| Component | Amount (μl) | Final concentration |
|---|---|---|
| 2× Gibson Assembly master mix | 10 | 1× |
| pCR-Blunt vector (25 ng/μl) | 4 | 5 ng/μl |
| T2A cassette PCR product from Step 22 (100 ng/μl) | 1 | 5 ng/μl |
| sgRNA cassette PCR product from Step 21(100 ng/μl) | 1 | 5 ng/μl |
| Upstream homologous arm PCR product from Step 23 (100 ng/μl) | 1 | 5 ng/μl |
| Downstream homologous arm PCR product from Step 23 (100 ng/μl) | 1 | 5 ng/μl |
| Water | 2 | |
| Total | 20 |
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