LOCUS       sequence-311322-        2788 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 2788)
  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 2788)
  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: pXW101Plux2-32Ptet2-gfp
FEATURES             Location/Qualifiers
     source          1..2788
                     /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             1073..1696
                     /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(1146..1165)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      1716..1787
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1803..1830
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    1845..1863
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1870..1888
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1914..1925
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1932..2645
                     /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(1962..1990)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2585..2605
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2668..2739
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2755..2782
                     /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 ggaaagtacc atatgtccag
     1081 attagataaa agtaaagtga ttaacagcgc attagagctg cttaatgagg tcggaatcga
     1141 aggtttaaca acccgtaaac tcgcccagaa gctaggtgta gagcagccta cattgtattg
     1201 gcatgtaaaa aataagcggg ctttgctcga cgccttagcc attgagatgt tagataggca
     1261 ccatactcac ttttgccctt tagaagggga aagctggcaa gattttttac gtaataacgc
     1321 taaaagtttt agatgtgctt tactaagtca tcgcgatgga gcaaaagtac atttaggtac
     1381 acggcctaca gaaaaacagt atgaaactct cgaaaatcaa ttagcctttt tatgccaaca
     1441 aggtttttca ctagagaatg cattatatgc actcagcgct gtggggcatt ttactttagg
     1501 ttgcgtattg gaagatcaag agcatcaagt cgctaaagaa gaaagggaaa cacctactac
     1561 tgatagtatg ccgccattat tacgacaagc tatcgaatta tttgatcacc aaggtgcaga
     1621 gccagccttc ttattcggcc ttgaattgat catttgcgga ttagaaaaac aacttaaatg
     1681 tgaaagtggg tcctaataat actagagcca ggcatcaaat aaaacgaaag gctcagtcga
     1741 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca
     1801 ctggctcacc ttcgggtggg cctttctgcg tttatatact agagtcccta tcagtgatag
     1861 agattgacat ccctatcagt gatagagata ctgagcacat attactagag attaaagagg
     1921 agaaatacta gatgcgtaaa ggagaagaac ttttcactgg agttgtccca attcttgttg
     1981 aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg
     2041 caacatacgg aaaacttacc cttaaattta tttgcactac tggaaaacta cctgttccat
     2101 ggccaacact tgtcactact ttcggttatg gtgttcaatg ctttgcgaga tacccagatc
     2161 atatgaaaca gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaaagaa
     2221 ctatattttt caaagatgac gggaactaca agacacgtgc tgaagtcaag tttgaaggtg
     2281 atacccttgt taatagaatc gagttaaaag gtattgattt taaagaagat ggaaacattc
     2341 ttggacacaa attggaatac aactataact cacacaatgt atacatcatg gcagacaaac
     2401 aaaagaatgg aatcaaagtt aacttcaaaa ttagacacaa cattgaagat ggaagcgttc
     2461 aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag
     2521 acaaccatta cctgtccaca caatctgccc tttcgaaaga tcccaacgaa aagagagacc
     2581 acatggtcct tcttgagttt gtaacagctg ctgggattac acatggcatg gatgaactat
     2641 acaaataata atactagagc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     2701 gcctttcgtt ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca
     2761 ccttcgggtg ggcctttctg cgtttata
//
