Prevalence of illegal restriction sites in Biobrick parts. (A) Restriction enzyme sites in the required BioBrick prefix and suffix sequences for RFC 10 are depicted above the expanded prefix and suffix sites with flanking homing endonuclease sites proposed in RFC 95. The four restriction enzyme sites EcoRI, XbaI, SpeI, and PstI contained within the BioBrick prefix and suffix must not be present within any part submitted in RFC 10 format. RFC 95 retains the Biobrick prefix and suffix and pSB1C3 plasmid backbone, but adds the homing endonuclease sites I-SceI and I-CeuI, which can be used for quality control. Recognition sites for the endonucleases are boxed and the cut sites are shown within the boxes. I-SceI and I-CeuI homing endonucleases tolerate some base substitutions in these sites, so the overall sequence degeneracy is roughly equivalent to that of a non-degenerate 10 to 12 bp restriction site . (B) The probabilities of encountering at least one of the four RFC 10 BioBrick restriction enzymes sites (colored) or at least one of the RFC 95 homing endonuclease sites (black) in random DNA sequences as a function of sequence length are shown. The impact of variable GC content in the part sequence is depicted for the BioBrick restriction enzymes. Performing quality control for part length with homing endonucleases would nearly eliminate the probability of an illegal site being observed in a gene-sized DNA sequence. BBF RFC 95 contains the equations used to calculate the curves . (C) The total number of DNA sequences in the Registry submitted in each year with a status of “Not Released” (lower) and the percentage of these parts that contain at least one RFC 10 illegal restriction site in their sequence (upper) is increasing with time, suggesting a significant and growing burden in adhering to this assembly standard. Data were collected for all parts submitted by July 29, 2013.