LOCUS       sequence-318582-        5702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  aTc-responsive single-layered circuit.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5702)
  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 5702)
  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: pXW117Ptet2-gfp
FEATURES             Location/Qualifiers
     source          1..5702
                     /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_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     RBS             124..135
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             142..765
                     /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(215..234)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      785..856
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      872..899
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    914..932
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    939..957
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             983..994
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1001..1714
                     /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(1031..1059)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1654..1674
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      1737..1808
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1824..1851
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      1933..2027
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        2100..2191
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2192..2260,2261..3052)
                     /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      3056..3095
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            3242..3350
                     /note="incP origin of transfer"
     CDS             complement(3411..4559)
                     /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      4952..5483
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(5610..5696)
                     /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 tctagagttg acagctagct cagtcctagg gattgtgcta gctactagag
      121 attaaagagg agaaatacta gatgtccaga ttagataaaa gtaaagtgat taacagcgca
      181 ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag
      241 ctaggtgtag agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac
      301 gccttagcca ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa
      361 agctggcaag attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat
      421 cgcgatggag caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc
      481 gaaaatcaat tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca
      541 ctcagcgctg tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc
      601 gctaaagaag aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct
      661 atcgaattat ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc
      721 atttgcggat tagaaaaaca acttaaatgt gaaagtgggt cctaataata ctagagccag
      781 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
      841 gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc ctttctgcgt
      901 ttatatacta gagtccctat cagtgataga gattgacatc cctatcagtg atagagatac
      961 tgagcacata ttactagaga ttaaagagga gaaatactag atgcgtaaag gagaagaact
     1021 tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt
     1081 ttctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat
     1141 ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt tcggttatgg
     1201 tgttcaatgc tttgcgagat acccagatca tatgaaacag catgactttt tcaagagtgc
     1261 catgcccgaa ggttatgtac aggaaagaac tatatttttc aaagatgacg ggaactacaa
     1321 gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg agttaaaagg
     1381 tattgatttt aaagaagatg gaaacattct tggacacaaa ttggaataca actataactc
     1441 acacaatgta tacatcatgg cagacaaaca aaagaatgga atcaaagtta acttcaaaat
     1501 tagacacaac attgaagatg gaagcgttca actagcagac cattatcaac aaaatactcc
     1561 aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtccacac aatctgccct
     1621 ttcgaaagat cccaacgaaa agagagacca catggtcctt cttgagtttg taacagctgc
     1681 tgggattaca catggcatgg atgaactata caaataataa tactagagcc aggcatcaaa
     1741 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     1801 acgctctcta ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttatatac
     1861 tagaagcggc cgctgcaggc atgcaagctt gcggccgcgt cgtgactggg aaaaccctgg
     1921 cgactagtct tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt
     1981 tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca ggggtcccca
     2041 ataattacga tttaaattag tagcccgcct aatgagcggg ctttttttta attcccctat
     2101 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     2161 aatgcttcaa taatattgaa aaaggaagag tatgagcatt cagcattttc gtgtggcgct
     2221 gattccgttt tttgcggcgt tttgcctgcc ggtgtttgcg catccggaaa ccctggtgaa
     2281 agtgaaagat gcggaagatc aactgggtgc gcgcgtgggc tatattgaac tggatctgaa
     2341 cagcggcaaa attctggaat cttttcgtcc ggaagaacgt tttccgatga tgagcacctt
     2401 taaagtgctg ctgtgcggtg cggttctgag ccgtgtggat gcgggccagg aacaactggg
     2461 ccgtcgtatt cattatagcc agaacgatct ggtggaatat agcccggtga ccgaaaaaca
     2521 tctgaccgat ggcatgaccg tgcgtgaact gtgcagcgcg gcgattacca tgagcgataa
     2581 caccgcggcg aacctgctgc tgacgaccat tggcggtccg aaagaactga ccgcgtttct
     2641 gcataacatg ggcgatcatg tgacccgtct ggatcgttgg gaaccggaac tgaacgaagc
     2701 gattccgaac gatgaacgtg ataccaccat gccggcagca atggcgacca ccctgcgtaa
     2761 actgctgacg ggtgagctgc tgaccctggc aagccgccag caactgattg attggatgga
     2821 agcggataaa gtggcgggtc cgctgctgcg tagcgcgctg ccggctggct ggtttattgc
     2881 ggataaaagc ggtgcgggcg aacgtggcag ccgtggcatt attgcggcgc tgggcccgga
     2941 tggtaaaccg agccgtattg tggtgattta taccaccggc agccaggcga cgatggatga
     3001 acgtaaccgt cagattgcgg aaattggcgc gagcctgatt aaacattggt aaaccgatac
     3061 aattaaaggc tccttttgga gccttttttt ttggacgacc cttgtccttt tccgctgcat
     3121 aaccctgctt cggggtcatt atagcgattt tttcggtata tccatccttt ttcgcacgat
     3181 atacaggatt ttgccaaagg gttcgtgtag actttccttg gtgtatccaa cggcgtcagc
     3241 cgggcaggat aggtgaagta ggcccacccg cgagcgggtg ttccttcttc actgtccctt
     3301 attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg taggccggcc
     3361 gcgatgcagg tggctgctga acccccagcc ggaactgacc ccacaaggcc ctagcgtttg
     3421 caatgcacca ggtcatcatt gacccaggcg tgttccacca ggccgctgcc tcgcaactct
     3481 tcgcaggctt cgccgacctg ctcgcgccac ttcttcacgc gggtggaatc cgatccgcac
     3541 atgaggcgga aggtttccag cttgagcggg tacggctccc ggtgcgagct gaaatagtcg
     3601 aacatccgtc gggccgtcgg cgacagcttg cggtacttct cccatatgaa tttcgtgtag
     3661 tggtcgccag caaacagcac gacgatttcc tcgtcgatca ggacctggca acgggacgtt
     3721 ttcttgccac ggtccaggac gcggaagcgg tgcagcagcg acaccgattc caggtgccca
     3781 acgcggtcgg acgtgaagcc catcgccgtc gcctgtaggc gcgacaggca ttcctcggcc
     3841 ttcgtgtaat accggccatt gatcgaccag cccaggtcct ggcaaagctc gtagaacgtg
     3901 aaggtgatcg gctcgccgat aggggtgcgc ttcgcgtact ccaacacctg ctgccacacc
     3961 agttcgtcat cgtcggcccg cagctcgacg ccggtgtagg tgatcttcac gtccttgttg
     4021 acgtggaaaa tgaccttgtt ttgcagcgcc tcgcgcggga ttttcttgtt gcgcgtggtg
     4081 aacagggcag agcgggccgt gtcgtttggc atcgctcgca tcgtgtccgg ccacggcgca
     4141 atatcgaaca aggaaagctg catttccttg atctgctgct tcgtgtgttt cagcaacgcg
     4201 gcctgcttgg cttcgctgac ctgttttgcc aggtcctcgc cggcggtttt tcgcttcttg
     4261 gtcgtcatag ttcctcgcgt gtcgatggtc atcgacttcg ccaaacctgc cgcctcctgt
     4321 tcgagacgac gcgaacgctc cacggcggcc gatggcgcgg gcagggcagg gggagccagt
     4381 tgcacgctgt cgcgctcgat cttggccgta gcttgctgga ctatcgagcc gacggactgg
     4441 aaggtttcgc ggggcgcacg catgacggtg cggcttgcga tggtttcggc atcctcggcg
     4501 gaaaaccccg cgtcgatcag ttcttgcctg tatgccttcc ggtcaaacgt ccgattcatt
     4561 caccctcctt gcgggattgc cccggaatta attccccgga tcgatccgtc gatcttgatc
     4621 ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc
     4681 caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc cataaaaccg
     4741 cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc tttgcgcttg
     4801 cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag caccgtttct
     4861 gcggactggc tttctacgtg gctgccattt ttggggtgag gccgttcgcg gccgaggggc
     4921 gcagcccctg gggggatggg aggcccgcgt tagcgggccg ggagggttcg agaagggggg
     4981 gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc agccctggtt aaaaacaagg
     5041 tttataaata ttggtttaaa agcaggttaa aagacaggtt agcggtggcc gaaaaacggg
     5101 cggaaaccct tgcaaatgct ggattttctg cctgtggaca gcccctcaaa tgtcaatagg
     5161 tgcgcccctc atctgtcagc actctgcccc tcaagtgtca aggatcgcgc ccctcatctg
     5221 tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac ttatccccag gcttgtccac
     5281 atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc cgatttgcga ggctggccag
     5341 ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg tcaacgccgc gccgggtgag
     5401 tcggcccctc aagtgtcaac gtccgcccct catctgtcag tgagggccaa gttttccgcg
     5461 aggtatccac aacgccggcg gccctacatg gctctgctgt agtgagtggg ttgcgctccg
     5521 gcagcggtcc tgatcccccg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     5581 acggcgcgcc cagctgtcta gggcggcgga tttgtcctac tcaggagagc gttcaccgac
     5641 aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc
     5701 ct
//
