LOCUS       sequence-178324-        5685 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses ThsR under the PltetO-1 promoter, sfGFP under PphsA, and 
            mCherry constitutively.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5685)
  AUTHORS   Daeffler KN, Galley JD, Sheth RU, Ortiz-Velez LC, Bibb CO, Shroyer 
            NF, Britton RA, Tabor JJ
  TITLE     Engineering bacterial thiosulfate and tetrathionate sensors for 
            detecting gut inflammation.
  JOURNAL   Mol Syst Biol. 2017 Apr 3;13(4):923. doi: 10.15252/msb.20167416.
  PUBMED    28373240
REFERENCE   2  (bases 1 to 5685)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Syst 
            Biol."; date: "2017-04-3"; volume: "13(4):923. doi"; pages: " 
            10.15252/msb.20167416"
COMMENT     Sequence Label: pKD184
FEATURES             Location/Qualifiers
     source          1..5685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      6..92
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(257..845)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(337..356)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(933..1027)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1048..1707)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1622..1641
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        1836..1870
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     RBS             1880..1891
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1899..2609
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2044..2062)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2312..2331
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2710..2728
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2749..2767,2768..2773,2774..2790,2791..2796,
                     2797..2822)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2749..2767
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2774..2792
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      3486..3557
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3573..3600
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             4066..4077
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4084..4797
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      4824..4895
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4911..4938
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4990..5024
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             5059..5685
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(5135..5154)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
       61 tgtcggtgaa cgctctcctg agtaggacaa atccgccgcc ctagacctag ggcgttcggc
      121 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
      181 ataacgcatg caagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
      241 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
      301 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
      361 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
      421 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
      481 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
      541 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
      601 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
      661 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
      721 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
      781 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
      841 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
      901 gttaagggat tttggtcatg actagtgctt ggattctcac caataaaaaa cgcccggcgg
      961 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac
     1021 aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg
     1081 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg
     1141 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac
     1201 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     1261 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     1321 ttcaccataa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     1381 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     1441 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg
     1501 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     1561 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
     1621 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     1681 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa
     1741 ctcaaaaaat acgaccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac
     1801 gggccggcca acgtctcatt ttcgccagat atcgatttat ggctagctca gtcctaggta
     1861 caatgctagc gaatccatta aagaggagaa aggtacccat ggtgagcaag ggcgaggagg
     1921 ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga
     1981 acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga
     2041 ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc
     2101 ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact
     2161 tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg
     2221 gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga
     2281 agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct
     2341 gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc gagatcaagc
     2401 agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc acctacaagg
     2461 ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg gacatcacct
     2521 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     2581 ccggcggcat ggacgagctg tacaagtaag ctttctaact aaaaaggcct cccaaatcgg
     2641 ggggcctttt ttattgataa caaaacgctc gtctaagaaa ccattattat catgacatta
     2701 acctataaaa ataggcgtat cacgaggccc tttcgtcttc acctcgagtc cctatcagtg
     2761 atagagattg acatccctat cagtgataga gatactgagc acatcagcag gacgcactga
     2821 cctattttag tttacttatt aatttattaa aattatatgc agcagcaaat caacggcccg
     2881 gtctacctgg tggatgatga tgaagccatt atcgactcca tcgatttttt gatggagggc
     2941 tacggttaca aactgaactc gtttaactgc ggcgatcgct ttttggcaga agtcgatctc
     3001 acccaggcag gatgtgtaat tctggatgcg cgtatgccag gcttaactgg tcctcaggtg
     3061 caacagctgc tgagcgacgc gaaaagcccg cttgcggtca tcttcctgac cggccatggc
     3121 gatgttccga tggcggttga tgcgttcaaa aatggcgcgt tcgatttctt tcaaaaacct
     3181 gtgccgggta gcttgctcag tcagtcaatt gccaaaggct tgacttattc aatcgatcaa
     3241 catctgaaac gtactaacca agcgttaatc gacacgctct cggaacgcga agctcaaatt
     3301 tttcaactgg tgattgcagg caacaccaac aaacagatgg ctaacgagct ttgcgtggct
     3361 attcgtacca ttgaggttca ccgtagcaaa ctgatgacca aactgggtgt taacaacctg
     3421 gctgaactgg ttaaactggc gccgctgctg gcacataaat ccgaataata atctagacca
     3481 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     3541 tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg
     3601 tttatagtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt ctggataatg
     3661 ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct ggttagctag
     3721 tcattcaagc attattatgc tgttttttga agtgaatgtg cggccatcta gccgcacatt
     3781 ttgcatctaa aacatgcagt catcagcaaa ataataaact tttccccaat atgtggttta
     3841 ccacaattta caggaattca ctcctgtggt ggtgcaaatt tgaactgtga attgcttcac
     3901 aaacgccgct atcgcaatgt cagtatgtgg tttaccacaa tatctaatat cactctgctc
     3961 aataacaatg atgaaaacct taggaagaag ttaattgtgt taaacagtta actaggggct
     4021 ttatctaacg ctctcctaag gacaactgtc attgggagat ttaacaaaga ggagaaatac
     4081 tagatgcgta aaggcgaaga gctgttcact ggtgtcgtcc ctattctggt ggaactggat
     4141 ggtgatgtca acggtcataa gttttccgtg cgtggcgagg gtgaaggtga cgcaactaat
     4201 ggtaaactga cgctgaagtt catctgtact actggtaaac tgccggtacc ttggccgact
     4261 ctggtaacga cgctgactta tggtgttcag tgctttgctc gttatccgga ccatatgaag
     4321 cagcatgact tcttcaagtc cgccatgccg gaaggctatg tgcaggaacg cacgatttcc
     4381 tttaaggatg acggcacgta caaaacgcgt gcggaagtga aatttgaagg cgataccctg
     4441 gtaaaccgca ttgagctgaa aggcattgac tttaaagaag acggcaatat cctgggccat
     4501 aagctggaat acaattttaa cagccacaat gtttacatca ccgccgataa acaaaaaaat
     4561 ggcattaaag cgaattttaa aattcgccac aacgtggagg atggcagcgt gcagctggct
     4621 gatcactacc agcaaaacac tccaatcggt gatggtcctg ttctgctgcc agacaatcac
     4681 tatctgagca cgcaaagcgt tctgtctaaa gatccgaacg agaaacgcga tcatatggtt
     4741 ctgctggagt tcgtaaccgc agcgggcatc acgcatggta tggatgaact gtacaaatga
     4801 tgataataat ctagaccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     4861 ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact ggctcacctt
     4921 cgggtgggcc tttctgcgtt tataggtacc cgtggatcct ctagagtcga cctgcaggca
     4981 tgcaagcttt ttacggctag ctcagtccta ggtatagtgc tagcccagcc agagaaacac
     5041 tctttaacag ggggtagtat gatgtctcgt ttagataaaa gtaaagtgat taacagcgca
     5101 ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag
     5161 ctaggtgtag agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac
     5221 gccttagcca ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa
     5281 agctggcaag attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat
     5341 cgcgatggag caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc
     5401 gaaaatcaat tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca
     5461 ctcagcgcag tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc
     5521 gctaaagaag aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct
     5581 atcgaattat ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc
     5641 atatgcggat tagaaaaaca acttaaatgt gaaagtgggt cttaa
//
