TABLE 2.

Primers used

NameRestriction site(s)Sequencea
Primers for engineering FRT cassettes
    FRTupNotI EcoRI, NotI GgaATTcTGTAGGCTGGAGCgGCcgCGAAGTTCC
    FRTdownSmaI SmaI GAGTGCTTGCGGCAGCGcccGGGGATCTTG
    FRTupSmaI SmaI TTGTGTAGcCcGGgGCTGCTTCGAAGTTCC
    FRTdownNotI NotI, HindIII CCTGAaaGCTTGCGGCcGCGTGAGGGGATCTTG
    MutLCP1 SnaBI CGCGTAAGGAAATCCATTAcGTACTATTTAAAAAACACAAAC
    MutLCP2 SnaBI GTTTGTGTTTTTTAAATAGTACgTAATGGATTTCCTTACGCG
    MutLCP3 SnaBI CGTCGCCCAATACGtAAACCGCCTCTCCC
    MutLCP4 SnaBI GGGAGAGGCGGTTTaCGTAttGGGCGACG
    MutPBR1 NotI CCGACCGCTTTGGCgGCCGCCCAGTCCTGC
    MutPBR2 NotI GCAGGACTGGGCGGCcGCCAAAGCGGTCGG
    NEWCAT-UP Ecl136II GGCGgAgcTcTTGAGTTATCGAGATTTTCAGGAGC
    NEWCAT-DWN SmaI, StuI TTATTCccGgGTAGCACtAGGCcTTTAAGGGCACC
    PLAC-1 StuI CAGGCATGCAgGCcTtGCGTAATC
    PLAC-3 StuI GAGGAgGCctgAcgtCGCCCAATAC
Replacement primersb
    ruvA deletion
        CiRuvA NotI ccgttccaagcggccgcaagagcgGTGAGCAAAATCGCTCGCCCTGAC
        CoRuvA SalI aaaaagtcgacAACAGCACGTCATGCGGTTCAAGG
        NiRuvA NotI cgctcttgcggccgcttggaacggTCTGAGTCTGCCTATCACATGACG
        NoRuvA PstI aaaaactgcagGTGATTACTCCAGCAATTTGATGC
    ruvB deletion
        CiRuvB4 NotI gttctgcagcggccgcgaattccgCCGCCAGAAATGCCGTAAGTC
        CoRuvB2 SalI cgcacgcatgtcgacCGCAATTTGCTGTCAATGACGATAAG
        NiRuvB3 NotI cggaattcgcggccgctgcagaacCATCCTTTACCTCATAACGCGGC
        NoRuvB3 SmaI aaggaaaaaagcccgggcAAGCGGGTCAGGAAGCG
    yacH deletion
        CiYacH NotI ccgttccaagcggccgcaagagcgCGTGGAGCAAGAAGACTGGAAAGG
        CoYacH SalI aaaaagtcgacATACCGAACCAGAAGTTGCATGGG
        NiYacH NotI cgctcttgcggccgcttggaacggGCAAATTAGCGCCAGCACATGGGG
        NoYacH PstI aaaaactgcagTACGGTATTCTTCTAGCACGACGG
    yacL deletion
        CiYacL NotI ccgttccaagcggccgcaagagcgGTCGTGGCGGCTTACCGCAATTTC
        CoYacL SalI aaaaagtcgacCAGCTTGAGTCCGATATCGTAACC
        NiYacL NotI cgctcttgcggccgcttggaacggCACTTCATGCCCCATGGACATACG
        NoYacL PstI aaaaactgcagGCGAATGTCTTCCTGGCTTCTGCG
    yacK deletion
        CiYacK NotI ccgttccaagcggccgcaagagcgGAAGATACGGGGATGATGTTAGGG
        CoYacK SalI aaaaagtcgacTCGCCGCTACGGCAGATAACTACC
        NiYacK NotI cgctcttgcggccgcttggaacggCGGCAAAGCCGAAGCCACACCCAG
        NoYacK PstI aaaaactgcagAGGTGTCGTAATTACTGAGGTCCC
    yacF deletion
        CiYacF NotI ccgttccaagcggccgcaagagcgTTTGCCATTCGTTTTATGCCGCTG
        CoYacF SalI aaaaagtcgacGGCGCGTCTTATCAGGCCTAAAGG
        NiYacF NotI cgctcttgcggccgcttggaacggAAACTCAATGCGCAGCCATGTACG
        NoYacF PstI aaaaactgcagCACCAGATCCAGCAAGCTACTTCC
    yacG deletion
        CiYacG NotI ccgttccaagcggccgcaagagcgGAAGAACCAAAGCAGTGACATTTG
        CoYacG SalI aaaaagtcgacCTGGAATATGAATTCCCGGACAGG
        NiYacG NotI cgctcttgcggccgcttggaacggCACCACCGTTTTCCCGCAGGTTGG
        NoYacG PstI aaaaactgcagGCAGCTTTGATTTACCTACATTGC
    yabB deletion
        CiYabB1 NotI ccgttccaagcggccgcaagagcgCTGTCGAGCATGAACCCGGTTGAG
        CoYabB SalI aaaaagtcgacCGGGTTGGGTCCATACGCATGTCC
        NiYabB1 NotI cgctcttgcggccgcttggaacggGTCGAGATTGACTAACGTTGCTCC
        NoYabB SmaI aaaaacccgggTGTTACATGCGGCGATGAATTGCC
    yabC deletion
        CiYabC NotI ccgttccaagcggccgcaagagcgGGGTTACCGATGACTGAAGAGCAG
        CoYabC SalI aaaaagtcgacAGCGCATTCTCTTCAAGGATCAGG
        NiYabC NotI cgctcttgcggccgcttggaacggCCCATCAATGTAGATGCCATCAGG
        NoYabC SmaI aaaaacccgggCACCATTGACATTTATCACCCGTG
  • a Restriction sites are underlined; sequences altered with respect to the template are in lowercase.

  • b For the replacement primers, the prefixes C and N indicate the 5′ and 3′ ends, respectively, of the deleted genes, while i and o stand for inner (outward-pointing) and outer (inward-pointing) primers.