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Circular relaxed plasmids with different S. solfataricus promoters fused to C-less cassettes were used as templates.
(C ) Sarkosyl control for the G-less cassette transcription.
Excision of the ARS-less cassette was allowed to proceed for four hours.
Residues underlined are C to G mutations in order to construct a C-less cassette.
The G-less cassette DNA template containing the adenovirus major late promoter is the pML(C2AT 19Δ-50 as reported (Szentirmay and Sawadogo, 1994) (a gift from Dr Manabu Mizuguchi).
The ARS-less cassette was created by integrating the 3083 bp PvuII fragment of pZeodir (described in the next paragraph) by homologous recombination.
ECs (10 nM) were formed at the end of the C-less cassette on the λPR- bgl template (A26 ECs) and then incubated with H-NS.
One way to do this is to use a G-less cassette with continuous labeling and direct analysis of transcripts as was done in Szertirmay and Sawadogo (1994).
For promoter-directed in vitro transcription, different promoters fused to a C-less cassette derived from a synthetic 390 nt G-less cassette (kindly provided by James Goodrich, CO) (Sawadogo and Roeder, 1985) were cloned into pGEM-T (Promega) (Supplementary file 4).
Immobilized templates (588 or 900 bp) carrying the HTLV-1 promoter linked to a G-less cassette (HTLV-1/G-less) were generated by PCR using an upstream biotinylated primer.
This model system is composed of a 588 bp (or 900 bp, see below) fragment carrying the natural HTLV-1 promoter linked to a G-less cassette immediately downstream of the TSS (Fig. 1a).
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