1 kb plus ladder

DNA templates used for in vitro transcription were subcloned into pUC19 that included a T7 RNA polymerase promoter sequence. DNA templates were PCR amplified with Q5 High Fidelity protocol (NEB) and transcribed by T7 RNA polymerase using T7 High Yield RNA Synthesis Kit (NEB). Monarch® PCR & DNA Cleanup kits were used and samples run on agarose gel electrophoresis to confirm products with a 1 kb Plus Ladder (NEB). Monarch® RNA Cleanup Kit was used and samples run on a Urea-TBE precast gel (Biorad) to confirm transcription. RNA concentration was determined on a BioTek Synergy H1 Microplate Reader using RNA nanodrop capabilities on a Take3 Multi-Volume plate. Sequences of all RNAs used in this study are listed in Supplementary Table 2.

Plasmid concentrations were determined with a spectrophotometer (NanoDrop, Thermo Fisher Scientific). A DNA mixture containing 125 fmol of each of the seven plasmids (4.4 μg DNA in total) was digested in a 50 μl reaction containing 50 U of the restriction enzyme BstBI (New England Biolabs). The digest was incubated at 50°C for 30 min. Next, 50 μl of 2X NEBuilder HiFi Assembly Master Mix was added. The reaction was briefly mixed and then returned to 50°C for 1 h. Following assembly, all DNAs were precipitated in 0.5 M ammonium acetate and 70% ethanol. Pelleted DNA was washed with 70% ethanol and stored at -20°C prior to electroporation.
For analysis of assembly efficiency, 8.8 μg of pooled plasmid DNA from each of the seven blocks was digested with BstBI. After 30 min at 50°C, one half of the digest was transferred to a second tube and combined with the enzyme components required for assembly. Both tubes were incubated at 50°C for an additional 60 min and then put on ice. An equal proportion of each mixture was analyzed on a 0.8% agarose gel run at 40 V for 11 h. Mono cut bacteriophage lambda DNA and 1 kb-plus ladder (New England Biolabs) were run as size markers.

RT-PCR reactions were performed with Phusion High Fidelity DNA Polymerase (NEB) according to the manufacturer’s recommendations, using cDNA as a template and primer pairs listed in Additional file 1: Table S4. PCR products were visualized using agarose gel electrophoresis supplemented with SafeView nucleic acid stain (NBS Biologicals) and a 1kb Plus ladder (NEB) as a size standard. PCR products were purified using the PureLink Quick Gel Extraction kit (Invitrogen) and sequenced by Eurofins Genomics.