Cloning of target DNA is a foundational technique for gene function studies, metabolic pathway engineering, and synthetic biology. However, convenient large-fragment cloning remains a major challenge. Based on the mechanism and operation complexity, DNA cloning methods can be divided into in vitro and in vivo techniques. In-Fusion Cloning is one of the most widely used in vitro cloning methods for its convenience, rapidity, broad applicability, and high throughput (Gibson, 2011). This method relies on in vitro homologous recombination (HR), which directly recognizes and joins DNA fragments with 15–20 bphomologous sequences, enabling efficient and seamless ligation via a 15 min in vitro incubation (Raman and Martin, 2014). However, In-Fusion Cloning struggles with the ligation of long fragments due to incomplete intermediates. Competent cell DH5α is broadly used for In-Fusion Cloning, which harbors exonucleases and is RecA-deficient to degrade assembly intermediates and inhibit HR after in vitro ligation (Bryant, 1988; Danilowicz et al., 2021). In contrast, the homologous recombinases of in vivo cloning systems, such as transformation-associated recombination (TAR) in yeast and RecET in Escherichia coli, exhibit higher efficiency in in vivo ligation of large DNA fragments, but require complex operations (Fu et al., 2012; Kouprina et al., 1998). For example, the RecET cloning system requires fresh RecET-expressing E. coli for in vivo ligation, with the entire procedure taking approximately two days to complete. In this system, RecE and RecT cooperate as a 5′–3′ exonuclease and single-stranded annealing protein pair, Redγ inhibits the exonuclease activity, and RecA facilitates homologous ssDNA invasion into dsDNA (Wang et al., 2017).