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Summary Advancement of DNA‐synthesis technologies has greatly facilitated the development of synthetic biology tools. However, high‐complexity DNA sequences containing tandems of short repeats are still notoriously difficult to produce synthetically, with commercial DNA synthesis companies usually rejecting orders that exceed specific sequence complexity thresholds. To overcome this limitation, we developed a simple, single‐tube reaction method that enables the generation of DNA sequences containing multiple repetitive elements. Our strategy involves commercial synthesis and PCR amplification of padded sequences that contain the repeats of interest, along with random intervening sequence stuffers that include type IIS restriction enzyme sites. GoldenBraid molecular cloning technology is then employed to remove the stuffers, rejoin the repeats together in a predefined order, and subclone the tandem(s) in a vector using a single‐tube digestion–ligation reaction. In our hands, this new approach is much simpler, more versatile and efficient than previously developed solutions to this problem. As a proof of concept, two different phytohormone‐responsive, synthetic, repetitive proximal promoters were generated and testedin plantain the context of transcriptional reporters. Analysis of transgenic lines carrying the synthetic ethylene‐responsive promoter10x2EBS‐S10fused to theGUSreporter gene uncovered several developmentally regulated ethylene response maxima, indicating the utility of this reporter for monitoring the involvement of ethylene in a variety of physiologically relevant processes. These encouraging results suggest that this reporter system can be leveraged to investigate the ethylene response to biotic and abiotic factors with high spatial and temporal resolution. 

Abstract Phytohormone ethylene regulates numerous aspects of plant physiology, from fruit ripening to pathogen responses. The molecular basis of ethylene biosynthesis and action has been investigated for over 40 years, and a combination of biochemistry, genetics, cell, and molecular biology have proven successful at uncovering the core machinery of the ethylene pathway. A number of molecular tools have been developed over the years that enable visualization of the sites of ethylene production and response in the plant. Genetically encoded biosensors take advantage of reporter proteins, i.e., fluorescent, luminescent, or colorimetric markers, to highlight the tissues that make, perceive, or respond to the hormone. This review describes the different types of biosensors currently available to the ethylene community and discusses potential new strategies for developing the next generation of genetically encoded ethylene reporters.