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    • 2. 发明申请
    • GENOMIC INTEGRATION OF DNA FRAGMENTS IN FUNGAL HOST CELLS
    • 基因组DNA片段在真菌宿主细胞中的基因整合
    • WO2018050666A1
    • 2018-03-22
    • PCT/EP2017/072967
    • 2017-09-13
    • NOVOZYMES A/S
    • KLITGAARD, Dorte, M.K.
    • C12N15/90
    • C12N15/902C07K14/37C07K2319/02C07K2319/036C12N9/0036C12N9/22C12N15/65C12N15/80C12N15/905
    • The instant invention relates to methods for the in vivo assembly and integration of a polynucleotide of interest at a specific chromosomal target site in a fungal host cell, said method comprising transforming the host cell with: iv. at least one polynucleotide fragment comprising an upstream flanking region and a 5' part of the polynucleotide of interest in that order; v. at least one polynucleotide fragment comprising a 3' part of the polynucleotide of interest and a downstream flanking region in that order; and, optionally vi. one or more additional polynucleotide fragments each comprising a 5' part and a 3' part of the polynucleotide of interest; wherein the 5' part and 3' part of each polynucleotide fragment overlap with that or those of another fragment with at least 20 bp, whereby the fragments cover the entire polynucleotide of interest, and wherein the flanking regions are of sufficient size and homology to effectuate homologous recombination with the specific target site for chromosomal integration.
    • 本发明涉及用于在特定染色体靶位点在真菌宿主细胞中体内组装和整合感兴趣多核苷酸的方法,所述方法包括用以下方法转化宿主细胞:iv。 至少一个多核苷酸片段,其按顺序包含上游侧翼区和5'部分目的多核苷酸; v。至少一个多核苷酸片段,所述多核苷酸片段按顺序包含目的多核苷酸的3'部分和下游侧翼区域; 并且可选地vi。 一个或多个另外的多核苷酸片段,每个包含目的多核苷酸的5'部分和3'部分; 其中每个多核苷酸片段的5'部分和3'部分与具有至少20bp的另一个片段的5'部分和3'部分重叠,由此片段覆盖整个目的多核苷酸,并且其中侧翼区域具有足够的大小和同源性以实现 与染色体整合的特定靶位点同源重组。