Figure 1. Embedding a watermark into a functional gene.

A) The 5′ end of HIV gag ORF optimized for Homo sapiens, HIVgag (optimized) with its amino acid and nucleotide sequence, and original codon ranking. The modified codon ranking and altered nucleotide sequence of HIVgag (message) shown below give the desired binary message in blue, spelling “GENE”. B) The ASCII symbols spelling “GENE” convert to the binary digits 100111, 100101, 101110, 100101, based on C) the modified ASCII table. A total of 64 typographic characters (Char) were chosen from the print characters 32 to 95 of the standard ASCII decimal code (ASCII Dec). Subtracting 32 from each value gave numbers ranging from 0 to 63 (Minus 32), which were converted into a 6-bit binary code (Binary). D) The sorted human codon usage table was used to incorporate this bitstream into the modified HIVgag (message) sequence depicted above. Only amino acids with ≥4 alternative codons were changed (red letters in HIVgag sequence at the top). Binary 1 represents codons ranking 1, 3 or 5 (odd); binary 0 is for codons ranking 2, 4 or 6 (even). To secure binary 0 at nucleotide position 43 the leucine codon ranking 4 was chosen since the 2^nd best codon would have created an undesirable SacI restriction site (GAGCTC). Embedding the four-letter text message required 12 silent substitutions (shaded grey in A) in the watermarked DNA sequence.