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1. 发明授权

US08883420B2 Process of clean cloning 有权
标题翻译：清洁克隆的过程
公开(公告)号：US08883420B2
公开(公告)日：2014-11-11
申请号：US13121019
申请日：2009-10-08
申请人： Sylvestre Marillonnet , Stefan Werner , Carola Engler , Romy Kandzia , Frank Thieme , Ernst Weber
发明人： Sylvestre Marillonnet , Stefan Werner , Carola Engler , Romy Kandzia , Frank Thieme , Ernst Weber
IPC分类号： C12Q1/68 , C12N15/66 , C12N15/64
CPC分类号： C12N15/64 , C12N15/66
摘要： A process of inserting a nucleic acid sequence of interest into an acceptor nucleic acid is provided. The process comprises amplifying by PCR a DNA comprising in the following order a sequence segment U, a nucleic acid sequence segment of known nucleotide sequence K2, and a nucleic acid sequence segment of known sequence K3. The process further comprises treating the linear double-stranded DNA molecules from the PCR amplification with an exonuclease to obtain a single-stranded overhang at the first end of the DNA and a single-stranded overhang comprising nucleic acid segments K2 and K3 at the second end of the DNA. The process additionally comprises annealing the product of the exonuclease treatment to a linearized double-stranded acceptor nucleic acid which has been designed to complement the single-stranded overhangs of the product of the exonuclease treatment.
摘要翻译：提供将感兴趣的核酸序列插入受体核酸的过程。该方法包括通过PCR扩增包含以下顺序的序列片段U，已知核苷酸序列K2的核酸序列片段和已知序列K3的核酸序列片段的DNA。该方法还包括用外切核酸酶从PCR扩增中处理线性双链DNA分子，以在DNA的第一端获得单链突出端，并且在第二末端包含包含核酸片段K2和K3的单链突出端的DNA。该方法还包括将外切核酸酶处理的产物退火至线性化双链受体核酸，其被设计为补充外切核酸酶处理产物的单链突出端。

2. 发明申请

US20110263024A1 PROCESS OF CLEAN CLONING 有权
标题翻译：清洁过程
公开(公告)号：US20110263024A1
公开(公告)日：2011-10-27
申请号：US13121019
申请日：2009-10-08
申请人： Sylvestre Marillonnet , Stefan Werner , Carola Engler , Romy Kandzia , Frank Thieme , Ernst Weber
发明人： Sylvestre Marillonnet , Stefan Werner , Carola Engler , Romy Kandzia , Frank Thieme , Ernst Weber
IPC分类号： C12N15/87
CPC分类号： C12N15/64 , C12N15/66
摘要： A process of inserting a nucleic acid sequence of interest into an acceptor nucleic acid, comprising the following steps: amplifying by PCR a DNA comprising in the following order a sequence segment U, a nucleic acid sequence segment of known nucleotide sequence K2 and a nucleic acid sequence segment of known sequence K3 using a forward primer defining a first end of the amplified DNA and a reverse primer defining a second end of the amplified DNA, said reverse primer terminating at its 3′-end in a nucleotide sequence of nucleic acid sequence segment K3; treating the linear double-stranded DNA molecules contained in the PCR product obtained in the previous step with an exonuclease to obtain a single-stranded overhang at the first end of the DNA and a single-stranded overhang comprising nucleic acid segments K2 and K3 at the second end of the DNA; and annealing the product of the previous step to a linearized double-stranded acceptor nucleic acid having at a first end thereof a single-stranded overhang complementary to the single-stranded overhang of the first end of the DNA and at a second end thereof a single-stranded overhang complementary to the single-stranded sequence segment K2 of the second end of the DNA.
摘要翻译：包括以下步骤：通过PCR扩增包含以下顺序的DNA序列的已知核苷酸序列K2的核酸序列片段和核酸的方法已知序列K3的序列片段使用限定扩增DNA第一末端的正向引物和限定扩增DNA第二末端的反向引物，所述反向引物在其核酸序列片段的核苷酸序列的3'末端终止 K3; 用外切核酸酶处理前一步骤中获得的PCR产物中包含的线性双链DNA分子，以在DNA的第一端获得单链突出端，并且在第一端包含核酸片段K2和K3的单链突出端 DNA的第二端; 并将前述步骤的产物退火到线性双链受体核酸，其在第一端具有与DNA的第一末端的单链突出部互补的单链突出端，并且在其第二端具有单链突出端与DNA的第二末端的单链序列片段K2互补的突出突出端。

3. 发明申请

US20130267021A1 SYSTEM AND METHOD OF MODULAR CLONING 审中-公开
公开(公告)号：US20130267021A1
公开(公告)日：2013-10-10
申请号：US13703123
申请日：2011-06-09
申请人： Ernst Weber , Stefan Werner , Carola Engler , Ramona Grützner , Sylvestre Marillonnet
发明人： Ernst Weber , Stefan Werner , Carola Engler , Ramona Grützner , Sylvestre Marillonnet
IPC分类号： C12N15/66
CPC分类号： C12N15/66 , C12N15/1093
摘要： System for producing a nucleic acid construct of interest, said system comprising: a set of n entry DNAs numbered 1 to n, n being an integer of at least 2, each of said n entry DNAs comprising in this order: (i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof; (ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and (iii) a cleavage site of a further type Ms restriction endonuclease recognition site followed by the recognition site of said cleavage site; the cleavage sites of the type IIs restriction endonuclease recognition sites of item (iii) of entry DNAs 1 to n−1 are complementary to the cleavage sites of the type IIs restriction endonuclease recognition sites of item (i) of entry DNAs 2 to n, respectively; the cleavage site of the type Ms restriction endonuclease recognition site of item (iii) of entry DNA n is complementary to the cleavage site of the type IIs restriction endonuclease recognition site of item (i) of entry DNA 1 for allowing annealing of complementary single-stranded overhangs formed by restriction at recognition site (i) of entry DNA 1 and at recognition site (iii) of entry DNA n; said system further comprising a destination vector comprising in this order: (I) a type Ms restriction endonuclease recognition site followed by the cleavage site thereof; (II) a vector backbone preferably comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III); (III) a further cleavage site of a type Ms restriction endonuclease recognition site followed by the recognition site of said cleavage site, and (IV) optionally, an insert between the recognition sites of item (III) and item (i); said cleavage sites of items (I) and (III) being different and non-complementary, said recognition sites of items (I) and (III) being preferably recognitions sites of the same endonuclease.

4. 发明申请

US20110055976A1 Method Of Protease Production In Plants 审中-公开
标题翻译：植物中蛋白酶生产的方法
公开(公告)号：US20110055976A1
公开(公告)日：2011-03-03
申请号：US12920847
申请日：2009-03-03
申请人： Romy Kandzia , Carola Engler , Sylvestre Marillonnet , Victor Klimyuk , Yuri Gleba
发明人： Romy Kandzia , Carola Engler , Sylvestre Marillonnet , Victor Klimyuk , Yuri Gleba
IPC分类号： C12N15/87 , C12N9/48 , C12N9/76 , C12N15/63 , A01H5/00 , C12N5/10
CPC分类号： C12N15/8257 , C07K14/415
摘要： A process of producing a protease in a plant or in plant cells, comprising (a) providing a plant comprising a heterologous nucleotide sequence comprising a coding sequence encoding a fusion protein, said fusion protein comprising: an apoplast or plastid signal peptide; a SUMO protein or a derivative of a SUMO protein; and a zymogen of said protease, and (b) expressing said fusion protein.
摘要翻译：一种在植物或植物细胞中产生蛋白酶的方法，包括（a）提供包含异源核苷酸序列的植物，所述异源核苷酸序列包含编码融合蛋白的编码序列，所述融合蛋白包含：质外体或质体信号肽; SUMO蛋白或SUMO蛋白的衍生物; 和所述蛋白酶的酶原，和（b）表达所述融合蛋白。

5. 发明授权

US07718848B2 Safe production of a product of interest in hybrid seeds 有权
标题翻译：安全生产杂交种子感兴趣的产品
公开(公告)号：US07718848B2
公开(公告)日：2010-05-18
申请号：US10559430
申请日：2004-06-04
申请人： Stefan Werner , Romy Kandzia , Serik Eliby , Sylvestre Marillonnet , Victor Klimyuk , Yuri Gleba
发明人： Stefan Werner , Romy Kandzia , Serik Eliby , Sylvestre Marillonnet , Victor Klimyuk , Yuri Gleba
IPC分类号： C12N15/82 , C12N15/34 , A01H1/02
CPC分类号： C12N15/8216 , C12N15/8217 , C12N15/8242 , C12N15/8257
摘要： A process of the production of a product of interest in an F1 seed obtained by a hybridization of a first and a second transgenic parental plant, said hybridization generating a genetic endowment in said F1 seed for said production by combining in said F1 seed first and second partial genetic endowments of said first and second transgenic parental plants, followed by isolating said product of interest from said F1 seed or a seedling thereof.
摘要翻译：通过第一和第二转基因亲本植物的杂交获得的F1种子中感兴趣的产品的生产过程，所述杂交通过在所述F1种子中组合产生用于所述生产的所述F1种子中的遗传禀赋，第一和第二所述第一和第二转基因亲本植物的部分遗传禀赋，然后从所述F1种子或其幼苗中分离所述目的产物。

6. 发明申请

US20060272051A1 Safe production of a product of interest in hybrid seeds 有权
标题翻译：安全生产杂交种子感兴趣的产品
公开(公告)号：US20060272051A1
公开(公告)日：2006-11-30
申请号：US10559430
申请日：2004-06-04
申请人： Stefan Werner , Romy Kandzia , Serik Eliby , Sylvestre Marillonnet , Victor Klimyuk , Yuri Gleba
发明人： Stefan Werner , Romy Kandzia , Serik Eliby , Sylvestre Marillonnet , Victor Klimyuk , Yuri Gleba
IPC分类号： A01H1/00 , C12N15/82 , A01H5/00 , C12N5/04
CPC分类号： C12N15/8216 , C12N15/8217 , C12N15/8242 , C12N15/8257
摘要： A process of the production of a product of interest in an F1 seed obtained by a hybridization of a first and a second transgenic parental plant, said hybridization generating a genetic endowment in said F1 seed for said production by combining in said F1 seed first and second partial genetic endowments of said first and second transgenic parental plants, followed by isolating said product of interest from said F1 seed or a seedling thereof.
摘要翻译：通过第一和第二转基因亲本植物的杂交获得的F1种子中感兴趣的产品的生产过程，所述杂交通过在所述F1种子中组合产生用于所述生产的所述F1种子中的遗传禀赋，第一和第二所述第一和第二转基因亲本植物的部分遗传禀赋，然后从所述F1种子或其幼苗中分离所述目的产物。

7. 发明授权

US08093458B2 Biologically safe transient protein expression in plants 有权
标题翻译：植物中生物安全的瞬时蛋白表达
公开(公告)号：US08093458B2
公开(公告)日：2012-01-10
申请号：US11631719
申请日：2005-07-07
申请人： Sylvestre Marillonnet , Carola Engler , Stefan Mühlbauer , Stefan Herz , Stefan Werner , Victor Klimyuk , Yuri Gleba
发明人： Sylvestre Marillonnet , Carola Engler , Stefan Mühlbauer , Stefan Herz , Stefan Werner , Victor Klimyuk , Yuri Gleba
IPC分类号： C12N15/82 , C12N15/63 , C12N15/09
CPC分类号： C12N15/8203 , C12N15/8205 , C12N15/8216 , C12N15/8257
摘要： A process of producing a protein of interest by expression of said protein of interest from a sequence of interest in a plant or in plant leaves, comprising: (a) transfecting said plant or said plant leaves by infiltrating said plant or said plant leaves with an Agrobacterium strain in the presence of a complementing factor, said Agrobacterium strain containing in T-DNA a heterologous DNA sequence having a sequence portion encoding a replicon, wherein said sequence encoding a replicon contains sequences necessary for replicon function of said replicon, said sequences being derived from a plant virus, and said sequence of interest to be expressed from said replicon, (b) optionally isolating said protein of interest from said plant or said plant leaves infiltrated in step (a), wherein said Agrobacterium strain is provided with a first genetic modification rendering said Agrobacterium strain defective for transfecting organisms with said T-DNA in the absence of said complementing factor.
摘要翻译：通过从植物或植物叶中感兴趣的序列表达所述目标蛋白质来产生感兴趣的蛋白质的方法，包括：（a）通过用所述植物或所述植物叶子渗透所述植物或所述植物叶来转染所述植物或所述植物叶农杆菌菌株在补充因子存在下，所述含有T-DNA的土壤杆菌菌株具有编码复制子的序列部分的异源DNA序列，其中编码复制子的所述序列含有所述复制子的复制子功能所必需的序列，所述序列得自从所述复制子表达的所述感兴趣的序列，（b）任选地从所述植物或所述在步骤（a）中渗透的所述植物叶分离所述目的蛋白质，其中所述农杆菌菌株被提供有第一遗传在没有所述补充因子的情况下，使所述农杆菌菌株对所述T-DNA转染生物有缺陷。

8. 发明申请

US20080057563A1 Biologically Safe Transient Protein Expression in Plants 有权
标题翻译：植物中生物安全的瞬时蛋白表达
公开(公告)号：US20080057563A1
公开(公告)日：2008-03-06
申请号：US11631719
申请日：2005-07-07
申请人： Sylvestre Marillonnet , Carola Engler , Stefan Muhlbauer , Stefan Herz , Stefan Werner , Victor Klimyuk , Yuri Gleba
发明人： Sylvestre Marillonnet , Carola Engler , Stefan Muhlbauer , Stefan Herz , Stefan Werner , Victor Klimyuk , Yuri Gleba
IPC分类号： A01H5/00 , C12Q1/68
CPC分类号： C12N15/8203 , C12N15/8205 , C12N15/8216 , C12N15/8257
摘要： A process of producing a protein of interest by expression of said protein of interest from a sequence of interest in a plant or in plant leaves, comprising: (a) transfecting said plant or said plant leaves by infiltrating said plant or said plant leaves with an Agrobacterium strain in the presence of a complementing factor, said Agrobacterium strain containing in T-DNA a heterologous DNA sequence having a sequence portion encoding a replicon, wherein said sequence encoding a replicon contains sequences necessary for replicon function of said replicon, said sequences being derived from a plant virus, and said sequence of interest to be expressed from said replicon, (b) isolating said protein of interest from said plant or said plant leaves infiltrated in step (a), wherein said Agrobacterium strain is provided with a first genetic modification rendering said Agrobacterium strain defective for transfecting organisms with said T-DNA in the absence of said complementing factor.
摘要翻译：通过从植物或植物叶中感兴趣的序列表达所述目标蛋白质来产生感兴趣的蛋白质的方法，包括：（a）通过用所述植物或所述植物叶子渗透所述植物或所述植物叶来转染所述植物或所述植物叶农杆菌菌株在补充因子存在下，所述含有T-DNA的土壤杆菌菌株具有编码复制子的序列部分的异源DNA序列，其中编码复制子的所述序列含有所述复制子的复制子功能所必需的序列，所述序列得自（b）从所述植物或所述在步骤（a）中渗透的所述植物叶分离所述感兴趣的蛋白质，其中所述农杆菌菌株被提供有第一种遗传修饰物使得所述农杆菌菌株在缺乏所述补体因子的情况下缺陷以用所述T-DNA转染生物体。

9. 发明申请

US20070300330A1 Two-Component Rna Virus-Derived Plant Expression System 有权
标题翻译：双组分Rna病毒来源植物表达系统
公开(公告)号：US20070300330A1
公开(公告)日：2007-12-27
申请号：US10586998
申请日：2005-01-19
申请人： Sylvestre Marillonnet , Carola Engler , Victor Klimyuk , Yuri Gleba
发明人： Sylvestre Marillonnet , Carola Engler , Victor Klimyuk , Yuri Gleba
IPC分类号： C12N15/82 , A01H5/00 , C12N15/11
CPC分类号： C12N15/8216
摘要： A process for replicating or for replicating and expressing a sequence of interest in a plant, comprising: (i) an RNA replicon or a precursor thereof, said RNA replicon being derived from a plus-sense single stranded RNA virus and comprising at least one sequence of interest; and (ii) a helper replicon, or a precursor thereof, wherein said helper replicon is (a) incapable of systemic movement in said plant both in the presence and in the absence of said RNA replicon (i) and (b) capable of expressing in a plant one or more proteins necessary for systemic movement of said RNA replicon (i), whereby said RNA replicon (i) is capable of replicating or replicating and expressing said sequence of interest in said plant, but unable to move systemically in said plant in the absence of said one or more proteins expressed by said helper replicon (ii).
摘要翻译：一种在植物中复制或表达感兴趣的序列的方法，包括：（i）RNA复制子或其前体，所述RNA复制子衍生自正义单链RNA病毒，并且包含至少一个序列出于兴趣; 辅助复制子或其前体，其中所述辅助复制子是（a）在所述RNA复制子（i）和（b）能够表达的存在和不存在下，在所述植物中不能全身运动在植物中，所述RNA复制子（i）的系统运动所必需的一种或多种蛋白质，其中所述RNA复制子（i）能够在所述植物中复制或复制和表达所述感兴趣的序列，但不能在所述植物中系统地移动在不存在由所述辅助复制子（ii）表达的所述一种或多种蛋白质的情况下。

10. 发明授权

US09267143B2 RNA virus-derived plant expression system 有权
标题翻译： RNA病毒来源植物表达系统
公开(公告)号：US09267143B2
公开(公告)日：2016-02-23
申请号：US10578962
申请日：2004-11-10
申请人： Sylvestre Marillonnet , Carola Engler , Victor Klimyuk , Yuri Gleba
发明人： Sylvestre Marillonnet , Carola Engler , Victor Klimyuk , Yuri Gleba
IPC分类号： C12N15/86 , A61K38/00 , C12N7/00 , C12N5/04 , C12N15/82
CPC分类号： C12N15/8203 , C12N15/8216
摘要： A process of expressing a sequence of interest in a plant, plant part, or plant cell culture, comprising: (a) providing a plant, plant part, or plant cell culture containing in cell nuclei a heterologous DNA having a sequence encoding an RNA replicon operably linked or linkable to a transcription promoter, wherein said sequence encoding an RNA replicon contains (i) sequences for replicon function of said RNA replicon, said sequences being derived from a sequence of a plant RNA virus, (ii) a sequence of interest, whereby said sequences for replicon function exhibit at selected localities of said sequences of said plant RNA virus function conservative differences from said sequence of said plant RNA virus, said differences causing an increased frequency of replicon formation compared to an RNA replicon not exhibiting said differences; and (b) causing expression of said sequence of interest.
摘要翻译：在植物，植物部分或植物细胞培养物中表达感兴趣的序列的方法，包括：（a）提供在细胞核中含有编码RNA复制子的序列的异源DNA的植物，植物部分或植物细胞培养物其可编码RNA复制子的所述序列包含（i）所述RNA复制子的复制子功能的序列，所述序列衍生自植物RNA病毒的序列，（ii）感兴趣的序列，其中所述复制子功能的序列在所述植物RNA病毒的所述序列的选定位置处表现出与所述植物RNA病毒的所述序列的保守差异，所述差异与不显示所述差异的RNA复制子相比导致复制子形成的频率增加; 和（b）引起所述感兴趣序列的表达。

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