FastDigest BpiI (IIs class)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	A modified protocol using the FastDigest versions (Thermo Fisher Scientific, USA) of the BsaI and BpiI restriction enzymes were used with an initial 10 min digestion, 20–50 cycles of digestion and ligation (37 °C for 2 min, 16 °C for 5 min), followed by a final digestion step and a heat inactivation step, was used for most assemblies, instead of the standard MoClo protocol.

Protocol tips
A modified protocol using the FastDigest versions (Thermo Fisher Scientific, USA) of the BsaI and BpiI restriction enzymes were used with an initial 10 min digestion, 20–50 cycles of digestion and ligation (37 °C for 2 min, 16 °C for 5 min), followed by a final digestion step and a heat inactivation step, was used for most assemblies, instead of the standard MoClo protocol.

Publication protocol

"PCR amplifications were conducted using KAPA HiFi HotStart ReadyMix (Roche Diagnostic, CH) or Phusion High-Fidelity DNA Master Mix (Thermo Fisher Scientific, USA), for primers see Additional file 1. The MoClo modular cloning system [53] was employed for construction of all expression cassettes (Figs. 1, 6a; Tables 1, 2 for more details see Additional file 1). Flanking regions of approximately 800 bps were designed for integration of the expression cassettes at the locus of the deleted penicillin cluster of DS68530 by in vivo homologous recombination. Internal BsaI, BpiI and in most cases also DraIII recognition sites of the DNA elements were removed during the cloning. A modified protocol using the FastDigest versions (Thermo Fisher Scientific, USA) of the BsaI and BpiI restriction enzymes were used with an initial 10 min digestion, 20–50 cycles of digestion and ligation (37 °C for 2 min, 16 °C for 5 min), followed by a final digestion step and a heat inactivation step, was used for most assemblies, instead of the standard MoClo protocol.

The endogenous elements for the expression cassettes constructed in this study, were amplified by PCR from genomic DNA of P. chrysogenum DS54468. The amdS selection cassette used was described previously [54]. The 138 bps Sc_ura3 CP amplified from genomic DNA of S. cerevisiae CEN. PK, is slightly longer compared to the version used by Pachlinger et al. [17]. The DsRed-SKL gene was amplified from the pJAK109 plasmid [54] while the promoter of A. nidulans ribosomal protein S8 (AN0465.2, referred to as 40S) was amplified from pDSM-JAK108 [39]. The gndA promoter (Sequence ID: AM270223.1 32820 to 32040) from A. niger CBS 513.88 was ordered as a synthetic DNA from IDT. The GFP was amplified from the pSpCas9-2A-GFP plasmid, kindly provided by Feng Zhang via Addgene (Plasmid #48138 [55]). The pAC-7-QFBDAD plasmid, used for amplification of the QF DBD was kindly provided by Christopher Potter, via Addgene (Plasmid #46096 [25]). The plasmid pVG2.2 used as a template for the VP16 AD was a gift from Vera Meyer [12]. The 94 bps long An_NirA CP (identical to the sequence used by Pachlinger et al. [17]) as well as the cassettes containing 1 or 5xQUAS sequences, were ordered as oligos that were annealed before assembly to level 0 vectors, the initial building blocks used in the MoClo system. The repetitions of the QUAS elements were designed to contain some variability as the genetic stability of P. chrysogenum strains was an initial concern. The 11xQUAS carrying plasmid was constructed with the assembly of three units of annealed oligos (see Additional file 1). The design was for creating a 15xQUAS containing part, but this was proven to be difficult for E. coli to assemble, as some QUAS sequences were looped out during the construction. The sequence of the actual 11xQUAS part constructed can be found in Additional file 1."

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