Step 1 Prepare vector

Linearize pYPKpw with EcoRV resulting in the linearized vector.

Step 2 PCR of first tp

Carry out a PCR with primers 577, 567 and template pYPKa_Z_PGItp resulting in the PCR product 1231bp_PCR_prod

5GTTCTGATCCTCGAGCATCTTAAGAATTC...CTCACTAGTGACCTGCAGCCGAC3
                                 ||||||||||||||||||||||| tm 59.0 (dbd) 70.7
                                3gagtgatcactggacgtcggcTG5
5gttctgatcctcgagcatcttaagaattc3
 ||||||||||||||||||||||||||||| tm 56.1 (dbd) 69.4
3CAAGACTAGGAGCTCGTAGAATTCTTAAG...GAGTGATCACTGGACGTCGGCTG5


Taq (rate 30 nt/s) 35 cycles             |1231bp
95.0°C    |95.0°C                 |      |SantaLucia 1998
|_________|_____          72.0°C  |72.0°C|SaltC 50mM
| 03min00s|30s  \         ________|______|
|         |      \ 56.0°C/ 0min36s| 5min |
|         |       \_____/         |      |
|         |         30s           |      |4-12°C

Step 3 Gene PCR

Carry out a PCR with primers 468, 467 and template pYPKa_A_ScXKS1 resulting in the PCR product 1892bp_PCR_prod

5GTCGAGGAACGCCAGGTTGCCCACT...TCTGTGCAGACAAACGCATCAGGAT3
                             ||||||||||||||||||||||||| tm 59.0 (dbd) 73.8
                            3agacacgtctgtttgcgtagtcctaAATTTA5
5gtcgaggaacgccaggttgcccact3
 ||||||||||||||||||||||||| tm 64.8 (dbd) 79.7
3CAGCTCCTTGCGGTCCAACGGGTGA...AGACACGTCTGTTTGCGTAGTCCTA5


Taq (rate 30 nt/s) 35 cycles             |1892bp
95.0°C    |95.0°C                 |      |SantaLucia 1998
|_________|_____          72.0°C  |72.0°C|SaltC 50mM
| 03min00s|30s  \         ________|______|
|         |      \ 57.0°C/ 0min56s| 5min |
|         |       \_____/         |      |
|         |         30s           |      |4-12°C

Step 4 PCR of last tp

Carry out a PCR with primers 568, 578 and template pYPKa_E_FBA1tp resulting in the PCR product 969bp_PCR_prod

5GTGCCATCTGTGCAGACAAACG...ACTTATGAATGTGGCAATGAGACAAGAAC3
                          ||||||||||||||||||||||||||||| tm 56.5 (dbd) 69.5
                         3tgaatacttacaccgttactctgttcttg5
5GTGCcatctgtgcagacaaacg3
 |||||||||||||||||||||| tm 57.1 (dbd) 71.5
3CACGGTAGACACGTCTGTTTGC...TGAATACTTACACCGTTACTCTGTTCTTG5


Taq (rate 30 nt/s) 35 cycles             |969bp
95.0°C    |95.0°C                 |      |SantaLucia 1998
|_________|_____          72.0°C  |72.0°C|SaltC 50mM
| 03min00s|30s  \         ________|______|
|         |      \ 56.0°C/ 0min29s| 5min |
|         |       \_____/         |      |
|         |         30s           |      |4-12°C

Step 5 Yeast transformation

Mix the four linear DNA fragments and transform a Saccharomyces cerevisiae ura3 mutant with the mixture. The fragments will be assembled by in-vivo homologous recombination:

 -|pYPKpw|124
|         \/
|         /\
|         124|1231bp_PCR_prod|50
|                             \/
|                             /\
|                             50|1892bp_PCR_prod|37
|                                                \/
|                                                /\
|                                                37|969bp_PCR_prod|242
|                                                                  \/
|                                                                  /\
|                                                                  242-
|                                                                     |
 ---------------------------------------------------------------------

Step 6 Diagnostic PCR confirmation