Fig. 2. Synthetic HPRT1 and HPRT1R loci are active in yeast.

a,b, Sequencing tracks for ATAC-seq, H3K4me3 CUT&RUN, RNA-seq and CAGE-seq reads aligned to the synthetic HPRT1 locus as an episome (Epi) (a) or integrated on chromosome XI (b). c,d, Sequencing tracks of ATAC-seq, H3K4me3 CUT&RUN, RNA-seq and CAGE-seq reads aligned to the synthetic HPRT1R locus as an episome (c) or integrated on chromosome XI (d). The synthetic locus regions (HPRT1 and HPRT1R) are shaded in b,d. The HPRT1 coding sequence is indicated in a,b and the relative position corresponding to the reversed coding sequence is indicated in c,d. For loci integrated into chromosome XI (b,d), approximately 50 kb of flanking yeast genome is shown upstream and downstream of the integrated synthetic loci with annotated yeast genes indicated. RNA-seq and CAGE-seq tracks are stranded, displayed with reverse strand reads inverted and below forward strand reads. Sequencing tracks are shown for one replicate for each genomic context. e,f, Metaplots of ATAC-seq (e) and H3K4me3 CUT&RUN (f) signal at the TSS (defined by experimental CAGE-seq peaks) ±0.5 kb for HPRT1 and HPRT1R episomal assemblons as well as the yeast (Sc) genome. Shaded region shows standard error. g, Example strategy for insertion of the Sphis5 coding sequence at two experimentally identified TSSs. RNA-seq and CAGE-seq tracks are shown, as well as CAGE-seq peaks. The Sphis5 coding sequence (green arrow) is inserted with the 5′ untranslated region at the 5′ boundary of the CAGE-seq peak. Fwd, forward; rev, reverse. h, Spot assays for yeast with Sphis5 integration on the HPRT1 or HPRT1R episome, and their parental strains, on SC–Leu–His medium. For Sphis5 insertion strains, the number indicates the position of the Sphis5 insertion along the synthetic locus.