Drug Cassette Kit

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

Last Update: Monday, 14-Nov-2016 14:57:44 PST
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Eric Kofoid eckofoid at ucdavis.edu