LOCUS       sequence-247901-        4723 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 4723)
  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 4723)
  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..4723
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..106
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(127..145,146..151,152..168,169..174,175..200)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    127..145
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    152..170
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     terminator      949..1020
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1036..1063
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             1316..1327
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1334..2047
                     /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      2074..2145
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2161..2188
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        2240..2274
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             2309..2935
                     /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(2385..2404)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      2941..3027
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(3192..3780)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3272..3291)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3868..3962)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3983..4642)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     4557..4576
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 ggaacctctt acgggccggc caacgtctca ttttcgccag atatcgacgt ctaagaaacc
       61 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac
      121 ctcgagtccc tatcagtgat agagattgac atccctatca gtgatagaga tactgagcac
      181 atcagcagga cgcactgacc taagcgtaaa ataagtaaat tacacctata caagtatgcc
      241 acatcacatt gttattgttg aagatgagcc ggttacccag gcgcgattac aatcctactt
      301 cactcaggag gggtataccg tttccgttac ggcgagcggt gctgggctac gggaaattat
      361 gcagaatcag tcggtagatt taattctgct ggatatcaac ttacccgatg aaaatggcct
      421 gatgttaacc cgcgccctgc gagaacgctc aacggtgggg attattctgg ttaccggacg
      481 cagcgatcgg attgaccgta ttgttgggct ggaaatgggc gcagacgatt acgtcaccaa
      541 accgctggaa ctgcgcgaac tggtagtacg ggtgaaaaat ctgctctggc gaatcgacct
      601 cgcgcgacaa gctcaaccgc acactcagga caactgctat cgctttgccg gttattgcct
      661 gaatgtgtcg cgccatacgc tggagcggga tggcgagccg attaaactga cccgcgcaga
      721 gtatgaaatg ttggtggcat ttgtgacgaa tccgggcgaa attctcagcc gtgaacgtct
      781 gctacgtatg ctttctgcgc gtcgggtgga aaaccctgac ctgcgcaccg tcgatgtgtt
      841 aattcgtcgt ttacgtcata aactcagcgc ggatttactg gtgacgcaac atggtgaagg
      901 ttatttctta gccgctgatg tgtgctaata ataatctaga ccaggcatca aataaaacga
      961 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc
     1021 tactagagtc acactggctc accttcgggt gggcctttct gcgtttatag taaatcactg
     1081 cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg cgccgacatc
     1141 ataacggttc tggcaaatat tctgaaatga gctggttagc tagtcacaga gggttttact
     1201 cattctgttc atatctgttc atattctgcc gtaagccgtt catcctgacc agtgccgctg
     1261 ttcatatttg ctcattaaga tcgcttcact aaaccataat tctacagggg ttattaaaga
     1321 ggagaaatac tagatgcgta aaggcgaaga gctgttcact ggtgtcgtcc ctattctggt
     1381 ggaactggat ggtgatgtca acggtcataa gttttccgtg cgtggcgagg gtgaaggtga
     1441 cgcaactaat ggtaaactga cgctgaagtt catctgtact actggtaaac tgccggtacc
     1501 ttggccgact ctggtaacga cgctgactta tggtgttcag tgctttgctc gttatccgga
     1561 ccatatgaag cagcatgact tcttcaagtc cgccatgccg gaaggctatg tgcaggaacg
     1621 cacgatttcc tttaaggatg acggcacgta caaaacgcgt gcggaagtga aatttgaagg
     1681 cgataccctg gtaaaccgca ttgagctgaa aggcattgac tttaaagaag acggcaatat
     1741 cctgggccat aagctggaat acaattttaa cagccacaat gtttacatca ccgccgataa
     1801 acaaaaaaat ggcattaaag cgaattttaa aattcgccac aacgtggagg atggcagcgt
     1861 gcagctggct gatcactacc agcaaaacac tccaatcggt gatggtcctg ttctgctgcc
     1921 agacaatcac tatctgagca cgcaaagcgt tctgtctaaa gatccgaacg agaaacgcga
     1981 tcatatggtt ctgctggagt tcgtaaccgc agcgggcatc acgcatggta tggatgaact
     2041 gtacaaatga tgataataat ctagaccagg catcaaataa aacgaaaggc tcagtcgaaa
     2101 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact
     2161 ggctcacctt cgggtgggcc tttctgcgtt tataggtacc cgtggatcct ctagagtcga
     2221 cctgcaggca tgcaagcttt ttacggctag ctcagtccta ggtatagtgc tagcccagcc
     2281 agagaaacac tctttaacag ggggtagtat gatgtctcgt ttagataaaa gtaaagtgat
     2341 taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact
     2401 cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa ataagcgggc
     2461 tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact tttgcccttt
     2521 agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta gatgtgcttt
     2581 actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag aaaaacagta
     2641 tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac tagagaatgc
     2701 attatatgca ctcagcgcag tggggcattt tactttaggt tgcgtattgg aagatcaaga
     2761 gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc cgccattatt
     2821 acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct tattcggcct
     2881 tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt cttaaggcat
     2941 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     3001 gtgaacgctc tcctgagtag gacaaatccg ccgccctaga cctagggcgt tcggctgcgg
     3061 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3121 gcatgcaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3181 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3241 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3301 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3361 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3421 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3481 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3541 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3601 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     3661 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3721 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3781 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3841 gggattttgg tcatgactag tgcttggatt ctcaccaata aaaaacgccc ggcggcaacc
     3901 gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag
     3961 tccaagcgag ctcgatatca aattacgccc cgccctgcca ctcatcgcag tactgttgta
     4021 attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg
     4081 ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg
     4141 cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat
     4201 tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac
     4261 cataacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt
     4321 cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa
     4381 cactatccca tatcaccagc tcaccgtctt tcattgccat acgaaattcc ggatgagcat
     4441 tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta
     4501 cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa
     4561 ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat
     4621 atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa
     4681 aaaatacgac cggtagtgat cttatttcat tatggtgaaa gtt
//
