design: Generation of a large pool of uniquely barcoded plasmids was performed using an optimized type II-S endonuclease (Golden Gate) cloning strategy {Engler, 2014 #123} modified from Coradetti et al. {Coradetti, 2018 #117}. The drug resistance cassette in the modified pGI2 (Abbott, Ianiri et al. 2013) ATMT vector used in Coradetti et al. {Coradetti, 2018 #117} (pDP11) was replaced with a hygromycin resistance cassette that expresses well in Sordariomycete species. A HindIII recognition site in the region between the two divergent SapI/LguI recognition sites was added just inside the right border of the T-DNA in the pDP11 vector. We used these two divergent SapI/LguI recognition sites to introduce the random barcodes. A population of random barcodes was generated by synthesizing the oligonucleotide GATGTCCACGAGGTCTCTNNNNNNNNNNNNNNNNNNNNCGTACGCTGCAGGTCGAC and amplifying it using the primers TCACACAAGTTTGTACAAAAAAGCAGGCTGGAGCTCGGCTCTTCGCCCGATGTCCACGAGGTCTCT and CTCAACCACTTTGTACAAGAAAGCTGGGTGGATCCGCTCTTCAATTGTCGACCTGCAGCGTACG. We combined 4 ug of plasmid DNA with 140 ng of barcode PCR fragments in a 50 ul reaction with 5 ul 10x T4 ligase buffer (ThermoFisher), 2.5 ul T4 ligase (ThermoFisher), and 2.5 ul LguI (ThermoFisher). We incubated the reaction for 5 m at 37 oC, then 25 cycles of 2 m at 37oC, followed by 5 m at 16oC. The enzymes were heat inactivated for 10 m at 65oC. We let the product cool to 37oC, added 2.5 ul FastDigest Buffer (ThermoFisher), 2.5 ul Tango Buffer (ThermoFisher), 3.13 ul 2mg/ml bovine serum albumin (NEB), 12.5 ul FastDigest HindIII (ThermoFisher), and 12.5 ul LguI (ThermoFisher), and incubated the reaction at 37oC for 5 h to digest any uncut vector, then cooled to 4oC. The reaction products were purified using the Zymo DNA clean and concentrator-5 kit (Zymo Research) and eluted into 15 ul of ddH2O. To ensure a large diversity in the plasmid library, we pooled the products of 18 barcoded plasmid cloning reactions. We then transformed 25 ul of NEB 10-beta electrocompetent E. coli (NEB) with 5 ul of the Golden Gate cloning reaction product according to the manufacturers instructions in 48 independent transformations and selected for transformed E. coli using kanamycin. We pooled all transformants and estimated the diversity of the barcoded vector pool by sequencing plasmid barcodes on an Illumina HiSeq 4000 as described in Coradetti et. al {Coradetti, 2018 #117}, except those primers included dual indexes to prevent index swapping on the Illumina HiSeq 4000 {Costello, 2018 #184}. The true pool size was estimated by the relative proportion of barcodes with 1 or 2 counts as described in the script Multicodes.pl {Wetmore, 2015 #49}. We estimate our barcoded plasmid pool contains ~800 million uniquely barcoded plasmids