Primers for hisG::URA3::hisG
Forward primer 5" AGGGAACAAAAGCTGGGTAC 3"
Reverse primer 5" CTATAGGGCGAATTGGAGCT 3"

Plasmids for Selectable Markers
Plasmid for amplifying Kan: pKan-GenMX4. seq (GCK), product size: ~1.4kb.
Plasmid for amplifying Clonat: pAG25-ClonatMX4. seq (GCK), product size: ~1.2kb.
Plasmid for amplifying HB: pAG32-hphMX4. seq (GCK), product size: ~1.5kb.
Plasmid for amplifying hisG::URA3::hisG: pMPY-Zap seq (GCK) product size ~2.2kb

PCR condition:
1. 95°C for 5 minutes
2. 95°C for 30 seconds
3. 57°C for 1 minute
4. 72°C for 3 minutes
5. Repeat steps 2 to 4 for 30 cycles
6. 72°C for 5 minutes
7. 4°C Forever


Primers for colony PCR to confirm gene knockout using Kan, Clonat, and HB.
For Kan:
Forward: KanMX4F1: 5’ ATTCTCACCGGATTCAGTCGT 3’
Reserve: KanMXR1: 5’ AATCCGGTGAGAATGGCAAA 3’
For Clonat:
Reverse: NatMX4R1: 5’ ATTCGTCGTCCGATTCGT 3’
For H-B:
Forward: hphMX4F1: 5’ TACACAAATCGCCCGCAGAA 3’
Reverse: hphMX4R1: 5’ TCGGTTTCAGGCAGGTCTT 3’


You need to design a PCR primer for your gene of interest to make it work.