TABLE 7.

Primers used in this study

ID	Description	Sequence (5′ to 3′)a
0001	16S rRNA fragment to confirm culture purity	ACGGCTACCTTGTTACGACTT
0002	16S rRNA fragment to confirm culture purity	ACGGCTACCTTGTTACGACTT
0224	pDGO145 backbone	GATATCGCCTCGTGATACGC
0225	pDGO145 backbone	CAGCTGCTAATAGTAGTGAAAAAATCAG
0226	PgapDH-cat-hpt selection cassette from pDGO145	CTGAACTACTGGGCCAGGTATG
0227	PgapDH-cat-hpt selection cassette from pDGO145	ATCGTGGGAATAGGCATGG
0557	Internal region of malE with an overhang matching the pDGO145 backbone	gcggcattatccctgattttttcactactattagcagctgGTTTATCTGGTTCGGGAAGTCG
0558	Internal region of malE with an overhang matching the PgapDH-cat-hpt cassette	cctccccatgctttaatacatacctggcccagtagttcagCGAAGATATATCCGCTCCGAGATG
0559	5′-flank to malE with an overhang matching the PgapDH-cat-hpt cassette	tccgggaaaacaaatcaaccTTGATTCTACCTCCAAATTA
0560	5′-flank to malE with an overhang matching the pDGO145 backbone	acattaacctataaaaataggcgtatcacgaggcgatatcCTGTGGGAAAAGTTATAGGT
0561	3′-flank to malE with an overhang matching the PgapDH-cat-hpt cassette	cctcgggcaaaaaaatcttttccatgcctattcccacgatAAGCCCAATATAATTACTGTCGC
0562	3′-flank to malE with an overhang matching the pyk gene from T. saccharolyticum	gtatagtgaatataaaatagCCGGAGTTGCAAGAATATAAAAC
0563	pyk gene from T. saccharolyticum with an overhang matching the 3′-flank of malE	ttatattcttgcaactccggCTATTTTATATTCACTATACCTTTGTAGACCA
0552	pyk gene from T. saccharolyticum with an overhang matching the Peno promoter	atatgaagggagaatggagaATGCGTAGAACTAAGATAATATGCACG
0553	Peno promoter with an overhang matching pyk	attatcttagttctacgcatTCTCCATTCTCCCTTCATATAGC
0564	Peno promoter with an overhang matching the 5′-flank of malE	taatttggaggtagaatcaaGGTTGATTTGTTTTCCCGGAA
1001	5′-flank to gogat with an overhang matching the pDGO145 backbone	attttgtttcccataggcgcgccgatTCCCCCCTCATCATGGGGAATATATGTGGG
1002	5′-flank to gogat with an overhang matching its 3′-flank	tattttgcagaatacatgaggcggccgcTAGCCGAGAATATGGCCAAAGGAGGT
1003	3′-flank to gogat with an overhang matching its 5′-flank	tagcggccgcCTCATGTATTCTGCAAAATACATTTTTT
1004	3′-flank to gogat with an overhang matching the PgapDH-cat-hpt cassette	attaattttttaaATACTTTCGCTTCTCCTCTATGAGCT
1005	PgapDH-cat-hpt cassette with an overhang matching the 3′-flank of gogat	agaggagaagcgaaagtatTTAAAAAATTAATTATTTTTTATCTAAACTATTGAA
1006	PgapDH-cat-hpt cassette with an overhang matching the int region of gogat	tccggaggatgtcggagagttTTATGAATACATTTCAGGTTTCAAAACGCC
1007	int region of gogat with an overhang matching the PgapDH-cat-hpt	aatgtattcataaAACTCTCCGACATCCTCCGGAAAACC
1008	int region of gogat with an overhang matching the pDGO145 backbone	tatacactccgctagcgcggatccgattTGAAAGAAGGTCAAATAAGGATACCGTCGGG
0033	Sequencing of hpt region in pJY19; confirmation of PgapDH-cat-hpt selection cassette removal	GCTATCTTTACAGGTACATCATTCTGTTTGTG
0034	Sequencing of hpt region in pJY19; confirmation of PgapDH-cat-hpt selection cassette removal	TTTCATCAAAGTCCAATCCATAACCC
0050	Sequencing of int region in pJY19	CCAGACGAAAAAGTTTTGAC
0051	Sequencing of int region in pJY19	TATGTCACGCTTACATTCAC
0284	Sequencing of int to hpt region in pJY19	GTTAGAGCGGCATTATCCCT
0555	Sequencing of backbone to 5′-flank in pJY19	GAGCGGATACATATTTGAATG
0556	Sequencing of 5′-flank to Peno-pyk region in pJY19	AGTTGGTATGAATTTATTCC
0565	Sequencing of 5′-flank to backbone in pJY19	TCTATTCCATACATGCCG
0566	Sequencing of 3′-flank to PgapDH region in pJY19	GCATATAAGATCCGCTTTC
0567	Sequencing of 3′-flank to pyk region in pJY19 and AVM064	GAAGCCGTAAGTGTGGATG
0568	Sequencing of 5′-flank to pyk in AVM064	ATCCATCGTAAATTCCGAAG
0281	Sequencing of PgapDH to 3′-flank region in pJY19	AAGAAAACAGACGCGCCC
0283	Sequencing of pyk to 3′-flank region in pJY19 and AVM064	ATCATCGTTGCTCAAAAAACTG
0159	Sequencing of tdk region in pJY19	GGTACGAATGTATAAGATGGTGC
0267	Sequencing of tdk region in pJY19	TTCTCCTGCCACATGAAG
0286	Sequencing of tdk region in pJY19	GCATGTTGTCGCCGTTATGA
0232	Sequencing of pyk region in pJY19	ATGCGTAGAACTAAGATAATATGCACG
0275	Sequencing of pyk region in pJY19 and AVM064	GAGATATAGGCGCAACTGCC
0276	Sequencing of pyk region in pJY19 and AVM064	GCGACAGGTATTCCTGCCGA
0277	Sequencing of pyk region in pJY19 and AVM064; confirmation of Peno-pyk insertion in AVM064	GGAATCGACATGATTGCAGCG
0278	Sequencing of pyk region in pJY19 and AVM064	TTTCGACACCCAAATCGCCG
0279	Sequencing of pyk to Peno-5′-flank in pJY19 and AVM064	CAGGCCCTTTTGTATCAAGC
1009	Sequencing of gogat region in pNJ22::GOGAT_del	ACGCACGAAAAGCCCTCTAG
1010	Sequencing of gogat region in pNJ22::GOGAT_del	AGGCGAGTCGGATAAATTTC
1011	Sequencing of gogat region in pNJ22::GOGAT_del	AATAAAAACGGTGAGTTTCC
1012	Sequencing of gogat region in pNJ22::GOGAT_del	GACGGAGAGTTAGGTTATTGGG
1013	Sequencing of gogat region in pNJ22::GOGAT_del	ACAGAAGAAGAGTTGAAGGAAAAG
1014	Sequencing of gogat region in pNJ22::GOGAT_del	CATCCTCCGGAAAACCTACC
0285	Confirmation of Pcbp-tdk selection marker removal	ACGTTATATTGCTTGCCGGG
0289	Confirmation of Pcbp-tdk selection marker removal	AAGACTCCTTTGCTCCAACC
0569	Confirmation of malE deletion, Peno-pyk insertion	CTTGTAGTATCCAATCCTGTTGACA
0472	Confirmation of malE deletion, Peno-pyk insertion	GTTGTAATGGTAAGCTGTTGCG
0544	Confirmation of malE deletion	GAGACATAGGACCTGAAGCC
0570	Confirmation of malE deletion	GCTCTTACATCAAGCGCACC
0238	Confirmation of hpt deletion	CACTTTCTTGTTGGCTCTGGCAGC
0239	Confirmation of hpt deletion	CCGGAGATGAGGCTTTTGTTGAGAAC
0003	Confirmation of nfnAB deletion	TCATCCACCCACGGTACT
0004	Confirmation of nfnAB deletion	GGGGGAAATGTATAAGAGGGGA
0681	Confirmation of gogat deletion	AGAACATATTAAAACCCCGGCA
0682	Confirmation of gogat deletion	TGGGTAAAATGGGTTGCTCC
1015	Confirmation of gogat deletion	CTATCTTTTTGTTTATCTTCATTACACATC
1016	Confirmation of gogat deletion	GATTTGCAGGTTATGGAATCTATC
1017	Confirmation of gogat deletion	CAGACAATTTTCTATGACAATATCTTTATC
1018	Confirmation of gogat deletion	TACAGGTTTGATAAAAGATATAAACGG
1019	Confirmation of gogat deletion	TCTATTTCATGTTGCCCGGG
1020	Confirmation of gogat deletion	AACCATACTTCCGAATACATTGGG

^aThe annealing sequences are represented by uppercase letters. The overhang sequences used for the Gibson assembly are represented by lowercase letters.