MGC HUMAN IRAV PLATE - FAQs

Are cDNA clones generated in pCMVSPORT6 (e.g. many of the IMAGE clones, non-Ultimate ORF full-length cDNA clones or clones generated by the SuperScript Plasmid system) fully Gateway-compatible?

These clones are not fully Gateway compatible. While the inserts are flanked by attB sites and can be transferred via BP reaction into Donor vectors, they are less than suitable for subsequent transfer into destination (DEST) vectors for protein expression, for the following reasons:

1. The inserts generally include a stop codon; hence C-terminal tagged DEST vectors are useless unless the intent is to express the insert in its native form (untagged).
2. The clones contain 5' and 3' untranslated regions of undetermined length, i.e. inserts were not specifically cloned into the Gateway reading frame. Even if the gene is in frame with a tag, the untranslated region would result in potentially several extra amino acids between the tag and the insert.
3. The 5' untranslated regions of the inserts may not contain a ribosome binding sequence (RBS) located 5-13 bases upstream of the ATG (a requirement for bacterial expression). While this does not apply to N-terminal tagged DEST vectors, which contain an RBS and ATG, it does apply to the use of C-terminal tagged vectors or vectors for native protein expression since they have no provision for an RBS upstream of the ATG.

In short, cDNA clones generated in pCMVSport6 can be transferred into mammalian DEST vectors for untagged native expression, or N-terminal tagged expression if the insert is found to already be in the Gateway reading frame (caveat - untranslated region will code for several amino acids). But for most other purposes, inserts should be amplified by PCR with primers directed at the ATG and stop codon regions and cloned into the appropriate pENTR/D-TOPO vector, or restriction-cloned in frame into the appropriate supercoiled pENTR vector.