Molecular Biology Reagents and Protocols

Deoxyribonucleoside triphosphates (dNTPs), Taq DNA polymerase with 10 × ThermoPol buffer, MgSO₄, GC enhancer, Q5 High-Fidelity DNA polymerase with 5 × Q5 reaction buffer, RNase inhibitor, Cas9 Nuclease (S. pyogenes) with NEBuffer r3.1, Proteinase K, Monarch DNA Gel Extraction Kit, and the Monarch Plasmid DNA Miniprep Kit were purchased from New England Biolabs. The SYBR Gold Nucleic Acid Gel Stain, Turbo DNase I, guanosine 5′-monophosphate (GMP), T4 RNA ligase with 10 × reaction buffer, and CloneJet PCR cloning kit were obtained from ThermoFisher Scientific. T7 RNA polymerases were purchased either from New England Biolabs (T7 RNA polymerase and HiScribe T7 High Yield RNA Synthesis Kit) or ThermoFisher Scientific (T7 RNA polymerase with 5× Transcription Buffer). pCp-Cy5 dye was purchased from Jena Bioscience. Gel filtration columns, illustra NAP-25, were acquired from Cytiva. All unmodified oligodeoxyribonucleotides were purchased from Integrated DNA Technologies (IDT, Leuven). All other chemicals were obtained either from VWR or Sigma-Aldrich.