LOCUS       sequence-356574-        5487 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc-inducible expression of NarL with C-terminal mNeonGreen fusion. 
            Also contains mCherry under NarL-controlled promoter PdcuS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5487)
  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 5487)
  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: pRB113.2
FEATURES             Location/Qualifiers
     source          1..5487
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     518..537
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     772..790
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(810..828,829..834,835..851,852..857,858..883)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    810..828
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    835..853
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      2366..2437
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2453..2480
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             2696..3406
                     /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(2841..2859)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3109..3128
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3412..3498
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3512..3569
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     promoter        3687..3721
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             3756..4382
                     /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(3832..3851)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      4388..4474
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(4639..5228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4719..4738)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(5316..5410)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(5431..5487,1..603))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAKCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 gacatggaag ccatcacaaa cggcatgatg aacctgaatc gccagcggca tcagcacctt
       61 gtcgccttgc gtataatatt tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt
      121 ggccacgttt aaatcaaaac tggtgaaact cacccaggga ttggctgaga cgaaaaacat
      181 attctcaata aaccctttag ggaaataggc caggttttca ccataacacg ccacatcttg
      241 cgaatatatg tgtagaaact gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa
      301 cgtttcagtt tgctcatgga aaacggtgta acaagggtga acactatccc atatcaccag
      361 ctcaccgtct ttcattgcca tacgaaattc cggatgagca ttcatcaggc gggcaagaat
      421 gtgaataaag gccggataaa acttgtgctt atttttcttt acggtcttta aaaaggccgt
      481 aatatccagc tgaacggtct ggttataggt acatttagca actgactgaa atgcctcaaa
      541 atgttcttta cgatgccatt gggatatatc aacggtggta tatccagtga tttttttctc
      601 cattttagct tccttagctc ctgaaaatct cgataactca aaaaatacga ccggtagtga
      661 tcttatttca ttatggtgaa agttggaacc tcttacgggc cggccaacgt ctcattttcg
      721 ccagatatcg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
      781 atcacgaggc cctttcgtct tcacctcgat ccctatcagt gatagagatt gacatcccta
      841 tcagtgatag agatactgag cacatcagca ggacgcactg accgggccca agttcactta
      901 aaaaggagat caacaatgaa agcaattttc gtactgaaac atcttaatca tgcctaggaa
      961 gttttctaat gagtaatcag gaaccggcta ctatcctgct gattgacgat cacccgatgc
     1021 tgcgaactgg cgtaaaacag cttatcagta tggcaccaga tatcaccgtg gttggcgaag
     1081 cgagtaatgg cgaacagggt attgaactgg cggagtctct tgatcccgat ctgatcctgt
     1141 tagatctcaa tatgcccggc atgaacggtc tggaaacgct ggataaactg cgcgaaaagt
     1201 ccctctcagg gcgcattgtg gtattcagcg tctctaacca tgaagaagat gtggtcaccg
     1261 cactgaaacg cggcgcggat ggctatctgt taaaagatat ggaaccggaa gatctgctga
     1321 aagcattgca tcaggcagct gctggcgaaa tggtattaag cgaagcatta acgcctgttc
     1381 tggccgccag cttgcgcgct aaccgtgcca ctactgagcg cgatgttaac cagttaaccc
     1441 cacgcgagcg cgatattctc aagctgattg cccagggttt gccgaacaag atgattgccc
     1501 gccgcctgga tatcaccgaa agcacagtaa aagtgcacgt caagcacatg ctgaagaaaa
     1561 tgaagctcaa gtctcgcgtg gaagcagcgg tatgggtgca tcaggagcgc attttcggtg
     1621 gcggtggctc tggaggtggt gggtccatgg tttcgaaagg tgaggaggat aacatggcgt
     1681 cgctgcctgc tacacatgaa ctgcatattt tcggcagcat caacggagtt gatttcgaca
     1741 tggtgggcca aggcaccggc aacccgaacg acggctatga agaattgaat ttgaaatcta
     1801 ccaagggcga cttgcaattt agtccgtgga ttctggtacc gcatattggt tatggttttc
     1861 atcagtatct gccctaccca gacggaatga gcccatttca ggcggctatg gtggatggca
     1921 gtggctatca ggtccatcgc accatgcaat tcgaggatgg ggcgagcctg accgtgaatt
     1981 atcggtacac ttacgaaggt tcacatatta aaggtgaagc acaagttaaa ggcaccggct
     2041 tcccggcgga tggcccagtg atgaccaaca gcctgacggc ggcggactgg tgtcgtagca
     2101 aaaagacgta tcccaatgat aaaacaatta tttctacctt taaatggagt tacaccacgg
     2161 gtaacggtaa acgctaccgt agcacggctc gcaccacata cacctttgcg aaacccatgg
     2221 ccgcgaacta tctgaaaaat cagccgatgt atgtgtttcg caaaacggaa ctgaaacatt
     2281 cgaaaactga actcaatttc aaagagtggc aaaaagcctt caccgacgta atgggcatgg
     2341 atgaactgta caaatgacca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     2401 ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc
     2461 ttcgggtggg cctttctgcg tttatagtaa atcactgcat aattcgtgtc gctcaaggcg
     2521 cactcccgtt ctgagagtct cttcccctat atagccaatc attcttactc cctttgaatt
     2581 acccgcctca tcagagataa tgcttaagaa gggcccaagt tcacttaaaa aggagatcaa
     2641 caatgaaagc aattttcgta ctgaaacatc ttaatcatgc ctaggaagtt ttctaatggt
     2701 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     2761 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     2821 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     2881 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     2941 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     3001 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     3061 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     3121 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     3181 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     3241 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     3301 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     3361 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaaggca tcaaataaaa
     3421 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     3481 ctcctgagta ggacaaatcc gccgccctag atccggcaaa aaagggcaag gtgtcaccac
     3541 cctgcccttt ttctttaaaa ccgaaaagat tacttcgcgt tgataatgtt ttttgcgccg
     3601 acatcataac ggttctggca aatattctga aatgagctgc tcgtacccgt ggatcctcta
     3661 gagtcgacct gcaggcatgc aagcttttta cggctagctc agtcctaggt atagtgctag
     3721 cccagccaga gaaacactct ttaacagggg gtagtatgat gtctcgttta gataaaagta
     3781 aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc
     3841 gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata
     3901 agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt
     3961 gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat
     4021 gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa
     4081 aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag
     4141 agaatgcatt atatgcactc agcgcagtgg ggcattttac tttaggttgc gtattggaag
     4201 atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc
     4261 cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat
     4321 tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtctt
     4381 aaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     4441 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ccctagacct agggcgttcg
     4501 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4561 ggataacgca tgcaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4621 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4681 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4741 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4801 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4861 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4921 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4981 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5041 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     5101 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5161 caccgctggt agcggtggtt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5221 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5281 cacgttaagg gattttggtc atgactagtg cttggattct caccaataaa aaacgcccgg
     5341 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc
     5401 aacaggagtc caagcgagct cgatatcaaa ttacgccccg ccctgccact catcgcagta
     5461 ctgttgtaat tcattaagca ttctgcc
//
