LOCUS       sequence-311321-        2786 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Two-layered AHL-responsive circuit containing a TetR-based inverter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2786)
  AUTHORS   Wan X, Pinto F, Yu L, Wang B
  TITLE     Synthetic protein-binding DNA sponge as a tool to tune gene 
            expression and mitigate protein toxicity.
  JOURNAL   Nat Commun. 2020 Nov 24;11(1):5961. doi: 10.1038/s41467-020-19552-9.
  PUBMED    33235249
REFERENCE   2  (bases 1 to 2786)
  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: "Nat 
            Commun."; date: "2020-11-24"; volume: "11(1):5961. doi"; pages: " 
            10.1038/s41467-020-19552-9"
COMMENT     Sequence Label: pXW101Plux2-33Ptet2-gfp
FEATURES             Location/Qualifiers
     source          1..2786
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     promoter        1..35
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     RBS             47..58
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      862..933
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      949..976
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             1071..1694
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /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="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(1144..1163)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      1714..1785
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1801..1828
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    1843..1861
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1868..1886
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1912..1923
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1930..2643
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1960..1988)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2583..2603
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2666..2737
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2753..2780
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
ORIGIN
        1 tttacagcta gctcagtcct aggtattatg ctagctacta gagattaaag aggagaaata
       61 ctagatgaaa aacataaatg ccgacgacac atacagaata attaataaaa ttaaagcttg
      121 tagaagcaat aatgatatta atcaatgctt atctgatatg actaaaatgg tacattgtga
      181 atattattta ctcgcgatca tttatcctca ttctatggtt aaatctgata tttcaatcct
      241 agataattac cctaaaaaat ggaggcaata ttatgatgac gctaatttaa taaaatatga
      301 tcctatagta gattattcta actccaatca ttcaccaatt aattggaata tatttgaaaa
      361 caatgctgta aataaaaaat ctccaaatgt aattaaagaa gcgaaaacat caggtcttat
      421 cactgggttt agtttcccta ttcatacggc taacaatggc ttcggaatgc ttagttttgc
      481 acattcagaa aaagacaact atatagatag tttattttta catgcgtgta tgaacatacc
      541 attaattgtt ccttctctag ttgataatta tcgaaaaata aatatagcaa ataataaatc
      601 aaacaacgat ttaaccaaaa gagaaaaaga atgtttagcg tgggcatgcg aaggaaaaag
      661 ctcttgggat atttcaaaaa tattaggttg cagtgagcgt actgtcactt tccatttaac
      721 caatgcgcaa atgaaactca atacaacaaa ccgctgccaa agtatttcta aagcaatttt
      781 aacaggagca attgattgcc catactttaa aaattaataa cactgatagt gctagtgtag
      841 atcactacta gagccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt
      901 cgttttatct gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg
      961 ggtgggcctt tctgcgttta tatactagag acctgtagga tcgtacaggt ttacgcaaga
     1021 aaatggtttg ttactttcga ataaatacta gagtcacaca ggactaccat atgtccagat
     1081 tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc ggaatcgaag
     1141 gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca ttgtattggc
     1201 atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta gataggcacc
     1261 atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta
     1321 aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac
     1381 ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta tgccaacaag
     1441 gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt actttaggtt
     1501 gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca cctactactg
     1561 atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc
     1621 cagccttctt attcggcctt gaattgatca tttgcggatt agaaaaacaa cttaaatgtg
     1681 aaagtgggtc ctaataatac tagagccagg catcaaataa aacgaaaggc tcagtcgaaa
     1741 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact
     1801 ggctcacctt cgggtgggcc tttctgcgtt tatatactag agtccctatc agtgatagag
     1861 attgacatcc ctatcagtga tagagatact gagcacatat tactagagat taaagaggag
     1921 aaatactaga tgcgtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa
     1981 ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca
     2041 acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg
     2101 ccaacacttg tcactacttt cggttatggt gttcaatgct ttgcgagata cccagatcat
     2161 atgaaacagc atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact
     2221 atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat
     2281 acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt
     2341 ggacacaaat tggaatacaa ctataactca cacaatgtat acatcatggc agacaaacaa
     2401 aagaatggaa tcaaagttaa cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa
     2461 ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac
     2521 aaccattacc tgtccacaca atctgccctt tcgaaagatc ccaacgaaaa gagagaccac
     2581 atggtccttc ttgagtttgt aacagctgct gggattacac atggcatgga tgaactatac
     2641 aaataataat actagagcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     2701 ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc
     2761 ttcgggtggg cctttctgcg tttata
//
