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. 2023 Sep 8;18(9):e0290097. doi: 10.1371/journal.pone.0290097

Fig 2. A universal entry generator.

Fig 2

(A) The Universal Entry Generator plasmid (pUEG) comprises a small backbone conferring resistance to ampicillin in bacteria. It contains a cloning site consisting of a pair of divergent PaqCI/AarI sites flanked by overlapping convergent Eco31I recognition sequences. The plasmid carries a bacterial expression cassette for the chromoprotein AmilCP that renders deep-purple colonies. (B) A sequence containing an illegal Eco31I recognition sequence can be amplified in two fragments using oligonucleotide primers with overhangs (black dashed lines) that (i) introduce a mutation to destroy the illegal site, (ii) add PaqCI/AarI recognition and fusion sites to allow one-step digestion–ligation into the Universal Entry Generator plasmid, and (iii) add the desired GreenGate overhangs (green numbers) that will define the type of GreenGate entry clone. (C) When the resulting amplicons are cloned into pUEG with one-step digestion–ligation, the new standard part will be flanked by a pair of convergent Eco31I recognition sequences capable of releasing the part with the desired GreenGate overhangs (green).