Btgzi

The transcript of graA and six artificial synthetic RNAs named 500–2, 1000–1, 1000–2, 1000–3, 1000–4, and 1000–5 [33 (link)] were used in this study. All synthetic RNAs had 500-nucleotide (500–2) or 1000-nucleotide (1000–1 to 1000–5) diverse sequences [33 (link)], and each had between three-guanine bases at the 5′ end and 30-base polyadenylated tail at the 3′ end. The pMD19 plasmid encoding the graA gene was digested with HindIII (New England Biolabs, Ipswich, MA). pUC19 plasmids encoding synthetic RNA (500–2, 1000–1, 1000–2, 1000–3, and 1000–4) and the pUC19 plasmid encoding 1000–5 synthetic RNA were digested with BbsI and BtgZI, respectively (New England Biolabs). Digested fragments were purified with a PCR purification kit and then in vitro transcription was carried out with MEGAscript T7 Kit (Life Technologies) according to the manufacturer’s instruction. Transcribed RNAs were purified with RNA Clean & Concentrator™-5 (Zymo Research, Orange, CA, USA) and their concentration was determined using Qubit RNA Assay Kit (Life Technologies).

Golden Gate assemblies were performed using T4 DNA ligase (30 WU) from Thermo Scientific^TM with the accompanied buffer. BsaI, BbsI, BsmBI and BtgZI were purchased from NEB, AarI from Thermo Scientific^TM. Entry parts were diluted to 20 or 40 nM stock concentrations and 2 or 1 µl were used per 15 µl reaction, respectively. For all enzymes, 0.5 µl were used per reaction, except for AarI where 1.5 µl were necessary because of its lower activity. BsaI and AarI are active in ligase buffer, but for AarI the accompanying oligonucleotides were supplemented for optimal efficiency. For BbsI half of the ligase buffer was replaced by NEBuffer 2.1 and for BsmBI half was replaced by NEBuffer 3.1.
The general assembly protocol was 37 °C, 30 min; 16 °C, 30 min; (37 °C, 3 min; 16 °C, 5 min) × 15; 37 °C, 10 min; 50 °C, 10 min; 80 °C, 10 min. The exception was BsmBI, where 55 °C were required for the first incubation step and ligase was only added afterwards. 7.5 µl of the final reaction were used for E. coli transformation.

The restriction enzymes AcuI, BaeI,
BbvI, MboII, BtgzI and T4
DNA ligase were obtained from New England Biolabs (Ipswich, MA, USA). T4
polynucleotide kinase (PNK) was from Fermentas Thermo Scientific (Grand Island,
NY, USA).