TABLE 1.
List of the primers and DNA substrates used for cloning, site-directed mutagenesis, strand transfer, and 3′ processing
| Step | Primer | Sequence (5′→3′)a |
|---|---|---|
| Cloning | Mac_NdeI | 5′-GCGGCAGCCATATGTTCTTGGAAAAGATAGAGCCAG-3′ |
| Mac_BamHI | 5′-TTAGCAGCCGGATCCCTATGCCACCTCTCTAGCCTCTC-3′ | |
| Site-directed mutagenesis | E92Q sense | 5′-GCAGAGGTAATTCCACAACAGACAGGAAGACAGAC-3′ |
| E92Q antisense | 5′-GTCTGTCTTCCTGTCTGTTGTGGAATTACCTCTGC-3′ | |
| T97A sense | 5′-CACAAGAGACAGGAAGACAGGCAGCACTATTTCTGTTAAAA-3′ | |
| T97A antisense | 5′-TTTTAACAGAAATAGTGCTGCCTGTCTTCCTGTCTCTTGTG-3′ | |
| F185H sense | 5′-AATGGCAGTTCATTGCATGAATCATAAAAGAAGGGGAGGAATAGGG-3′ | |
| F185H antisense | 5′-CCCTATTCCTCCCCTTCTTTTATGATTCATGCAATGAACTGCCATT-3′ | |
| DNA substrates used for strand transfer assay | Donor LTR DNA sense (primer A) | 5AmMC12-ACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-3′ |
| Donor LTR DNA antisense (primer B) | 5′-ACTGCTAGAGATTTTCCACACTGACTAAAAG-3′ | |
| Target DNA sense (primer C) | 5′-TGACCAAGGGCTAATTCACT-3Bio | |
| Target DNA antisense (primer D) | 5′-AGTGAATTAGCCCTTGGTCA-3Bio | |
| DNA substrates used for 3′ processing | LTR-3′-sense (primer E) | 5AmMC12-ACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-BioTEG |
| LTR-3′-antisense (primer F) | 5′-ACTGCTAGAGATTTTCCACACTGACTAAAAG-3′ |
a
“5AmMC12” refers to a reactive amino group attached to the 5′ end of the oligonucleotides using a 12-carbon linker, and “BioTEG” refers to a modified 3′ end with a biotin tag (3Bio) attached via a triethylene glycol spacer. BamHI and NdeI restriction sites are in boldface in the sequences of the Mac_NdeI and Mac_BamHI cloning primers. All primers were designed using QuikChange Primer Design software (Agilent Technologies).