Novel nucleic acid molecules

The inventors have engineered novel DNA constructs for the expression of the tRNApyl genes in eukaryotic cells, especially mammalian cells, under new and improved promoter systems. The tRNApyl gene sequence possesses two internal regions that resemble an eukaryotic A box and B box, with the B box-like region more closely resembling a functional B box. Although previous attempts at reconstituting a consensus A- Box and B-box were unsuccessful as reported in Hancock et al (2010) and Mukai et al. (US 8,168,407), the inventors have now surprisingly found that the tRNApyl sequence can be altered to enable a functioning intragenic promoter and obtain a tRNApyl able to mediate efficient amber suppression in combination with WT pylRS. Such new tRNApyl gene can be used to generate highly active and stable cell lines for the incorporation of nnAAs into cells. The inventors have also found that the new modified tRNApyl gene containing a functional intragenic promoter element can be further improved by placing them downstream of the 5’ regulatory elements of genes expressed under type 4 promoters, thereby reconstituting a functional type 4 promoter element containing both extragenic and intragenic elements. The inventors have also surprisingly found that the WT tRNApyl gene can be expressed under transcriptional control of a tRNAglu gene and/or a tRNAasp gene, when said tRNAglu gene and/or tRNAasp gene is placed upstream of the tRNApyl gene and altered to lack the transcription termination sequence in order to effectively form a bicistronic message. DNA constructs bearing tandem repeats of novel tRNApyl genes of the invention have shown to lead to increased amber suppression.