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dc.contributor.authorReece-Hoyes, John S.
dc.contributor.authorWalhout, Albertha J. M.
dc.date2022-08-11T08:10:59.000
dc.date.accessioned2022-08-23T17:27:23Z
dc.date.available2022-08-23T17:27:23Z
dc.date.issued2018-01-02
dc.date.submitted2018-02-20
dc.identifier.citation<p>Cold Spring Harb Protoc. 2018 Jan 2;2018(1):pdb.prot094938. doi: 10.1101/pdb.prot094938. <a href="https://doi.org/10.1101/pdb.prot094938">Link to article on publisher's site</a></p>
dc.identifier.issn1559-6095 (Linking)
dc.identifier.doi10.1101/pdb.prot094938
dc.identifier.pmid29295905
dc.identifier.urihttp://hdl.handle.net/20.500.14038/49848
dc.description.abstractThis 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.
dc.language.isoen_US
dc.relation<p><a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=29295905&dopt=Abstract">Link to Article in PubMed</a></p>
dc.relation.urlhttps://doi.org/10.1101/pdb.prot094938
dc.subjectGenetic Phenomena
dc.subjectGenetics and Genomics
dc.subjectGenetic Structures
dc.subjectInvestigative Techniques
dc.subjectLaboratory and Basic Science Research
dc.subjectMedical Sciences
dc.subjectMolecular Biology
dc.subjectSystems Biology
dc.titleGenerating an Open Reading Frame (ORF) Entry Clone and Destination Clone
dc.typeJournal Article
dc.source.journaltitleCold Spring Harbor protocols
dc.source.volume2018
dc.source.issue1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/sysbio_pubs/121
dc.identifier.contextkey11595674
html.description.abstract<p>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.</p>
dc.identifier.submissionpathsysbio_pubs/121
dc.contributor.departmentProgram in Molecular Medicine
dc.contributor.departmentProgram in Systems Biology
dc.source.pagespdb.prot094938


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