Figure 1

Detailed outline of the restriction site free cloning (RSFC) strategy. a Recognition sites of various type IIS REs. The cleavage patterns are indicated as red lines. b Schematic workflow of restriction site free cloning. After removal of a stuffer fragment using type IIS REs, a single PCR product can be ligated into all vectors in a seamless, sequence independent fashion. The strategy is shown for four vectors but can be extended to as many as desired. c Design of the MlyI stuffer fragment for blunt end ligations. The MlyI recognition sequence is written in italics, the entire cleavage pattern is underlined. Variable bp are denoted as ‘N’. Upstream sequences may include promoters, N-terminal tags and signal sequences, downstream sequences may include C-terminal tags and stop codons. d Design of the BmrI stuffer fragment for TA cloning. Same explanation as (c), in addition the incorporation of the dA and dT residues for TA cloning via Start- and Stop/Tyr-codons are shown (red). By varying the last nucleotide ‘X’ of the Stop/Tyr codon, either translation can be terminated or a C-terminal tag linked in frame. A dA-tailed PCR product suitable for ligation is also shown.