Most bacterial species carry their genetic information on a single chromosome. However, members of the Vibrionaceae family (e.g. V. cholerae, V. natriegens) have two chromosomes of uneven size. The two chromosomes are each replicated from a single replication origin (ori). Interestingly, the replication of chromosome 2 (chr2) starts delayed ensuring that both chromosomes terminate replication in synchrony. It was found that the replication of the so called crtS (chr2 replication triggering site) on chr1 provides the signal for the replication initiation at ori2. Integration of a single crtS into the E. coli chromosome stabilizes the maintenance of an ori2- based plasmid (synVicII). We found that additional crtS-sites increase the copy number of the synVicII gradually. To identify the minimal spacing of two crtS to remain functionality, we integrated crtS pairs separated by 100 bps up to 8 kbps in E. coli strains harboring a synVicII. Indirect copy number measurements suggested a minimum spacing to maintain the functionality of two adjacent crtS- sites. In addition, we designed an approach to identify the variable and the interchangeable bases of roughly 80 bp long crtS by systematically introducing up to four N throughout the whole sequence making use of DNA oligo libraries. A genetic selection system would then separate respective mutant libraries to more or less functional crtS-sequences. As a first step, we were able to show that a differentiation of functional and non-functional crtS-sequences is possible in a competitive setting using E. coli harboring a synVicII carrying an ampicillin resistance marker. We found that at increasing ampicillin concentrations, an E. coli harboring a functional crtS sequence can overgrow an E. coli with a non- functional crtS- sequence. Application of this screen to crtS mutant libraries combined with next-generation-sequencing readout should allow complete dissection of the functional crtS sequence space as next step to uncover its mechanism of action.