Extended Data Fig. 6: Insertion and characterization of Sphis5 at transcription start sites in synthetic HPRT1 and HPRT1R loci.

a, Identification of Sphis5 insertion sites based on experimental RNA-seq and CAGE-seq. Sequencing tracks for HPRT1 (top, blue) and HPRT1R (bottom, pink) are shown with forward reads on the top and reverse reads underneath and inverted. Sphis5 insertion sites are indicated with black dashed lines across the sequencing tracks. The precise insertion position, in base-pairs, and direction of transcription is indicated below the sequencing tracks. b, Example strategy for insertion of the Sphis5 coding sequence at a third experimentally-identified transcription start site. RNA-seq and CAGE-seq sequencing tracks are shown, as well as CAGE-seq peaks. The Sphis5 coding sequence (green arrow) is inserted with the 5’UTR at the 5’ boundary of the CAGE-seq peak. c, PCR verification of on-target Sphis5 insertion. PCRs were performed using a forward primer outside of the Sphis5 coding sequence and a reverse primer inside the Sphis5 coding sequence (as indicated by red arrows in b) for each insertion position. PCRs were performed on the parental yeast strain with just the HPRT1 or HPRT1R episome (Epi, top panel) and on the derivative clones with Sphis5 inserted at the indicated position, in base-pairs (bottom panel). d, Spot assays of parental HPRT1 and HPRT1R episome-containing yeast strains, and their derivative Sphis5 insertion strains, with the position of the Sphis5 insertion is indicated in base-pairs. Yeast were spotted on YPD, SC–Leu, and SC–Leu–His with 3-amino-1,2,4-triazole (3-AT), a competitive inhibitor of the Sphis5 gene product, added at the indicated concentrations. e, Spot assays of the HPRT1 strain with Sphis5 inserted at 13493 bp and derivatives in which transcription factors were knocked out by URA3 integration. Yeast were spotted on SC–Leu–Ura and SC–Leu–His with 3-AT added at the indicated concentrations. f, Insertion of the Sphis5 coding sequence into the yeast genome at the YKL162C-A locus, not adjacent to a transcription start site, as a negative control. g, PCR verification of on-target Sphis5 insertion into the genome using a primer pair as indicated by red arrows in f. h, Spot assays of the WT parental yeast strain, that strain with Sphis5 inserted into the genome, with the HPRT1 episome, and with Sphis5 inserted into the HPRT1 episome at 13493 bp. Yeast were spotted on YPD, SC–Leu, and SC–His. Residual background growth of the strains with Sphis5 inserted into the genome and in cells with the HPRT1 episome (lacking Sphis5) reflects the fact that yeast cells contain large stores of vacuolar amino acids which allow for a limited number of cell divisions on selective medium; the slightly higher growth in the former presumably reflects exceedingly low levels of Sphis5 transcription.