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Fig. 1 | BMC Systems Biology

Fig. 1

From: GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris

Fig. 1

Assembly strategy and hierarchical backbone levels of the cloning systems GoldenMOCS and GoldenPiCS. In the microorganism-independent general platform GoldenMOCS, DNA products (synthetic DNA, PCR products or oligonucleotides) are integrated into BB1 by a BsaI Golden Gate Assembly and fusion sites Fs1, Fs2, Fs3 and Fs4. Fusion sites are indicated as colored boxes with corresponding fusion site number or letter. Basic genetic elements contained in backbone 1 (BB1) can be assembled in recipient BB2 by performing a BpiI GGA reaction. The transcription units in BB2 are further used for BsaI assembly into multigene BB3 constructs. Single transcription units can be obtained by direct BpiI assembly into recipient BB3 with fusion sites Fs1-Fs4. Fusion sites determine module and transcription unit positions in assembled constructs. Thereby, fusion sites Fs1 to Fs4 are used to construct single expression cassettes in BB2 and are required between promoter (Fs1-Fs2), CDS (Fs2-Fs3) and terminator (Fs3-Fs4). Fusion sites FsA to FsI are designed to construct BB3 plasmids and separate the different expression cassettes from each other. The FSs are almost randomly chosen sequences and only FS2 has a special function, because it includes the start codon ATG. GoldenPiCS additionally includes module-containing BB1s specific for P. pastoris: 20 promoters, 1 reporter gene (eGFP) and 10 transcription terminators, and recipient BB3 vectors containing different integration loci for stable genome integration in P. pastoris and suitable resistance cassettes (Additional file 2)

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