The circular vector-fragment-annealed DNA is then transformed into Escherichia coli. LIC-compatible vectors contain specifically designed segments into which the incoming fragments are cloned. LIC-compatible vectors have recently been described for various experimental frameworks, including the high-throughput production of recombinant human proteins for crystal structure determination in bacteria, the generation of intron-containing hairpin RNA constructs for RNAi in plants, and the rapid construction of vectors for targeted mutagenesis in mycobacteria. Next to cloning efficiency, the detection of proteins expressed in heterologous hosts represents a further experimental challenge. Although high-throughput protein expression has been described for e.g. E. coli, insect cells, mammalian cells and for in vitro systems, rapid and cheap detection of recombinantly expressed proteins is still a time-consuming factor and remains a major bottleneck for multi-parallel expression of large numbers of different proteins. Recently, infrared fluorescent protein has been engineered as a new reporter protein, derived from a bacterial phytochrome. IFP covalently incorporates biliverdin, a natural product of heme catabolism involved in aerobic respiration, and becomes infrared fluorescent with excitation and emission maxima at 684 nm and 708 nm, respectively. Successful expression of IFP has been reported for E. coli, human embryonic kidney cells and mice. Recently, we demonstrated that IFP also functions as an excellent reporter for protein expression in Leishmania tarentolae, a unicellular eukaryotic protozoan for recombinant protein production. Generally, vectors for heterologous protein expression are only partly standardized, which complicates strategies for expression of proteins in multiple hosts. Here, we decided to AMN107 inquirer combine the benefits of LIC and IFP for protein expression in multiple expression systems in high-throughput. We chose to generate LICcompatible vectors for protein expression in Escherichia coli and Pichia pastoris. According to recent data 80% of all recombinant proteins are currently expressed in these two organisms. However, as these expression systems are often inadequate for expression of eukaryotic proteins, the use of alternative and less frequently used systems has been recommended. We therefore included the yeast Kluyveromyces lactis and the protozoan Leishmania tarentolae as two additional, eukaryotic expression hosts in our setup. Finally, the LICcompatible cloning system was also established for in vitro protein expression. To demonstrate the capacity of our platform we generated ten LIC-compatible vectors for oriented insertion of open reading frames and then built 54 constructs for the expression of eight different plant and two fungal proteins, including transcription factors and enzymes, in the five production systems.