Synthesis and assembly of full-length cyanophage A-4L genome

Artificial cyanophages are considered to be an effective biological method to control harmful cyanobacterial bloom. However, no synthetic cyanophage genome has been constructed and where its obstacles are unclear. Here, we survey a stretch of 16 kb length sequence of cyanophage A-4L that is unclonable in Escherichia coli. We test 12 predicted promoters of cyanophage A-4L which were verified all active in E. coli. Next, we screen for eight ORFs that hindered the assembly of intermediate DNA fragments in E. coli and describe that seven ORFs in the 16 kb sequence could not be separately cloned in E. coli. All of unclonable ORFs in high-copy-number plasmid were successfully cloned using low-copy-number vector, suggesting that these ORFs were copy-number-dependent. We propose a clone strategy abandoned the promotor and the start codon that could be applied for unclonable ORFs. Last, we de novo synthesized and assembled the full-length genome of cyanophage A-4L. This work deepens the understanding of synthetic cyanophages studies.