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    Engineering nicking endonucleases from type IIs restriction endonucleases
    6.
    发明授权
    Engineering nicking endonucleases from type IIs restriction endonucleases 有权
    来自II型限制性内切核酸酶的工程化切口内切核酸酶

    公开(公告)号：US06395523B1

    公开(公告)日：2002-05-28

    申请号：US09872861

    申请日：2001-06-01

    申请人： Huimin Kong ,  Caroline Besnier ,  Yan Xu

    发明人： Huimin Kong ,  Caroline Besnier ,  Yan Xu

    IPC分类号： C12N900

    CPC分类号： C12N9/22

    摘要： The present invention relates to methods to engineer nicking endonucleases from existing Type IIs restriction endonucleases, and the production of the engineered nicking endonucleases. Two engineering methods are disclosed. One involves inactivating the dimerization function of a Type IIs restriction enzyme using site-directed mutagenesis approach. The other involves replacing the cleavage domain of a Type IIs restriction enzyme with the cleavage domain from a natural occurring nicking endonuclease, N.BstNBI.

    摘要翻译： 本发明涉及从现有的II型限制性内切核酸酶工程化切口内切核酸酶的方法，以及工程化切口内切核酸酶的产生。 公开了两种工程方法。 一个涉及使用定点诱变方法使II型限制酶的二聚功能失活。 另一个涉及用来自天然存在的切口内切核酸酶N.BstNBI的切割结构域替换II型限制酶的切割结构域。

    Method for cloning and producing the SwaI restriction endonuclease
    7.
    发明授权
    Method for cloning and producing the SwaI restriction endonuclease 有权
    用于克隆和产生SwaI限制性内切核酸酶的方法

    公开(公告)号：US06245545B1

    公开(公告)日：2001-06-12

    申请号：US09299378

    申请日：1999-04-27

    申请人： Huimin Kong ,  Lauren S. Higgins ,  Michael A. Dalton

    发明人： Huimin Kong ,  Lauren S. Higgins ,  Michael A. Dalton

    IPC分类号： C12N922

    CPC分类号： C12N9/22

    摘要： The present invention relates to the recombinant DNA which encodes the SwaI restriction endonuclease, modification methylase, and the production of SwaI restriction endonuclease from the recombinant DNA. Related expression vectors, pHKUV5 which features a strong, constitutive UV5 promoter without the Lac repressor binding site and pHKT7 which contains a powerful controllable T7 promoter and a low copy number origin of replication, are also disclosed.

    摘要翻译： 本发明涉及编码SwaI限制性内切核酸酶，修饰甲基化酶和从重组DNA产生SwaI限制性内切核酸酶的重组DNA。 还公开了相关表达载体pHKUV5，其特征在于不含Lac阻遏物结合位点的强的组成型UV5启动子和含有强大的可控T7启动子和低拷贝数复制起点的pHKT7。

    Method for cloning and producing the DraIII restriction endonuclease
    8.
    发明授权
    Method for cloning and producing the DraIII restriction endonuclease 有权
    用于克隆和产生DraIII限制性内切核酸酶的方法

    公开(公告)号：US6048719A

    公开(公告)日：2000-04-11

    申请号：US235246

    申请日：1999-01-22

    申请人： Huimin Kong ,  Lauren S. Higgins ,  Michael A. Dalton

    发明人： Huimin Kong ,  Lauren S. Higgins ,  Michael A. Dalton

    IPC分类号： C12N9/22 ,  C12N15/55

    CPC分类号： C12N9/22

    摘要： The present invention relates to the recombinant DNA which encodes the DraIII restriction endonuclease modification methylase, and the production of DraIII restriction endonuclease from the recombinant DNA. Related expression vectors, pHKUV5 vector which features a strong, constitutive UV5 promoter without the Lac repressor binding site and pHKT7 vector which contains a powerful controllable T7 promoter and a low copy number origin of replication, are also disclosed.

    摘要翻译： 本发明涉及编码DraIII限制性内切核酸酶修饰甲基化酶的重组DNA，以及从重组DNA产生DraIII限制性内切核酸酶。 还公开了相对表达载体，其具有强的组成型UV5启动子的pHKUV5载体，其不含Lac阻遏物结合位点和含有强大的可控T7启动子和低拷贝数复制起点的pHKT7载体。

    Method for cloning and producing the SnaBI restriction endonuclease and purification of the recombinant SnaBI restriction endonuclease
    9.
    发明授权
    Method for cloning and producing the SnaBI restriction endonuclease and purification of the recombinant SnaBI restriction endonuclease 有权
    用于克隆和产生SnaBI限制性内切核酸酶并纯化重组SnaBI限制性内切核酸酶的方法

    公开(公告)号：US6025179A

    公开(公告)日：2000-02-15

    申请号：US143776

    申请日：1998-08-31

    申请人： Keith D. Lunnen ,  Huimin Kong ,  Geoffrey G Wilson

    发明人： Keith D. Lunnen ,  Huimin Kong ,  Geoffrey G Wilson

    IPC分类号： C12N15/09 ,  C12N1/15 ,  C12N1/19 ,  C12N1/21 ,  C12N5/10 ,  C12N9/10 ,  C12N9/16 ,  C12N9/22 ,  C12N15/52 ,  C12N15/55

    CPC分类号： C12N15/52 ,  C12N9/1007 ,  C12N9/22

    摘要： The methylase selection method was used to clone the SnaBI methylase gene (snaBIM) from Sphaerotilus natans (ATCC 15291). An active SnaBI methylase was cloned in E. coli using pSnaBI-2, a pUC19 derivative containing two SnaBI sites. Because methylase and restriction genes are usually located alongside each other in a restriction-modification systems, efforts were made to clone the downstream DNA by inverse PCR. Inverse PCR cloned the missing portion of the SnaBI endonuclease and also identified a control, or C, protein. The two open reading frames snaBIR (ORF1) and snaBIC (ORF2) converged toward the SnaBI methylase gene (ORF). Attempts to establish a snaBIR-recombinant plasmid expressing the SnaBI endonuclease in E.coli modified with SnaBI methylase failed. Overexpression of the SnaBI endonuclease in E. coli required the use of the heterospecific BsaAI methylase.

    摘要翻译： 甲基化酶选择方法用于从Sphaerotilus natans（ATCC 15291）克隆SnaBI甲基化酶基因（snaBIM）。 使用含有两个SnaBI位点的pUC19衍生物pSnaBI-2将活性SnaBI甲基化酶克隆到大肠杆菌中。 因为甲基化酶和限制性基因通常在限制性修饰系统中彼此并列，所以努力通过反向PCR克隆下游DNA。 反向PCR克隆了SnaBI内切核酸酶的缺失部分，并鉴定了对照或C蛋白。 snaBIR（ORF1）和snaBIC（ORF2）两个开放阅读框向SnaBI甲基化酶基因（ORF）收敛。 在SnaBI甲基化酶修饰的大肠杆菌中建立表达SnaBI内切核酸酶的snaBIR重组质粒的尝试失败。 大肠杆菌中SnaBI内切核酸酶的过表达需要使用异源特异性BsaAI甲基化酶。