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. 2020 Jul 20;86(15):e00902-20. doi: 10.1128/AEM.00902-20

TABLE 4.

Details for gene cloning of the pyr gene cluster

Gene	Primer and sequence^a	Restriction endonuclease^b	Vector/plasmid for:
Gene	Primer and sequence^a	Restriction endonuclease^b	In vitro expts	In vivo expts
pyrA	M1, AACCCATATGAAGTTCGGAGCTATTAACG	NdeI	pET21b (Ap) with N-His of pET28b/pMNX28-NHis	pCDFDuet (Sm)/pMNXCDF7
pyrA	M2, TCGGGATCCTCAGATGAGGAACGACTTCG	BamHI	pET21b (Ap) with N-His of pET28b/pMNX28-NHis	pCDFDuet (Sm)/pMNXCDF7
pyrE	FMNRF, GAGCAAAGCCATGGTCAGCTCAAACACC	NcoI	pACYCDuet1 (Cm)/pFrd (C-His tag)	pACYCDuet1 (Cm)/pFrd (C-His tag)
pyrE	FMNRHisR, GGTAGACAAGCTTGGTACAGACGCCGA	HindIII	pACYCDuet1 (Cm)/pFrd (C-His tag)	pACYCDuet1 (Cm)/pFrd (C-His tag)
pyrB	Spadh1NF, CAGAGAGGGAACCATGGGCACTGCAC	NcoI	pET21b (Ap) with N-His tag of pETM11/pSdh1M11	pRSFDuet (Km)/pSdh1Rsf
pyrB	Spadh1R, CTCCGGGTTAAGCTTCAGCGCTTG	HindIII	pET21b (Ap) with N-His tag of pETM11/pSdh1M11	pRSFDuet (Km)/pSdh1Rsf
pyrC	AmhdNheF, CTGCCAGCTAGCGTGAGGCGCATG	NheI	pET21b (Ap)/pAmh (C-His tag)	pET21b (Ap)/pAmh (C-His tag)
pyrC	AmhdHNR, GCATCTTTGCTCCTCTCGAGCCAGTTGC	XhoI	pET21b (Ap)/pAmh (C-His tag)	pET21b (Ap)/pAmh (C-His tag)
pyrD	Sdh2NdeF, TTTCATATGACTAAATTCTCTACC	NdeI	pET21b (Ap)/pSDH2
pyrD	Sdh2HindR, AATGAAGCTTAACCGGCGTGGG	HindIII	pET21b (Ap)/pSDH2

^a

The restriction endonuclease site is underlined.

^b

Used for cloning.