Table S1. List of primers used in this study

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Table S1. List of primers used in this study Name KanMx-F2 KanMx-A2 FEN1-DG-S FEN1-DG-A SUR4-DG-S SUR4-DG-A CaARG4-R1130 CaARG4-F61 CaHIS1-DR CaHIS1-ter CaFEN1-US1 CaFEN1-UA1 CaFEN1-DS2 CaFEN1-DA2 CaFEN1-DG-S CaFEN1-DG-R1 CaFEN12-US1 CaFEN12-UA1 CaFEN12-DS2 CaFEN12-DA2 Sequence (5-3 ) a CTATGGAACTGCCTCGGTG TTGATGGTCGGAAGAGGC GTTGGTGAAACTCTCGAGC GCGCTAAAATAACGCCAG TTACGCATTTGGCTTGTC TCGTGGCAAGTAAAGTGG GCCAACATATCCATAGTTAAAGC GGTGCCACTGATCCATTG AACTTCTGTCTCCTCATCCTCC CTACCACCTTGATGTACACACC CAATCATCGCACATAAAACC GACCTGCAGCGTACGAAGATACTTGTGGGAACTCAGTTGG CTCGAATTCATCGATGATATCAGAGAGAGGTATGACGGTTGTTG GGTGATACATTTTTCGGAG CTCAATAGTCATCGACACG GTGGTAGTCAAACCACTCCAC ATAATGGAAGAGGGAAGGC GACCTGCAGCGTACGAAGGTAAAAGAAATTGCAGACCAG CTCGAATTCATCGATGATATCAGACCAACCATGTCAAGATGAG GTCATGTAGTTCCTGCTACC

CaFEN12-DG-S GAAGGATATGGAACATTCG CaFEN12-DG-R1 TCCATACTGCTCATGTTGAAG a Underlined sequences are homologous to HAH2 cassette for fusion of PCR amplified upstream or downstream flanking regions to HAH2.

Fig. S1. Diagnostic PCR to confirm deletion of FEN1 gene in putative deletant obtained from Euroscarf. Primers specific to regions flanking deleted FEN1 ORF were used in combination with primers specific for KanMX deletion cassette. The size of the PCR products obtained with forward primer (FEN1-DG-S) upstream of deleted FEN1 ORF and reverse primer from KanMX cassette (KanMx-A2), and forward primer of KanMX cassette (KanMx-F2) and reverse primer (FEN1-DG-A) downstream of deleted FEN1 ORF are comparable to the expected size of the PCR products, i.e., 1039 bp and 690 bp respectively, confirming the strain as true deletant of FEN1 gene.

Fig. S2. Diagnostic PCRs to confirm deletion of SUR4 gene in putative deletant obtained from Euroscarf. Primers specific to regions flanking deleted SUR4 ORF were used in combination with primers specific for KanMX deletion cassette. The size of the PCR products obtained with forward primer (SUR4-DG-S) upstream of deleted SUR4 ORF and reverse primer from kanamycin cassette (KanMx-A2 ), and forward primer from kanamycin cassette (KanMx-F2) and reverse primer (SUR4-DG-A) downstream of deleted SUR4 ORF are comparable to expected size of products, i.e., 938 bp and 738 bp, respectively, confirming the strain as true deletant of SUR4 gene.

Fig. S3. Deletion of both alleles of a target gene after a single transformation with HAH2 cassette. As is seen with UAU1 cassette, as much as half of the Arg + His + segregants can retain a wild-type copy of the target gene, besides the two deleted alleles, e.g, due to trisomy. Such undesired segregants have to be identified by diagnostic PCR and discarded. If the target gene is essential, then all the segregants will retain a wild-type copy of the gene.

Fig. S4. Eviction of HAH2 and HIS1 markers by recombination between LoxLE and LoxRE sites (depicted as red boxes) by Cre recombinase.

Fig. S5. Sequence of the CaFEN1 locus after eviction of markers by recombination at LoxLE and LoxRE. The complete ORF including 130bp upstream of start codon and 25bp downstream of stop codon has been deleted. The deleted region was PCR amplified with flanking primers (CaFEN1-DG-S and CaFEN1- DG-R1), and sequenced (with primer CaFEN1-DG-R1). The orange bar indicates the sequence of the scar left behind after the recombination, which includes the recombined LoxLE/RE (red bar) and the flanking primer binding regions from HAH2. Rest of the sequence corresponds to the upstream and downstream regions of CaFEN1

Fig. S6. Sequence of the CaFEN12 locus after eviction of markers by recombination at LoxLE and LoxRE. The complete ORF including 84bp upstream of start codon and 15bp downstream of stop codon has been deleted. The deleted region was PCR amplified with flanking primers (CaFEN12-DG-S and CaFEN12-DG-R1) and sequenced (with primer CaFEN12-DG-R1). The orange bar indicates the sequence of the scar left behind after the recombination, which includes the recombined LoxLE/RE (red bar) and the flanking primer binding regions from HAH2. Rest of the sequence corresponds to the upstream and downstream regions of CaFEN12

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