LOCUS       sequence-317677-        6710 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 6710)
  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 6710)
  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..6710
                     /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             1150..1773
                     /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(1223..1242)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      1793..1864
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1880..1907
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    1922..1940
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1947..1965
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             1991..2002
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2009..2722
                     /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(2039..2067)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2662..2682
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2745..2816
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2832..2859
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      2941..3035
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        3108..3199
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3200..3268,3269..4060)
                     /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      4064..4103
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            4250..4358
                     /note="incP origin of transfer"
     CDS             complement(4419..5567)
                     /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      5960..6491
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(6618..6704)
                     /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 aagtaccata tgtccagatt agataaaagt aaagtgatta acagcgcatt agagctgctt
     1201 aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg cccagaagct aggtgtagag
     1261 cagcctacat tgtattggca tgtaaaaaat aagcgggctt tgctcgacgc cttagccatt
     1321 gagatgttag ataggcacca tactcacttt tgccctttag aaggggaaag ctggcaagat
     1381 tttttacgta ataacgctaa aagttttaga tgtgctttac taagtcatcg cgatggagca
     1441 aaagtacatt taggtacacg gcctacagaa aaacagtatg aaactctcga aaatcaatta
     1501 gcctttttat gccaacaagg tttttcacta gagaatgcat tatatgcact cagcgctgtg
     1561 gggcatttta ctttaggttg cgtattggaa gatcaagagc atcaagtcgc taaagaagaa
     1621 agggaaacac ctactactga tagtatgccg ccattgttac gacaagctat cgaattattt
     1681 gatcaccaag gtgcagagcc agccttctta ttcggccttg aattgatcat ttgcggatta
     1741 gaaaaacaac ttaaatgtga aagtgggtcc taataatact agagccaggc atcaaataaa
     1801 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     1861 tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt atatactaga
     1921 gtccctatca gtgatagaga ttgacatccc tatcagtgat agagatactg agcacatatt
     1981 actagagatt aaagaggaga aatactagat gcgtaaagga gaagaacttt tcactggagt
     2041 tgtcccaatt cttgttgaat tagatggtga tgttaatggg cacaaatttt ctgtcagtgg
     2101 agagggtgaa ggtgatgcaa catacggaaa acttaccctt aaatttattt gcactactgg
     2161 aaaactacct gttccatggc caacacttgt cactactttc ggttatggtg ttcaatgctt
     2221 tgcgagatac ccagatcata tgaaacagca tgactttttc aagagtgcca tgcccgaagg
     2281 ttatgtacag gaaagaacta tatttttcaa agatgacggg aactacaaga cacgtgctga
     2341 agtcaagttt gaaggtgata cccttgttaa tagaatcgag ttaaaaggta ttgattttaa
     2401 agaagatgga aacattcttg gacacaaatt ggaatacaac tataactcac acaatgtata
     2461 catcatggca gacaaacaaa agaatggaat caaagttaac ttcaaaatta gacacaacat
     2521 tgaagatgga agcgttcaac tagcagacca ttatcaacaa aatactccaa ttggcgatgg
     2581 ccctgtcctt ttaccagaca accattacct gtccacacaa tctgcccttt cgaaagatcc
     2641 caacgaaaag agagaccaca tggtccttct tgagtttgta acagctgctg ggattacaca
     2701 tggcatggat gaactataca aataataata ctagagccag gcatcaaata aaacgaaagg
     2761 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact
     2821 agagtcacac tggctcacct tcgggtgggc ctttctgcgt ttatatacta gaagcggccg
     2881 ctgcaggcat gcaagcttgc ggccgcgtcg tgactgggaa aaccctggcg actagtcttg
     2941 gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc
     3001 tcggttgccg ccgggcgttt tttattggtg agaatccagg ggtccccaat aattacgatt
     3061 taaattagta gcccgcctaa tgagcgggct tttttttaat tcccctattt gtttattttt
     3121 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3181 atattgaaaa aggaagagta tgagcattca gcattttcgt gtggcgctga ttccgttttt
     3241 tgcggcgttt tgcctgccgg tgtttgcgca tccggaaacc ctggtgaaag tgaaagatgc
     3301 ggaagatcaa ctgggtgcgc gcgtgggcta tattgaactg gatctgaaca gcggcaaaat
     3361 tctggaatct tttcgtccgg aagaacgttt tccgatgatg agcaccttta aagtgctgct
     3421 gtgcggtgcg gttctgagcc gtgtggatgc gggccaggaa caactgggcc gtcgtattca
     3481 ttatagccag aacgatctgg tggaatatag cccggtgacc gaaaaacatc tgaccgatgg
     3541 catgaccgtg cgtgaactgt gcagcgcggc gattaccatg agcgataaca ccgcggcgaa
     3601 cctgctgctg acgaccattg gcggtccgaa agaactgacc gcgtttctgc ataacatggg
     3661 cgatcatgtg acccgtctgg atcgttggga accggaactg aacgaagcga ttccgaacga
     3721 tgaacgtgat accaccatgc cggcagcaat ggcgaccacc ctgcgtaaac tgctgacggg
     3781 tgagctgctg accctggcaa gccgccagca actgattgat tggatggaag cggataaagt
     3841 ggcgggtccg ctgctgcgta gcgcgctgcc ggctggctgg tttattgcgg ataaaagcgg
     3901 tgcgggcgaa cgtggcagcc gtggcattat tgcggcgctg ggcccggatg gtaaaccgag
     3961 ccgtattgtg gtgatttata ccaccggcag ccaggcgacg atggatgaac gtaaccgtca
     4021 gattgcggaa attggcgcga gcctgattaa acattggtaa accgatacaa ttaaaggctc
     4081 cttttggagc cttttttttt ggacgaccct tgtccttttc cgctgcataa ccctgcttcg
     4141 gggtcattat agcgattttt tcggtatatc catccttttt cgcacgatat acaggatttt
     4201 gccaaagggt tcgtgtagac tttccttggt gtatccaacg gcgtcagccg ggcaggatag
     4261 gtgaagtagg cccacccgcg agcgggtgtt ccttcttcac tgtcccttat tcgcacctgg
     4321 cggtgctcaa cgggaatcct gctctgcgag gctggccgta ggccggccgc gatgcaggtg
     4381 gctgctgaac ccccagccgg aactgacccc acaaggccct agcgtttgca atgcaccagg
     4441 tcatcattga cccaggcgtg ttccaccagg ccgctgcctc gcaactcttc gcaggcttcg
     4501 ccgacctgct cgcgccactt cttcacgcgg gtggaatccg atccgcacat gaggcggaag
     4561 gtttccagct tgagcgggta cggctcccgg tgcgagctga aatagtcgaa catccgtcgg
     4621 gccgtcggcg acagcttgcg gtacttctcc catatgaatt tcgtgtagtg gtcgccagca
     4681 aacagcacga cgatttcctc gtcgatcagg acctggcaac gggacgtttt cttgccacgg
     4741 tccaggacgc ggaagcggtg cagcagcgac accgattcca ggtgcccaac gcggtcggac
     4801 gtgaagccca tcgccgtcgc ctgtaggcgc gacaggcatt cctcggcctt cgtgtaatac
     4861 cggccattga tcgaccagcc caggtcctgg caaagctcgt agaacgtgaa ggtgatcggc
     4921 tcgccgatag gggtgcgctt cgcgtactcc aacacctgct gccacaccag ttcgtcatcg
     4981 tcggcccgca gctcgacgcc ggtgtaggtg atcttcacgt ccttgttgac gtggaaaatg
     5041 accttgtttt gcagcgcctc gcgcgggatt ttcttgttgc gcgtggtgaa cagggcagag
     5101 cgggccgtgt cgtttggcat cgctcgcatc gtgtccggcc acggcgcaat atcgaacaag
     5161 gaaagctgca tttccttgat ctgctgcttc gtgtgtttca gcaacgcggc ctgcttggct
     5221 tcgctgacct gttttgccag gtcctcgccg gcggtttttc gcttcttggt cgtcatagtt
     5281 cctcgcgtgt cgatggtcat cgacttcgcc aaacctgccg cctcctgttc gagacgacgc
     5341 gaacgctcca cggcggccga tggcgcgggc agggcagggg gagccagttg cacgctgtcg
     5401 cgctcgatct tggccgtagc ttgctggact atcgagccga cggactggaa ggtttcgcgg
     5461 ggcgcacgca tgacggtgcg gcttgcgatg gtttcggcat cctcggcgga aaaccccgcg
     5521 tcgatcagtt cttgcctgta tgccttccgg tcaaacgtcc gattcattca ccctccttgc
     5581 gggattgccc cggaattaat tccccggatc gatccgtcga tcttgatccc ctgcgccatc
     5641 agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca accttaccag
     5701 agggcgcccc agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagct
     5761 atcgccatgt aagcccactg caagctacct gctttctctt tgcgcttgcg ttttcccttg
     5821 tccagatagc ccagtagctg acattcatcc ggggtcagca ccgtttctgc ggactggctt
     5881 tctacgtggc tgccattttt ggggtgaggc cgttcgcggc cgaggggcgc agcccctggg
     5941 gggatgggag gcccgcgtta gcgggccggg agggttcgag aagggggggc accccccttc
     6001 ggcgtgcgcg gtcacgcgca cagggcgcag ccctggttaa aaacaaggtt tataaatatt
     6061 ggtttaaaag caggttaaaa gacaggttag cggtggccga aaaacgggcg gaaacccttg
     6121 caaatgctgg attttctgcc tgtggacagc ccctcaaatg tcaataggtg cgcccctcat
     6181 ctgtcagcac tctgcccctc aagtgtcaag gatcgcgccc ctcatctgtc agtagtcgcg
     6241 cccctcaagt gtcaataccg cagggcactt atccccaggc ttgtccacat catctgtggg
     6301 aaactcgcgt aaaatcaggc gttttcgccg atttgcgagg ctggccagct ccacgtcgcc
     6361 ggccgaaatc gagcctgccc ctcatctgtc aacgccgcgc cgggtgagtc ggcccctcaa
     6421 gtgtcaacgt ccgcccctca tctgtcagtg agggccaagt tttccgcgag gtatccacaa
     6481 cgccggcggc cctacatggc tctgctgtag tgagtgggtt gcgctccggc agcggtcctg
     6541 atcccccgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggcgcgccca
     6601 gctgtctagg gcggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata
     6661 aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct
//
