Helix server fiu11/11/2022 Thus, in this study, we tried to isolate such a high copy number shuttle vector derived from the pHM1519-replicon. However, shuttle vectors with high copy number are required for some protein expression in C. glutamicum strains had been estimated to be around 40 copies per cell (unpublished result). coli shuttle vector pPK4 provides kanamycin tolerance to both host strains, and it is useful for DNA manipulation in C. Using pHM1519, a variety of shuttle vectors containing another replicon driven in Escherichia coli have been constructed, a representative of which is pPK4. pCG1- and pCGR2-derived mutant shuttle plasmids exhibiting about 20–30 copies per chromosome were constructed by weakening the promoter activity of the small antisense RNA transcript. They characterized a small RNA encoded in the antisense strand sequence of DNA encoding an initiator Rep protein and indicated that the antisense RNA negatively controls plasmid replication through an interaction between the small RNA and the mRNA of Rep. reported antisense RNA-mediated plasmid copy number control in pCG1 family plasmids. glutamicum ATCC 13058 was assumed to be around 140 copies in a previous study, but it was reported that the copy numbers of pCG100 and pSR1 are about 30 per chromosome. Plasmids are normally maintained with a defined number of copies in growing populations of bacteria, and plasmid copy number is defined as the ratio of plasmid DNA molecules to chromosomal DNA in the host cell. RepA of pHM1519 is composed of 485 amino acid residues including the conserved Yuxk motif of replication initiator proteins for rolling circle DNA replication. From genetic and molecular analyses of pSR1 and pCG100, it was determined that the autonomous replication region of pHM1519 is located in a 1.9-kbp DNA fragment containing the repA gene, which encodes essential replication protein RepA. In particular the complete nucleotide sequences of pHM1519 (3,070 bp) and pCG1 (3,069 bp, GenBank: AB027714.1) were determined showing only one base difference between them, and pHM1519 is now classified as a member of the pCG1 family of corynebacterial plasmids. glutamicum strains and named pCG1, pSR1, pCG100, and so on. Plasmids very similar to pHM1519 were independently isolated from other C. A small cryptic plasmid pHM1519 was originally found in Corynebacterium glutamicum ATCC 13058.
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