The MGC platform manages 2 human ORFeome library. The version 5.1 which represents 15483 human ORF clones corresponding to 12794 human genes, and version 8, containing 12 230 human clones (11 149 non-redundant ORF). The clones exclude the 5’ and 3’ untranslated region, and the ORFs are in-frame with the attB recombination sites. The termination codons have also been removed to allow for the addition of a 3’ tag to the ORF of interest. The ORFs have been cloned into the pDONR223 vector (Spectinomycin resistant). To know if your gene of interest is cloned into the hORFome, connect to the website (http://horfdb.dfci.harvard.edu/hv5/... for the version 5.1, or on http://horfdb.dfci.harvard.edu/inde... for the version 8 ) and follow the handbook instructions (How to find an ORF clone?).
The Gateway technology is based on site-specific recombination reactions derived from bacteriophage lambda. This method permits the directional recombination of two DNA fragments of carrying Att sites, and it is used here as a high-throughput cloning method. Clones from the ORFeome (Entry vectors) are recombined with a variety of destination vectors carrying a large choice of tags, and adapted to express the ORF in the organism of interest (from E. Coli to mammals). Entry vectors are created by recombining a PCR product that contains the sequence of interest (flanked by two attB recombination sites included into the PCR primers), and a donor vector (pDONR carrying two recombination sites attP).