The TelN/tos-assisted precise targeting of chromosome segments (TAPE)

[Display omitted] •We first apply the TelN/tos system to target genomic segments in E. coli host.•We successfully cloned the targeted bacterial DNA fragment up to 156 kb.•The TAPE method takes no more than five days to directly obtain the large DNA sequence.•The TAPE method has no preferences on genome sequence.•The results showed a considerable improvement of cloning efficiency.•The TAPE method provides a powerful tool to support the study on synthetic biology. Performing genomic large segmentation experiments will promote the annotation of complex genomic functions and contribute to the synthesis of designed genomes. It is challenging to obtain and manipulate large or complex DNA sequences with high efficiency. This study aims to develop an effective method for direct cloning of target genome sequences from different species. The TelN/tos system and a linear plasmid vector were first used to directly clone the large genomic segments in E. coli. For the in vitro cloning reaction, two telomeric sites were developed using TelN protelomerase at the end of the linear plasmid vector. The target DNA sequence can be easily hooked with the homology arms and maintained as a linear artificial chromosome with arbitrary restriction sites in a specific E. coli strain. Using the linear cloning strategy, we successfully cloned the bacterial DNA fragment of 156 kb, a yeast genomic fragment of 124 kb and mammalian mitochondrial fragment of 16 kb. The results showed a considerable improvement in cloning efficiency and demonstrated the important role of vector ratio in the cloning process. Due to the high efficiency and stability, TAPE is an effective technique for DNA cloning and fundamental molecular biotechnology method in synthetic biology.