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    Generating an Open Reading Frame (ORF) Entry Clone and Destination Clone

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    Authors
    Reece-Hoyes, John S.
    Walhout, Albertha J. M.
    UMass Chan Affiliations
    Program in Molecular Medicine
    Program in Systems Biology
    Document Type
    Journal Article
    Publication Date
    2018-01-02
    Keywords
    Genetic Phenomena
    Genetics and Genomics
    Genetic Structures
    Investigative Techniques
    Laboratory and Basic Science Research
    Medical Sciences
    Molecular Biology
    Systems Biology
    
    Metadata
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    Link to Full Text
    https://doi.org/10.1101/pdb.prot094938
    Abstract
    This protocol describes using the Gateway recombinatorial cloning system to create an Entry clone carrying an open reading frame (ORF) and then to transfer the ORF into a Destination vector. In this example, BP recombination is used to clone an ORF from a cDNA source into the Donor vector pDONR 221. The ORF from the resulting Entry clone is then transferred into the Destination vector pDEST-15; the product (the Destination clone) will express the ORF as an amino-terminal GST-fusion. The technique can be used as a guide for cloning any other DNA fragment of interest-a promoter sequence or 3' untranslated region (UTR), for example-with substitutions of different genetic material such as genomic DNA, att sites, and vectors as required. The series of constructions and transformations requires 9-15 d, not including time that may be required for sequence confirmation, if desired/necessary.
    Source

    Cold Spring Harb Protoc. 2018 Jan 2;2018(1):pdb.prot094938. doi: 10.1101/pdb.prot094938. Link to article on publisher's site

    DOI
    10.1101/pdb.prot094938
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/49848
    PubMed ID
    29295905
    Related Resources

    Link to Article in PubMed

    ae974a485f413a2113503eed53cd6c53
    10.1101/pdb.prot094938
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