to create the building blocks that make up the synthetic promoters oligonucleotide pairs , each with a 5 phosphate , were annealed by being boiled and then slowly cooled to room temperature ( see supplementary information for building block sequences ) . 15 l of 50 m double stranded building blocks were then ligated with 200u of t4 dna ligase ( new england biolabs ) for 2 hours at 16c . the ligation products were then purified using a microcon ym-100 column from millipore ( billerica , ma ) to reduce the number of short promoters . 15 ng of purified ligation product were then ligated into the bamh1 site of the integrating reporter plasmid pjg102 ( 20 ng ) and transformed into e. coli . transformants were scraped into luria broth plus carbinecillan , grown overnight and then maxipreped using the genelute hp plasmid maxiprep kit from sigma ( st . 130 g of the maxiprep was digested with bgli , bamh1 , sal1 and ecor1 ( 200u each ) and transformed into yeast as described in 22 . colonies growing on medium lacking uracil were picked into 96 well plates and trp colonies were then identified by replica plating onto medium lacking tryptophan . we observed that some building blocks were represented slightly more than others , even though they were added at equal molar concentrations . the relative abundance of each building block in each library scaled similarly to the melting temperature of the building block .