**8. Experimental procedure for donor DNA plasmid construction by MultiSite Gateway technology**

Here, we describe our protocol for the donor DNA plasmid construction by the MultiSite Gateway technology.-

#### **8.1. PCR amplification of arm DNA fragments (1000bp)-**

*8.1.1. The first-round PCR-*

Primers (forward and reverse): arm DNA-specific oligonucleotides (35 mer)-

Primer stocks (50 or 100 μM) are diluted to 10μM prior to use.-

Template: human genomic DNA, purified with a Blood & Cell Culture DNA Mini Kit (QIAGEN).-

Template stock is diluted to 50 ng/μl prior to use.-

PCR amplification is performed with PrimeSTAR GXL DNA Polymerase kit (Takara).-

5X PrimeSTAR GXL buffer: 10μl

dNTP Mixture (2.5mM each): 4μl

 Forward primer (10μM): 1μl

 Reverse primer (10μM): 1μl

Template (50 ng/μl): 1μl

Sterile distilled water: 32μl

PrimeSTAR GXL DNA Polymerase: 1μl

PCR conditions

Initial step: 3s at 98°C (denaturation)-

25cycles:-

10s at 98°C (denaturation)-

15s at 55°C (annealing)-

1–2min/kb at 68°C (extension)-

Hold: 4°C (storage)-

The PCR reaction mixture is purified with a QIAquick PCR Purification Kit (QIAGEN) to remove the PCR primers.-

*8.1.2. The second-round PCR-*

Forward primer for the left arm:-

5′-GGGG-attB1(ACAAGTTTGTACAAAAAAGCAGGCT)-(NN)-(template-specific sequence)-3´-

Reverse primer for the left arm:-

5′-GGGG-attB5r (ACAACTTTTGTATACAAAGTTG)T-(template-specific sequence)-3´-

Forward primer for the right arm:-

5′-GGGG-attB3 (ACAACTTTGTATAATAAAGTTG)-(NN)-(template-specific sequence)-3´-

Reverse primer for the right arm:-

5′-GGGG-attB2 (ACCACTTTGTACAAGAAAGCTGGGT)A-(template-specific sequence)-3´-

The first-round primer sequences are used as each template-specific sequence.-

If AA, AG, or GA is present 5′ of the template-specific sequence, then NN (except for AA, AG, or GA) is added in order to avoid the generation of a stop codon, as described in the MultiSite Gateway User Manual.-

5X PrimeSTAR GXL buffer: 10μl

dNTP Mixture (2.5mM each): 4μl

 Forward primer (100μM): 0.5μl

 Reverse primer (100μM): 0.5μl

Template (5 ng/μl): 2μl

Sterile distilled water: 32μl

PrimeSTAR GXL DNA Polymerase: 1μl

PCR conditions

Initial step: 3s at 98°C (denaturation)-

10cycles:-

10s at 98°C (denaturation)-

1 min/kb at 68°C (annealing/extension)-

Hold: 4°C (storage)-

### **8.2. PCR amplification of the gene of interest (cDNA of the gene and polyadenylation region) or the antibiotic resistance gene (promoter, antibiotic resistance gene, and polyadenylation region)-**

Template: plasmid DNA-

Forward primer for the gene of interest:-

5′-GGGG-attB5 (ACAACTTTGTATACAAAAGTTG)-(NN)-Kozak sequence and start codon (ACCATG)-template-specific sequence)-3´-

Reverse primer for the gene of interest:-

5′-GGGG-attB4 (ACAACTTTGTATAGAAAAGTTGGGT)G-(template-specific sequence)-3´-

Forward primer for the antibiotic resistance gene:-

5′-GGGG-attB4r (ACAACTTTTCTATACAAAGTTG)-(NN)-(template-specific sequence)-3´-

Reverse primer for the antibiotic resistance gene:-

5′-GGGG-attB3r (ACAACTTTATTATACAAAGTTG)T-(template-specific sequence)-3´-

If AA, AG, or GA is present 5′ of the template-specific sequence, then NN is added as above-mentioned.-

5X PrimeSTAR GXL buffer: 10μl

dNTP Mixture (2.5mM each): 4μl

 Forward primer (100μM): 0.5μl

 Reverse primer (100μM): 0.5μl

Template: 10ng-

PrimeSTAR GXL DNA Polymerase: 1μl

Sterile distilled water: to final reaction volume of 50μl

PCR conditions

Initial step: 3s at 98°C (denaturation)-

30–35cycles:-

10s at 98°C (denaturation)-

min/kb at 68°C (annealing/extension)-

Hold: 4°C (storage)-

If the template plasmid DNA contains a kanamycin resistance gene, which also exists in the pDONR entry vector, then the PCR reaction mixture is treated with the *Dpn*I enzyme to digest the template DNA.-

The PCR products are purified by phenol/chloroform extraction and ethanol precipitation and are resuspended in TE buffer (10–50μl).-
