PMC:4502370 / 11300-13113
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4502370","sourcedb":"PMC","sourceid":"4502370","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502370","text":"The following DNA fragments with flanking BamHI sites at both 5′ and 3′ ends were amplified by PCR using DTS1 or DTS2 plasmids as templates: (i) the RPO26 ORF and intron (544 bp) plus 419 bp and 239 bp of 5′ and 3′ flanking genomic DNA; (ii) the MLC2 ORF (492 bp) plus 443 bp and 240 bp of 5′ and 3′ flanking genomic DNA; (iii) the PZF1 ORF (1.3 kbp) plus 455 bp and 244 bp of 5′ and 3′ flanking genomic DNA; (iv) the SKI3 ORF (3.0 kbp) plus 442 bp and 231 bp of 5′ and 3′ flanking genomic DNA; (v) the AZF1 ORF (2.7 kbp) plus 441 bp and 303 bp of 5′ and 3′ flanking genomic DNA; (vi) the TRS33 ORF plus 463 bp and 276 bp of 5′ and 3′ flanking genomic DNA; and (vii) the YOR114w ORF plus 475 bp and 232 bp of 5′ and 3′ flanking genomic DNA. A DNA fragment containing the RPO31 ORF (4.4 kbp) plus 426 bp of upstream (5′) and 230 bp of downstream (3′) chromosomal DNA was amplified by PCR from the DTS2 plasmid using primers that introduced SalI sites at both the 5′ and 3′ ends. The RPO26 intron was removed cleanly from its genomic fragment via two-stage overlap extension PCR to generate the cDNA (RPO26*) with its genomic DNA flanks and BamHI terminal restriction sites. The PCR products were then restricted at the terminal sites and inserted into yeast expression vector YEp24 (2 μ URA3). The restricted fragments of RPO26, RPO26*, and RPO31 were also inserted into yeast expression vector pRS415 (CEN LEU2). RPO26 was inserted into BamHI-cut pRS316 (CEN URA3) to yield pRS316-RPO26 for use in the plasmid shuffle assays described below. The plasmid inserts were sequenced completely to exclude the acquisition of unwanted changes during PCR amplification and cloning. Plasmids p360-TGS1 (CEN URA3TGS1) and pUN100-TGS1 (CEN LEU2TGS1) used as positive controls were described previously (Hausmann et al. 2008).","tracks":[{"project":"2_test","denotations":[{"id":"25911228-18775984-43354267","span":{"begin":1807,"end":1811},"obj":"18775984"}],"attributes":[{"subj":"25911228-18775984-43354267","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93d7","default":true}]}]}}