Synthetic secondary chromosomes are valuable tools to study chromosome maintenance systems. Important construction rules for synthetic chromosomes can be derived from their design, assembly and characterization. Furthermore, synthetic secondary chromosomes can be used to genetically modify microorganisms for biotechnology. For all applications, it is important that the synthetic secondary replicon employed is well described and characterized. In this work the synthetic secondary chromosome synVicII was established and characterized in the monochromosomal bacterium E. coli. The natural occurring secondary chromosome II of Vibrio cholerae, a bacterium which possesses two different sized chromosomes, was used as template for synVicII. The replication origin of V. cholerae chromosome II oriII, the initiator gene rctB and the specific chromosome II segregation system parABII were successfully integrated into synVicII. Transformation of synVicII proved that the synthetic secondary chromosome is able to replicate in E. coli. For the efficient assembly, modification and transfer of synVicII, different elements were inserted in synVicII: For example, an integrated oriT allows the conjugal transfer of synVicII, a yeast replication origin and a selection marker allows the assembly in yeast and a Flp-FRT-cassette allows the removal of elements only needed for construction after transfer to final strain. SynVicII was developed to be compatible with ModularCloning, which allows the fast and efficient integration of new DNA sequences. In the second part of this work synVicII was investigated regarding typical chromosome characteristics such as stability, copy number and genetic integrity. With a newly developed flow-cytometry based stability assay it was shown that synVicII is more stable than an oriC-based replicon, synEsc. A plate counting test confirmed these results and further demonstrated that synVicII is more unstable than a 100 % stable synF-plasmid. With a developed directed evolution experiments, new stable synVicII variants were identified. synVicII revealed in E. coli a comparable low copy number relative to oriC, the replication origin of the E. coli chromosome, which was shown by qPCR and microarray analysis. Copy number analysis also indicated that synVicII replicates in E. coli similarly to chromosome II in V. cholerae. Thus, synVicII probably starts DNA replication later than the E. coli chromosome. Southern Blot experiments demonstrated that synVicII in comparison to synEsc does not integrate into the E. coli chromosome. Besides secondary chromosomes, tertiary chromosomes could also be applied in biotechnology. Therefore, the diversity of secondary chromosomes of Vibrionaceae was investigated. Marker frequency analysis indicated that the eleven analyzed members of Vibrionaceae initiate DNA replication of the secondary chromosomes later than that of the primary chromosome; replication of both chromosomes is terminated simultaniously. Nine new synthetic secondary chromosomes with different Vibrionaceae replication origins were assembled and their potential as tertiary chromosome was initially tested. Conjugation experiments showed that all used Vibrionaceae replication origins are not compatible with each other. Taken together and based on the characteristics so far, synVicII revealed great potential to be applied in basic research and biotechnology.