. 2020 Aug 20;10:14018. doi: 10.1038/s41598-020-71051-5

Table 1.

Primers used for PCR.

Primers used for generation of pcDNA5/FRT_puro vector
Fragment	Primer	Sequene (5′–3′)	Amplicon size (bp)
pcDNA5 backbone	PuroR_p5_fwd	cacgaccccatgGGCTGGATGATCCTCCAGCG	3,834
pcDNA5 backbone	SV40/FRT_p5_rev	gacacgtacgtacgtGGCGAACGTGGCGAGAAAGG	3,834
Puromycin resistance	SV40/FRT_PuroR_fwd	ttccttggccACCGAGTACAAGCCCACGG	669
Puromycin resistance	p5_PuroR_rev	tcatccagccCATGGGGTCGTGCGCTCC	669
SV40 promotor-FRT region	p5_SV40/FRT_fwd	gccacgtacgtacgtGTCAGTTAGGGTGTGGAAAG	395
SV40 promotor-FRT region	PuroR_SV40/FRT_rev	tcggtggccaagGAAGTTCCTATACTTTCTAGAG	395

Primers used for site-directed mutagenesis
Vector	Primer	Sequence (5′–3′)	Amplicon size (bp)
pcDNA5/FRT_hygro	SDM_hygro_fwd	GTATAGGAACTTCCTTGGC-AAAAAGCCTGAACTCACC	N/A
pcDNA5/FRT_hygro	SDM_hygro_rev	GGTGAGTTCAGGCTTTTT-GCCAAGGAAGTTCCTATAC	N/A
pcDNA5/FRT_puro	SDM_puro_fwd	CATGGCAGAAGTTCCTATTCCGAAGTTCC	N/A
pcDNA5/FRT_puro	SDM_puro_rev	AATAGGAACTTCTGCCATGGTAGCCTCC	N/A

Primers used for cloning of CYP2C19
Reaction	Primer	Sequence (5′–3′)	Amplicon size (bp)
Reverse transcription	CYP2C19_GSP_rev	GAGGAAAGAGAGCTGCAGGG	N/A
Introduction of restriction sites	CYP2C19_HindIII_fwd	AAGAGGAGaagcttACCATGGATCCTTTTGTGGTCCTTG	1516
Introduction of restriction sites	CYP2C19_EcoRV_rev	CATCTGTgatatcTCAGACAGGAATGAAGCACAGC	1516

Primers used for Validation PCRs
Reaction	Primer	Sequence (5′–3′)	Amplicon size (bp)
Integration PCR 1	P_SV40	AGCTGTGGAATGTGTGTCAGTTAGG	559
Integration PCR 1	P_{puro_r}	CGACGCGCGTGAGGAAGAGTTCTTG	559
Integration PCR 2	P_{uni_f}	CGTTCGCCACGTACGTACGTGTCAG	489
Integration PCR 2	P_{hyr_r}	CTTCGCCCTCCGAGAGCTGCATCAG	489
Multiple Integration PCR A	P_{uni_f}	CGTTCGCCACGTACGTACGTGTCAG	564
Multiple Integration PCR A	P_{puro_r}	CGACGCGCGTGAGGAAGAGTTCTTG	564
Multiple Integration PCR B	P_{FRT_f}	AATCGGGGGCTCCCTTTAGGGTTCC	313
Multiple Integration PCR B	P_{puro_r}	CGACGCGCGTGAGGAAGAGTTCTTG	313
Multiple Integration PCR C	P_{FRT_f}	AATCGGGGGCTCCCTTTAGGGTTCC	238
Multiple Integration PCR C	P_{hyr_r}	CTTCGCCCTCCGAGAGCTGCATCAG	238

Primers used for quantitative RT-PCR
Gene	Primer	Sequence (5′-3′)	Amplicon size (bp)
CYP2C19	P_{CYP2C19_f}	CCTGATCAAAATGGAGAAGGAAAAG	99
CYP2C19	P_{CYP2C19_r}	TCTGTCCCAGCTCCAAGTAAG	99
HPRT1	P_{HPRT1_f}	TGACACTGGCAAAACAATGCA	94
HPRT1	P_{HPRT1_r}	GGTCCTTTTCACCAGCAAGCT	94
OCT1	P_{OCT1_f}	TGTCACCGAAAAGCTGAGCC	96
OCT1	P_{OCT1_r}	TCCGTGAACCACAGGTACATC	96

The 5′-hybrid part of primers used for generation of overlapping amplicons for creation of the pcDNA5/FRT_puro vector is shown by nucleotides in small capital letters. The unique sequence introduced into the generated vector is underscored. The newly introduced base into the pcDNA5/FRT_puro vector for frame shift generation is marked in bold, the nucleotide substitution for the correction of the FRT site is highlighted by an italic, bold letter. The position of the single nucleotide deletion introduced into the pcDNA5/FRT_hygro vector is shown by a hyphen. Endonuclease restriction sites are indicated by lower case letters.