LOCUS       sequence-239100-        4722 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of torR under PLtetO-1, output promoter PtorCAD129.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4722)
  AUTHORS   Schmidl SR, Ekness F, Sofjan K, Daeffler KN, Brink KR, Landry BP, 
            Gerhardt KP, Dyulgyarov N, Sheth RU, Tabor JJ
  TITLE     Rewiring bacterial two-component systems by modular DNA-binding 
            domain swapping.
  JOURNAL   Nat Chem Biol. 2019 Jul;15(7):690-698. doi: 
            10.1038/s41589-019-0286-6. Epub 2019 May 20.
  PUBMED    31110305
REFERENCE   2  (bases 1 to 4722)
  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"; doi: 
            "10.1038/s41589-019-0286-6"; journalName: "Nat Chem Biol"; date: 
            "2019-07"; volume: "15"; issue: "7"; pages: "690-698"
COMMENT     Sequence Label: pKD250.2
FEATURES             Location/Qualifiers
     source          1..4722
                     /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(256..844)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(336..355)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(932..1026)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(1047..1706)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1621..1640
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1707..1809)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     1875..1893
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1914..1932,1933..1938,1939..1955,1956..1961,
                     1962..1987)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    1914..1932
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1939..1957
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      2736..2807
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2823..2850
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             3103..3114
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3121..3834
                     /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      3861..3932
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3948..3975
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        4027..4061
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             4096..4722
                     /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(4172..4191)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
ORIGIN
        1 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
       61 tgtcggtgaa cgctctcctg agtaggacaa atccgccgcc ctagacctag gcgttcggct
      121 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
      181 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      241 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      301 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
      361 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
      421 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
      481 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
      541 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
      601 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
      661 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
      721 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
      781 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
      841 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
      901 ttaagggatt ttggtcatga ctagtgcttg gattctcacc aataaaaaac gcccggcggc
      961 aaccgagcgt tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca
     1021 ggagtccaag cgagctcgat atcaaattac gccccgccct gccactcatc gcagtactgt
     1081 tgtaattcat taagcattct gccgacatgg aagccatcac agacggcatg atgaacctga
     1141 atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg
     1201 ggggcgaaga agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag
     1261 ggattggctg agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt
     1321 tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg
     1381 tattcactcc agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg
     1441 tgaacactat cccatatcac cagctcaccg tctttcattg ccatacgaaa ttccggatga
     1501 gcattcatca ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc
     1561 tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga
     1621 gcaactgact gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg
     1681 gtatatccag tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac
     1741 tcaaaaaata cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg
     1801 tgccgatcaa cgtctcattt tcgccagata tcgacgtcta agaaaccatt attatcatga
     1861 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcacctc gagtccctat
     1921 cagtgataga gattgacatc cctatcagtg atagagatac tgagcacatc agcaggacgc
     1981 actgacctaa gcgtaaaata agtaaattac acctatacaa gtatgccaca tcacattgtt
     2041 attgttgaag atgagccggt tacccaggcg cgattacaat cctacttcac tcaggagggg
     2101 tataccgttt ccgttacggc gagcggtgct gggctacggg aaattatgca gaatcagtcg
     2161 gtagatttaa ttctgctgga tatcaactta cccgatgaaa atggcctgat gttaacccgc
     2221 gccctgcgag aacgctcaac ggtggggatt attctggtta ccggacgcag cgatcggatt
     2281 gaccgtattg ttgggctgga aatgggcgca gacgattacg tcaccaaacc gctggaactg
     2341 cgcgaactgg tagtacgggt gaaaaatctg ctctggcgaa tcgacctcgc gcgacaagct
     2401 caaccgcaca ctcaggacaa ctgctatcgc tttgccggtt attgcctgaa tgtgtcgcgc
     2461 catacgctgg agcgggatgg cgagccgatt aaactgaccc gcgcagagta tgaaatgttg
     2521 gtggcatttg tgacgaatcc gggcgaaatt ctcagccgtg aacgtctgct acgtatgctt
     2581 tctgcgcgtc gggtggaaaa ccctgacctg cgcaccgtcg atgtgttaat tcgtcgttta
     2641 cgtcataaac tcagcgcgga tttactggtg acgcaacatg gtgaaggtta tttcttagcc
     2701 gctgatgtgt gctaataata atctagacca ggcatcaaat aaaacgaaag gctcagtcga
     2761 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca
     2821 ctggctcacc ttcgggtggg cctttctgcg tttatagtaa atcactgcat aattcgtgtc
     2881 gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg
     2941 caaatattct gaaatgagct ggttagctag tcacagaggg ttttactcat tctgttcata
     3001 tctgttcata ttctgccgta agccgttcat cctgaccagt gccgctgttc atatttgctc
     3061 attaagatcg cttcactaaa ccataattct acaggggtta ttaaagagga gaaatactag
     3121 atgcgtaaag gcgaagagct gttcactggt gtcgtcccta ttctggtgga actggatggt
     3181 gatgtcaacg gtcataagtt ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt
     3241 aaactgacgc tgaagttcat ctgtactact ggtaaactgc cggtaccttg gccgactctg
     3301 gtaacgacgc tgacttatgg tgttcagtgc tttgctcgtt atccggacca tatgaagcag
     3361 catgacttct tcaagtccgc catgccggaa ggctatgtgc aggaacgcac gatttccttt
     3421 aaggatgacg gcacgtacaa aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta
     3481 aaccgcattg agctgaaagg cattgacttt aaagaagacg gcaatatcct gggccataag
     3541 ctggaataca attttaacag ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc
     3601 attaaagcga attttaaaat tcgccacaac gtggaggatg gcagcgtgca gctggctgat
     3661 cactaccagc aaaacactcc aatcggtgat ggtcctgttc tgctgccaga caatcactat
     3721 ctgagcacgc aaagcgttct gtctaaagat ccgaacgaga aacgcgatca tatggttctg
     3781 ctggagttcg taaccgcagc gggcatcacg catggtatgg atgaactgta caaatgatga
     3841 taataatcta gaccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     3901 gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg
     3961 gtgggccttt ctgcgtttat aggtacccgt ggatcctcta gagtcgacct gcaggcatgc
     4021 aagcttttta cggctagctc agtcctaggt atagtgctag cccagccaga gaaacactct
     4081 ttaacagggg gtagtatgat gtctcgttta gataaaagta aagtgattaa cagcgcatta
     4141 gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc gtaaactcgc ccagaagcta
     4201 ggtgtagagc agcctacatt gtattggcat gtaaaaaata agcgggcttt gctcgacgcc
     4261 ttagccattg agatgttaga taggcaccat actcactttt gccctttaga aggggaaagc
     4321 tggcaagatt ttttacgtaa taacgctaaa agttttagat gtgctttact aagtcatcgc
     4381 gatggagcaa aagtacattt aggtacacgg cctacagaaa aacagtatga aactctcgaa
     4441 aatcaattag cctttttatg ccaacaaggt ttttcactag agaatgcatt atatgcactc
     4501 agcgcagtgg ggcattttac tttaggttgc gtattggaag atcaagagca tcaagtcgct
     4561 aaagaagaaa gggaaacacc tactactgat agtatgccgc cattattacg acaagctatc
     4621 gaattatttg atcaccaagg tgcagagcca gccttcttat tcggccttga attgatcata
     4681 tgcggattag aaaaacaact taaatgtgaa agtgggtctt aa
//
