Table 3.

Homologous recombination frequencies of pTSCNeo^r, TSCNeo^r, pTDCNeo^r, and TDCNeo^r transfectants^a

Introduced DNA	No. of transformants:			HR frequency (%)
	Subjected to analyses	With single-crossover HR	With double-crossover HR
pTSCNeor (circular)	20	0		0
TSCNeor (linear)	20	2		10
pTDCNeor (circular)	15		0	0
TDCNeor (linear)	15		3	20

^aVectors for pTSCNeo^r, TSCNeo^r, pTDCNeo^r, and TDCNeo^r were separately transfected into T. aureum ATCC 34304 cells by particle bombardment, followed by incubation on PD agar plates containing 2 mg/ml of G418 at 25°C until colonies of transformants formed. Transformants were then cultured in GY liquid medium containing 1 mg/ml of G418 at 25°C for 5 days and genomic DNA was prepared. Homologous recombination (HR) frequencies were determined by genomic PCR with primers, as follows: Sneo F Sneo R for detection of vector integration, Ta18S F1 Sneo R and Sneo F/Ta18S R for detection of single-crossover homologous recombination by pTSCNeo^r and TSCNeo^r, and Ta18S F2 Sneo R and Sneo F/Ta18S R for detection of double-crossover homologous recombination by pTDCNeo^r and TDCNeo^r. Annealing sites of primers are shown in Fig. S6A and B in the supplemental material.