The sequences of the watermarked genes,
promoters (800 bp) and terminators (300 bp) were ordered from
GeneArt(Thermo Fisher, Regensburg, Germany). For compatibility with Golden
Gate Cloning, the sequences were ordered flanked with BsaI and BsmBI
restriction sites. The promoters and terminators were delivered by
GeneArt subcloned in the entry vector pUD565 and for the watermarked
genes the subcloning into pUD565 was done in house using BsmBI Golden
Gate cloning. An exception was made for
pTDH3,
pPGK1,
tPGK1,
tENO2, and
tADH1 which were amplified from genomic DNA of CEN.PK113–7D
using primers with flanks containing BsaI restriction sites listed
in
Table S7.
Subsequently, the assembly
of the promoter, gene and terminator was done in the preassembled
vector pGGKd012 using Golden Gate cloning as described in the previous
section. pGGKd012 was assembled from the Yeast toolkit
51 (link) plasmids pYTK-002, 047, 072, 078, 081, and 083
(
Table S4).
Correct plasmid assembly
was verified by enzyme digestion with
either BsaI, BsmBI (New England Biolabs) or
FastDigest enzymes (Thermo
Fisher Scientific) following the manufacturer’s instructions.
Boonekamp F.J., Dashko S., Duiker D., Gehrmann T., van den Broek M., den Ridder M., Pabst M., Robert V., Abeel T., Postma E.D., Daran J.M, & Daran-Lapujade P. (2020). Design and Experimental Evaluation of a Minimal, Innocuous Watermarking Strategy to Distinguish Near-Identical DNA and RNA Sequences. ACS Synthetic Biology, 9(6), 1361-1375.