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S3 Text. List of all primers used for mutant generation.

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S3 Text. List of all primers used for mutant generation. Primer name, description, and sequence are listed. Underline – first/last 126bp of the gtr6 ORF at the 5’ end.

Primer Use Sequence

TetF 5’ end primer for amplification of the tetracycline resistance gene cassette

CCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAAT TAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCT

AAAGCCACGTTGTGTCTCAAAATCTCT TetR 3’ end primer for amplification of the

tetracycline resistance gene cassette

ATGCATGGCTTTCTACGGGTTCGCTGCGAGTCTTGCCACGCCGAGCACCTG GTCGCTTTCAGAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGAC

AGTCAGGTCGAGGTGGCCCGGCTCCAT CHK5Tet 5’ flanking of antibiotic cassette

pAT04, pAT03 and pAT03a plasmids TGGCCTTTTTGCGTTTCTAC CHK3Tet 3’ flanking of antibiotic cassette

pAT04, pAT03 and pAT03a plasmids GCTATGTGCAACGGGAATTT pUC19-Gib-F 5’ Gibson primer for pUC19

amplification TCCCCGGGTACCGAGCTC

pUC19-Gib-R 3’ Gibson primer for pUC19

amplification CAGGCATGCAAGCTTGGC

Gtr6-Gib-F 5’Gibson primer for gtr6 amplification

ACGCCAAGCTTGCATGCCTGATGAAAATTGGATTGACAAAATTTAATTAT C

Gtr6-Gib-R 3’Gibson primer for gtr6

amplification ACCCTCCGGCTTAAATACCAATAGTATAAACACTCTTAATTTTATC Hyg-Gib-F 5’Gibson primer for hygromycin-

FRT amplification TGGTATTTAAGCCGGAGGGTTGCGTTTG Hyg-Gib-R 3’Gibson primer for hygromycin-

FRT amplification TCGAGCTCGGTACCCGGGGAGTCGCAGCACAGCTCGCT Gtr6-Hyg-5

5’ primer for amplification of chimeric gtr6-hygromycin-FRT

cassette

ATGAAAATTGGATTGACAAAATTTAATTATCCGGAAATTAGATGTGTTAC AACTTCGCAGGAAAATGACTATATAAATTTAAAAAAATATAATATTTATT

ATTATTTTAATAATATACCA

Gtr6-Hyg-3

3’ primer for amplification of chimeric gtr6-hygromycin-FRT

cassette

AAGCATCTTTCATATAAGCAGCATGTAAAATTTTCATATAAAGACCGGAA AAGTTAAACTTATATATTATAACTAAATATTAGCACCTACGACATCATTAT TAGGTACATTAATAACAAGATAACATAGTCGCAGCACAGCTCGCTGGTCC

AGAACTGAT Gtr6-Hyg Internal 5 Primer internal of WT gtr6 gene GCTTGTGGTTGCCCTGTAAT Gtr6-Hyg Flanking 3 3’ flanking of hygromycin-FRT AAATCTAAACCATGCCAACCA

1128 5’ flanking of gtr6 TAGATGAATGGTTGGATTGAGTT

1129 3’ flanking of gtr6 CTGAGGAAGCATCTTTCATATAAG

Gtr6::kanF Amplification of kan cassette

ATGAAAATTGGATTGACAAAATTTAATTATCCGGAAATTAGATGTGTTAC AACTTCGCAGGAAAATGACTATATAAATTTAAAAAAATATAATATTTATT ATTATTTTAATAATATACCAGTCATTAAAGCCACGTTGTGTCTCAAAATC Gtr6::kanR Amplification of kan cassette

TTAAATACCAATAGTATAAACACTCTTAATTTTATCATTATAATAATCAAT AGAATGTTCTTTTTTAATACGATTCAATGATTCAACACCTTTATTAATAAC

AACATTAGGATTTTCATATGCACTCATTATTCCCTCCAGGTATTAGAA MutF Outside For insertion verification of kan

cassette TAGATGAATGGTTGGATTGAGTT

MutR Outside For insertion verification of kan

cassette ATTGCCCGACATTATCGC

MutF Kan For insertion verification of kan

cassette TGCCTCGGTGAGTTTTCTCC

MutR Kan For insertion verification of kan

cassette CTGAGGAAGCATCTTTCATATAAG

pSC1-Gib F Anneals tp pSC1 ATGAAAATTGGATTGACAAAATTTAATTATCCGGAAATTAGATG

pSC1-Gib R Anneals to pSC1 AGCTGTTTCCTGTGTGAAATTG

Gtr6prom-Gib F Gtr6 promoter ATTTCACACAGGAAACAGCTTAAGGAGATTAAGTAATAGTAAAGTTATAAT AATATTAG

Gtr6prom-Gib R Gtr6 promoter TTTGTCAATCCAATTTTCATATAGTAACCTTAAAAAATAGTTAAATCAC

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