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Table 3 List of primers used for constructing and verifying gene knockouts and gene cloning

From: Genetic analysis of the roles of agaA, agaI, and agaS genes in the N-acetyl-D-galactosamine and D-galactosamine catabolic pathways in Escherichia colistrains O157:H7 and C

Namea

Strainb

Sequence (5' to 3')

Primers for gene knockouts

5agaA

Both

GGCGTTGATGTAATGGATGACGCGCCGGATGTACTCGACAATGGTGTAGGCTGGAGCTGCTTC

3agaA

Both

CTGCCGCATCAACAGACAGCGTACTGCCCGCCAG CCACCATTATTCCGGGGATCCGTCGACC

5nagA

Both

TAGCGGAACTGCCGCCAGAGATCGAACAACGTTCACTGAAAATGGTGTAGGCTGGAGCTGCTTC

3nagA

Both

AGGATGATATGTGGACCGGCAGCGACGATGTCGCTGCTTTATTATTCCGGGGATCCGTCGACC

5nagB

Both

AATCCGCCAACGGCTTACATTTTACTTATTGAGGTGAATAATGGTGTAGGCTGGAGCTGCTTC

3nagB

Both

AAATATTGCCCTGAGCAAGGAGCCAGGGCAGGGATAACAAATTATTCCGGGGATCCGTCGACC

5agaI

Both

TGTGCTCTCTATTGTTTGTTTCCGCATTCGGCATTTTGTAAATGGTGTAGGCTGGAGCTGCTTC

3agaI

EDL933

ATAAGTTAATTTAAACATTTTGAGCAATTTTTCATCTGGATTATTCCGGGGATCCGTCGACC

3agaI

E. coli C

GGCGACCCGCGGTTTTTAACATCTCATGTTGCTGTGTTCTATTATTCCGGGGATCCGTCGACC

5agaS

EDL933

TGCGGATCATCCTGACCGGAGCCGGAACCTCGGCATTTATATGGTGTAGGCTGGAGCTGCTTC

5agaS

E. coli C

CTGCGGATCATCCTGACCGGAGCCGGAACGTCGGCATTTATATGGTGTAGGCTGGAGCTGCTTC

3agaS

Both

AGGATGATATGTGGACCGGCAGCGACGATGTCGCTGCTTTATTATTCCGGGGATCCGTCGACC

5agaR

E. coli C

ACGCAGCGTTGCGAAAGCTGCCGTTGAGTTGATTCAGCCAATGGTGTAGGCTGGAGCTGCTTC

3agaR

E. coli C

CTGACGCCGCGCTCCAGATCGATCGCATCTACACCAAGAAATTATTCCGGGGATCCGTCGACC

Primers for PCR and sequencing for verification of gene knockouts

FagaA

Both

ATGACACACGTTCTGCGCGCCAG

RagaA

Both

TCAAAACGAAGCTAATTGACCCTG

FnagA

Both

ATGTGGACCATCAGCTGTCTGC

RnagA

Both

TTCTTTGATCAGCCCGCGTTCGA

FnagB

EDL933

TATCGCAAATTAAACGAGTGTCT

RnagB

Both

GTTCAGTGAACGTTGTTCGATCTCT

FnagB

E. coli C

TATCGCAAATTAAACGCGTGTCT

RagaI

Both

TGACATTCGTTTGCCATCGACAGTAC

FagaI

EDL933

GACTTTGCTGCGCCAGGGGGCGAGT

RagaI

E. coli C

TGAGCAAATTTTTCATCTGGTTAGG

FagaS

Both

CATCCAGCAATCCTTTTGCTTC

RagaS

EDL933

TAGATCTCTTCCAGCGCGATATGTT

RagaS

E. coli C

TAGATCTCTTCCAGCGCGATGTGTT

FagaR

E. coli C

ATGAGTAATACCGACGCTTCAGGT

RagaR

E. coli C

ACCAGAATCACTTCAACCCCAGCC

Primers for cloning genes

5nagAHindIII

Both

GCATAAGCTTACATTTTACTTATTGAGGTGAATAATGTATGCATTAACCCAGGGCCGGATC

3nagASmaI

Both

GCATCCCGGGTTATTGAGTTACGACCTCGTTACCGTTAA

5agaAHindIII

EDL933

GCATAAGCTTCAGTAATCTGAACTGGAGAGGAAAATGTCCGGTCGAGGAAGGGATATGACA

5agaAHindIII

E. coli C

GCATAAGCTTCAGTAATCTGAACTGGAGAGGAAAATGTCCGGTCGAGGAAGGAATATGACA

3agaAPstI

Both

GCATCTGCAGTCAAAACGAAGCTAATTGACCCTGAATCC

5agaIHindIII

E. coli C

GCATAAGCTTGTTCATCAGACTAAGGATTGAGTTATGGAACGAGGCACTGCGTCTGGTGG

3agaISmaI

E. coli C

GCATCCCGGGTTAAGGTGTTAATTAAACAAATAAAGTTC

5nagBHindIII

E. coli C

GCATAAGCTTACATTTTACTTATTGAGGTGAATA

3nagBSmaI

E. coli C

GCATCCCGGGTTACAGACCTTTGATATTTTCTGC

5agaSHindIII

EDL933

GCATAAGCTTGTTCATCAGACTAAGGATTGAGTT

3agaSPst1

EDL933

GCATCTGCAGTTATGCCTGCCACGGATGAATGATTACGC

3agaYPst1

EDL933

GCATCTGCAGTTATGCTGAAATTCGAATTCGCTG

5agaSDHindIII

E. coli C

TAGCATAAGCTTATGCCAGAAAATTACACCCCT

3agaSDEcoR1

E. coli C

TAGCATGAATTCTTACAAAATGCCGAATGCGGA

5agaBDHindIII

E. coli C

GCATAAGCTTGTTCATCAGACTAAGGATTGAGTTATGACCAGTCCAAATATTCTCTTAAC

3agaBDSmaI

E. coli C

GCATCCCGGGTTACAAAATGCCGAATGCGGAACAAACAA

  1. a The primer names indicate the genes that are targeted for construction and verification of knockout mutants and for cloning. The number, 5, and the letter, F, preceding the name of the gene indicate forward primers and the number, 3, and letter, R, preceding the name of the gene indicates reverse primers. Restriction enzymes used for cloning a gene are stated in the primer name following the name of the gene.
  2. b The strain name indicates the primer used for that particular strain and when the same primer is used for both strains it is indicated as both.