Table 1. Summary of the genes tested. Wildtype gene sequences optimized for expression in different organisms¹ were used for embedding various messages.

Protein	Protein Length	Optimization [Encoding]	Message	Message Length		CAI		GC		Rel. Expression (msg : opt)	Assay System
					Δ opt : msg	Opt	msg	opt	msg
GST-T7RNAP	1134 aa	E. coli [msg]	GENEART AG, GERMANY/THE GENE OF YOURCHOICE/MARCH 19TH 2008/WAGNER & LISS….	83 char 498 bit	351 bp 10%	0.90	0.85	50%	47%	1.05±0.21 0.80	E. coli
GFP	239 aa	S. cerevisiae [msg]	AEQUOREA VICTORIA.	18 char 108 bit	52 bp 7%	0.95	0.86	35%	34%	0.87±0.20 0.37	S. cerevisiae
GFP	239 aa	A. thaliana [msg]	AEQUOREA VICTORIA.	18 char 108 bit	69 bp 10%	0.92	0.89	44%	44%	0.94±0.39 0.67 1.03±0.22 0.91	N. benthamiana in vitro - wheat
HIVgag	513 aa	H. sapiens [msg]	GENE DESIGNED BY MARCUS GRAF/GENEART 2008.	45 char 270 bit	167 bp 11%	0.99	0.90	65%	59%	1.03±0.05 0.40	HEK 293
EMG1	252 aa	H. sapiens [msg]	GENEART AG PAT US1234567	24 char 144 bit	92 bp 12%	0.79	0.87	64%	59%	0.92±0.12 0.35	HEK 293
EMG1	252 aa	H. sapiens[msg enc]	:JQWF&G%DY%$4Y#′XE%87G;K Pwd “Secret” →GENEART AG PAT US1234567	24 char 144 bit	93 bp 12%	0.79	0.87	64%	59%	0.67±0.27 0.19	HEK 293
GFP	239 aa	H. sapiens [msg]	AEQUOREA VICTORIA.	18 char 108 bit	63 bp 9%	0.97	0.91	59%	57%	1.12±0.31 0.61 1.04±0.25 0.89	HEK 293 in vitro - rabbit
GFP	239 aa	H. sapiens[msg enc]	4JT′T&8F#(NWGTU[FB Pwd “Secret” →AEQUOREA VICTORIA.	18 char 108 bit	68 bp 9%	0.97	0.90	59%	55%	1.00±0.22 0.91 1.07±0.37 0.88	HEK 293 in vitro - rabbit
GFP	239 aa	H. sapiens[msg + cut]	AEQUOREA VICTORIA.	18 char 108 bit	63 bp 9%	0.97	0.91	59%	57%	0.92±0.07 0.20 0.91±0.26 0.54	HEK 293 in vitro - rabbit
GFP	239 aa	H. sapiens[msg long]	GREEN FLUORESCENT PROTEIN GENEART 2008	38 char 228 bit	126 bp 18%	0.97	0.83	59%	48%	0.81±0.17 0.23 0.71±0.43 0.33	HEK 293 in vitro - rabbit

[msg]  =  Only codons for amino acids with ≥4 alternative codons (AGPTVLRS) were used for data storage. [msg enc]  =  Text message was keyed with the password “Secret” using the polyalphabetic Vigenère cipher. [msg + cut]  =  the codon usage table for Homo sapiens, used for encryption, was added in a 35 bp sequence after the stop codons of the ORF. [msg long]  =  additional codons for amino acids (CDEFHIKNQY) were used for message embedding, doubling the capacity of the container gene. Δ opt:msg  =  total number and percentage of altered nucleotides resulting from message deposition. CAI and GC  =  codon adaptation index and GC content of optimized and watermarked genes. Rel. Expression  =  mean relative expression ratio between watermarked and optimized genes ± standard deviation and students t-test p-value. Assay systems included expressing constructs in vivo in E. coli BL 21, S. cerevisiae strain AH109, human HEK293 cells, and in vitro with wheat germ and rabbit reticulocyte lysates; protein expression was determined by Western blots and ELISA.