgccctgggg 8760
ctcgcttcct cctcgtgtca cctgaagatc atcaccaatg atgtctgccc ggtgcgggcc 8820
ggggatcccg agcgtccgct ggccgcgggg atacacggtc tggcccggtc catcgtcaag 8880
gagtaccccc ggctcaaggt cagctgcatc gacatcgcga ccgaggagct cagccgcccg 8940
gaagaggcgc tgatcagcgc cgtgatcgcc gagcctggtc gcctgcgcgg caaggaggtg 9000
gccctgcgag gcggcaagcg cttccagcgc tcgatggccg ccctgccgct ggcgccgccc 9060
gcggccgagc cgttccgcca gggcggcgtc tacctggtgc tgggcggcgc cagcggcctc 9120
ggctacctgt tcagccagca cctcgcagag gtccatggcg cccggctcgt gtggctcggc 9180
cgtcgcccgc ccggcgacga cattcgagcg aacatcagcg acgtcgaggc gcgcgggggc 9240
aaggtcctct acctccaggc ggacgccggc gacccgacct ccctgcgcgc ggctgtcgcg 9300
cgcgccaagg cgcacttcgg cgccctccac ggggtcgtcc attccgccgt cgtcctcggc 9360
gaccatccca tcgccacgac cgatgaggcc acgttcaccg ccggagtccg cgccaagatc 9420
accggcagcc tcgccctcca ccaggccgtc gccggtgagc cgctcgattt cttcctctat 9480
ttcggttcga tcgcctccta cctgaacaac ggcggggcca gcgcgtacgc cgccggttgc 9540
accttccagg acaggtacgc gctcttccac cgcgcgcacg cgccctaccc ggtcaggatc 9600
atcaactggg gatactgggg caaggtcggc gcggtcgccc gcaccgccga tgtccatgat 9660
cagcagttcg gcgccatcgg ggtcggcgcc atcgcgcccg cggacgggat ggaggccgtg 9720
cgccgcgtcc tcgcgcagcg tgtaccccag gtggtggccg tgcagctcac gcgcgagccc 9780
acggacctct tcggctacga gctgagccac atgacgaccg tctacccgga gcgcttcgag 9840
ccgctgctcg tccggagcgt gccgcgcatc cagcccgagc tcggcgccgt ccgcgcgctg 9900
ctgagctgcc agacctcgtt cgacaaactg gagcgcttca gcgaggatct gctgctgagc 9960
gcgttccagg acatgggcgc cttccggacg ggcggccgcg agtccgcggc agccctgcgc 10020
gagcggctgg ggatcgcccc ccgctacagc cggctctacg attcactgct cgcgatcctc 10080
gagggagccg ggtacctccg tatcgaaggg gacggcgtgc tcatcagcga ccgggtgacg 10140
cgcgagcagc gcgacattca ccggcagatg ctgcagctcg ccgccctgcc ggagatcgag 10200
ccgtacgtcc gcctgctctg ggcgtgctac cagcgctacc ccgagctcct ccgcgcgcag 10260
gtggcggcga ccgacgtgct cttcccgcag ggctcgatgg agctgatggg ccggctctac 10320
aagggcaact tcaccgccga ccatttcaat gagctggtca tcaagagcct gctctcgttc 10380
ctggatgctc gcctcgcgcg gctgcaaaag ggcgagaaga tcgcgatcct cgaggtgggg 10440
gccggcaccg gcggcaccag cgcgtccgtg ctcaaggcgc tcgatcccta cggggcccat 10500
atcgagtact tctacaccga catctcccgc gccttcacgc agtacggaaa gcgccagtac 10560
ggcccgagcc accccttcgt caccttccag ccgctcaacc tggaagaaga cgtggtggcg 10620
caggggtact ccgcagcgcg cttcgacgtg gtgctggggg cgaacgtcgt tcacgccacc 10680
aggaacctgc gcaacaccct gcagagcatc aagagcctcc tcaaggccaa cggctggctg 10740
atcctcaacg agatgactcg cgtcgtccac ttcctcaccc tctctgcggg tctcctggac 10800
ggctggtggc tgttcgagga cgagatagag cgcatgaagt ggtccccgct gctcagcgcc 10860
tcgatgtgga agggcctgct cgaggaagag ggattcggcc gcgtcgcgcc gatcgatcac 10920
agcgacggcg ccgcctcctg ggacatccag agcgtgatcc tcgccgagag cgacggcgtg 10980
gtccgcgggc gacgccccga gcacgtcgcc tcccgtccgg agccgtccgc cgcggcgccc 11040
gcgcccgcga cgcccgcgcc cgcggcggtc gcgccggccc ccgtcgttcc cgccgcggag 11100
caggtcgcga gccctcagcc aatgtccttg cgcgccatcg aggacaggat cctcgagggt 11160
ctcgcgcaaa cgctgcagct caacaggtcc gagctcgacc cggacgtgcc cttcacgacg 11220
ttcggcgtcg actcgatctt cgccgtggag gtcgccggcg tcgtcggccg cgagctcggc 11280
ctcgagctga ggaccacggc cctctacaac catccaaccg cgcgcgcgct cgccgcgcac 11340
atcgcggccg acttcgctcc cgtacaggcg gtcgccgccc ccgcgacggg aacggcgccg 11400
gcggcgcagc cgcagcgggc acaggctcag ccggcgcagc ccccgccggc gcagccgcgc 11460
acgcccgtcg agccgtcgat gccggctcac cggccggcat ctccgcggcc cgacgccgtc 11520
gcgcaggtcc gacaggtcac gatggatgcg ctcgccgagg cgctggccat cgatgcgcga 11580
gagctcgaca tgagcggtaa cccggcagag tacggactgg acgcgcagca ggcggtcgcg 11640
gcctcgaacc gcatcaatca ggtcctcggg acgagcgtca ccgccacgga gatcctccgg 11700
tgcgaggcgc tcgaccagct cgtggaccac ctcgtcgcgt ccctgcccgc gccccgtgga 11760
gccaccgaga cgcgcgcccc catcgtcgcg gcgccccccg cgccgacgcc gccaccagcg 11820
ctcgccgcgc ggcctgtccg cagcatggac atcgcggtgg taggcatgtc cggccggctc 11880
cccggcgccg agaccgtcgc cgacttctgg cggaatctgt gcaatgggca cgacgcgatc 11940
ggcgaggttc cgcccgagcg ctggcccctc gacgggtttt acgatcccga tcccgacgcc 12000
gccgcgcgca gctacagcaa atggggcggg ttcctgagcg gcatcggcga ctttgacccg 12060
ctcttcttcg gcatctcgcc gcgcgaggcg gagctcaccg atccccagca acgcctcttc 12120
ctccaggaag cctggaaggc cctcgaggac gccgggtaca gcgccgaagc cctgaacggg 12180
cgccggtgct gcgtcttcgt ggggtgcaag gacggagact atgtcaacaa gctcgacgcg 12240
tcggcggatc cttcctaccg gctcatcggg aacacgctgt ccatcctgtc ggcgcgcatc 12300
tcgtacttcc tcaacctcaa ggggccgagc gtcccgatcg acaccgcctg ctcgtcgtcg 12360
ctcgtggcga ttcacctggc ctgccagagc ctgatcagcg gcgccagcga gctcgccgtg 12420
gccgggggag tcgccctcat gaccaccccg atcagccacg tcatgctcag caagaccggc 12480
atgctgtccc ccacgggcag atgccgcacc ttcgacgact ccgccgatgg gctggtcccg 12540
gcggaaggcg tggcggcggt cgtcctgaag cccctcgacg ccgcgctgcg cgaccgcaac 12600
cacatctacg gcgtcatccg tggctccgag gcgaaccagg acggcaagag caacgggatc 12660
acggcgccca gcaccccctc gcaggcagcc ctcgagatcg aggtctaccg caagctcgac 12720
gttcacccgg agaccatcgg ttacatcgag gcccacggca ccggcaccaa gctgggcgac 12780
cccatcgaga tccacgcgct cacggatgcg ttcgccgcct tcaccgacaa gaagcggttc 12840
tgcccggtcg gctcggtgaa gaccaacatc ggccacacgc tggccgcgtc gggcgtggcc 12900
tccctcatca aggtgctctg ctgcctgaag caccgcacgc tcgtgccgtc gctccactac 12960
gaccggccga gccggcatat cgacttcgac gccagcccct tttacgtcaa caccgcgaca 13020
agggactgga tccccgccgg cgaccacccg cgccgggcgg ccatcagctc ctttggcatg 13080
agcggcacca acgtacacct ggtcgtcgag gaggccccgg cagaggcgga ggtcacggag 13140
cccacggtgg ccccttacac cctcgttccc ctctcggcga aggcgccggg gtcgctccac 13200
cggaaggtgg tggatctgct cgcctggctc gacgccggcg gcagcgaccg cgagctgggc 13260
gacatcggat ataccctcgg ggtcggacgg acgcacttcc ccttgcggct cgccttcgtg 13320
gcgcgcgaca cgcgggatct gcgcgaccag ctcgcggcgt ggctcgcgcg ctacccgacc 13380
gcggacgacg cgccggcgcc ggccgggcag ccggatcccg ccttcgagca gctggctggc 13440
cacctggtga aggagctccg cgacgcgcct ccagcgcgcg ccgacgcata ccgcgagaag 13500
ctgcaggcgg tggccaacgt gtacgcgacg aggcacgacc tcgaatggac cgcgctgtat 13560
gccggtcagg cgcgacgcct gctgtctctg cccacgtacc cgttcaatgg ccgccggtac 13620
tgggtgaacg agcccctgcg cagcggcgcc gagcaagaga cgacgctcgc ggcaagcccc 13680
gctccggcgc agcgaccgga gcccgcgccg gccgctcgcc cgtcgacagg ggcaggcgcg 13740
gaggcaaggc tgccggagcg cgcggaccag cacgcggcct cgatcctcta tttccggccg 13800
tcctgggagc ccgcggccgc cgagccggcg accgatcagc tccgcggtcc ggtcctgctc 13860
ttcgacaccg acgagggggt gcgtgagcgg ctgagagacc gctgcggtcc cgtcctcctc 13920
gtcaagccgg gcgccgagtt ccgcgagctg ggcgacggga gctacgagat cgcccctgac 13980
gaggagtcga gctatcgccg cctcgtcgat gcctgcgggc ggcgaggcct gctgccgcgc 14040
cacgtcgtgc acctgtggcc gctcactcga gctcccgcgg cgggcggcgc gacagccccg 14100
ttcttccagg cgacctctct gtgccgcgcg ctcgccgccc atctcccggc ccacggcggc 14160
gaggtcactg gcatcctgta cgcctacagg cggcgcggtg accggctgga ctcggcccat 14220
gcggccatgg gcgggctggc cgagagcctc cggctcgacg ttccgcacct ccgcctgagg 14280
gcgctcggcc tcgccccgca gccgctggac agcgccgcgc tgacagacat cctcctcgcc 14340
gagatggccg ccccccacga gggcgcggtc cgctacgaag ggcgagagcg gcagatccag 14400
cgcgcccggc cgtggcggcc cagcgaggag gcgaaggcgc ctctccgcag ccagggggtt 14460
tacctgatca ccggcggcgc cggcggcctc ggccgggtgt tcgcagagca cctcgctcgc 14520
cgcttccagg ccaggctggt cctttgcggg cgctctcccc tgacctcggc cggcgaggat 14580
ctgctccgcc gcctcacgca gctgggcgcg gaggtcgcct acatccgggc tgacatcgcc 14640
gatcgcgagg acgtgtttgc cctgctgggg cgcgtcgagg cccggttcgg cgcgctccat 14700
ggcgtcatcc acagcgccgg cgtcacggcc gacgccaacc tgcggaacaa gggtcgcgag 14760
cagatggccg cggtgctcgc gcccaagctg ctcggcgccc tgcacctgga cgacgccacc 14820
cgccaccgag agctggactt cttcgccctg ttctcctcca tgaccgccgt cctcggcaac 14880
atgggccaga cggactacgg ctacgcgaac agcttcctgg accacttcgc ggcgtggcgc 14940
gaggccgagc ggcagggcgg ccgccgcgcc ggaaagacag tgtccatcaa ctggccgctc 15000
tggcgagaag gcggcatgag cgtctcgcag gagatgcagg cgctgctggc gtccgccttc 15060
ggcatgaccg cgctcgatag cgaggcgggc gtcgacgcct tcacgcgcgc cgtggcctcg 15120
gcgtacccgc aggtcctcgt cctggccggc gatgaggcca ggatccatcg cagcctgggg 15180
ctcgccgggc cgacggcgcc cgccggcgcg ccgcgccccg cggcctcgcg ggcgacaggg 15240
gccaccgtgg aggcccgcgc ggaggcgccg tccagcgccg ccgctgctcg gaccgcgctg 15300
gcggagcggg tcagggcgct cttgctgcag gcggtctcca gggtgctgaa gctcacgccc 15360
gaagagctga gctacgagac gccgctgatg gaatatggcc tggagtccat caacgtcatc 15420
gtcctcgcca atcacctgaa ccgcacgtac ggcctcgccc tcacgccggc gcgcttcttc 15480
gagcacgaga cgctcgcctc gctcggcgcc tttctttgcg aggcgtacgg agatcacctg 15540
gcccagcgcc tcggcgtcac gccagcgccg gcggtcgagc tcccggccgc tgctgccgag 15600
gccccggagc ccgagcggcc ggcgccggcg cccgcggcct cgagcgcgcg ggagccccgg 15660
cgccccgagc cggccgtgcc cgctgtcagc gccggcggcg agccgggcgc ctcttcacgc 15720
gacgagcccg tcgccatcat cggcatcagc ggggcgctgc cggggtcgag cgatctgaac 15780
gcgttctggg agcacctcga ggccggtcgg agcctcgtct ccgagctgcc cggagaccgc 15840
tgggactggc gcgctcacga cagcggcgag ccgaaccgca aggggctgcg ctggggcagc 15900
ttctacgagg acatggacaa gttcgatccc atgttcttcg ggctctctcc caaggaggcc 15960
gagctgatgg atccgcagca ccgggtcttt ctgcagaccg tgtggagagc catcgaggac 16020
gccgggtacg gcccctccgc gctgagccag agcaacaccg gcgtcttcgt gggcgctgcc 16080
gcggccgact acctcgatct gctgaacgga caccggaccg aggcgtacgc cctcaccggc 16140
acgacgcact cgatcctggc gaaccgcatc tcgttcctgc tcaacctgcg cgggccgagc 16200
gagccgatca acacggcgtg ctccagcgcg ctcatcgcga tccaccgcgc cgtggaggcc 16260
atccattccg gctcttgcga tctggccatc gccggcgggg tcaacgccat cctcagcccc 16320
accaccgcgc tcgccatcgc gaaggcgggc atgctcagcc cggacgggaa gtgcaagacg 16380
ttcgacaaga gcgccaacgg gtacgtgcgc ggcgaaggcg ccggcgccct gctcctcaag 16440
ccgctccgcc gcgcgctcgc cgacggcgac catgtctatg cggtcatcaa gggcagcgcc 16500
gagaaccacg gcgggcgcgc caactcgctc accgcgccca acccgcgcgc ccaggccgat 16560
ctcatcgtcg cggcgtttcg caaggccggc gtcgatcccg cgacggtcag ctacatcgag 16620
acgcacggca ccggcacggc gctgggcgac ccgatcgaga tcaacggcct caagatggcc 16680
ttcgagcggc tctacgaggc ccacggccgg cccgcgcccg cggcgcccca ctgcgcgctc 16740
ggctcggtca agaccaacat cggccacctg gaggcggccg cggggatccc cagcgtcttc 16800
aaggtcctcc tggcgatgaa gcaccgcaag ctgcccggga gcctgcacct cgacgacctg 16860
aacccctata tcgagctcga gggcagcccc ttccgcatcg tcacgcgcac ggaggagtgg 16920
aagcccgccc tggacgggga cgggcgcgct ctcccgctgc gcgccggggt cagctcgttc 16980
ggcgtcggcg gctccaacgc ccatctggtg ctcgagtcgt tcgacgcgga cagctccgga 17040
ggctcgcccg cggccgaggg gcggcgcggc cctcacctca tcgtcctctc cgccagagac 17100
gaggagcgcc tgaacgacgc gatcgacgcg ctcgtcgccc acctccgcgg caccgctccg 17160
gagatgcgac cctcgctgga gcgcatctcc tatacgctgc tcaccggtcg tgacgtgatg 17220
agcgcgcggc tcgcctgcgt ggcggccgac acggaggagc tcatcgactt gctctcccgc 17280
caccgggccg gccagggctc gatcgggctc ttcaccgggc aggacgacgc gccgcacgcc 17340
gcgacgccga tgctcatcga gggggaggaa ggcaggcagt tcgtggaggc gctcgtccgc 17400
aaccgcaagc tgccgcagct cgcccggctg tgggccgccg ggctcacgcg cctcgactgg 17460
tctcccctct tcggcggcgc ccgcgtgagg cgcgcgcctc tgcccaccta tcccttcgcc 17520
agagagcggt actgggtgcc cgtcgatgaa ggcaagggcc gcgcgggcca gaacggcgtc 17580
catcctccgg cggcgagcgc ccctccgccg gcgagcgccg ccgccgcgcc gcacccgatg 17640
atcgacgccg agctctccag cccggatggg ctcgtgtacc gcaaggacct cgacgccggg 17700
gtcttctacc tgagggatca cgtcgtcgcg ggcaacatca tcctgccggg cgtgggtcac 17760
ctggagctcg ctcgcgccgc cggcgagctc gcgggcggcc ggccggtccg cgtgatccgc 17820
gacgtcatgt ggatcaagcc catcctgctc gacgggccgc ggcacgaggt ccgggtcgcc 17880
atcacccctg acaagcaggg agtcgagtac cagatccgcc acgagggcga gggccccgcc 17940
gcgctctact cgcgcgggag gctcgcctac gagccgccca cggacggccg cggcgccccg 18000
ccccggtacg atctcgaggc catacgctcc cgctgccggg agctcaggga tcacgaagcg 18060
ttctatcgcg ggtaccggga ggccggcttt cattacggcc cctcgttccg ggtcaaccag 18120
gaggtgcgcg gcaacgagcg ggagtcgctg ggcacgctgg tcttgccgga tcacctgcgc 18180
catgagttct cccggttcgg actgcacccc tccctgctgg acgcctcgtt gcaagccatc 18240
accgggatcc ggctcgacgt cggccgcgag gcgccgtccc tgagcatccc gttcgccctc 18300
ggccagctcg agatcctggg gccgttgccc ccggtctgcc acgcgtacgc gaccctgggg 18360
tcgcggcgcg gcgagggcgc gcgcgaggtc ctcaagttca atgtggccat cgtcgacgag 18420
acgggccggg ccctggtgcg catcaccgac ttcagcgcgc gcgccttcaa gcaggagcag 18480
ggccgcgcgc ccgccgcgcc cgccgcgccc gccgcgcagc cgctcagcta ctaccacgcc 18540
gcctggaccc aaagagcgct ttgatcaccg agggaacttt catgtccagc aacctccgcc 18600
ccacagacac gatcctcgtc ttcctgccgg aaggagcggc gtccggcggg ctcgacgagc 18660
aactgaaggc gcagctctcc ggtgcgcacc ggccgttctt cgtccggccc gcggagcgct 18720
tcacgtcgct cgatccgcgc acctacggca tcaacccggc tgacccggag gaccaccggc 18780
ggctgttctc ggcgctggag cagcatcacg ccctgcccac gcacatcctg cacgcgggca 18840
actgcgtcgg cggcggcgcc ggggcggccg gggaggacga cgcgttcgcg accctgcgag 18900
agcggctgga cgaggagctc gggcggggcc tttattcgat ggtcgcgctg gtccaggcca 18960
agctggcggc gaacccgtcc ggcgccaccc gctgcgtgtt cgcgttcacc gccgacgaga 19020
agcgccctcg ccctcatcac gaggccgtga gcggcctcgc cagggccctc acgacggtcg 19080
atcaccgctt cgagctggcg acggtgcaga tggaccgctg cgacgcggcc acagtcgcgc 19140
gccggctcat cgacgagctg acctcccctc atcaccgcaa tggcggcgag gtgcgctaca 19200
gggacgggca ccggtacagc cacgagatcc agccgttcga ggccgctccg cgcgctccgg 19260
agcccacggc cgacctgccg ctgcgcgcgg acggcgtgta cctcgtgacg ggcggctcgg 19320
gcggcctggg gatgctgttc gcccggcatc tcgcgagcac ctaccgcgcc cgcctggcgc 19380
tgagcggccg cgctccgctc gacgacgaaa ggcgcgccat gctcgccgag ctggcgtcgc 19440
tcggcggtcg cgctgtgtac gtgcaagccg acgtgggcga cgcggcggac acccgtcgcc 19500
tgatcgccgc cgtcgattcg gagttcggcc gcctcgacgg catcttccac tgcgcgggcg 19560
tcgcggaccg caccccgctc gccagggcca ccctcgcgga tttcgagcgg gtcctgcgtc 19620
ccaaggtcca cggcacgctc cacctcgatc tggagacgcg cgatcgagag ctcgacgtct 19680
tcgtcctgtt ctcctcgatc tcggcgctgg tcggcgactt cggcgccggc agctactccg 19740
cggcgaactt cttcctcgac cgcttcgccg aggcgcgcga gcacctgcgg cgcagcggcc 19800
tgcgcgccgg acagacgctg tcggtcaact ggcccctctg gcaggacggg ggcatgaagc 19860
tgcaggagca ggacaaggct ctgtacttcg agttctccgg catgggcgcg ctcgaggccg 19920
cccaggggat cgcggccttc gaggacgccc tccgggccgg gcgcccccag ctgctcgtga 19980
tgagcggcga ccgcaggaag atcgatcgca tcctgcaggc gcgcgagcag cggccggagc 20040
ctccgccagg cgaggagcgc cgacggcccg acgccgaggg cgccgcgacg ccgcgctcgg 20100
accgccggag cgccgccgcg ctcccgaagt ccgccgcgag ccagggtggc ccagccaggc 20160
cggcccctcg ggccgcgctg cagcgcgagc agctcgcggc cctgacccgg gattacctgc 20220
gccggatgct ctcgcacgcc accaagctgc ccgtggagaa gatccacgcg gacagggacc 20280
tcgaggacta cggcatcaac tccctcatga tcatggagtt gaactcgctg ctcgacaggg 20340
atttcgactc gctgccgcgc accctcttct tcgagtacaa gagccttgcc gagctggccg 20400
ctttcttcgt caacgagcac gaggcgcggc tccagcagct cctcggcgcg cccccggcgg 20460
cggcgccgcc cggcgaggat cacccgtcgg cggaggagag cgcgacagga gatgtcctgg 20520
atgcagggcc ggagcccacg ccgcccgcgc ccgccgcgcc cggacaggag gacctcggcg 20580
tcgcggtgat cgggttcggc ggccgcttcc cgcaggcaga cgatctcgac gcgttctgga 20640
gggtcctcag ctccggcgtc gattgcatca ccgagatccc gagcgagcgc tgggactggc 20700
gcagctacca cgacgcgacc ccggggacgc cggggaagag ctactgcaag tggggcggct 20760
tcatcagcga tgtggatcgc ttcgacccgc tcttcttccg cctgtctccc cgcgccgcgc 20820
acagcatgga ccctcaggag cggctcttcc tgaaggtggc ctgggagacc ctggagcacg 20880
cggggtacac cgtcgatcgg ctggcgcgcg ggccggaggc gccgaggggc gcaggccagc 20940
gcaaccgggt gggcgtcttc gcgggcgtca tgtggggcga ctacggcaag cacgggcacg 21000
acgagctcca caagggcaat cccgtgatcg cgagcgccga ctactcgtcg atcgccaacc 21060
gcgtctccta cgcgctcaac ctgcacggcc cgagcatcgc cttcgatacg gcgtgctcgt 21120
cctcgctggt cgccatccac ctcgcctgcg agagcctcag gcggggcgag tgcgactacg 21180
ccatcgccgg cggcgtgagc ctctcgctgc acccctccaa gtacctccag atgagcaacc 21240
tcaaggccct gagcgccgag ggcaagtgcc gcagcttcgg cgccgggggc gccgggtacg 21300
tgcccggcga gggcgcgggc gcgctcctcc tcaagccgct gcgccgggcc atcgaggacg 21360
gcgactacat ccacgccgtc atccggggca ccgccgtgaa ccacgacggc aagaccaacg 21420
ggtacacggt gccgagcccg aacgcccagg ccgaggtcat ctcggaagcg ctgcgccagg 21480
gcgacatcga cgcgcgcacg gtcagctacg tggaggctca cgggacaggg accgagctgg 21540
gcgacccgat cgaggtcgcc ggcctgacca agagctatcg ccgcgacacg aaggacaggc 21600
agttttgcgc cctcggatcg gcgaagtcca acatcggcca cctcgagggc gcggccggcg 21660
ccgtgggcgt gatcaaggtg ctcttgcagc tgaagcacag gcagatcgcg ccgtcgctgc 21720
actcgcagca gctgaacccc agcatcgatt tcgcgagctc gcctttctgg gtgccccagc 21780
aactcagcgc gtgggagcga ccgcgcctcg ccgggccgga cggcgcccgg gagatcccgc 21840
gaagggcggg cgtcagctcc ttcggcgccg gcggcgccaa cgcgcacgtc gtgctggagg 21900
agtgggagaa cccgccgcgc gcgggggcag gccgggacga ggcgctcgtc gtgctctcgg 21960
cgatgagcga ggagcgcctg cgggcctacg ccggcaagct cgccgcctcc ctgagccggg 22020
ccgacggcga cgtggccgcc gccgagctcc gcgatctcga gcgcgtcgcg tacaccttgc 22080
agaccgggcg tgaggccctg gagtcacggc tcgccatcat cgccgccgac caccggcagc 22140
tcatcgccga tctgcaggcc tacagcgaag gccgccaggg cggcgagcca tcccgcgtgt 22200
tccacggcac ggtcaagccg tacgagctgc ccgagctcgg ggaggcggag cgggccgccc 22260
tcgacgaggc cacggcgagc cacgatctga ccacgatcgc gcggcgatgg gtcgcgggag 22320
ccgcgatcga ctggcgccgc ctctatccct ctccgcctcc ctacccgctg gccctgccca 22380
cgtacccttt cgcgcgagac cgctactgga tacccgtggt cgcggagcga ccggcggcct 22440
ccggggtcgc gagggctctc cacccgttcc ttgacaccaa cgtatccacc ctgggcgagc 22500
tggccttcga gaagaccttc tccagcgccg accccgtgct ccgggaccat gtggtcgccg 22560
gccggcaggt gctgccagcg gcggtgtacc tggagatggc ccgcgccgcc ggccaccacg 22620
cggggcgcgc gggcgtctcc agcatccacg acgccgtgtg ggcgaggccc gtcatcgccg 22680
cgggcgagcg cgtcacgctg cgcatcagcc tcgcctcgga gcgagaggcc gtcgtctacc 22740
gtatctactc gcaggccgag ggtcagtccg ttgtccacgg ccacggatac ctcgccacgg 22800
agccccccga gggcgctcgc cccgctgtgt cgctccaggc gctgctggac cgctgccctc 22860
ggcagatcgc gggcgacgcg ctctatcgct tcttcgaggg cctggggatc cactacgggc 22920
ccgcgttccg gcccgtgcag gcgctccact gcggggagcg ggaagcggtc gccctgctgc 22980
ggatgcccga cgccgccgcg gcgggcggcg acgaggaagg gctgaacccg tctctcctgg 23040
acggcgccct gcaggcgatc gctcacctcg ggttcgatca cgagctcgag ccctcggtcc 23100
tgcgcctgcc cttcgccctc ggccggctcg tgatccggcg gcctctcacc gcggcgtcgt 23160
gctacgcgca cgcggtcctc acgcaggact cccgggctgg cggggagcgg gtcctgaagt 23220
tccgtatcga tgtgttcgac ccgggcggcg ctgtcctggt cgagatcatc gattacagcg 23280
tgcgggtcgt ggcgcgcggc gcgctcggcc agcccgtgcc ccaggcagcc caggcggagc 23340
gagcggcgcc cgcccacacc ctctggtaca agccggtctg ggaagcgacg cccgtcgcct 23400
ccgggcacgc agccgccgcg gcgggagagc tgccggagcg gatcctggtc ctcggccggg 23460
aggacgagct gacctcgcgc ctcgtcgacg cgctgagccg ggtgcgcccc acgcgccggc 23520
tctcggcagg gacgacgttc ggagagctcg acccgcaggg ctaccgggtg gatccggcgg 23580
atccgagcca tatccggcgc gctctcgagg cgctcgcgcg cgacggccgg tggtccggcg 23640
gcagcctcgg gatcgtccac ctctggcgcc atggcgccgg cgccgaggaa gcgctcaccg 23700
cgggggtcca cgcgctgctc cacctggtcc agggcctcgg cgcgctgggc gccacgcagc 23760
gcgtccgctg cctgtctgtc cttggccacc gcgacggcat cgccgatccg cgcgacgagg 23820
cgctggccgg cttcgccgcc gcgctcgccc cggcgacccc gcaggtcgag atcgtcacgg 23880
tgcaggcgga gccggcccgg ctcggcgccc aggagctgct cgacatcgtg tcgagcgagc 23940
tcggcgcccg cgacacaggg gccgggagcg agatccgtta tacctcctcg accgcccggt 24000
ggacacgcgc gctgcggccg ctcgcggaag cgccggcacg gcccgagggc gccgcgccgc 24060
tgaggaccgg cggcgtttac ctgatcaccg gcggctgcgg ccacctgggc tcgatcttcg 24120
cgcgccacct cgccgggcgc cacggcgcgc ggctcgtcct cagcggccgt tcgccgagcg 24180
acgccgagaa ggacgcgctg atccgggaga tccgcggcct gggcggcgac gctgtctacg 24240
ttcaagccga cgtgtgcgac gcggaggccg cgcgggcgct ggtgcagacc gcagagcggc 24300
gcttcggcgg gctccacggc atcttccacg ccgccggcac ggacaaggcg ccgcccatcg 24360
cccaggccga cgccgcctcc ttcgccaggg tcctcgggcc caaggtccag ggcaccttga 24420
acctggacgc cgccagccgc cacctcgcca ccctcgacct cttcgtgctg ttctcgtcga 24480
tcgccgcggt catgggcgac ttcggcgccg gctgctacgc gtacgcgaac gcgttcatgg 24540
accgcttcgc cgcgggccgc gaagcgcagc gcgcgcaagg gcaccgtcac ggcaagacgc 24600
tgtcgatcaa ctggccgctg tgggccggag agggcatgag cctgcccgcg gggcagagcg 24660
agctttactt cgatgtggca ggcatgcgcg cgctggatcc ggcgctcgga ctggacctct 24720
tcgcccgggc cctgaccgcg ggcgcgccgc agctcctcgt ggcccacggg atccccgagc 24780
ggatgcggcg ggtgatcgag cggaggaacc cgcgcccggc cgcgaccgcg accgccgcga 24840
ccgccgcgac cgccgcgacc gcgaccgccg cgaccgcgac cgcggtcgcc agcgacgctg 24900
ccgccggtgg gcggcacctc gcggaggccg tcgaggagta cctcaagggc cacttcgccg 24960
cggtcttctc gatgggcgtc gaccagatcg acgcgcaaac gagcctggaa gactacggca 25020
tcgactcgat catgatcgtg gagctccaca cgcgcctcga tcgggacatg gctccgctgc 25080
cgcgcacgac cttcttcgag ctccggacca tccgcgcgct cgccgaccac ctcgtcaagg 25140
tgcgcggcgc ggagatgcgc caggtgctcg gcctcgaccg gccggagaag gcgccgcctc 25200
cctcgagcat cgacgcgcct gcgccgcgcg aacgccaagg agcgccggcc tcgctccccg 25260
cggtggagcc gcgcccgccc gccggcgcgt cgcgggacga ggccgcgctc gccggggtgg 25320
ctcgccagcc cgacagcgcc gccgccgggc ccggcgcggc cctcgcggac gacgacatcg 25380
ccgtcatcgg catgagcggc cggtacccga tggcgcccga tctcgacgcg ttctgggcca 25440
acctcaaggc ggggcgcgac tgcatcgagg agatccccgc ggagcggtgg gatcaccgcc 25500
ggtacttcga tcccgagccg ggcaccgagg ggaagagtta ctgctcgtgg ggcgggttca 25560
tcgacgacat cgacaagttc gatccgcact tcttccatat ctcgccgaag caggtcgcca 25620
cgatggaccc gcaagagcgg ctcttcctgg agaccgcgtg ggccacgctg gagcacggcg 25680
ggtacgcgcg cgtgaacgag gaggcagctc cgatcggggt gttcgcgggg gtcatgtggg 25740
acgactacgg cctcctcggg ctggagcagg ccgcgctcgg caatcacgtg ccggccggct 25800
ccgaccatgc ctcgatcgcc aaccgggtct cgtacgtgat gaacctgagg ggcccgagcc 25860
tcaccgtgtc gacggcgtgc tcctcgtcgc tcctggcggt gcacctcgcg gtggagagcc 25920
tgaggcgcgg cgagtgcgcg atggccatcg cgggcggcgt caacctgtcc attcacccca 25980
gcaagtacac ccggctatgc cagctccaga tgctcgcgcc ggacggccgc tgccggagct 26040
tcggcgccgg cgggaagggg tacgtgcccg gagagggcgt gggcgcagtg ctgctcaagc 26100
ccttgaagag cgccgtggct gacggcgaca cgatctacgc ggtgatcaag ggcagcgccg 26160
tcaaccacgg aggcaagacc aacgggtaca ccgtgccgaa ccccagggcg caggccgacg 26220
tcatcggccg cgccctcgag cgcgccggcg tcgacgcgcg cacggtcagc tacgtcgagg 26280
cccacggcac cggcacctcg ctgggagatc ccatcgaggt cggcgggctc gacgagagct 26340
tcaagcgcta caccggcgac agccagttct gcgcgctggg atcggtgaag tcgaacatcg 26400
gccacctgga gtgcgccgcg gggatcgcgg cgatcacgaa ggtcgcgctc cagctgcacc 26460
accggcagct cgtgccgtcc ctgcacgcgg aggccctcaa tccaaacatc gacttcgagc 26520
gcacgccctt ccacgttcag cgcacgctcg gcgcgtggcg ccgccccgag gtgcccgacg 26580
gcggggcgac cgtggtgtac ccgcgccgcg cgggcatcag ctcgttcggc gcgggcggga 26640
ccaacgtcca cgtcgtcctg gaagagtacc agggcccggc gccggtcgcg gaggccggag 26700
ggcccgagcc ggcgctcgtc gtgctctcgg cgcacaccga ggaacggctg cgcgcccatg 26760
ccgagcgact gctccgcttc ttgcacagtg tagaggcaga tgcagataca gacgcagacg 26820
cagagcccac gtcgctcccg gcctccgcgc cgggcctgcc cgacgccgag cagctccgga 26880
tcgcgctgcg agacctcatc gcgcgccatc tggagatcga tcccggcgag atcgacatgg 26940
aggtcgcgct gagcgagctc ggcctcgagg cgctcgatct gacgctcctc gcagagcaga 27000
tcgagcgtcg cttcggcgtt ccggtgagcc gccagcagct gaccggccag gccacgccgg 27060
ccgggctctc gcggctcctg gtgcagggca gtacggcgcc gggggcggcg caccgccgcg 27120
cgccgcgccg ccgcggcgtg ctgctcgggg acgtcgccta cacgctgcag gtcggtcgcg 27180
agccccggca gcaccgcctc gcgctgctcg ccgccagcat ggacgagctc gtcgagcgcc 27240
tgggccggta ttgcgacggc gccgccatgg acgcgtcatg gtccttcacc ggtcaggcga 27300
cccgaaagcc tggcgcggcc gcgtcccggg agagcgccga gcgcgaggca gaccgcgtgc 27360
gcgccctgct cgagcagcag gacctgggcg cgctcggccg gctctgggtc accgggcgcc 27420
acgtcgactg gtccctgctc taccggagcg cgaagccgcg ccggatcgcc ttgccgacat 27480
accccttcgc gcgggagcgg tactggttcg ccgagtccgc agagctccgg cacgacaggc 27540
ccgctgcgca cgacgacgct cccgcgagga aagcgctgca ccccctcgtg ggccgcaaca 27600
cgtcgacctt ccgggagcag aggttcgcca cgaccttcac gggcgaggag gtgttcgtcg 27660
cccaccaccg gatccgcggc cgcgcgctgc tgcccggcac ggcctacctg gagatggcgc 27720
gcgcggccgg cgaactcgcg gccgagcgcc aggtgcgccg gatctcgggc gtcacgtggt 27780
cgaggccgat cgaggtgaac ggcctgcccg tcgacgccac catccacctc gagccgaccg 27840
acacccacgg agagttccgg gtctgcaccg aggacggggc ggtcatccac gcggagggcc 27900
gcatccactt cgagccagag cccctcgggg gcgagccggc cgtggatctg gccgccatca 27960
aggcgcgttg cgtcgagcat cgaaccaagg aagacaacta ccgcttcctg cgagagcgcg 28020
ggttcgagta cgggcctgcg ttccaggccg tggaggcctt tcatgacaac gagcgggaag 28080
ccctggccct gctcaccctg cccgagccct acttcagcgc cttccccgcg gggctgaacc 28140
cgctcctcct ggacgcggcc gtccacgccg gggtgctcca catgcgccgc gcggccgcgg 28200
gcgagggcgg cacgccggtg cctttctacc tcgacgagct ggtcctccac cgcccgctga 28260
cgagccgttg ttacgcccac ctcgaggtgc ggcggcccgc cgcaggagga gcccggggcg 28320
acgtcgcgct cgacatcacc ctgctcgacg agggcggcgt gcccctcgtg caggtcagag 28380
ggttcacggg tcgacggctc gacagcgcca atgcagcctc ggagcagaac agcctgctct 28440
tcttcgcgga cgggtggcag cccgccccgc tcgcgccggc ggagacgccg gatcgcgcgg 28500
cgatcaggag cgtgctcctc ctggcagaag acggcccgcg ggcgcgcgcg ttcgagcggc 28560
tgctccgcgg ccagggcacc gacctcgtgt gggtccgccc gagcaagacg cgccgggagg 28620
agagcgcgca gcgcgcggac gcgcgccgca gcggcgacca cgccggcacg ctcacgatcg 28680
acccctctcg cgccgaggac cacctcgcct tgctggcgga gctcaaggag cagggccgcc 28740
tgcccgacgg gatcgtccgc ctctgggatg cctcgctcga gggcgcaggc gcggccgacg 28800
caggagggca accggagcgc gtcgacgcgc tggaggagct ctttcacctc gtcggcgccc 28860
tcgggcgcgt cgctccggac ccgcaggcgc gcctgctcct cgcggttcac ggggagacgc 28920
cgcccctcgc gatcgaggcg gcctccgggt tctgcagatc cctcggcctc gtcatgcccg 28980
gcctccgcgc gagcacgatc cggtggagcg acagggcgcc ggagccgcac gcccgggagc 29040
tctgggccga gctcgtggcc gggagcgcgg cttccacctc gacggcgagc gctggcagga 29100
gcgcgggcga cgtctcgtac gacgaccgcg accgcctcgt gcgcgtggcc gtgcccacga 29160
ccctggcccc cgaggggaac gccggctctc ccccgctccg ccgggagggt gtctatctca 29220
tcaccggcgg ttgcggcgga ctcgggcacc tcgtcgctct tcacctggcg cagcgctacg 29280
gtgcgaaggt cgtcctcacc ggccgctccg cgctcgacga cgagaaggag cggcagctgg 29340
tccggctccg cgcggccggc ggcgagggcc tctaccacca ggccgacgcg gccgacgagg 29400
gcgccatggc cgccgcggtg cgcctcgcga agcggcgatt cggcgcgctg cacggggtga 29460
ttcacgccgc gggcgtgtcc gacaagcggc ctgtcaccga aaagacgtgg gcggagttcc 29520
acgccaacct gcgacccaag gtggagggca ccgccgtcct cgaccgggtc accgccggcg 29580
agcccctcga cttcttcgcg ctgttctcct ccacctccgc cttgctcggc gacttcggcg 29640
cctgcgacta cgccaccggg aaccggttcc aggtggccta tggcgcctac cgcgaggggc 29700
tgcggcagga aggccggcgg cggggcgtca ccctcgtcat gaactggccc ctgtggcgcg 29760
acggcggcat gggcggcagc gccgagtcgg agcagatcta cctgaagacc agcggcctcg 29820
attacctcga gacggacgtc ggtctcgcca ccttcgagcg catcgtccac gcgcggcggt 29880
ctcccatcac cgtgctctat ggaaagccct cacgggcggc cagggccctc ggcgtggagg 29940
cgcccccgcg cgcggcgagc gcgccagcgg cgccggcgcc cacggacacc gcggcgcccg 30000
cccgccgggc gccggagccg gagccggcgg gtccggtcga ggccacgccc gcggcgtcgc 30060
cgcaagcgca gctgcgcgag gtgatcatcg acgccatcgt cgacgtgctc caccagaagc 30120
gcggcgtcat cgcgccggac gtcaacatcg cagaatacgg gttcgactcc ctgtccatgg 30180
cgaagttcgc cggtgagctg aaccgccgcc tcggggtgaa gctgccgccg ctcgtgctct 30240
tcgagcacac cacggtgcgc gagatcgagg cctacctgga gcagagccac ggggccgagg 30300
tccgcgcccg gctgagccag cgcgccggcg aggccgcgcg ctccccggcg ccggccccga 30360
gcgccgctgc cccggcgcag gcgtcgccgg gcggcggctc ccggttcgcc agcgcgcctc 30420
gccccggcgc ggcgcgcccg tcgcctgacg gcgactcgag cagagacatc gccatcatcg 30480
gcgtcagcgg ccgctacccg aaggccggcg acctgcgcac gttctggtcg cggatcaagg 30540
gcggcgagag ctgcatcgag gagatccccg cagaccgctg ggacagggag cgctacttcg 30600
atccgcggaa ggagcggagc ggcacgacga cgagccagtg gggcggcttc ctcgatggag 30660
tcgaccagtt cgatcccctg ttcttcaaca tgaccccgaa ccgggctcgg ctcatggatc 30720
cgatgcagcg gctcttcctg gagagcgcct acgagacgat cgaggacgcc ggctacaccc 30780
gcgccagcct gtcggcgggc ggcggcaagg tcggcgtgta cgcgggcgcc atgtatcagc 30840
attacgccat gctcgccgga gacgaggcga cgcgcggcta cctgctcgcg acctgcggcg 30900
ccagcatcgc caatcatgtg gcgtatttcc tcaacctgca cgggccctgc atggcggtgg 30960
acaccgcgtg cgcgtcgtcc ctcaccgcca ttcacctcgc ctgcgagagc ctgctcctcg 31020
gtcgctgcga gatggccatc gccggagggg tcaacctctc catcatcccg cagaagtacg 31080
tgggcctcag cgagctccag ttcctgagcg gaagcgcgct cagccgcccc ttcggcgaca 31140
gcgacggcat ggtcccgggc gaaggcgtgg gtacggtgct gctgaagccc ctcgatcgcg 31200
ccgttcgcga ccgcgaccac atccacgcgg tcatcaaggc gagcgccgtc agccacggtg 31260
ggaccagcac ggggatgacc gtgccgaacc tcaaggccca ggcggagctg ttcgtcgagg 31320
cgctggagcg ggggggcatc gagcctcgca cgatcagcta cgtggaggcc gccgccaacg 31380
gctcggcgct cggcgacccg atcgaggtga acgcgctcac gagagcgttc cggcgcttca 31440
ccgccgacac gggcttctgc gcgctcggga ccgtcaagtc caacatcggg cacctggagg 31500
cggcctccgg catctcgcag ctcaccaagg tgttgctgca gctccagcac ggcgagctgg 31560
cgccgaccat caacagcgag ccccgcaatc cccacctcca gctcgacggg acgccgttcc 31620
gtgtccagga gcgcctggag gcatggcggc gacccgtcat tgacggccgg gaggtcccgc 31680
gccgcgcgtt ggtcaacgcc ttcggggccg gcggcggata cgccaccctg ctcgtcgagg 31740
agcaccgcca gccggcgcgg ctcgcggcgc cggcccacgc gcccgccggg cggcccgagg 31800
tcttcgtgct ctccgcgaag agccggaaga gcctgcgcga cctcgccgcc cggatgctgt 31860
ccttcttcga ggaggcgacg gccctccctc tcgaggacgt ggcgtacacc ctgcaagtgg 31920
gccgcgaggc catggaggag cgcatcgcgg tggtggcggc ctcgcgcgag gcgatcctga 31980
cggccctggg cgcctacgtc cgcgatcccg acgcccccgt gcctggcctg ttcagcggcc 32040
gggtcgatct cgacgaggcg caggcgggcg acgccgagag gccagctggc gagcgggttc 32100
gcgacctcga ggaagcggcg cgcctgtggg tgcgcggcgc cgtgatcgac tgggaggctt 32160
cgtatcccca ccgcgccgcg catcgcgtcc cattgccgac gtacccgttc gatcgccgga 32220
gctgctggct cgatccgctg ccggccgagc aggcgcccgc gcctcccgcg gcgttcacgc 32280
cagagccccg ccggcccccg gcgtcgcgcg cggagccgac cgcggctgaa gccccggatc 32340
tggagcgcta tctctgcgag cgcgtgacag cggcgctggg gctccaccgc ggcgagctct 32400
cggccgacac gccgcttcgc cgcttcgggc tggactcgat cacgaccgcg aagctcaagg 32460
tcaccctgga gggcggtctc gccatgacga ttccgatgga cgtcatgagc agggcccgca 32520
gcgtggcgga gctcgccgat cgcctcgcgg cgcggggggc acgcgcgccg cgggccgcgg 32580
cggaggacgt cgagatcccg gccggcgcgg cgctctggtc ccgatccgat cgcccccctc 32640
agaatggagc gctcaggtcc cagttcctgg cctctcatca caacctgacc ggcgtcgccg 32700
acgacgagct cgtccggctt tatgccagct tgcaagagga tacatgacga ccgagagacc 32760
ggtgagcagc agcgagttcg ccaggctgcc cacggaggag aagaagcgag tcctgctgcg 32820
cctgcgggag gagcgcgcct cgagcgtggc ggcccccgga gggcagaccg gcggccatcc 32880
gcgggacgcc gcgccgctcc gccccgtcat ctcggcgcgt ccaggtgacc gctttctccc 32940
cttcccgctg accccgatcc aggagtcctt cctggtcgcc aagcagctcg atctggggtc 33000
ggatcccgtg gggtgccaca tctacctgga gatcgaggag gcgggcctcg acgtgccgcg 33060
cctcgagcgc gcctgggaca ggctcgtcgc ccaccacgac atgctccgtg cctccgtctt 33120
cctcgacggc acccagaagg tgcacgagca cggagagccc cggcgttttc aggtcgacga 33180
tctgcgcgag ctgcgcggac cggagctcgc cgcccacctg gaagccgtgc gcgacagcat 33240
gtctcaccgg gtctacaggc ccggggcgtc gccgctccac gagatccgca tcagccgctg 33300
ccgcgacgac cgcagcctca tccacctcag catcgacgag tggatcgtgg acgcggcgag 33360
cgtcaacctc ctgctcgccc agtggtaccg cctctatcac gaccccgagg cggtcctgcc 33420
ccgctgcgag ctcaccttcc gcgactacgt cctggcgctc cgggccttcg agcaggcgcc 33480
cgcctacaag gcggatctcg cgtactggtg cgacaaactg gccagcatgc ccgcgggccc 33540
cgcgctcccg agcgccgagc cttcacaggc ccccgagggc cgcgccggcc acgcccgccg 33600
tcgcgtccac ggccggctgc cccgtgagcc gtggagcgcg ctcaaggaca ggtcgacgga 33660
gctcggcgtc tccccgactg ccctcctcct caccgtcttc tccgaggccc tcgccctcca 33720
ctgcccgccc gggccgttct ccctcacgct cacctatttc aatcgcccgc cgatccacgc 33780
ggacatcgag cgcctgctcg gccctctcat ctcggcccac cgcttcctcg tcgaacacct 33840
gcccggcctc cctctgcagg agaaggtgca gcgcaaccag cagcagctct ggcgcgacct 33900
ggaccacgac cgctccgaca gcatcagcgc gtcgcgcgcc ctcaaggcca ggcgcaacct 33960
gatcctcacg agccccatcg tcttcaccag cgtcatcagc aacgtgggca aggaggcaca 34020
gcggcagggg cgcagctggg cggatcagat cacccactcc gtcacccaga ccccgcaggt 34080
ctacctggat caccaggtct ccgagaagga cggcgacctg cacttcacct gggacgtcgt 34140
ggacgccgtc ttctcgcccg ggctcatcga cgcggtcttc gacgactaca tgcgcctgct 34200
gcgcgcgctc gcggcagagg accggctctg gacgtcgtcc cgtcttcgcg atgagctccg 34260
cgacctcctc ccccggctcc acggcggtcc cgagcggccc tcgccggccc cgcgcggcga 34320
cggcttccag atcgtcgctc ggccggagga gcgacaccgc aggtttcccc tgtcggacct 34380
gcaacaggcc tacttcgtgg gccgcaccgc gctcatgtcg aacggcggcg tgagctgcca 34440
gatgtaccag gacttcgagc tgcgcgcccc ggacgtcgcg aagctggagc gggcgtggca 34500
gcgcgtggtc gacacccacg agatgcttcg cgccgtcgtc cacagcgacg gcacgcagag 34560
catccgcgcc gaggcggtcc ggtacaccat ccaggtcgcc gactaccgcg gccattcgcc 34620
cgaggcccgc gccgcggcgc tggccgaggt gcgagaggcc atggtggtga aggtcttccc 34680
cctggacggc tggcccttct tcgacgtgcg gctctctctc acggagccgt ccagggccat 34740
cctgcatgtc agcatcgatc tgctcatcgc cgacgcggtc agcattcaca ccgtcttcaa 34800
gcagttcttc gcgctgtacc agcagcctga cgcgccgtgc tccgcgccgg cgctctcctt 34860
ccgcgactac cagctcgcgc tcaaggagta cgagcgcgcg cccgcgtacc aggtcggcgc 34920
ggagcactgg cgccgccggc tcacggacct ccccggcggt cccgagctcg gcctgcgcct 34980
gccggaggac ggcgaccgcc gcctcgagcg ccgcgagctg cacggcgtcc tgacgcgatg 35040
gtcgctgctc caggagaggg ccgcggcgct ccgtgtgtcg gccgagaccg tgctgctggg 35100
cgtctacatc gaggtcctgg gcagccgctc cagccggcat cccttcaccg tggtcgctgt 35160
ccgctgggat cggccgccgg tgcacccgga gatcgacgag gtcgtcggcg acttcacggc 35220
catcagctgg gtcgcctcgc cccaggggga caccttcgcc gagcgcctcc agcacctcga 35280
gctcaccctg gccgaggatc gcgcccaccg cctgatcagc ggcccccgca tgctccagca 35340
gctcgccagg agatcccgcc agcggcaatt cctcaccttc ccggtggtgt tcaccggcct 35400
cgcccccacc ctcaggggcg tgctccccga cagcgtcgcc ctggggcatc ggatcaccca 35460
gacgccccag gtcttcctgg acaacatcag cgtggaggtg ggcgactcgc tgcagctcca 35520
ctgggactcg gtgcagggcg tgttccccga ggggctcatc gagtccatgt tcgacgccta 35580
ctgccgcatc ctcgacctgc tcgcgcggga cggcgacgcg tggcaagagc cccggttcga 35640
tgcggtcctg cgtgggcccg ccgccgcgcc gctccccggg acagccgcct tcgagccggg 35700
ccgcgccgcc gtcctgccgc ccggggaggc gccgggcagc ggcgagcgct cgccgcgctc 35760
gtccaccgac gtccgtcacc tcacgagcct gcaccggctg atcgaggagc gcgcgctcgg 35820
ttgccccgat catccggcgg tggtcttcga gggcgaagag ctcacgtacc gcgagctcaa 35880
ccggcgcgcc aacaagacgg cgcgttacct ccggaagcac ggtgttggtc cggatcggct 35940
ggtgggcgtg ctcgccgagc gctcgctcga gatggtggtt ggcctgctcg ccatcctcaa 36000
ggccgggggc gcttacgtgc ccatcgaccc agcctaccct ctcgaccgca tcgagttcat 36060
cgccgaggac gccggtatct ccgtcctcct cacccaggag cgccaccggc tcccgggctt 36120
ccgcggcgcc cagctgtgcc tggacacgca gcgctccttg ctcgaaggcg aggcggagca 36180
cgatctcggt caaaccgccg ggccggagga tctcgcctac gtcatctaca cctccgggtc 36240
caccggcaag cccaaggggt gcatgatctc gcatctcgcg atctgcaacc gcctgatctg 36300
gatgcaggac gaataccggc tgcagccgac ggatcgcgtg ctccagaaga cgccctatac 36360
cttcgacgtc tccgtatggg agttcttcct gccgctcatc gcgggcgcca cgctggtcat 36420
ggccaggccg gagggccaca aggacgcggc ctacctggcc cgggtcatgg aggagcagcg 36480
gatcaccacg tgccatttcg tgccctccat gctcaatttc ttcctcagga gcccggtgct 36540
cccctcgcac ctgcgccagg tgttcacgag cggcgaggcg ctgccgtacg agctcgtgga 36600
gacgttcctc cgccgctcgg cggccaggct ccacaacctg tacgggccca cggaggccgc 36660
ggtcgacgtg acctactggc agtgcgagat ccggcccgat cgcaaggtgc cgatcggccg 36720
cgcgatcgac catgtcgagc tgtacatcct cgacgatgac ctgcggccgg tgccggcggg 36780
ggccgagggc gagctccaca tcggcggcgt ctgcctcgcc cgtggctacc tcaaccgccc 36840
cgagctcacg cgggagaagt tcatccagag cccgttcgac cccggcggtc gcctctacaa 36900
gaccggcgac agggcgcgtt acctggaaga cgggaacatc gagtttctcg gtcggctcga 36960
ctcccaggtc aagctgcgcg ggttccgcat cgagctcggc gagatcgagg ccgtgctgtg 37020
cgcccacgag gacgtgaggg acgcggtggt ggtcgtgcag gaggcgcaga ccgaggatcc 37080
ccggctcgtc gcctacgtgg tcgccggcga ccggcccttc cccggccccg gggcgctcag 37140
ggcttacctc aaggaccgcc tccccgagta catggtcccc aaccagttcg tgccgctgcc 37200
ggagctgccc gtgacggccc acggcaagct cgaccgcaag gcgctgccct ggccagcgcc 37260
ccgctccgcc gcggcggcag cggccccgca ggccgcagcg gcgccggagc cccccgcgcc 37320
cgccgcccct cccgtgccgg cggtcgaccc ggagccggcg gtccgcgacg agctccagcg 37380
cttcctcggc ggggcgctgc gcctcgagca tgtggacgcc gacgccgacc tcttcgacct 37440
cggggccaca tcgctcacgg tcgtccaggc gtcgcagcgc atccaggaat gcttcggcgt 37500
cgagctgccg gtcagcgtcg tcctcgccac gccgaccctc agcgccgtcg cccgtcacgt 37560
cgtcgggcaa ttgaccgccg gcgcgcgcgt gccttcggcc gcagcgccct cggccgcagc 37620
gccctcggcc gcagcgcccc caccgcccgc cgcgacgccc gcagctgccg tggcggcgcc 37680
cgcccgggcc cccgccccgg cagcggggcc gtccaccggc acggacgcgg aggccccgct 37740
caacttcttc tccaaggaag acagggatcg cctcaagcag cgagagctcc acctgcggaa 37800
cgatctcgcg ggcctcccgg ccgtggatct gctcgacgcg cccgcggccc cggaggtcta 37860
tcgcgagcgc gccagccggc acgattacca gcccaggccg atcccgctcg ccgccttctc 37920
gagcttgctc gccctcctca ggcgctatcc gagcggacag cgaacccagt tttgctaccc 37980
atccgccggc ggcacctacg cggtccagac gtatgtccat gtcaaggagg gcgcgatcga 38040
gggcctcgat cccggcctct attaccatca tccggagcgc aaccagctgg tgctcatcaa 38100
cgcgcgcttc gccatccgcc gcgcgcacca cttctattac aaccgggagc acttcgatcg 38160
cgccgggttc ggcctgttct tcatcgcgca gaccgacgcg ctcaggccca tctacggcga 38220
cagcagcttc accttcgccg cgatcgaggc aggatgcatg atccagctgc tcatgagcca 38280
tcaggccagg acgggcctgg gcctgtgccc catgggcggc ctcgatttcg acgcgatcag 38340
cgctgatttc aagctcggca gcgggcaccg ctacgtgctc agcatgctcg gcggccgcgt 38400
cgaccacgcc cgcggccccg cggacgaccg cgcgaagcct gggcagagcc cccgggatca 38460
cggcccgccc gcgctggccg ccgcgcccgc ggacaggcgc tcccctgcgc cggcggtcgc 38520
ttccgggtcg cgcgacgtcg ccgtcatcgg cctcgccggc cgctatcccg gcgccgagac 38580
gccccgcgac ctgtggcggc tgctcagcga gggcaggagc gccatcacca gggcacccgc 38640
ctcgcgcgcc ggcgccgccg gcgagggggg cgaccccggc tggggcggct tcctcccccg 38700
catcgacgcg ttcgacagcc tgttcttcaa catctcgccc gccgaggcgc ggcacatgga 38760
ccctcaggag cgcctgttcg tcgaggtggt ctgggagtgc ctggagaacg ccggatacac 38820
gcctcaggag ctcacgcgct cggctccccg ggtgggcgtc ttcgcgggcg tcatgtggag 38880
cgattaccag agcgtagggc tggaggcctg gcagcgggac gggcgcgccc aggcggtgac 38940
cctccactcc tcgatctgca atcgcatctc tcacctcttc gacttccagg ggccgagcgc 39000
ggcgatcgac acgtcctgct cctcggccct gaccgcgctg cacctggcct gccgcagcct 39060
ccagcgaggc gagtgcgacg tggccctcgt cggcggcgtc aacctcctcg gccacccttc 39120
ccatcgcgac ctgctcgccg cgctcaacct cacctccgga gacgacagga cccgcgcctt 39180
cggcgccggc ggcaccggct gggtgcccgg cgagggcgtc ggcgcggtgc tgctccggcg 39240
cctgcaggac gccgagcagc acggcgattt catccacggc gtcgtcaagg gcaccgcggt 39300
cgctcacgcc ggcaagacct cccggtacgg catgccgaac acgcaggcgc aggccggatc 39360
catccgcgcc gccctcgcgg acgcggagct cgccgcggag gacatcgatt acgtcgagtg 39420
cgcggcgacc ggctccggca tcgcggacgc cgcggaggtc agcgcgctcc ggcaggcgtt 39480
ccaggagcgg agccccgacg gcccgccctg cgccctcggc tcgatcaagc ccaacatcgg 39540
tcacctcgag tcggcctccg ggatatccca gctgatcaag gtcttgctgc agctcgagca 39600
cggccagatc gccccgacgc tgtactccga gccgcgcaac ccgttgatcc agctggaccg 39660
cacgcccttc cggatcaacc aggagctcgc gccctggccc ggcagcgccg gagccgcctc 39720
ctcgccgcgg cgcgcgctgg tcaacgcgtt cggcgccacc ggctcctcgg cgcacgccgt 39780
cgtggaggag tacggccccc gtcgccccgg cgcccctgcc gggcccgcgg gcccgcgcgt 39840
cttcgtgctg tccgcggaga cggcggagca gctggacacc cacgcccgcg cgctcgccga 39900
ccacctgcgc gacctgcagc gcgggtcgca gcctcccggc gccgcgccgc cggcggccac 39960
ggacgtcgcg tacaccctgc tggtgggccg ccgcgcgatg gacgagcggc tggccgtcgt 40020
cgcgagcgac ctcgacgagc tcgaggcccg cttgcgcgac cacctcgccg ggcgccgagg 40080
gccaggcggc gagcacgtct tccgcggccg cgccggcgcc cgcgccgagg cggcgccgcc 40140
ccccgacgcg ccgcccgcgg ccctggcgcg cgcgtgggtc cacggcgccc ccgtcgcctt 40200
ccaggacctg cacgggcccg gtccgcgccg ccgggtgcct ctccccacct accccttcgc 40260
tcgcccgtcc cactggctcg cgcggccccc gcagccggcg ggcgccgcca cgggcgccga 40320
gctcccggcc gcagagcccg cgccgcagcg ccgcgcggcc gaggacgccc ccgccgcccc 40380
gctcgcgccc accgcggatc ccgccctccg ccaggccgcg ctgcgcctcg tgtgcgcctg 40440
cttctccgag gccgccgaga tcccgcgcca gcgcctcgac cccgaggcgc ctctcgaccg 40500
ctacggcctc aactcgctgc tcgccgtcca gttcacccgg ctgctggagg cgcagctcgg 40560
cgcgctgccg aggacccttg tttacgagca caacaccctg acctccctcg ccgagggcct 40620
gatcgcccgc cacggcgacg cgctcctcgg acatctcggc cgcccgcgcg cggcccccgc 40680
gacgcgcgct ccggctctcc ccgcgcaggc ctccggcgcg tcgcgggccg cggaagcggc 40740
gctcccgagc gccgatatcg ccatcgtcgg cctgaccggc cgctatcccg gcgccgacac 40800
catcgacgcc ttctggcaga acctgcagca agggcgggac tgcgtgaccg aggtgcccga 40860
gggccgctgg gggcccgtcg ccgccggcct ccagggcagc gccgacgccg cgccccgccg 40920
gcgctggggc gggttcctcg gcgacgtcga ccggttcgat cccctcttct tcaacatctc 40980
gccgcgcgag gcggcggcga tggatcccca ggagcggctg ttcctgcaga ccgcctgggg 41040
cgccttcgag gacgcgggct acacccgcca gcggctcgcg gaggaccagg cgcggcaagg 41100
cgcgggcgtc ggcgtgttcg tcggcagcat gtaccagcac tacccgctgc tggcgcggga 41160
tccggccgcc gaggtgtcct cctcgttctg gtcgatcgcc aaccgcgtct cgtacttctt 41220
cgatctgcgg gggccgagct tcgccgtcga cgctgcctgc gcttcctcgc tcaccgcgat 41280
ccacctggcc tgcgagagcc tgcgccgcgg cgagagctgc ctcgcgctgg ccggcggcgt 41340
caacctccac ctgcaccccg acaagtacgc cgccctcgag cgcctggggc tcctgagcag 41400
cggcgccgcg agcaagagcc tcggcgacgg ggacggctac gtgcccggcg aggcggtcgg 41460
cgccgtcgtg ctcaagcccc tcgatcgcgc gctcgcggac aacgatcgta tctacggcgt 41520
catcaagggc agcttcacga gccacgctgg caggaccgtg ggctacgggg tccccagccc 41580
ggccgcccag gccgatctca tcgcgaccgc cctgcggcgg tccggcgttc accccgacac 41640
catcggttac atcgaggtgg cggccaacgg ctcctcggtc ggcgacgcca tcgagctcgc 41700
cggtctccag caggcgttcc gcaggttcac ggacaggaag cggttctgcg cggtgggctc 41760
ggtcaaatcc aacatcggtc acccggaggc cgcctcgggc atcgcccagc tcaccaaggt 41820
cctttgccag ctccagcaca agacgctggt gcccacgctc cacgcagagc cgctcaaccc 41880
cgacatcgcg ctggacgaca gccctttcta tgtccagagg gagctcggcc cgtggccggc 41940
gccgctcgac gaggagggag ggcgtccctg cccgcgccgc gcggcgctca gctcgttcgg 42000
ctccggcggg acgagcaccc atatcgtggt ggaggagtac gcggatcccg agggcgcggc 42060
gcagcccacg caggaggtcg ccggcggcgc gcccctcgag ccggctgcgt tcgtcctgcc 42120
cgtctccgct cgaacccggg agcagctctg cgcgctcgcg gccgcgctgg cgcacgacat 42180
cgagcgccgg atgcgcccgg gcagccatgg agagcgcccg ttgaccgacc gcgacctgcc 42240
cgccatcgcg cacacgctgc aggtcggaag ggaggccatg gccgagcgtc tggccgtggt 42300
gacaatgcgc ctcgtcgatc tcgtggccaa gctgaggcgg ttcgccggcg gcgacggcga 42360
cgtggaggat ctctacctgg gcagcgccgc cacgcccggt cccgggtcgc tgctcgacgg 42420
ccgtgaaggc gaggcgttcc tcgcgatcct cctcgaggac ggccggtatg acaagctggc 42480
ccgtctctgg gtgagcggcg cccccatcga ctggcggcgt ctccacggga ccgggcgggc 42540
gcccagaccc ctctcgctgc ccagctaccc cttcgcgagc gagcgcttct ggatcgccga 42600
gcggccgcgg cccctgcccc cgcgcgccga gcccccggcg ccgggccgcg gcgccgagcc 42660
cgcccccgcc ctcgacagcg tcgccgacgc ccgggggccc atcgagcagg aggtcacggc 42720
gatgctgtgc gacgtgctcc agctcgacgg caggcacgtc gagccggatc gagagttccg 42780
cgattacggc ctcgattcgc gcctctcggt cgccttcatg cgatcggtgc agcagcggtt 42840
cggccctcgc gtcgcgctca ccgctgcgca cgcccatcct accctgggcc ggctcacggc 42900
gtacctccac cggaccctcg cgaacggcca tggcgcgagc cgctccgcgc catccgccgt 42960
ggcgtctctg ccggcagcgc ccgccgggtc gattccgccc gtggggccgc gcgccccgag 43020
cgccccctcg cccggcgcgc ggcccgcgcc gcgcgacgtc acggcgccgc tcgcgcctgg 43080
cctcgatccg atggagctcg tcagcatcaa cccgagcggc gctcgccaga gctcgttctg 43140
ggtgcacggc gcgcccgggc tcgcgcagcc cttcgtccat ctctccgcgg ccctcggcgg 43200
cgactatccg ctcttcgcct tccaggcccg cggcatggac ggcagcgtca tgccattcac 43260
gagcatcgag gagaccgccg ctcactacat cgcgtgcatg cagcagcggc gctccacggg 43320
accctatttc ctgggagggc tgtcctccgg cggcatcatc gccttcgaga tggcgcgtca 43380
gctccagcaa aagggcgagg ccgtctcccg gcttgtcctg ctcgacacgt acccctccgt 43440
cggcggcatc atggagtcga ccccggagaa cagcgatccg acgttccaca acctgctgat 43500
ggccaactcc ttcctcagct tcaatctctc gggcgaggtc gccatcaggc ccgccgacgt 43560
cgccgacctc gcccccgagc accagatccc gcgcatcgtc cggctgatca aggagcggag 43620
cggcaccgcg ctcacgctcg atcagattta ccggcagctg accgggagca tcgccgtgta 43680
caggcacctg gatctcgcgc tgaagagcta cgagccccgg cctctcgacg cggtggacgt 43740
gctgttcttc cgggccgaaa atggcttctt cggcgggtcg aacccgctgg acctgccctt 43800
gctcgacgcg ctgtccggct acgatgccgt caccccctgg cgccagtggc tgaaggggag 43860
cctgcgcgtc gtggggctgc cgtgcgcgca cgtcgagatc atggatcctc cggcgctcga 43920
tcaggtcgtc gctcacctcc gggaagatct cgcgtgacgc gccacgcgcg ctcgccgctc 43980
gcgcggccca ggacgcgaac gcaatgggaa tcaaccatgg tcgacagggg cgacaacgcg 44040
acagcgcgac agcacgacac gacatgatgg aatgataaat ggtatttcga ttgacctcgg 44100
ctggagcgtg cgataagcga tcgcagtcgc agctcccagc cgacgaaggg acgatcccgg 44160
gcaccgcggt cgcatgtcgc tgcgaacgcc ttgaccggtg tgaaatcaga gctgcggcgc 44220
tcccccatcg cacagtccct gggcgctgga ggcgcgaagg ttcaacggcc gaaaggctcc 44280
ccacatacgg agttgctcga tggcatcgac gacagatcga aggcgtgaga ttcacgacga 44340
gttccccgag actcgcccgc tgccgcctcg cagcatggag tggcgcaagg cgatgcgcct 44400
ggccaagcag ctgaagaaga cgccgtacaa tccctcggtc tcctacgagc tggtgctctc 44460
cctcgacggg ggcgatttcg agcgtgtgtt ccaggacttc ctgggcgagc cgggcgcgcg 44520
cgacatgatc atcgagcagc cgaacctgat cgcgctcctc gccgaccggg cggcgctggc 44580
ggcgatggat gaaggcagtc tgggccggat ctacctggcc ttgacccagg aggacggtta 44640
caccgccgac ggcctcgccg acgtgcagga caagacccct ggcttcaatg agatcgcccc 44700
ggacccgatc cgccgctggc tctacaagcg caacgcggcg ctgcacgacg tctctcatgc 44760
gttcacgggg tacgggcgcg acagggctgg tgaggccgcg ctgaacatgt tcacgtcggc 44820
catctaccct caccgcatcg tgcgcttcta ctcggtgatc ggggcgctcg tcgcgccgcg 44880
cgatcgctat ctgcgcaacc tttcgtacat gtacgagacg tgggcgcgcg gccggcgcgc 44940
gcgcatcccg ctcagcgccc cgtgggagca gctgctcccg ctccagctca aggaagtatg 45000
ccggcgcctc cagatccagc ccgtggagga ggctcacccc agcgggatca tgcgtgaagc 45060
tacggtcggc ggtccctggg tccccgccag cgctgtccag ggcagcgcct aggccgcctc 45120
gcgagctcac gagaggcgtc gcccgggatc acgcaggtcg caggcacgag cagggctctc 45180
tcatctagga ggcgcttatg aaggccgtca tgtttccggg gcaggggtcg cagtcgccag 45240
ggatgggagg ggagctgttc ctggagttcc ctgccatcgt ggcccaggcg gacgaggtcc 45300
tcgggtactc catccgggag ctgtgcctgc aggaccctca ccagcagctg ggccagaccc 45360
agttcaccca gccggcgctc tacgtcgtca acgcgctgat gttctcgaag cgttgccagc 45420
gggaggcgcc gcccgatttc ctcgtcggcc acagcctcgg cgagtacaac gccctcctcg 45480
ccgcgggcgt gttcgacttc gagaccgggc tcaggctggt gaagaagcgc ggtgagctga 45540
tgagccaggc ccgcgacggc ggcatggccg ccgtgaccgg cctggacccg gagcgggcgc 45600
gcgagatcct ggcgcgggag ggcgccgagg cggtggacat cgccaacatc aacagtccat 45660
cccaggtggt gatcgccggg gcgaagcacg agatctcccg cttgcaagcc gccttcgagc 45720
gggccggggc gaagaggtat accgtgctgc gcgtgagcgc cgcgttccac tcccgcttca 45780
tgcggccggc gatggaggag ttccgccgct tctcggcggg ccatcgcttc gccccgccgg 45840
ccatccccgt gatctcgaac ctgaccgccc ggccgtaccg cgccgatcgc gtccgcgaca 45900
ccctgtgcga gcagatcgcg agcccggtcc ggtggtgcga gtcgatacgt tatctgatgg 45960
gcaagggggt gaaggatttc gcggagtgcg gtcacggggt cgtgctgacg ggcctttacg 46020
ctcagatccg gcgcgacgcc gggcccctgt tcgtcgagga cgacccgccc ggatcgcccc 46080
caggggacgg gccggaggcg cctcgagcgc ccgccgccgc tgccccctac gagccggcgc 46140
gcccgggcgc cgcggcgcct gtcaggaggg tgtcgcccgg gtcgctgggg agctcggcct 46200
tccgggagga ctacggcctg cgctacgcct acgtcgccgg atccatggtc gagggcatct 46260
cgtccagcga gctggtggtg cgcatgggca aggccgggct gctcggctat ctcgggacca 46320
aggggctcac cctggaggcg gtcgatcgag cgctccgctc catccagggc gagctccgcg 46380
gcggggggag ctacggcgtg agcttgtggt gcgatctcga cgcgccccgc ctcgagcggg 46440
aggctgtcga cctctacctg aagcacgatg tccagaacct cgaggcgatc gcctgcctgc 46500
aggtcactcc ggacctggtc cgcttccggc tggcgggcgc ccaccgcgac gggagcggac 46560
gggccgcggc gcgccggcgg gtgctcgcga gggtctcgca ccccgagatc gctcgggcgc 46620
tcatgagccc tgcgccggag cagatcctgg gccggctcgt ggaggagggc aggctcaccc 46680
gcgaggaggc ggcgctcggc cgggaattgc ccgtgagcga ggacatctgc gtgcacgccg 46740
actccggggg gcacaccgag ctcggctccg gcgcggcgct gatgccggtc atgctgcggc 46800
tgcgcgagga gatgacggcg cggcaccggt acagcaagcc gatccgcgtg ggcctgtccg 46860
gcggcatcgg cgccccggag gcggccgcct ccgcgttcgt gctcggcgcc gacttcatcg 46920
tcaccaactc catcaaccag tgctcgccgg aggctggcac cagcgaccgg gtgaaggaca 46980
tgctgcaggc cgcgaacgtg caagacacca cgcacgcgcc cgccggcgac atgctcgaca 47040
gggggaccaa ggtccaggtc ctcaagcggg gcgtgctgtt cccggcgcgg gccagcaggt 47100
tgcatgagct gtaccggcag cacgcgtcgc tcgacgttct cgacaagaag acgacggatc 47160
agctggagaa gagctatttc aagcgcgatc tcggcgaggt ctggcaggac acgcagtcct 47220
actggcagcg catgcacccg gaggagctgg ccagggcgga gcgcgacccg agacgcaaga 47280
tgtcccttgt cttcgggtgg tacttccgcc gcgcctcgga gctggcgcgg cggggggagg 47340
ccggccaggt cgattatcag gtgcagtgcg gccccgccat gggggccttc aatcaatggg 47400
tgagggacac ggatctggag agctggcgca gccgccacgt cgacgtgatc gcggagcgcc 47460
tgatgcaggc ctcggccgat ctcctggacc accgcctgcg cgcgctgtcg cggtaaaccg 47520
taaagagtcg aagcttcgac cggaggtcat cgtcatgctt gcaaaactca tgttgtctca 47580
ggcgcggaac ccgaggggtc tcggagggaa gatcacgtcc tttttcatga acaagggcaa 47640
ccaggacgtg aacgatttga cgctggagtt cctcgacgtc cagccgcacc atcacgtgct 47700
ggacctgggg ttcggcggtg gcctcacgtt cccgatcttg ctggacaagc tcaagggcgg 47760
gaagctctat ggcctggaga tgtcccggac gatggtcgag caagccgcga agaagtacgc 47820
gaggaacatc gacgacggca agctggaggt caaggagggt gtcgtcgaca ggatgggctt 47880
cagcgatggc cagttcgacc gcatcctcac ggtcaacacc gtctatttct ggccgaacct 47940
gggcaccggc ttcaaggaga tcgcgcgcgt cctgaagccg ggcggcaagg tggggctcgg 48000
ctacaggagc aagcagacgg tgctctcttt gggttacgag aagcacgggg tcaacgccat 48060
ctcggagagc gacgtggagt ccgccgcgag ggaggccggc ttgacggtcc tggagacgcg 48120
ctcccggaaa gggcgcttcg acgatcgcgt caccatcgcc cagcggagcg cgtagacggg 48180
cgaccgcgcg ccggccgggc gacgagcgcc tcggggccga cggcgccgcg agcggctcgt 48240
tcgccctcgc ggagctccgc ggccgcgccc ccgcgacgga ccggtgggtc ccacacggaa 48300
ccacctctc 48309
<210>2
<211>102
<212>DNA
<213>人工序列
<220>
<221> p15A-cm BstBI and AflII for dis427-F
<222>(1)…(102)
<400>2
aagccgtcac gggcgctctg gtctccctta gtagcaggac acgggccagg gctcggcctg 60
acagatttcc cgcgtttacc agttacggat cttaaggatc tc 102
<210>3
<211>102
<212>DNA
<213>人工序列
<220>
<221> p15A-cm BstBI and AflII for dis427-R
<222>(1)…(102)
<400>3
cgattgctcg ggggcgccgg agaccgccgg caggggcttc gatttccgcg ggtatctggc 60
gcgcatggcc gccacggaga cttattcggc cttgaattga tc 102
Claims (4)
1.一种Disorazole Z的生物合成基因簇,其特征在于:该基因簇命名为dis427,其包含Disorazole Z生物合成所必需的编码聚酮合成酶及非核糖体多肽合成酶的四个核心基因disA,disB,disC和disD,一个假设蛋白基因orf4和一个后修饰基因orf6;该基因簇来源于纤维堆囊菌Sorangium cellulosum So ce 427,其核苷酸序列如SEQ ID No.1所示。
2.一株高效异源表达Disorazole Z的工程菌株,其特征在于:该菌株命名为工程菌株DK1622::Km-Ptet-dis427,其基因型为:Myxococcus xanthus DK1622,kanamycinresistance,tetracycline inducible Ptet promoter,disA,disB,disC,orf4,disD andorf6,是利用黄色粘球菌Myxococcus xanthus DK1622为出发菌株,通过转座的方法在其基因组上整合了Disorazole Z的生物合成基因簇dis427获得。
3.权利要求2所述高效异源表达Disorazole Z的工程菌株的构建方法,步骤是:
(1)利用Red/ET DNA重组技术将Disorazole Z的生物合成基因簇(dis427)直接克隆至p15A-cm-tetR-tetO-hyg-ccdB载体上,构建得到质粒p15A-cm-dis427;
(2)在步骤(1)构建的质粒p15A-cm-dis427上插入反向筛选标记amp-ccdB,构建得到质粒p15A-cm-amp-ccdB-dis427;
(3)步骤(2)构建的质粒p15A-cm-amp-ccdB-dis427通过限制性内切酶PacI和PmeI酶切后与tetR-tetO PCR片段进行线线重组,构建得到质粒p15A-cm-tetR-tetO-dis427;
(4)在步骤(3)构建的质粒p15A-cm-tetR-tetO-dis427上插入转座元件,构建得到表达质粒p15A-tnpA-kan-tetR-tetO-dis427;
(5)将步骤(4)构建的表达质粒p15A-tnpA-kan-tetR-tetO-dis427电转至Myxococcusxanthus DK1622中,表达质粒在Myxococcus xanthus DK1622中表达转座酶将DisorazoleZ的生物合成基因簇dis427整合到Myxococcus xanthus DK1622的基因组上,得到能异源表达Disorazole Z的工程菌株,命名为工程菌株DK1622::Km-Ptet-dis427。
4.高效异源表达Disorazole Z的工程菌株DK1622::Km-Ptet-dis427在制备Disorazole Z中的应用。
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WO2019120132A1 (zh) * | 2017-12-18 | 2019-06-27 | 山东大学 | 一株高效异源表达Disorazole Z的工程菌株和构建该菌株的基因簇及其应用 |
CN112011587A (zh) * | 2020-08-07 | 2020-12-01 | 华东理工大学 | 一种可擦除并重写的活细胞传感记录系统及其应用 |
CN115094079A (zh) * | 2022-06-28 | 2022-09-23 | 上海交通大学 | T6ss大肠杆菌工程菌及其构建方法与应用 |
CN116904328A (zh) * | 2023-07-13 | 2023-10-20 | 山东大学 | 一种高表达啶南平a的工程菌及发酵培养基 |
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Cited By (5)
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WO2019120132A1 (zh) * | 2017-12-18 | 2019-06-27 | 山东大学 | 一株高效异源表达Disorazole Z的工程菌株和构建该菌株的基因簇及其应用 |
CN112011587A (zh) * | 2020-08-07 | 2020-12-01 | 华东理工大学 | 一种可擦除并重写的活细胞传感记录系统及其应用 |
CN115094079A (zh) * | 2022-06-28 | 2022-09-23 | 上海交通大学 | T6ss大肠杆菌工程菌及其构建方法与应用 |
CN115094079B (zh) * | 2022-06-28 | 2023-11-07 | 上海交通大学 | T6ss大肠杆菌工程菌及其构建方法与应用 |
CN116904328A (zh) * | 2023-07-13 | 2023-10-20 | 山东大学 | 一种高表达啶南平a的工程菌及发酵培养基 |
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