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T7 extension PCR

This protocols consists in 3 steps:

• Gene-specific PCR: amplifies the gene you want to put under the control of the T7 promoter adding RBS upstream and overhangs for the next PCR
• Extension PCR: adds the T7 promoter (or its variants) upstream the gene and overhangs for the Gibson assembly
• Final PCR: amplifies the fragments extending the gibson overhangs

Gene specific-PCR

Just a normal PCR to amplify the genes. The primers should include the RBS and overhangs with the extension primers

Extension PCR