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    • 11. 发明申请
      WO2014044782A1 CELL MODIFICATION METHOD USING ESSENTIAL GENES AS MARKERS AND OPTIONALLY RECYCLING THESE 审中-公开
    • CELL MODIFICATION METHOD USING ESSENTIAL GENES AS MARKERS AND OPTIONALLY RECYCLING THESE
    • 使用基因基因作为标记的细胞修饰方法和可循环的这些方法
    • WO2014044782A1
    • 2014-03-27
    • PCT/EP2013/069532
    • 2013-09-19
    • DSM IP ASSETS B.V.
    • LANGE, DE, Ilse , MEIJRINK, Bernard , ROUBOS, Johannes, Andries , OOI, Siew-loon
    • C12N15/65 , C12N15/80
    • C12N15/65 , C12N15/80 , C12N15/81
    • The invention relates to a method for modification of a host cell at a target locus, which method comprises: providing a host cell comprising, at a first locus, at least two site-specific recombination sites and a nucleic acid having an essential function or encoding a product having an essential function; introducing into the host cell, at the target locus, a further nucleic acid having the essential function or encoding for a product having the essential function; and carrying out recombination at the first locus via the at least two site-specific recombination sites, so that the nucleic acid having an essential function or encoding a product having an essential function is rendered non-functional, thereby to modify the host cell at the target locus. The invention also relates to a cell obtainable by a method of the invention.
    • 本发明涉及用于在靶标位点修饰宿主细胞的方法，该方法包括：提供宿主细胞，其在第一场所包含至少两个位点特异性重组位点和具有基本功能或编码的核酸 具有基本功能的产品; 在靶标位点向宿主细胞中引入具有基本功能或编码具有基本功能的产品的另一种核酸; 并通过至少两个位点特异性重组位点在第一位点进行重组，使得具有基本功能或编码具有基本功能的产物的核酸变得不起作用，从而修饰宿主细胞 目标位点。 本发明还涉及可通过本发明的方法获得的细胞。
    • 基本信息 添加关注 加入工作空间 PDF全文 PDF下载 全文下载 相似专利 双屏查看 权利要求书 说明书全文 Global Dossier Espacenet 法律信息 同族专利 专利引用
    • 12. 发明申请
      WO2013144257A1 CLONING METHOD 审中-公开
    • CLONING METHOD
    • 克隆方法
    • WO2013144257A1
    • 2013-10-03
    • PCT/EP2013/056623
    • 2013-03-27
    • DSM IP ASSETS B.V.
    • ROUBOS, Johannes, Andries , PEL, Herman Jan , MEIJRINK, Bernard , LOS, Alrik Pieter
    • C12N15/66 , C12N15/90
    • C12N15/1024 , C12N15/66 , C12N15/90 , C12N2800/30 , C12N2800/40 , C12N2800/50 , C12N2800/80
    • The present invention relates to a method for the preparation of two or more standardized expression cassettes, which method comprises: a. providing two or more sets of element sequences, each set of element sequences together comprising at least one functional expression cassette,each element sequence being flanked on both sides by a type IIs restriction endonuclease cleavage site followed by the recognition site thereof, the type IIs restriction endonuclease recognition sites and cleavage sites being selected so that the sets of element sequences may be assembled into a functional expression cassette;b providing at least two backbone entry vectors,each backbone entry vector comprising in this order (i) a restriction enzyme cleavage site with its recognition site and a first connector sequence (LF); (ii) a vector backbone comprising a selectable marker gene; and (iii) a second connector sequence (RF) and a restriction enzyme recognition site with its cleavage sequence, and; (iv) optionally, an insert between the recognition sites of (i) and (iii), the connector sequences, RF and LF, on any backbone entry vector being selected so that they can assemble with a LF or RF connector sequence respectively on the same or a different backbone entry vector; and c. assembling the two or more sets of element sequences as functional expression cassettes in the at least two backbone entry vectors, using a method based on the use of restriction enzyme digestion and ligation via the cleavage sites, thereby to prepare two or more standardized expression cassettes.
    • 本发明涉及一种制备两种或多种标准化表达盒的方法，该方法包括：a。 提供两组或更多组元件序列，每组元件序列一起包含至少一个功能性表达盒，每个元件序列在两侧通过II型限制性内切核酸酶切割位点，随后是其识别位点，II型限制 选择内切核酸酶识别位点和切割位点，使得可以将这组元件序列组装成功能性表达盒; b提供至少两个主要入口载体，每个主要入口载体按以下顺序包括：（i）限制性酶切割位点与 其识别位置和第一连接器序列（LF）; （ii）包含选择标记基因的载体骨架; 和（iii）具有其切割序列的第二连接体序列（RF）和限制酶识别位点， （iv）可选地，在（i）和（iii）的识别位点之间的插入物，连接器序列RF和LF在任何主干入口向量上被选择，使得它们可以分别与LF或RF连接器序列 相同或不同的主干入口向量; 和c。 使用基于限制酶消化和通过切割位点连接的方法将两组或更多组元件序列组装在至少两个主干进入载体中作为功能性表达盒，由此制备两个或多个标准化表达盒。
    • 基本信息 添加关注 加入工作空间 PDF全文 PDF下载 全文下载 相似专利 双屏查看 权利要求书 说明书全文 Global Dossier Espacenet 法律信息 同族专利 专利引用
    • 15. 发明申请
      WO2013135728A1 RECOMBINATION SYSTEM 审中-公开
    • RECOMBINATION SYSTEM
    • 重塑系统
    • WO2013135728A1
    • 2013-09-19
    • PCT/EP2013/055047
    • 2013-03-12
    • DSM IP ASSETS B.V.
    • WIESSENHAAN, Nathalie , KOLEN, Catharina Petronella Antonia Maria , MEIJRINK, Bernard , BOER, Viktor , ROUBOS, Johannes, Andries , ARENDSEN, Yvonne Johannes Odilia
    • C12N15/63 , C12N15/90 , C12N15/00 , C12N5/00
    • C12N15/102 , C12N15/902 , C12N2800/30
    • The present invention relates to a method for carrying out recombination at a target locus, which method comprises: providing two or more nucleic acids which, when taken together, comprise: (a) sequences capable of homologous recombination with sequences flanking the target locus; (b) two or more site-specific recombination sites; and (c) a sequence encoding a recombinase which recognizes the site-specific recombination sites, wherein the two or more nucleic acids are capable of homologous recombination with each other so as to give rise to a single nucleic acid, and wherein at least two of the two or more nucleic acids each comprise a sequence encoding a non-functional portion of the recombinase; and recombining the said two or more nucleic acids with each other and with the sequences flanking the target locus so that a contiguous nucleic acid sequence encoding a functional recombinase is inserted at the target locus, said recombinase-encoding sequence being flanked by at least two site-specific recombination sites and the said site-specific recombination sites being flanked by the sequences capable of homologous recombination with sequences flanking the target locus.
    • 本发明涉及在目标基因座上进行重组的方法，该方法包括：提供两种或多种核酸，当它们一起时，它们包含：（a）能够与靶基因座侧翼的序列同源重组的序列; （b）两个或多个位点特异性重组位点; 和（c）编码识别位点特异性重组位点的重组酶的序列，其中所述两个或更多个核酸能够彼此进行同源重组以产生单个核酸，并且其中至少两个 两个或多个核酸各自包含编码重组酶的非功能部分的序列; 并将所述两种或多种核酸彼此重组，并与靶基因座侧翼的序列重组，使得编码功能重组酶的连续核酸序列插入靶基因座，所述重组酶编码序列侧接至少两个位点 特异性重组位点和所述位点特异性重组位点的侧翼是能够与靶基因座侧翼的序列同源重组的序列。
    • 基本信息 添加关注 加入工作空间 PDF全文 PDF下载 全文下载 相似专利 双屏查看 权利要求书 说明书全文 Global Dossier Espacenet 法律信息 同族专利 专利引用
    • 16. 发明申请
      WO2013076280A1 NUCLEIC ACID ASSEMBLY SYSTEM 审中-公开
    • NUCLEIC ACID ASSEMBLY SYSTEM
    • 核酸组装系统
    • WO2013076280A1
    • 2013-05-30
    • PCT/EP2012/073532
    • 2012-11-23
    • DSM IP ASSETS B.V.
    • ROUBOS, Johannes, Andries , MEIJRINK, Bernard , KERKMAN, Richard , DULK, DEN, Ben
    • C12N15/10 , C12N15/52 , C40B40/06 , C40B40/08
    • C12N15/1082 , C12N15/1027 , C12N15/1093 , C12N15/52 , C12N15/80 , C12N15/905
    • The present invention relates to a method for the preparation of a library of host cells, a plurality of which comprise an assembled polynucleotide at a target locus, which method comprises: (a) providing a plurality of polynucleotides comprising two or more polynucleotide subgroups, wherein: (i) a plurality of polynucleotides in each polynucleotide subgroup comprises sequence encoding a peptide or polypeptide and/or a regulatory sequence; (ii) a plurality of peptides or polypeptides encoded by, or a plurality of regulatory sequences comprised within, each polynucleotide subgroup share an activity and/or function; (iii) at least one polynucleotide subgroup comprises at least two non-identical polynucleotide species; (iv) a plurality of polynucleotides of each polynucleotide subgroup comprises sequence enabling homologous recombination with a plurality of polynucleotides from one or more other polynucleotide subgroups; and (v) a plurality of polynucleotides in two polynucleotide subgroups comprise a nucleotide sequence enabling homologous recombination with a target locus in host cells; and (b) assembling the plurality of polynucleotides at the target locus by homologous recombination in vivo in host cells, thereby to generate a library of host cells, a plurality of which comprise an assembled polynucleotide at the target locus. The assembled polynucleotides may be recovered, thereby to prepare a library of nucleic acids.
    • 本发明涉及一种用于制备宿主细胞文库的方法，其多个在靶位点包含组装的多核苷酸，该方法包括：（a）提供多个包含两个或多个多核苷酸亚基的多核苷酸，其中 （i）每个多核苷酸亚组中的多个多核苷酸包含编码肽或多肽和/或调节序列的序列; （ii）每个多核苷酸亚组中由多个或多个编码的多肽或多肽调节序列共享活性和/或功能; （iii）至少一个多核苷酸亚组包含至少两个不相同的多核苷酸种类; （iv）每个多核苷酸亚组的多个多核苷酸包括能够与来自一个或多个其它多核苷酸亚组的多个多核苷酸进行同源重组的序列; 和（v）两个多核苷酸亚组中的多个多核苷酸包含能够与宿主细胞中的靶基因座进行同源重组的核苷酸序列; 和（b）通过在宿主细胞中的体内同源重组在靶位点组装多个多核苷酸，从而产生宿主细胞文库，其多个包含靶标位点处的组装的多核苷酸。 可以回收组装的多核苷酸，从而制备核酸文库。
    • 基本信息 添加关注 加入工作空间 PDF全文 PDF下载 全文下载 相似专利 双屏查看 权利要求书 说明书全文 Global Dossier Espacenet 法律信息 同族专利 专利引用
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