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. 2020 Jul 25;13(6):1793–1806. doi: 10.1111/1751-7915.13609

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© 2020 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd

This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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Fig. 2 — Construction of SEVA 3.1 vectors. The Ep/Pp insert primers anneal on the standard BioBrick prefix and suffix, respectively, amplifying any BioBrick. The AR‐F/AR‐R and Ori‐F/Ori‐R primers sets anneal on the SEVA transcriptional terminators (T0 and T1, respectively) and on the origin of transfer (OriT), amplifying any AR and Ori module, respectively, when pSEVA is used as template. The amplified parts contain two BsaI recognition sites (red loop or line) required for SevaBrick Assembly. The AR‐F and the Ori‐R primers carry overhang sequences that reconstruct the BioBrick prefix and suffix after SevaBrick Assembly. In addition, they carry nucleotides of the upstream and downstream BioBrick terminators which are essential sequences of the new MCS 2.0.