Open reading frames coding for 5 amino acid residues or less are difficult for E. coli to process (2,3). Such small genes, or minigenes, lead to premature release of the very small peptide from the ribosome, with the last amino acid residue still covalently attached to its tRNA, such that the tRNA is not readily available for protein synthesis. If the pMiniT 2.0 vector recircularizes without an insert, it will cause lethal inhibition of protein synthesis and no colony will result. If the pMiniT 2.0 vector carries an insert, a colony will grow.