LOCUS       sequence-317145-        6671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Two-layered AHL-responsive circuit carrying an ECF11-based 
            amplifier.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6671)
  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 6671)
  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-Pecf11-gfp
FEATURES             Location/Qualifiers
     source          1..6671
                     /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"
     terminator      1752..1823
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1839..1866
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             1952..1963
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1970..2683
                     /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(2000..2028)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2623..2643
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2706..2777
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2793..2820
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    2827..2847
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      2902..2996
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        3069..3160
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3161..3229,3230..4021)
                     /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      4025..4064
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            4211..4319
                     /note="incP origin of transfer"
     CDS             complement(4380..5528)
                     /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      5921..6452
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(6579..6665)
                     /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 ctactagatg atgagcgata gtccgcagaa actgggtcgt aatgaatgga atgcatatat
     1201 ggataaagtg aaagccaaag atcgtgaagc ctttgccttt gtgtttcgtt tttatgcacc
     1261 gaaactgaaa cagttcgcct ataaacatgt tggcaatgaa caggttgcaa tggaaatggt
     1321 tcaagaaacc atggcaaccg tttggcagaa agcacatctg tatgatggta aaaaaagcgc
     1381 actgagcacc tggatttata ccattattcg taacctgtgc tttgacctgc tgcgtaaaca
     1441 gaaaggtaaa gaactgcata ttcacagcga cgatatttgg ccgagcgaat attatccgcc
     1501 tgatatggtt gatcattata gtccggaaca ggatatgctg aaagaacagg tggttaaatt
     1561 tctggatatc ctgccgaaaa atcagcgtga tgttctgcaa gcagtttatc tggaagaact
     1621 gccgcatcag caggttgcag aactgtttga tattccgctg ggcaccgtta aaagccgtct
     1681 gcgtctggca gttgaaaaac tgcgtcatag catgcatacc gaacagctgt aataatacta
     1741 gagccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
     1801 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
     1861 tctgcgttta tatactagag gcctccacac cgctcgtcac atcctgtgat ccactcttca
     1921 tcccgctacg taacacctct tactagagat taaagaggag aaatactaga tgcgtaaagg
     1981 agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
     2041 gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa aacttaccct
     2101 taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg tcactacttt
     2161 cggttatggt gttcaatgct ttgcgagata cccagatcat atgaaacagc atgacttttt
     2221 caagagtgcc atgcccgaag gttatgtaca ggaaagaact atatttttca aagatgacgg
     2281 gaactacaag acacgtgctg aagtcaagtt tgaaggtgat acccttgtta atagaatcga
     2341 gttaaaaggt attgatttta aagaagatgg aaacattctt ggacacaaat tggaatacaa
     2401 ctataactca cacaatgtat acatcatggc agacaaacaa aagaatggaa tcaaagttaa
     2461 cttcaaaatt agacacaaca ttgaagatgg aagcgttcaa ctagcagacc attatcaaca
     2521 aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc tgtccacaca
     2581 atctgccctt tcgaaagatc ccaacgaaaa gagagaccac atggtccttc ttgagtttgt
     2641 aacagctgct gggattacac atggcatgga tgaactatac aaataataat actagagcca
     2701 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     2761 tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg cctttctgcg
     2821 tttatatact agtagcggcc gctgcaggca tgcaagcttg cggccgcgtc gtgactggga
     2881 aaaccctggc gactagtctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc
     2941 atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccag
     3001 gggtccccaa taattacgat ttaaattagt agcccgccta atgagcgggc ttttttttaa
     3061 ttcccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3121 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagcattc agcattttcg
     3181 tgtggcgctg attccgtttt ttgcggcgtt ttgcctgccg gtgtttgcgc atccggaaac
     3241 cctggtgaaa gtgaaagatg cggaagatca actgggtgcg cgcgtgggct atattgaact
     3301 ggatctgaac agcggcaaaa ttctggaatc ttttcgtccg gaagaacgtt ttccgatgat
     3361 gagcaccttt aaagtgctgc tgtgcggtgc ggttctgagc cgtgtggatg cgggccagga
     3421 acaactgggc cgtcgtattc attatagcca gaacgatctg gtggaatata gcccggtgac
     3481 cgaaaaacat ctgaccgatg gcatgaccgt gcgtgaactg tgcagcgcgg cgattaccat
     3541 gagcgataac accgcggcga acctgctgct gacgaccatt ggcggtccga aagaactgac
     3601 cgcgtttctg cataacatgg gcgatcatgt gacccgtctg gatcgttggg aaccggaact
     3661 gaacgaagcg attccgaacg atgaacgtga taccaccatg ccggcagcaa tggcgaccac
     3721 cctgcgtaaa ctgctgacgg gtgagctgct gaccctggca agccgccagc aactgattga
     3781 ttggatggaa gcggataaag tggcgggtcc gctgctgcgt agcgcgctgc cggctggctg
     3841 gtttattgcg gataaaagcg gtgcgggcga acgtggcagc cgtggcatta ttgcggcgct
     3901 gggcccggat ggtaaaccga gccgtattgt ggtgatttat accaccggca gccaggcgac
     3961 gatggatgaa cgtaaccgtc agattgcgga aattggcgcg agcctgatta aacattggta
     4021 aaccgataca attaaaggct ccttttggag cctttttttt tggacgaccc ttgtcctttt
     4081 ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt
     4141 tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac
     4201 ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca
     4261 ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgt
     4321 aggccggccg cgatgcaggt ggctgctgaa cccccagccg gaactgaccc cacaaggccc
     4381 tagcgtttgc aatgcaccag gtcatcattg acccaggcgt gttccaccag gccgctgcct
     4441 cgcaactctt cgcaggcttc gccgacctgc tcgcgccact tcttcacgcg ggtggaatcc
     4501 gatccgcaca tgaggcggaa ggtttccagc ttgagcgggt acggctcccg gtgcgagctg
     4561 aaatagtcga acatccgtcg ggccgtcggc gacagcttgc ggtacttctc ccatatgaat
     4621 ttcgtgtagt ggtcgccagc aaacagcacg acgatttcct cgtcgatcag gacctggcaa
     4681 cgggacgttt tcttgccacg gtccaggacg cggaagcggt gcagcagcga caccgattcc
     4741 aggtgcccaa cgcggtcgga cgtgaagccc atcgccgtcg cctgtaggcg cgacaggcat
     4801 tcctcggcct tcgtgtaata ccggccattg atcgaccagc ccaggtcctg gcaaagctcg
     4861 tagaacgtga aggtgatcgg ctcgccgata ggggtgcgct tcgcgtactc caacacctgc
     4921 tgccacacca gttcgtcatc gtcggcccgc agctcgacgc cggtgtaggt gatcttcacg
     4981 tccttgttga cgtggaaaat gaccttgttt tgcagcgcct cgcgcgggat tttcttgttg
     5041 cgcgtggtga acagggcaga gcgggccgtg tcgtttggca tcgctcgcat cgtgtccggc
     5101 cacggcgcaa tatcgaacaa ggaaagctgc atttccttga tctgctgctt cgtgtgtttc
     5161 agcaacgcgg cctgcttggc ttcgctgacc tgttttgcca ggtcctcgcc ggcggttttt
     5221 cgcttcttgg tcgtcatagt tcctcgcgtg tcgatggtca tcgacttcgc caaacctgcc
     5281 gcctcctgtt cgagacgacg cgaacgctcc acggcggccg atggcgcggg cagggcaggg
     5341 ggagccagtt gcacgctgtc gcgctcgatc ttggccgtag cttgctggac tatcgagccg
     5401 acggactgga aggtttcgcg gggcgcacgc atgacggtgc ggcttgcgat ggtttcggca
     5461 tcctcggcgg aaaaccccgc gtcgatcagt tcttgcctgt atgccttccg gtcaaacgtc
     5521 cgattcattc accctccttg cgggattgcc ccggaattaa ttccccggat cgatccgtcg
     5581 atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc
     5641 agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc
     5701 ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct
     5761 ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc
     5821 accgtttctg cggactggct ttctacgtgg ctgccatttt tggggtgagg ccgttcgcgg
     5881 ccgaggggcg cagcccctgg ggggatggga ggcccgcgtt agcgggccgg gagggttcga
     5941 gaaggggggg cacccccctt cggcgtgcgc ggtcacgcgc acagggcgca gccctggtta
     6001 aaaacaaggt ttataaatat tggtttaaaa gcaggttaaa agacaggtta gcggtggccg
     6061 aaaaacgggc ggaaaccctt gcaaatgctg gattttctgc ctgtggacag cccctcaaat
     6121 gtcaataggt gcgcccctca tctgtcagca ctctgcccct caagtgtcaa ggatcgcgcc
     6181 cctcatctgt cagtagtcgc gcccctcaag tgtcaatacc gcagggcact tatccccagg
     6241 cttgtccaca tcatctgtgg gaaactcgcg taaaatcagg cgttttcgcc gatttgcgag
     6301 gctggccagc tccacgtcgc cggccgaaat cgagcctgcc cctcatctgt caacgccgcg
     6361 ccgggtgagt cggcccctca agtgtcaacg tccgcccctc atctgtcagt gagggccaag
     6421 ttttccgcga ggtatccaca acgccggcgg ccctacatgg ctctgctgta gtgagtgggt
     6481 tgcgctccgg cagcggtcct gatcccccgc agaaaaaaag gatctcaaga agatcctttg
     6541 atcttttcta cggcgcgccc agctgtctag ggcggcggat ttgtcctact caggagagcg
     6601 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     6661 atttgatgcc t
//
