T7 primer

A fragment of the tk gene spanning the original location of the gene-disrupting oligonucleotide was amplified from 500 ng of genomic DNA isolated from tk-positive clones using primers AW100 (5′-TAATACGACTCACTATAGGGTTGCGCCCTCGCCGGCAGC-3′) and AW133 (5′-CAGGAAACAGCTATGACCCGGTGGGGTATCGACAGAGT-3′). AW100 is composed of nucleotides 600-618 of the coding sequence of the HSV-1 tk gene (numbering according to [63 (link)]), with a T7 forward universal primer appended to the 5′ end of the primer. AW133 is composed of nucleotides 1786-1767 of the HSV-1 tk gene with an M13 reverse universal primer appended to the 5′ end of the primer. PCR was accomplished using Ready-To-Go PCR beads (Cytiva) and a “touchdown” protocol as previously described [67 (link)]. PCR products were then sequenced using a T7 primer by Eton Bioscience, Inc. (Research Triangle Park, NC).

A segment of the tk-neo fusion gene spanning the I-SceI site was amplified from 500 ng of genomic DNA isolated from G418^R clones using primers AW85 (5’-TAATACGACTCACTATAGGGCCAGCGTCTTGTCATTGGCG-3’) and AW91 (5’-GATTTAGGTGACACTATAGCCAAGCGGCCGGAGAACCTG-3’). AW85 is composed of nucleotides 308–327 of the coding sequence of the herpes tk gene, numbering according to [59 (link)] with a T7 forward universal primer appended to the 5’ end of the primer. AW91 is composed of 20 nucleotides from the noncoding strand of neomycin gene mapping 25 through 44 bp downstream from the neomycin start codon, with an Sp6 primer appended to the 5’ end of the primer. The position of the PCR primers are indicated in Fig. 1. PCR was carried out using Ready-To-Go PCR beads (GE Healthcare) and a “touchdown” PCR protocol as previously described [60 (link)]. PCR products were then sequenced using a T7 primer by Eton Bioscience, Inc. (Research Triangle Park, NC).