LOCUS       sequence-317676-        6708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Two-layered AHL-responsive circuit containing a TetR-based inverter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6708)
  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 6708)
  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..6708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..31
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    56..77
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        78..112
                     /label=BBa_J23101
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.7"
     RBS             124..135
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      939..1010
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1026..1053
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             1148..1771
                     /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(1221..1240)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      1791..1862
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1878..1905
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    1920..1938
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1945..1963
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1989..2000
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2007..2720
                     /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(2037..2065)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2660..2680
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2743..2814
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2830..2857
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      2939..3033
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        3106..3197
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3198..3266,3267..4058)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     terminator      4062..4101
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            4248..4356
                     /note="incP origin of transfer"
     CDS             complement(4417..5565)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      5958..6489
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(6616..6702)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaattaaag cggataacaa tttcacacag gaggccgcct aggccgcggc cgcgcgaatt
       61 cgcggccgct tctagagttt acagctagct cagtcctagg tattatgcta gctactagag
      121 attaaagagg agaaatacta gatgaaaaac ataaatgccg acgacacata cagaataatt
      181 aataaaatta aagcttgtag aagcaataat gatattaatc aatgcttatc tgatatgact
      241 aaaatggtac attgtgaata ttatttactc gcgatcattt atcctcattc tatggttaaa
      301 tctgatattt caatcctaga taattaccct aaaaaatgga ggcaatatta tgatgacgct
      361 aatttaataa aatatgatcc tatagtagat tattctaact ccaatcattc accaattaat
      421 tggaatatat ttgaaaacaa tgctgtaaat aaaaaatctc caaatgtaat taaagaagcg
      481 aaaacatcag gtcttatcac tgggtttagt ttccctattc atacggctaa caatggcttc
      541 ggaatgctta gttttgcaca ttcagaaaaa gacaactata tagatagttt atttttacat
      601 gcgtgtatga acataccatt aattgttcct tctctagttg ataattatcg aaaaataaat
      661 atagcaaata ataaatcaaa caacgattta accaaaagag aaaaagaatg tttagcgtgg
      721 gcatgcgaag gaaaaagctc ttgggatatt tcaaaaatat taggttgcag tgagcgtact
      781 gtcactttcc atttaaccaa tgcgcaaatg aaactcaata caacaaaccg ctgccaaagt
      841 atttctaaag caattttaac aggagcaatt gattgcccat actttaaaaa ttaataacac
      901 tgatagtgct agtgtagatc actactagag ccaggcatca aataaaacga aaggctcagt
      961 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc
     1021 acactggctc accttcgggt gggcctttct gcgtttatat actagagacc tgtaggatcg
     1081 tacaggttta cgcaagaaaa tggtttgtta ctttcgaata aatactagag tcacacagga
     1141 ctaccatatg tccagattag ataaaagtaa agtgattaac agcgcattag agctgcttaa
     1201 tgaggtcgga atcgaaggtt taacaacccg taaactcgcc cagaagctag gtgtagagca
     1261 gcctacattg tattggcatg taaaaaataa gcgggctttg ctcgacgcct tagccattga
     1321 gatgttagat aggcaccata ctcacttttg ccctttagaa ggggaaagct ggcaagattt
     1381 tttacgtaat aacgctaaaa gttttagatg tgctttacta agtcatcgcg atggagcaaa
     1441 agtacattta ggtacacggc ctacagaaaa acagtatgaa actctcgaaa atcaattagc
     1501 ctttttatgc caacaaggtt tttcactaga gaatgcatta tatgcactca gcgctgtggg
     1561 gcattttact ttaggttgcg tattggaaga tcaagagcat caagtcgcta aagaagaaag
     1621 ggaaacacct actactgata gtatgccgcc attattacga caagctatcg aattatttga
     1681 tcaccaaggt gcagagccag ccttcttatt cggccttgaa ttgatcattt gcggattaga
     1741 aaaacaactt aaatgtgaaa gtgggtccta ataatactag agccaggcat caaataaaac
     1801 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     1861 tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat atactagagt
     1921 ccctatcagt gatagagatt gacatcccta tcagtgatag agatactgag cacatattac
     1981 tagagattaa agaggagaaa tactagatgc gtaaaggaga agaacttttc actggagttg
     2041 tcccaattct tgttgaatta gatggtgatg ttaatgggca caaattttct gtcagtggag
     2101 agggtgaagg tgatgcaaca tacggaaaac ttacccttaa atttatttgc actactggaa
     2161 aactacctgt tccatggcca acacttgtca ctactttcgg ttatggtgtt caatgctttg
     2221 cgagataccc agatcatatg aaacagcatg actttttcaa gagtgccatg cccgaaggtt
     2281 atgtacagga aagaactata tttttcaaag atgacgggaa ctacaagaca cgtgctgaag
     2341 tcaagtttga aggtgatacc cttgttaata gaatcgagtt aaaaggtatt gattttaaag
     2401 aagatggaaa cattcttgga cacaaattgg aatacaacta taactcacac aatgtataca
     2461 tcatggcaga caaacaaaag aatggaatca aagttaactt caaaattaga cacaacattg
     2521 aagatggaag cgttcaacta gcagaccatt atcaacaaaa tactccaatt ggcgatggcc
     2581 ctgtcctttt accagacaac cattacctgt ccacacaatc tgccctttcg aaagatccca
     2641 acgaaaagag agaccacatg gtccttcttg agtttgtaac agctgctggg attacacatg
     2701 gcatggatga actatacaaa taataatact agagccaggc atcaaataaa acgaaaggct
     2761 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctctactag
     2821 agtcacactg gctcaccttc gggtgggcct ttctgcgttt atatactaga agcggccgct
     2881 gcaggcatgc aagcttgcgg ccgcgtcgtg actgggaaaa ccctggcgac tagtcttgga
     2941 ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc
     3001 ggttgccgcc gggcgttttt tattggtgag aatccagggg tccccaataa ttacgattta
     3061 aattagtagc ccgcctaatg agcgggcttt tttttaattc ccctatttgt ttatttttct
     3121 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3181 attgaaaaag gaagagtatg agcattcagc attttcgtgt ggcgctgatt ccgttttttg
     3241 cggcgttttg cctgccggtg tttgcgcatc cggaaaccct ggtgaaagtg aaagatgcgg
     3301 aagatcaact gggtgcgcgc gtgggctata ttgaactgga tctgaacagc ggcaaaattc
     3361 tggaatcttt tcgtccggaa gaacgttttc cgatgatgag cacctttaaa gtgctgctgt
     3421 gcggtgcggt tctgagccgt gtggatgcgg gccaggaaca actgggccgt cgtattcatt
     3481 atagccagaa cgatctggtg gaatatagcc cggtgaccga aaaacatctg accgatggca
     3541 tgaccgtgcg tgaactgtgc agcgcggcga ttaccatgag cgataacacc gcggcgaacc
     3601 tgctgctgac gaccattggc ggtccgaaag aactgaccgc gtttctgcat aacatgggcg
     3661 atcatgtgac ccgtctggat cgttgggaac cggaactgaa cgaagcgatt ccgaacgatg
     3721 aacgtgatac caccatgccg gcagcaatgg cgaccaccct gcgtaaactg ctgacgggtg
     3781 agctgctgac cctggcaagc cgccagcaac tgattgattg gatggaagcg gataaagtgg
     3841 cgggtccgct gctgcgtagc gcgctgccgg ctggctggtt tattgcggat aaaagcggtg
     3901 cgggcgaacg tggcagccgt ggcattattg cggcgctggg cccggatggt aaaccgagcc
     3961 gtattgtggt gatttatacc accggcagcc aggcgacgat ggatgaacgt aaccgtcaga
     4021 ttgcggaaat tggcgcgagc ctgattaaac attggtaaac cgatacaatt aaaggctcct
     4081 tttggagcct ttttttttgg acgacccttg tccttttccg ctgcataacc ctgcttcggg
     4141 gtcattatag cgattttttc ggtatatcca tcctttttcg cacgatatac aggattttgc
     4201 caaagggttc gtgtagactt tccttggtgt atccaacggc gtcagccggg caggataggt
     4261 gaagtaggcc cacccgcgag cgggtgttcc ttcttcactg tcccttattc gcacctggcg
     4321 gtgctcaacg ggaatcctgc tctgcgaggc tggccgtagg ccggccgcga tgcaggtggc
     4381 tgctgaaccc ccagccggaa ctgaccccac aaggccctag cgtttgcaat gcaccaggtc
     4441 atcattgacc caggcgtgtt ccaccaggcc gctgcctcgc aactcttcgc aggcttcgcc
     4501 gacctgctcg cgccacttct tcacgcgggt ggaatccgat ccgcacatga ggcggaaggt
     4561 ttccagcttg agcgggtacg gctcccggtg cgagctgaaa tagtcgaaca tccgtcgggc
     4621 cgtcggcgac agcttgcggt acttctccca tatgaatttc gtgtagtggt cgccagcaaa
     4681 cagcacgacg atttcctcgt cgatcaggac ctggcaacgg gacgttttct tgccacggtc
     4741 caggacgcgg aagcggtgca gcagcgacac cgattccagg tgcccaacgc ggtcggacgt
     4801 gaagcccatc gccgtcgcct gtaggcgcga caggcattcc tcggccttcg tgtaataccg
     4861 gccattgatc gaccagccca ggtcctggca aagctcgtag aacgtgaagg tgatcggctc
     4921 gccgataggg gtgcgcttcg cgtactccaa cacctgctgc cacaccagtt cgtcatcgtc
     4981 ggcccgcagc tcgacgccgg tgtaggtgat cttcacgtcc ttgttgacgt ggaaaatgac
     5041 cttgttttgc agcgcctcgc gcgggatttt cttgttgcgc gtggtgaaca gggcagagcg
     5101 ggccgtgtcg tttggcatcg ctcgcatcgt gtccggccac ggcgcaatat cgaacaagga
     5161 aagctgcatt tccttgatct gctgcttcgt gtgtttcagc aacgcggcct gcttggcttc
     5221 gctgacctgt tttgccaggt cctcgccggc ggtttttcgc ttcttggtcg tcatagttcc
     5281 tcgcgtgtcg atggtcatcg acttcgccaa acctgccgcc tcctgttcga gacgacgcga
     5341 acgctccacg gcggccgatg gcgcgggcag ggcaggggga gccagttgca cgctgtcgcg
     5401 ctcgatcttg gccgtagctt gctggactat cgagccgacg gactggaagg tttcgcgggg
     5461 cgcacgcatg acggtgcggc ttgcgatggt ttcggcatcc tcggcggaaa accccgcgtc
     5521 gatcagttct tgcctgtatg ccttccggtc aaacgtccga ttcattcacc ctccttgcgg
     5581 gattgccccg gaattaattc cccggatcga tccgtcgatc ttgatcccct gcgccatcag
     5641 atccttggcg gcaagaaagc catccagttt actttgcagg gcttcccaac cttaccagag
     5701 ggcgccccag ctggcaattc cggttcgctt gctgtccata aaaccgccca gtctagctat
     5761 cgccatgtaa gcccactgca agctacctgc tttctctttg cgcttgcgtt ttcccttgtc
     5821 cagatagccc agtagctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc
     5881 tacgtggctg ccatttttgg ggtgaggccg ttcgcggccg aggggcgcag cccctggggg
     5941 gatgggaggc ccgcgttagc gggccgggag ggttcgagaa gggggggcac cccccttcgg
     6001 cgtgcgcggt cacgcgcaca gggcgcagcc ctggttaaaa acaaggttta taaatattgg
     6061 tttaaaagca ggttaaaaga caggttagcg gtggccgaaa aacgggcgga aacccttgca
     6121 aatgctggat tttctgcctg tggacagccc ctcaaatgtc aataggtgcg cccctcatct
     6181 gtcagcactc tgcccctcaa gtgtcaagga tcgcgcccct catctgtcag tagtcgcgcc
     6241 cctcaagtgt caataccgca gggcacttat ccccaggctt gtccacatca tctgtgggaa
     6301 actcgcgtaa aatcaggcgt tttcgccgat ttgcgaggct ggccagctcc acgtcgccgg
     6361 ccgaaatcga gcctgcccct catctgtcaa cgccgcgccg ggtgagtcgg cccctcaagt
     6421 gtcaacgtcc gcccctcatc tgtcagtgag ggccaagttt tccgcgaggt atccacaacg
     6481 ccggcggccc tacatggctc tgctgtagtg agtgggttgc gctccggcag cggtcctgat
     6541 cccccgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg cgcgcccagc
     6601 tgtctagggc ggcggatttg tcctactcag gagagcgttc accgacaaac aacagataaa
     6661 acgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcct
//
