LOCUS       sequence-356570-        5486 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc-inducible expression of NarL with N-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 5486)
  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 5486)
  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: pRB117.2
FEATURES             Location/Qualifiers
     source          1..5486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(33..51,52..57,58..74,75..80,81..106)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    33..51
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    58..76
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      1589..1660
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1676..1703
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             1919..2629
                     /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(2064..2082)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2332..2351
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      2635..2721
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2735..2792
                     /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        2910..2944
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             2979..3605
                     /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(3055..3074)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      3611..3697
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(3862..4450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3942..3961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(4538..4632)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4653..5312)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAKCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5227..5246
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     join(5481..5486,1..13)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgtatcacga ggccctttcg tcttcacctc gatccctatc agtgatagag attgacatcc
       61 ctatcagtga tagagatact gagcacatca gcaggacgca ctgaccgggc ccaagttcac
      121 ttaaaaagga gatcaacaat gaaagcaatt ttcgtactga aacatcttaa tcatgcctag
      181 gaagttttct aatggtttcg aaaggtgagg gggataacat ggcgtcgctg cctgctacac
      241 atgaactgca tattttcggc agcatcaacg gagttgattt cgacatggtg ggccaaggca
      301 ccggcaaccc gaacgacggc tatgaagaat tgaatttgaa atctaccaag ggcgacttgc
      361 aatttagtcc gtggattctg gtaccgcata ttggttatgg ttttcatcag tatctgccct
      421 acccagacgg aatgagccca tttcaggcgg ctatggtgga tggcagtggc tatcaggtcc
      481 atcgcaccat gcaattcgag gatggggcga gcctgaccgt gaattatcgg tacacttacg
      541 aaggttcaca tattaaaggt gaagcacaag ttaaaggcac cggcttcccg gcggatggcc
      601 cagtgatgac caacagcctg acggcggcgg actggtgtcg tagcaaaaag acgtatccca
      661 atgataaaac aattatttct acctttaaat ggagttacac cacgggtaac ggtaaacgct
      721 accgtagcac ggctcgcacc acatacacct ttgcgaaacc catggccgcg aactatctga
      781 aaaatcagcc gatgtatgtg tttcgcaaaa cggaactgaa acattcgaaa actgaactca
      841 atttcaaaga gtggcaaaaa gccttcaccg acgtaatggg catggatgaa ctgtacaaag
      901 gtggcggtgg ctctggaggt ggtgggtcca tgagtaatca ggaaccggct actatcctgc
      961 tgattgacga tcacccgatg ctgcgaactg gcgtaaaaca gcttatcagt atggcaccag
     1021 atatcaccgt ggttggcgaa gcgagtaatg gcgaacaggg tattgaactg gcggagtctc
     1081 ttgatcccga tctgatcctg ttagatctca atatgcccgg catgaacggt ctggaaacgc
     1141 tggataaact gcgcgaaaag tccctctcag ggcgcattgt ggtattcagc gtctctaacc
     1201 atgaagaaga tgtggtcacc gcactgaaac gcggcgcgga tggctatctg ttaaaagata
     1261 tggaaccgga agatctgctg aaagcattgc atcaggcagc tgctggcgaa atggtattaa
     1321 gcgaagcatt aacgcctgtt ctggccgcca gcttgcgcgc taaccgtgcc actactgagc
     1381 gcgatgttaa ccagttaacc ccacgcgagc gcgatattct caagctgatt gcccagggtt
     1441 tgccgaacaa gatgattgcc cgccgcctgg atatcaccga aagcacagta aaagtgcacg
     1501 tcaagcacat gctgaagaaa atgaagctca agtctcgcgt ggaagcagcg gtatgggtgc
     1561 atcaggagcg cattttctga ccaggcatca aataaaacga aaggctcagt cgaaagactg
     1621 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc
     1681 accttcgggt gggcctttct gcgtttatag taaatcactg cataattcgt gtcgctcaag
     1741 gcgcactccc gttctgagag tctcttcccc tatatagcca atcattctta ctccctttga
     1801 attacccgcc tcatcagaga taatgcttaa gaagggccca agttcactta aaaaggagat
     1861 caacaatgaa agcaattttc gtactgaaac atcttaatca tgcctaggaa gttttctaat
     1921 ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     1981 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     2041 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     2101 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     2161 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     2221 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     2281 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     2341 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     2401 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     2461 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     2521 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     2581 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaag gcatcaaata
     2641 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     2701 gctctcctga gtaggacaaa tccgccgccc tagatccggc aaaaaagggc aaggtgtcac
     2761 caccctgccc tttttcttta aaaccgaaaa gattacttcg cgttgataat gttttttgcg
     2821 ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgctcgtacc cgtggatcct
     2881 ctagagtcga cctgcaggca tgcaagcttt ttacggctag ctcagtccta ggtatagtgc
     2941 tagcccagcc agagaaacac tctttaacag ggggtagtat gatgtctcgt ttagataaaa
     3001 gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa
     3061 cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa
     3121 ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact
     3181 tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta
     3241 gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag
     3301 aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac
     3361 tagagaatgc attatatgca ctcagcgcag tggggcattt tactttaggt tgcgtattgg
     3421 aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc
     3481 cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct
     3541 tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt
     3601 cttaaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     3661 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccgccctaga cctagggcgt
     3721 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3781 aggggataac gcatgcaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3841 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3901 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3961 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4021 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4081 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4141 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4201 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4261 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     4321 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4381 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4441 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     4501 ctcacgttaa gggattttgg tcatgactag tgcttggatt ctcaccaata aaaaacgccc
     4561 ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta
     4621 tcaacaggag tccaagcgag ctcgatatca aattacgccc cgccctgcca ctcatcgcag
     4681 tactgttgta attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga
     4741 acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg
     4801 aaaacggggg cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc
     4861 acccagggat tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc
     4921 aggttttcac cataacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg
     4981 tcgtggtatt cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa
     5041 caagggtgaa cactatccca tatcaccagc tcaccgtctt tcattgccat acgaaattcc
     5101 ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta
     5161 tttttcttta cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta
     5221 catttagcaa ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca
     5281 acggtggtat atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc
     5341 gataactcaa aaaatacgac cggtagtgat cttatttcat tatggtgaaa gttggaacct
     5401 cttacgggcc ggccaacgtc tcattttcgc cagatatcga cgtctaagaa accattatta
     5461 tcatgacatt aacctataaa aatagg
//
