These strains contain our collection of universal drug cassettes and are available on request. The sequence of the tetracycline cassette is given below. Its expression is dependent on tetracycline or appropriate analog. Others were derived from the chloramphenicol cassette (also below) by replacing aaaC1 with the indicated gene, and are constitutive. All are flanked by universal caps, UL1 & UR1, allowing the same primer pair to amplify any of them. Also included is TT22970, as a source of the lambda red plasmid, pKD46.
Chloramphenicol: TT23216 sty(LT2) $DEL leu-21 proB1657::Tn10 $F'128 lacIq lacI33(fs) lacIZ(omega fusion) lacA4646:UNI-cmr-RES(gammadelta)(sw)
Rifampicin: TT23430 sty(LT2) terY2::UNI-rif(sw)
Kanamycin: TT23664 sty(LT2) $DEL21(leu) proB1657::Tn10 $F' pro+ lacIq lacI33(fs) lacIZ(omega fusion) dinB62::UNI-kan(sw)
Gentamycin: TT24691 sty(LT2) eutR::UNI-gent(sw) metA22 metE551 trpD2 ilv-452 leu- pro-(leaky) hsdLT6 hsdSA29 hsdB strA120
Zeocin: TT24692 sty(LT2) eutR::UNI-zeo(sw) metA22 metE551 trpD2 ilv-452 leu- pro-(leaky) hsdLT6 hsdSA29 hsdB strA120
Spectinomycin: TT24693 sty(LT2) eutR::UNI-spec(sw) metA22 metE551 trpD2 ilv-452 leu- pro-(leaky) hsdLT6 hsdSA29 hsdB strA120
Tetracycline: TT25401 sty(LT2) $CRR2061[zfa-9223::kan*zfa-9228::UNI-tet*Peut] eut-38::MudA
λ red: TT22970 sty(LT2) $pKD46 araC bla oriR101 repA101ts lambda red (gam+ bet+ exo+) $COM grow at 30°!; lambda red under Para control; Datsenko & Wanner PNAS 97 6640-6645 2000
Sequence of universal CmR-cassette template.
Made by amplifying pACYC184 with TP928 (ul1-cmr), which binds upstream
of cat promoter and introduces the UL1 universal binding site at the
upstream end of the amplified DNA, and TP929 (ur1-cmr), which binds
downstream of putative transcriptional stop following aaaC1 and
introduces the UR1 universal binding site at the downstream end of the
amplified DNA.
UL1 1 21
promoter 214 240
potential msg start 247
aaaC1 (camR) 307 966
UR1 1204 1224
1 CACCAAACAC CCCCCAAAAC CAAGCCACTG GAGCACCTCA AAAACACCAT
51 CATACACTAA ATCAGTAAGT TGGCAGCATC ACCCGACGCA CTTTGCGCCG
101 AATAAATACC TGTGACGGAA GATCACTTCG CAGAATAAAT AAATCCTGGT
151 GTCCCTGTTG ATACCGGGAA GCCCTGGGCC AACTTTTGGC GAAAATGAGA
201 CGTTGATCGG CACGTAAGAG GTTCCAACTT TCACCATAAT GAAATAAGAT
251 CACTACCGGG CGTATTTTTT GAGTTATCGA GATTTTCAGG AGCTAAGGAA
301 GCTAAAATGG AGAAAAAAAT CACTGGATAT ACCACCGTTG ATATATCCCA
351 ATGGCATCGT AAAGAACATT TTGAGGCATT TCAGTCAGTT GCTCAATGTA
401 CCTATAACCA GACCGTTCAG CTGGATATTA CGGCCTTTTT AAAGACCGTA
451 AAGAAAAATA AGCACAAGTT TTATCCGGCC TTTATTCACA TTCTTGCCCG
501 CCTGATGAAT GCTCATCCGG AATTCCGTAT GGCAATGAAA GACGGTGAGC
551 TGGTGATATG GGATAGTGTT CACCCTTGTT ACACCGTTTT CCATGAGCAA
601 ACTGAAACGT TTTCATCGCT CTGGAGTGAA TACCACGACG ATTTCCGGCA
651 GTTTCTACAC ATATATTCGC AAGATGTGGC GTGTTACGGT GAAAACCTGG
701 CCTATTTCCC TAAAGGGTTT ATTGAGAATA TGTTTTTCGT CTCAGCCAAT
751 CCCTGGGTGA GTTTCACCAG TTTTGATTTA AACGTGGCCA ATATGGACAA
801 CTTCTTCGCC CCCGTTTTCA CCATGGGCAA ATATTATACG CAAGGCGACA
851 AGGTGCTGAT GCCGCTGGCG ATTCAGGTTC ATCATGCCGT CTGTGATGGC
901 TTCCATGTCG GCAGAATGCT TAATGAATTA CAACAGTACT GCGATGAGTG
951 GCAGGGCGGG GCGTAATTTT TTTAAGGCAG TTATTGGTGC CCTTAAACGC
1001 CTGGTGCTAC GCCTGAATAA GTGATAATAA GCGGATGAAT GGCAGAAATT
1051 CGAAAGCAAA TTCGACCCGG TCGTCGGTTC AGGGCAGGGT CGTTAAATAG
1101 CCGCTTATGT CTATTGCTGG TTTACCGGTT TATTGACTAC CGGAAGCAGT
1151 GTGACCGTGT GCTTCTCAAA TGCCTGAGGC CAGTTTGCTC AGGCTCTCCC
1201 CGTGTGGTGT GGTGTGGTTG TGTG
Sequence of universal Tn10-derived tetRA cassette.
UL1 1 21
tetR (23 646)
tetA 728 1933
UR1 (1934 1954)
1 CACCAAACAC CCCCCAAAAC CTTTAAGACC CACTTTCACA TTTAAGTTGT
51 TTTTCTAATC CGCATATGAT CAATTCAAGG CCGAATAAGA AGGCTGGCTC
101 TGCACCTTGG TGATCAAATA ATTCGATAGC TTGTCGTAAT AATGGCGGCA
151 TACTATCAGT AGTAGGTGTT TCCCTTTCTT CTTTAGCGAC TTGATGCTCT
201 TGATCTTCCA ATACGCAACC TAAAGTAAAA TGCCCCACAG CGCTGAGTGC
251 ATATAATGCA TTCTCTAGTG AAAAACCTTG TTGGCATAAA AAGGCTAATT
301 GATTTTCGAG AGTTTCATAC TGTTTTTCTG TAGGCCGTGT ACCTAAATGT
351 ACTTTTGCTC CATCGCGATG ACTTAGTAAA GCACATCTAA AACTTTTAGC
401 GTTATTACGT AAAAAATCTT GCCAGCTTTC CCCTTCTAAA GGGCAAAAGT
451 GAGTATGGTG CCTATCTAAC ATCTCAATGG CTAAGGCGTC GAGCAAAGCC
501 CGCTTATTTT TTACATGCCA ATACAATGTA GGCTGCTCTA CACCTAGCTT
551 CTGGGCGAGT TTACGGGTTG TTAAACCTTC GATTCCGACC TCATTAAGCA
601 GCTCTAATGC GCTGTTAATC ACTTTACTTT TATCTAATCT AGACATCATT
651 AATTCCTAAT TTTTGTTGAC ACTCTATCAT TGATAGAGTT ATTTTACCAC
701 TCCCTATCAG TGATAGAGAA AAGTGAAATG AATAGTTCGA CAAAGATCGC
751 ATTGGTAATT ACGTTACTCG ATGCCATGGG GATTGGCCTT ATCATGCCAG
801 TCTTGCCAAC GTTATTACGT GAATTTATTG CTTCGGAAGA TATCGCTAAC
851 CACTTTGGCG TATTGCTTGC ACTTTATGCG TTAATGCAGG TTATCTTTGC
901 TCCTTGGCTT GGAAAAATGT CTGACCGATT TGGTCGGCGC CCAGTGCTGT
951 TGTTGTCATT AATAGGCGCA TCGCTGGATT AGTTATTGCT GGCTTTTTCA
1001 AGTGCGCTTT GGATGCTGTA TTTAGGCCGT TTGCTTTCAG GGATCACAGG
1051 AGCTACTGGG GCTGTCGCGG CATCGGTCAT TGCCGATACC ACCTCAGCTT
1101 CTCAACGCGT GAAGTGGTTC GGTTGGTTAG GGGCAAGTTT TGGGCTTGGT
1151 TTAATAGCGG GGCCTATTAT TGGTGGTTTT GCAGGAGAGA TTTCACCGCA
1201 TAGTCCCTTT TTTATCGCTG CGTTGCTAAA TATTGTCACT TTCCTTGTGG
1251 TTATGTTTTG GTTCCGTGAA ACCAAAAATA CACGTGATAA TACAGATACC
1301 GAAGTAGGGG TTGAGACGCA ATCGAATTCG GTATACATCA CTTTATTTAA
1351 AACGATGCCC ATTTTGTTGA TTATTTATTT TTCAGCGCAA TTGATAGGCC
1401 AAATTCCCGC AACGGTGTGG GTGCTATTTA CCGAAAATCG TTTTGGATGG
1451 AATAGCATGA TGGTTGGCTT TTCATTAGCG GGTCTTGGTC TTTTACACTC
1501 AGTATTCCAA GCCTTTGTGG CAGGAAGAAT AGCCACTAAA TGGGGCGAAA
1551 AAACGGCAGT ACTGCTCGAA TTTATTGCAG ATAGTAGTGC ATTTGCCTTT
1601 TTAGCGTTTA TATCTGAAGG TTGGTTAGAT TTCCCTGTTT TAATTTTATT
1651 GGCTGGTGGT GGGATCGCTT TACCTGCATT ACAGGGAGTG ATGTCTATCC
1701 AAACAAAGAG TCATGAGCAA GGTGCTTTAC AGGGATTATT GGTGAGCCTT
1751 ACCAATGCAA CCGGTGTTAT TGGCCCATTA CTGTTTACTG TTATTTATAA
1801 TCATTCACTA CCAATTTGGG ATGGCTGGAT TTGGATTATT GGTTTAGCGT
1851 TTTACTGTAT TATTATCCTG CTATCGATGA CCTTCATGTT AACCCCTCAA
1901 GCTCAGGGGA GTAAACAGGA GACAAGTGCT TAGTGTGGTG TGGTGTGGTT
1951 GTGT