TABLE S1. Strains, plasmids, and primers.
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Strain
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Description
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Reference/Source
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4220
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Methicillin Sensitive Restriction Deficient S. aureus
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W. Shafer
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Newman
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Methicillin Susceptible Clincal S. aureus Isolate
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W. Shafer
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COL
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Methicillin Resistant Clinical S. aureus Isolate
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W. Shafer
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SF8300
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Methicillin Resistant Clinical S. aureus Isolate
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(1)
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LAC
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Methicillin Resistant Clinical S. aureus Isolate
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NARSA
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ATCC 29970
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S. haemolyticus human skin isolate
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ATCC
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ATCC 15305
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S. saprophyticus urine isolate
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ATCC
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AR0355
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S. aureus Newman menD::ErR (NWMN_0913)
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(2)
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AR0407
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S. aureus COL ∆ccpA::SpR (SACOL1786)
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(3)
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AR0455
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S. aureus COL ldh1::ErR/ldh2::KnR/∆ddh::SpR (SACOL0222, SACOL2618, SACOL2535)
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(4)
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AR0744
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S. aureus COL ∆pfkA (SACOL1746)
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(5)
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AR0763
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S. aureus COL ∆pyc::ErR (SACOL1123)
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This study
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AR0766
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S. aureus COL ∆pckA::KnR (SACOL1838)
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This study
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AR1179
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S. aureus Newman ∆pckA::KnR (NWMN_1681)
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This Study
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AR0769
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S. aureus COL ∆pyk::ErR (SACOL1745)
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This study
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AR1180
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S. aureus Newman ∆pyk::ErR (NWMN_1592)
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This Study
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AR0891
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S. aureus LAC ∆pyk::ErR
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(6)
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AR0772
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S. aureus COL ∆gapB::KnR (SACOL1735)
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This study
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AR0777
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S. aureus COL ∆gapA (SACOL0838)
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This study
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AR0905
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S. aureus COL ∆ackA::KnR (SACOL1760)
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Spahich (Unpublished)
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AR1148
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S. aureus COL ∆pfkA + pNV061
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This study
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AR1149
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S. aureus COL ∆pyk + pNV041
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This study
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AR1150
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S. aureus COL ∆pckA + pNV056
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This study
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AR1151
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S. aureus COL ∆gapB + pNV058
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This study
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Plasmid
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Description
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Reference/Source
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pBT2ts
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E. coli/S. aureus shuttle vector
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(7)
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pBTE
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1.2kb ermB (ErythromycinR) allele cloned into SmaI of pBT2ts
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(4)
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pBTK
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1.4kb aph-A3 (KanamycinR) allele cloned ino SmaI of pBT2ts
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(4)
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pBTS
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1.3kb aad9 (SpectinomycinR) allele cloned into SmaI of pBT2ts
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(4)
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pNV014
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5' and 3' homology regions of pyc cloned into pBTE to yield ∆pyc::ErR
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This study
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pNV018
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5' and 3' homology regions of pckA cloned into pBTK to yield ∆pckA::KnR
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This study
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pNV026
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5' and 3' homology regions of pyk cloned into pBTE to yield ∆pyk::ErR
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This study
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pNV030
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5' and 3' homology regions of gapB cloned into pBTK to yield ∆gapB::KnR
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This study
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pNV032
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5' and 3' homology regions of gapA were amplified via overlap extension PCR and cloned into EcoRI of pBT2ts
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This study
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pNV034
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5' and 3' homology regions of pfkA were amplified via overlap extension PCR and cloned into EcoRI of pBT2ts
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This study
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pTR61
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ldh1::ErR in pBT2ts (ErR in internal NdeI site in ldh1)
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(8)
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pTR75
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ldh2::KnR in pBT2ts (KnR in an engineered SalI site)
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(8)
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pJF103
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5' and 3' homology regions of ccpA cloned into pBTS to yield ∆ccpA::SpR
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(3)
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pNAS24
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5' and 3' homology regions of ackA cloned into pBTK to yield ∆ackA::KnR
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Spahich (Unpublished)
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pNV061
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pfkA cloned into the NdeI/BamHI sites of pOS1-plgt
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This study
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pNV041
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pyk cloned into theBamHI site of pOS1-plgt
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This study
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pNV056
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pckA + 418bp upstream of the start site cloned into the BamHI site of pOS1
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This study
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pNV058
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gapB + 201bp upstream of the start site cloned into the BamHI site of pOS1
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This study
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Primer
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Sequence
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Use
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pckA-5.1A
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tggtagaattcCAAGTGTCACATCCTCAGC
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Deletion
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pckA-5.1B
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tggtagaattcTGTTTCAGTGTATGTGTCTAC
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Deletion
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pckA-3.1A
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gtaggatccGCTGAAAAAGGTAGCTTCAAC
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Deletion
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pckA-3.1B
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gtaggatccTAGTATGAAACGTGGTGTGC
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Deletion
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pfkA-5.1A
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tggtagaattcTGCAAGAAAGACCTACGACC
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Deletion
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pfkA-5.1B
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TCCACCACTAGTTAAAACTGCCAAGGTGAAACAGCTAAGGG
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Deletion
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pfkA-3.1A
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CCCTTAGCTGTTTCACCTTGGCAGTTTTAACTAGTGGTGGA
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Deletion
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pfkA-3.1B
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tggtagaattcCCAGCAGCAGTTTCACCAG
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Deletion
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pyk-5.1A
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tggtagaattcGCAGTTTTAACTAGTGGTGG
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Deletion
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pyk-5.1B
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tggtagaattcGCTGGTCCAATTGTACATAC
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Deletion
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pyk-3.1A
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gtaggatccACGATTGATGCTGCTCAAGG
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Deletion
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pyk-3.1B
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gtaggatccAGAAGGTGTTTGATCTTGAGC
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Deletion
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gapB-5.1A
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tggtagaattcGATTATCGTCATGCTGATGG
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Deletion
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gapB-5.1B
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tggtagaattcCTTCCAATTCTACCCATACC
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Deletion
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gapB-3.1A
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gtaggatccCTGAGCAAATTGGAGCAC
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Deletion
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gapB-3.1B
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gtaggatccCGCCTTTAACAATCCAATCG
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Deletion
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pckA-5.1A
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tggtagaattcCAAGTGTCACATCCTCAGC
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Deletion
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pckA-5.1B
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tggtagaattcTGTTTCAGTGTATGTGTCTAC
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Deletion
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pckA-3.1A
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gtaggatccGCTGAAAAAGGTAGCTTCAAC
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Deletion
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pckA-3.1B
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gtaggatccTAGTATGAAACGTGGTGTGC
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Deletion
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gapA-5.1A
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tggtagaattcCGAACATATGGATATGACTGAACG
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Deletion
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gapA-5.1B
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GCTAAGTATGCTAATGTACGAACTGCTAAACGACCAATTCTACC
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Deletion
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gapA-3.1A
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GGTAGAATTGGTCGTTTAGCAGTTCGTACATTAGCATACTTAGC
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Deletion
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gapA-3.1B
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tggtagaattcATGGTACTACAGTGATTTTGGC
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Deletion
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pyc-5.1A
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ggtagaattcGTAACGGTGGCGTATTTGG
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Deletion
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pyc-5.1B
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tggtagaattcGCAATTTCTCCACGGTTAGC
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Deletion
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pyc-3.1A
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gtaggatccGTGACACAATAGCGACAGG
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Deletion
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pyc-3.1B
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gtaggatccGTTACCAAAACCGGATCAGC
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Deletion
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pfkA-lgt
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tggcacatatgAAGAAAATTGCAGTTTTAACTAGTGG
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Complementation
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pfkA.1B
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tggtaggagtccGCTGGTCCAATTGTACATAC
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Complementation
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pyk.2A
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tggtaggatccCAAGGTGAAACAGCTAAGGG
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Complementation
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pyk.1B
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tggtaggatccCCTTTCCATTGTTGATAAATGG
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Complementation
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gapB.1A
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tggtaggatccTTGAAAAGCCGAATTACGGAG
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Complementation
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gapB.1B
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tggtaggatccTCCGAAGTCAGAGTTAGGC
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Complementation
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pckA.1A
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tggtaggatccTCCTTCTGTAACAGACTCTG
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Complementation
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pckA.1B
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tggtaggatccCCCGTTCACACGGGTGG
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Complementation
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1. Diep BA, Gill SR, Chang RF, Phan TH, Chen JH, Davidson MG, Lin F, Lin J, Carleton HA, Mongodin EF, Sensabaugh GF, Perdreau-Remington F. 2006. Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. The Lancet 367:731–739.
2. Joshi GS, Spontak JS, Klapper DG, Richardson AR. 2011. Arginine catabolic mobile element encoded speG abrogates the unique hypersensitivity of Staphylococcus aureus to exogenous polyamines. Mol Microbiol 82:9–20.
3. Crooke AK, Fuller JR, Obrist MW, Tomkovich SE, Vitko NP, Richardson AR. 2013. CcpA-Independent Glucose Regulation of Lactate Dehydrogenase 1 in Staphylococcus aureus. PLoS ONE 8:e54293.
4. Fuller JR, Vitko NP, Perkowski EF, Scott E, Khatri D, Spontak JS, Thurlow LR, Richardson AR. 2011. Identification of a Lactate-Quinone Oxidoreductase in Staphylococcus aureus that is Essential for Virulence. Front. Cell. Inf. Microbio. 1:1–15.
5. Mike LA, Dutter BF, Stauff DL, Moore JL, Vitko NP, Aranmolate O, Kehl-Fie TE, Sullivan S, Reid PR, DuBois JL, Richardson AR, Caprioli RM, Sulikowski GA, Skaar EP. 2013. Activation of heme biosynthesis by a small molecule that is toxic to fermenting Staphylococcus aureus. Proceedings of the National Academy of Sciences 110:8206–8211.
6. Kumar NS, Dullaghan EM, Finlay BB, Gong H, Reiner NE, Selvam JJP, Thorson LM, Campbell S, Vitko N, Richardson AR, Zoraghi R, Young RN. 2014. Discovery and optimization of a new class of pyruvate kinase inhibitors as potential therapeutics for the treatment of methicillin-resistant Staphylococcus aureus infections. Bioorganic & Medicinal Chemistry 22:1708–1725.
7. Brückner R. 1997. Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS Microbiol. Lett. 151:1–8.
8. Richardson AR, Libby SJ, Fang FC. 2008. A Nitric Oxide-Inducible Lactate Dehydrogenase Enables Staphylococcus aureus to Resist Innate Immunity. Science 319:1672–1676.
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