Table 1.

Primer sequences and plasmid templates used to obtain the experimental DNA substrates by PCR amplification

DNA substrate	Template	Primer sequences
Direct FRT sites	PL451(direct)	5′-DigN –GCTCACTCATTAGGCACCC
1168 bp DNA		5′-Bio- CGAAATTCTACCGGGTAGG
Inverted FRT sites	PL451(inverted)	5′-DigN –GCTCACTCATTAGGCACCC
1168 bp DNA		5′-Bio- CGAAATTCTACCGGGTAGG
Single FRT site	PL451(single)	5′-DigN –GCTCACTCATTAGGCACCC
1168 bp DNA		5′-Bio- CGCAGCTGTGCTCGACGTT
1168 bp DNA	pBR322	5′-DigN –GGCTGGCTGGTTTATTGC
		5′-Bio- GCACAGATGCGTAAGGAG
Single FRT site	PL451(direct)	5′-DigN –GCTCACTCATTAGGCACCC
549 bp DNA		5′-Bio- CAGCCATCTGTTGTTTGCC

PL451 (41) is the parent plasmid, which was engineered to harbor a pair of FRT sites spaced ∼600 bp apart in either the direct or the inverted orientation.

All DNA substrates used in the TPM assays were prepared by PCR.

The primer pairs for the amplification reactions were labeled at the 5′ ends with digoxigenin (DigN) in one case and biotin (Bio) in the other. A DNA substrate lacking FRT was prepared from pBR322 DNA as the template for PCR.