T7 enzyme

To generate a DNA substrate for chromatin reconstitution, a PCR product consisting of a 601 positioning sequence ^{17 (link)}, six LexA binding sites, five Gal4 DNA binding sites, and a viral E4 promoter was cloned into pGEM3z/601 vector backbone via isothermal assembly. PCR with a biotinylated reverse primer was used to add a biotin tag to the 3′ end of the 1.1-kb DNA substrate. RepA was PCR amplified from HeLa genomic DNA with Phusion polymerase, followed by PCR cleanup with the E.Z.N.A. Cycle Pure Kit (Omega Biotek). The RepA DNA template for in vitro transcription was generated by adding a 5’ T7 promoter sequence and 3’ RAT tag sequence using tailed PCR primers. 5’ RAT tagged RNA derived from the antisense sequence of the firefly luciferase mRNA (anti-Luc) and the PP7-LexA fusion protein were prepared as previously described^{18 (link)}. The Gal4-VP16 fusion protein was prepared as previously described ^{19 (link)}. In vitro transcription of all RNA was performed using the T7 enzyme (NEB) for 4 h at 37°C. RNA was treated with DNase1 (Ambion) for 15 min, and RNA was purified with the RNeasy kit (QIAGEN)