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

Cold Spring Harb Protoc. 2018 Jan 2;2018(1). doi: 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.

MeSH terms

  • Cloning, Molecular / methods*
  • DNA, Complementary / genetics*
  • Gene Expression
  • Genetic Vectors*
  • Open Reading Frames*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombination, Genetic*

Substances

  • DNA, Complementary
  • Recombinant Fusion Proteins