LOCUS       sequence-356593-        5432 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  IPTG-inducible expression of NarX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5432)
  AUTHORS   Butcher RJ, Tabor JJ
  TITLE     Real-time detection of response regulator phosphorylation dynamics 
            in live bacteria.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Aug 30;119(35):e2201204119. doi: 
            10.1073/pnas.2201204119. Epub 2022 Aug 22.
  PUBMED    35994658
REFERENCE   2  (bases 1 to 5432)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2022-08-30"; volume: "119(35):e2201204119. 
            doi"; pages: " 10.1073/pnas.2201204119. Epub 2022 Aug 22"
COMMENT     Sequence Label: pKD317.8
FEATURES             Location/Qualifiers
     source          1..5432
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     439..458
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     655..672
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        711..788
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             789..1871
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(808..827)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      1877..1963
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(2136..2141,2142..2157,2158..2164)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2172..2188
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2177..2199
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     terminator      4100..4171
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4187..4214
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             4414..5205
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     terminator      5217..5311
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      join(5425..5432,1..538)
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
ORIGIN
        1 gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg cagggcggtt
       61 tttcgaaggt tctctgagct accaactctt tgaaccgagg taactggctt ggaggagcgc
      121 agtcaccaaa acttgtcctt tcagtttagc cttaaccggc gcatgacttc aagactaact
      181 cctctaaatc aattaccagt ggctgctgcc agtggtgctt ttgcatgtct ttccgggttg
      241 gactcaagac gatagttacc ggataaggcg cagcggtcgg actgaacggg gggttcgtgc
      301 atacagtcca gcttggagcg aactgcctac ccggaactga gtgtcaggcg tggaatgaga
      361 caaacgcggc cataacagcg gaatgacacc ggtaaaccga aaggcaggaa caggagagcg
      421 cacgagggag ccgccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      481 ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc tatggaaaaa
      541 cggctttgcc gcggccctct cacttccctg ttaagtatct tcctggcatc ttccgggaaa
      601 tctccgcccc gttcgtaagc catttccgct cgccgcagtc gaacgaccga gcgtagcgag
      661 tcagtgagcg aggaagcgga atatatcccg aagcggcatg catttacgtt gacaccatcg
      721 aatggtgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
      781 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
      841 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
      901 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
      961 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     1021 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     1081 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     1141 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     1201 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     1261 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     1321 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     1381 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     1441 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     1501 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     1561 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     1621 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     1681 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     1741 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     1801 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     1861 gcgggcagtg aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt
     1921 tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgc cctagaccta
     1981 gggcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac gtaaatcact
     2041 gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat
     2101 cataacggtt ctggctaata ttctgaaatg agctgttgac aattaatcat cggctcgtat
     2161 aatgtgtgga attgtgagcg gataacaatt tcacacaggg cccaagttca cttaaaaagg
     2221 agatcaacaa tgaaagcaat tttcgtactg aaacatctta atcatgcctg ggactttttc
     2281 tgatgcttaa acgttgtctc tctccgctca ccctggttaa tcaggttgcg cttattgtgt
     2341 tgctttctac tgctattgga ctggcaggga tggcggtttc tggctggctg gtgcaaggcg
     2401 ttcagggcag cgcccatgcg atcaacaaag cgggatcgct gcgcatgcaa agttaccgtc
     2461 tgttggcggc agtgccatta agcgagaaag acaagccctt aattaaagag atggaacaaa
     2521 cggcatttag cgccgagttg actcgagcag cagaacgaga cggacaactg gcgcaattac
     2581 agggtttaca agattactgg cgtaatgaac tgatccctgc gctgatgcgt gcacaaaacc
     2641 gagaaacggt gtcagcggat gtcagccagt ttgttgccgg gcttgatcaa ctggtatctg
     2701 gttttgaccg caccacggaa atgcgcatcg agacagtggt actggtccat cgggtaatgg
     2761 cggtatttat ggcactttta ctggtgttca ctattatctg gttgcgggcg cgactgctac
     2821 aaccgtggcg gcaactgctg gcaatggcga gtgccgtcag tcatcgcgat tttacccaac
     2881 gcgcaaacat cagcgggcgc aacgaaatgg cgatgcttgg aactgcgttg aacaatatgt
     2941 ctgcagaact ggccgaaagt tatgccgtac ttgagcagcg ggttcaggag aaaaccgccg
     3001 ggctggagca taaaaatcag atcctctctt ttttatggca ggctaaccgc cgtttgcatt
     3061 cccgcgcccc gctgtgtgaa cgcctgtcac ctgtactcaa cggcttacag aatttaaccc
     3121 tgctacgtga tatcgaattg cgggtgtatg acactgatga tgaagagaat catcaggagt
     3181 ttacctgcca gccagatatg acttgtgatg ataaaggctg ccagctctgc ccgcgcggcg
     3241 tattacccgt tggtgatcgc ggcacgaccc tgaagtggcg gctggctgac tctcatacgc
     3301 agtacggtat tttgctggcg accctgccgc aggggcgtca tcttagccat gatcaacaac
     3361 aactggtgga taccctggtt gaacaactca ccgccacgct ggcgctggat cgccatcagg
     3421 aacgtcagca acagttgatc gtgatggaag agcgtgccac cattgcgcgc gaactgcatg
     3481 attctattgc ccaatctctc tcttgcatga agatgcaggt gagttgttta cagatgcagg
     3541 gcgatgcgct gccagaaagc agccgcgaac tgttaagtca gatccgtaac gaactgaatg
     3601 catcctgggc gcagttgcgt gaattgctca ccacattccg cttgcagctc accgagcctg
     3661 gattacgtcc ggcgctggag gcgagttgcg aagagtacag cgccaaattt ggcttcccgg
     3721 tgaagctgga ttatcaattg ccgcctcgcc tggtgccttc gcatcaggca atccacttgt
     3781 tgcaaattgc ccgtgaggca ttaagtaacg ccctcaaaca ttcgcaagcg agtgaagtcg
     3841 tggtgacggt ggcgcaaaac gataatcagg tcaaactgac cgtccaggat aacggctgcg
     3901 gcgtgcctga aaatgccatc cgcagcaatc actacggcat gataataatg cgcgatcgtg
     3961 cgcaaagttt acgaggcgat tgccgcgtcc gccgtcgtga atcaggtggc accgaagtgg
     4021 tggtcacctt tattcccgaa aaaactttca cagacgtcca aggagatacc catgagtaat
     4081 aataatctag accaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     4141 ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct caccttcggg
     4201 tgggcctttc tgcgtttata ggtacccggg gatcctctag agtcgacctg caggcatgca
     4261 agcttagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt tgggaagccc
     4321 tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag gggatcaaga
     4381 tctgatcaag agacaggatg aggatcgttt cgtatgaggg aagcggtgat cgccgaagta
     4441 tcgactcaac tatcagaggt agttggcgtc atcgagcgcc atctcgaacc gacgttgctg
     4501 gccgtacatt tgtacggctc cgcagtggat ggcggcctga agccacacag tgatattgat
     4561 ttgctggtta cggtgaccgt aaggcttgat gaaacaacgc ggcgagcttt gatcaacgac
     4621 cttttggaaa cttcggcttc ccctggagag agcgagattc tccgcgctgt agaagtcacc
     4681 attgttgtgc acgacgacat cattccgtgg cgttatccag ctaagcgcga actgcaattt
     4741 ggagaatggc agcgcaatga cattcttgca ggtatcttcg agccagccac gatcgacatt
     4801 gatctggcta tcttgctgac aaaagcaaga gaacatagcg ttgccttggt aggtccagcg
     4861 gcggaggaac tctttgatcc ggttcctgaa caggatctat ttgaggcgct aaatgaaacc
     4921 ttaacgctat ggaactcgcc gcccgactgg gctggcgatg agcgaaatgt agtgcttacg
     4981 ttgtcccgca tttggtacag cgcagtaacc ggcaaaatcg cgccgaagga tgtcgctgcc
     5041 gactgggcaa tggagcgcct gccggcccag tatcagcccg tcatacttga agctagacag
     5101 gcttatcttg gacaagaaga agatcgcttg gcctcgcgcg cagatcagtt ggaagaattt
     5161 gtccactacg tgaaaggcga gatcaccaag gtagtcggca aataagagct cgcttggact
     5221 cctgttgata gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg
     5281 ttgccgccgg gcgtttttta ttggtgagaa tccaagcact agtaacaact tatatcgtat
     5341 ggggctgact tcaggtgcta catttgaaga gataaattgc actgaaatct agtaatattt
     5401 tatctgatta ataagatgat cttcttgaga tc
//
