LOCUS       sequence-356600-        5580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc-inducible expression of TorR with C-terminal mNeonGreen fusion. 
            Also contains mCherry under TorR-controlled promoter PtorCAD129.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5580)
  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 5580)
  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: pRB116.2
FEATURES             Location/Qualifiers
     source          1..5580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(20..608)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(100..119)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(696..790)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(811..1470)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAKCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1385..1404
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     1639..1657
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1677..1695,1696..1701,1702..1718,1719..1724,
                     1725..1750)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1677..1695
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1702..1720
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      3275..3346
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3362..3389
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             3657..4367
                     /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(3802..3820)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4070..4089
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      4373..4459
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4473..4530
                     /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        4648..4682
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             4717..5343
                     /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(4793..4812)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      5349..5435
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
       61 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
      121 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
      181 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
      241 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
      301 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
      361 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
      421 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
      481 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
      541 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
      601 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
      661 cacgttaagg gattttggtc atgactagtg cttggattct caccaataaa aaacgcccgg
      721 cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac tggatctatc
      781 aacaggagtc caagcgagct cgatatcaaa ttacgccccg ccctgccact catcgcagta
      841 ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac
      901 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa
      961 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     1021 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag
     1081 gttttcacca taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     1141 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     1201 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg
     1261 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     1321 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca
     1381 tttagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac
     1441 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga
     1501 taactcaaaa aatacgaccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     1561 tacgggccgg ccaacgtctc attttcgcca gatatcgacg tctaagaaac cattattatc
     1621 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca cctcgatccc
     1681 tatcagtgat agagattgac atccctatca gtgatagaga tactgagcac atcagcagga
     1741 cgcactgacc gggcccaagt tcacttaaaa aggagatcaa caatgaaagc aattttcgta
     1801 ctgaaacatc ttaatcatgc ctaggaagtt ttctaatgcc acatcacatt gttattgttg
     1861 aagatgagcc ggttacccag gcgcgattac aatcctactt cactcaggag gggtataccg
     1921 tttccgttac ggcgagcggt gctgggctac gggaaattat gcagaatcag tcggtagatt
     1981 taattctgct ggatatcaac ttacccgatg aaaatggcct gatgttaacc cgcgccctgc
     2041 gagaacgctc aacggtgggg attattctgg ttaccggacg cagcgatcgg attgaccgta
     2101 ttgttgggct ggaaatgggc gcagacgatt acgtcaccaa accgctggaa ctgcgcgaac
     2161 tggtagtacg ggtgaaaaat ctgctctggc gaatcgacct cgcgcgacaa gctcaaccgc
     2221 acactcagga caactgctat cgctttgccg gttattgcct gaatgtgtcg cgccatacgc
     2281 tggagcggga tggcgagccg attaaactga cccgcgcaga gtatgaaatg ttggtggcat
     2341 ttgtgacgaa tccgggcgaa attctcagcc gtgaacgtct gctacgtatg ctttctgcgc
     2401 gtcgggtgga aaaccctgac ctgcgcaccg tcgatgtgtt aattcgtcgt ttacgtcata
     2461 aactcagcgc ggatttactg gtgacgcaac atggtgaagg ttatttctta gccgctgatg
     2521 tgtgcggtgg cggtggctct ggaggtggtg ggtccatggt ttcgaaaggt gaggaggata
     2581 acatggcgtc gctgcctgct acacatgaac tgcatatttt cggcagcatc aacggagttg
     2641 atttcgacat ggtgggccaa ggcaccggca acccgaacga cggctatgaa gaattgaatt
     2701 tgaaatctac caagggcgac ttgcaattta gtccgtggat tctggtaccg catattggtt
     2761 atggttttca tcagtatctg ccctacccag acggaatgag cccatttcag gcggctatgg
     2821 tggatggcag tggctatcag gtccatcgca ccatgcaatt cgaggatggg gcgagcctga
     2881 ccgtgaatta tcggtacact tacgaaggtt cacatattaa aggtgaagca caagttaaag
     2941 gcaccggctt cccggcggat ggcccagtga tgaccaacag cctgacggcg gcggactggt
     3001 gtcgtagcaa aaagacgtat cccaatgata aaacaattat ttctaccttt aaatggagtt
     3061 acaccacggg taacggtaaa cgctaccgta gcacggctcg caccacatac acctttgcga
     3121 aacccatggc cgcgaactat ctgaaaaatc agccgatgta tgtgtttcgc aaaacggaac
     3181 tgaaacattc gaaaactgaa ctcaatttca aagagtggca aaaagccttc accgacgtaa
     3241 tgggcatgga tgaactgtac aaatgaccag gcatcaaata aaacgaaagg ctcagtcgaa
     3301 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac
     3361 tggctcacct tcgggtgggc ctttctgcgt ttatagtaaa tcactgcata attcgtgtcg
     3421 ctcaaggcgc actcccgttc tgcagagggt tttactcatt ctgttcatat ctgttcatat
     3481 tctgccgtaa gccgttcatc ctgaccagtg ccgctgttca tatttgctca ttaagatcgc
     3541 ttcactaaac cataattcta caggggttat tgggcccaag ttcacttaaa aaggagatca
     3601 acaatgaaag caattttcgt actgaaacat cttaatcatg cctaggaagt tttctaatgg
     3661 tgagcaaggg cgaggaggat aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc
     3721 acatggaggg ctccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     3781 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc ctgcccttcg
     3841 cctgggacat cctgtcccct cagttcatgt acggctccaa ggcctacgtg aagcaccccg
     3901 ccgacatccc cgactacttg aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     3961 tgaacttcga ggacggcggc gtggtgaccg tgacccagga ctcctccctg caggacggcg
     4021 agttcatcta caaggtgaag ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc
     4081 agaagaagac catgggctgg gaggcctcct ccgagcggat gtaccccgag gacggcgccc
     4141 tgaagggcga gatcaagcag aggctgaagc tgaaggacgg cggccactac gacgctgagg
     4201 tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca
     4261 tcaagttgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgaacgcg
     4321 ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtaaggc atcaaataaa
     4381 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     4441 tctcctgagt aggacaaatc cgccgcccta gatccggcaa aaaagggcaa ggtgtcacca
     4501 ccctgccctt tttctttaaa accgaaaaga ttacttcgcg ttgataatgt tttttgcgcc
     4561 gacatcataa cggttctggc aaatattctg aaatgagctg ctcgtacccg tggatcctct
     4621 agagtcgacc tgcaggcatg caagcttttt acggctagct cagtcctagg tatagtgcta
     4681 gcccagccag agaaacactc tttaacaggg ggtagtatga tgtctcgttt agataaaagt
     4741 aaagtgatta acagcgcatt agagctgctt aatgaggtcg gaatcgaagg tttaacaacc
     4801 cgtaaactcg cccagaagct aggtgtagag cagcctacat tgtattggca tgtaaaaaat
     4861 aagcgggctt tgctcgacgc cttagccatt gagatgttag ataggcacca tactcacttt
     4921 tgccctttag aaggggaaag ctggcaagat tttttacgta ataacgctaa aagttttaga
     4981 tgtgctttac taagtcatcg cgatggagca aaagtacatt taggtacacg gcctacagaa
     5041 aaacagtatg aaactctcga aaatcaatta gcctttttat gccaacaagg tttttcacta
     5101 gagaatgcat tatatgcact cagcgcagtg gggcatttta ctttaggttg cgtattggaa
     5161 gatcaagagc atcaagtcgc taaagaagaa agggaaacac ctactactga tagtatgccg
     5221 ccattattac gacaagctat cgaattattt gatcaccaag gtgcagagcc agccttctta
     5281 ttcggccttg aattgatcat atgcggatta gaaaaacaac ttaaatgtga aagtgggtct
     5341 taaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     5401 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gccctagacc tagggcgttc
     5461 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5521 gggataacgc atgcaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
//
