FastDigest AjuI

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	Uracil excision compatible vectors were generated by PCR as described by 34 using the settings outlined above or by restriction digest using the enzymes Fast Digest AjuI (Thermo Scientific) and Nb.

Protocol tips
Uracil excision compatible vectors were generated by PCR as described by 34 using the settings outlined above or by restriction digest using the enzymes Fast Digest AjuI (Thermo Scientific) and Nb.

Publication protocol

PCR and uracil-excision cloning Uracil-excision compatible PCR products were amplified with 28 cycles in 50 µl reaction mixtures using proof-reading PfuX7 polymerase33 318 . USER Fusion was performed as previously described 20 with minor modifications. Uracil-excision cloning cassettes were designed following the principles described by3 . Uracil excision compatible vectors were generated by PCR as described by 34 using the settings outlined above or by restriction digest using the enzymes Fast Digest AjuI (Thermo Scientific) and Nb.BtsI (New England Biolabs). Purified PCR products of inserts and vector were mixed in a total insert to vector ratio of 6:1 (volume). When more than one insert is cloned simultaneously, equimolar amounts of each insert was maintained. The reaction mixture was buffered with 5x Phusion HF buffer (Invitrogen) and 1 U of USERTM enzyme mix (New England Biolabs) were added. The reaction mixture was incubated for 20 min at 37 °C, followed by 20 min at 25 °C 329 before transformation of chemically competent E. coli cells. All primers used in this study are summarized in Table S1

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