Metabolic pathway engineering and genetic circuitry often rely on the assembly of a large number of DNA fragments. Site-directed recombination via integrases is already utilized for cloning, e.g. in the Gateway™ System, since the integration reaction of the attachment sites *attP* and *attB* to *attL* and *attR* is highly directional. But in comparison to tyrosine integrases, serine integrases (SIs) do not require additional proteins other than the recombination directionality factor (RDF), which inhibits integration but is essential for excision, the reverse reaction. Serine Integrase Recombinational Assembly (SIRA) allows up to six fragments to be assembled by one SI in a single reaction, making use of six pairs of orthogonal *att* sites with different central dinucleotide sequences. For the assembly of larger constructs or rearrangements, more SIs and their cognate RDFs are needed. To date, only 10 RDFs have been functionally characterised and range from 7.5 to 28.2 kDa. A major problem in identifying RDFs is their sequence diversity, making it hard to use purely a bioinformatics approach. We focus on the discovery of RDFs matching known bacteriophage- and prophage-derived integrases by an *in vivo* co-expression assay in the presence of a reporter plasmid containing the corresponding *att* sites. Once identified, heterologously produced RDFs are tested for activity and specificity *in vitro*. Our aim is to develop a set of orthogonal SI and cognate RDFs that are ideally active in the same buffer conditions. This toolbox for rapid and precise DNA module rearrangements *in vitro* and *in vivo* would enable large Synthetic Biology applications like the construction and optimisation of pathways for novel antibiotics, genetic memory storage or artificial chromosomes.