DNA sequence ATGGCAAGCTTGGCACCTAGCGATCGTACCGGATTCGATGGCAAGCTTAGGCTAGCTAGCTAGGGTTCTAGA

DNA sequence (SpCas9, 20 nt guide + NGG PAM, both strands) ATGGCAAGCTTGGCACCTAGCGATCGTACCGGATTCGATGGCAAGCTTAGGCTAGCTAGCTAGGGTTCTAGA

DNA sequence (scan for internal Type IIS sites that break assembly) ATGGGTCTCAGGAGACGTCTCTGCTCTTCAAGCTTGGATCCGAATTCCAGGTCTCAA

Local alignment, match +2, mismatch −1, gap −2. Sequences capped at 120 nt.

Template DNA sequence ATGGCACCTAGCGATCGTACCGGATTCGATGGCAAGCTTAGGCTAGCTAGCTAGGGTTCTAGACCTGAACAGGTTGCTCGACGTGAACCTGGACACGGTTGACCATGCCAAGCTTCCAAGGATGGACCTCAAGGTCACGATCTGGAATCCGGTACAAATGGCACCTTCAGAAGCTGGTCCTGATCAAGGAGGCCTTAGGAATCCACCTGGAAGCTGATCCGTACTGGGGAACCTTCAGACGTACCGGATTCGATGGCAAGCTTAGGCTAGCTAGCTAGGGTTCTAG

Primer3-style design, length 18–27 (opt 20), Tm 57–63 °C (opt 60), GC 40–60%, 3′ GC clamp. Nearest-neighbour Tm (SantaLucia, 50 mM Na⁺, 0.25 µM primer). Nothing is uploaded.