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Compatible Gateway Destination vectors
BCCM/LMBP also offers a range of compatible Gateway destination vectors both with and without C- or N-terminal markers, as well as some multisite Gateway destination vectors. This way, you can create the expression vectors that best suit your experimental setup.
Recent technological advances provide the means to efficiently scan the human genome to identify genes associated with diseases. However, the subsequent functional characterisation of the human genome remains a bottleneck in elucidating pathways and developing therapeutics. Several tools (RNAi, TALEN, CRISPR) are available to systematically modulate gene expression loss-of-function experiments. In order to perform complementary gain-of-function gene studies, genome-wide cDNA or protein-encoding open reading frame (ORF) libraries are required.
The human ORFeome version 8.1 was created at the Center for Cancer Systems Biology (CCSB) of the Dana-Farber Cancer Institute and encompasses all previous human ORFeome releases. It was based primarily on the ORFs that are available in the Mammalian Gene Collection (MGC). Expansion of this ORFeome library is still ongoing, and it is the intention to include mutant genes as well.
hORFeome v8.1 represents a central resource of over 12,000 single-colony human ORFs cloned in pDONR223 (Figure 1). This set of ORFs ranges in size from 75 to more than 10,000 base pairs. For some genes, multiple splice variants are present. The entire library has been sequence-verified via Illumina sequencing.
These ORF entry plasmids are accessible as individual plasmids and can be readily transferred to Gateway compatible destination vectors for various functional studies. Upon ordering, the sequence of individual clones will be validated.
Figure 1: representative circular map for all the hORFeome v8.1 clones.
Each ORF consists of the entire coding sequence including the initiation and termination codon and excluding the 5’ and 3’ mRNA untranslated regions (UTRs). The termination codon of the ORFs is incomplete (Figure 2), allowing the addition of any C-terminal tag or marker of your choice.
Figure 2: representation of an ORF and flanking attL sites. The termination codon lacks the 3rd nucleotide (TR = TA/TG).
The ORFeome v8.1 library was described in Yang et al., Nat. Methods 8 (2011), 659-661 [PMID: 21706014].
Detailed information can also be found on http://horfdb.dfci.harvard.edu/.