Gibson assembly, ligase cycling reaction, Restriction-free cloning and In-Fusion assembly are prominent examples of in vitro methods relying on recombinant thermo-stable enzymes, such as exonucleases, polymerases and ligases.  Such seamless assembly cloning methods are generally based on in vitro or in vivo recombination events of short homologous regions flanking double-, or partially single-stranded linear DNA molecules and allow for a one-pot, one-step assembly of multiple DNA fragments to form a desired circular plasmid. While conventional type II restriction enzyme- and DNA-ligase-dependent cloning has provided valuable possibilities for the manufacturing of recombinant genetic material in the past, more avant-garde techniques recently overcame inherent drawbacks of conventional cloning, such as the introduction of seams, or the limited availability of unique restriction enzyme sites. The molecular toolbox required for such design approaches must include suitable cloning methods for convenient and reliable assembly of functional biological elements on a genetic level. Inspired by electrical engineering, which combines well-specified and characterized electronic elements to construct devices with defined performance, modern synthetic biological engineering aims to follow the same principles of rational design, simulation, construction and verification.   The emerging field of synthetic biology seeks for efficient ways to engineer novel genetic circuits and switches.

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