Linearize pYPKpw with EcoRV resulting in the linearized vector.
Perform the following 4 PCR reactions:
primers 577, 778 and pYPK0_TEF1tp_SsXYL1_TDH3tp => 2524bp_PCR_prod
primers 775, 778 and pYPK0_TDH3tp_SsXYL2_PGItp => 2924bp_PCR_prod
primers 775, 778 and pYPK0_PGItp_ScXKS1_FBA1tp => 3567bp_PCR_prod
primers 775, 578 and pYPK0_FBA1tp_ScTAL1_PDC1tp => 3009bp_PCR_prod
Mix the DNA fragments and transform a S. cerevisiae ura3 mutant. The DNA fragments will be assembled by in-vivo homologous recombination:
-|pYPKpw|124 | \/ | /\ | 124|2524bp_PCR_prod|711 | \/ | /\ | 711|2924bp_PCR_prod|1013 | \/ | /\ | 1013|3567bp_PCR_prod|643 | \/ | /\ | 643|3009bp_PCR_prod|242 | \/ | /\ | 242- | | ---------------------------------------------------------------------------------------------