LOCUS       sequence-473111-        6161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repression of gene VIII by Plux/tet hybrid promoter .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6161)
  AUTHORS   Kusumawardhani, H., Zoppi, F., Avendano, R. et al.
  TITLE     Engineering intercellular communication using M13 phagemid and 
            CRISPR-based gene regulation for multicellular computing in 
            Escherichia coli
  JOURNAL   Nat Commun 16, 3569 (2025)
REFERENCE   2  (bases 1 to 6161)
  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 
            16, 3569 (2025)"
COMMENT     Sequence Label: pHK401
FEATURES             Location/Qualifiers
     source          1..6161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(79..870)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDDGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(871..962)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             complement(2033..2656)
                     /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     2564..2583
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      2830..2857
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    3975..3993
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4054..4068
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     RBS             4069..4080
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4081..4794
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     protein_bind    4795..4814
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        5147..5181
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             5310..5332
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5339..5560
                     /codon_start=1
                     /gene="VIII"
                     /product="pVIII"
                     /label=M13 gene VIII
                     /note="major coat protein"
                     /translation="MKKSLALKASVAVATLVPMLSFAAEGDDPAKAAFNSLQASATEYI
                     GYAWAMVVVIVGATIGIKLFKKFTSKAS"
     primer_bind     complement(5836..5854)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5850..5897
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      5906..5992
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6084..6111
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 acgaccctgc cctgaaccga cgaccgggtc atcgtggccg gatcttgcgg cccctcggct
       61 tgaacgaatt gttagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg
      121 gacaaattct tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata
      181 agcctgtcta gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca
      241 gtcggcagcg acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga
      301 caacgtaagc actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt
      361 taaggtttca tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc
      421 cgccgctgga cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag
      481 atcaatgtcg atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc
      541 tccaaattgc agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac
      601 aatggtgact tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa
      661 aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag
      721 caaatcaata tcactgtgtg gcttcaggcc gtcatccact gcggagccgt acaaatgtac
      781 ggccagcaac gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt
      841 cgatacttcg gcgatcaccg cttccctcat actcttcctt tttcaatatt attgaagcat
      901 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
      961 aatagctagc tcactcggtc gctacgctcc gggcgtgaga ctgcggcggg cgctgcggac
     1021 acatacaaag ttacccacag attccgtgga taagcagggg actaacatgt gaggcaaaac
     1081 agcagggccg cgccggtggc gtttttccat aggctccgcc ctcctgccag agttcacata
     1141 aacagacgct tttccggtgc atctgtggga gccgtgaggc tcaaccatga atctgacagt
     1201 acgggcgaaa cccgacagga cttaaagatc cccaccgttt ccggcgggtc gctccctctt
     1261 gcgctctcct gttccgaccc tgccgtttac cggatacctg ttccgccttt ctcccttacg
     1321 ggaagtgtgg cgctttctca tagctcacac actggtatct cggctcggtg taggtcgttt
     1381 gctccaagct gggctgtaag caagaactcc ccgttcagcc cgactgctgc gccttatccg
     1441 gtaactgttc acttgagtcc aacccggaaa agcacggtaa aacgccactg gcagcagcca
     1501 ttggtaactg ggagttcgca gaggatttgt ttagctaaac acgcggttgc tcttgaagtg
     1561 tgcgccaaag tccggctaca ctggaaggac agatttggtt gctgtgctct gcgaaagcca
     1621 gttaccacgg ttaagcagtt ccccaactga cttaaccttc gatcaaacca cctccccagg
     1681 tggttttttc gaaggttaag tcagttgggg aactgcttaa ccttcgatca aaccacctcc
     1741 ccaggtggtt ttttcgttta cagggcaaaa gattacgcgc agaaaaaaag gatctcaaga
     1801 agatcctttg atcttttcta ctgaaccgct ctagatttca gtgcaattta tctcttcaaa
     1861 tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc
     1921 cgcgcccacc ggaaggagct gactgggttg ctcctaggat gcagtgactc gcacatttcc
     1981 ccgaaaagtg ccacctgaat tcgaaatccc tgaaactgag actgtagaaa atttaggacc
     2041 cactttcaca tttaagttgt ttttctaatc cgcatataat caattcaagg ccgaataaga
     2101 aggctggctc tgcaccttgg tgatcaaata attcgatagc ttgtcgtaat aatggcggca
     2161 tactatcagt agtaggtgtt tccctttctt ctttagcgac ttgatgctct tgatcttcca
     2221 atacgcaacc taaagtaaaa tgccccacag cgctgagtgc atataatgca ttctctagtg
     2281 aaaaaccttg ttggcataaa aaggctaatt gattttcgag agtttcatac tgtttttctg
     2341 taggccgtgt acctaaatgt acttttgctc catcgcgatg acttagtaaa gcacatctaa
     2401 aacttttagc gttattacgt aaaaaatctt gccagctttc cccttctaaa gggcaaaagt
     2461 gagtatggtg cctatctaac atctcaatgg ctaaggcgtc gagcaaagcc cgcttatttt
     2521 ttacatgcca atacaatgta ggctgctcta cacctagctt ctgggcgagt ttacgggttg
     2581 ttaaaccttc gattccgacc tcattaagca gctctaatgc gctgttaatc actttacttt
     2641 tatctaatct ggacattttt ttcctcctgg atccttaatt tttaaagtat gggcaatcaa
     2701 ttgctcctgt taaaattgct ttagaaatac tttggcagcg gtttgttgta ttgagtttca
     2761 tttgcgcatt ggttaaatgg aaagtgacag tacgctcact gcgccgtcac tgcgtctttt
     2821 actcacactg gctcaccttc gggtgggcct ttctgcgttt atatactaga gagagaatat
     2881 aaaaagccag attattaatc cggctttttt attatttgaa ttcgaaatcc ctgaaactga
     2941 gactgtagaa aatttattaa tttttgaagt acggacagtc gatagcgccg gtcaggattg
     3001 ctttggagat ggattggcag cgattcgtgg tgttcagttt catttgcgcg ttcgtcagat
     3061 gaaacgtaac ggtacgttcg gaacaaccca ggattttact aatatcccaa gagcttttgc
     3121 cttcacaggc ccacgccagg cattcttttt cgcgtttggt caggtcattg ttacttttgt
     3181 tgttagcgat gttgatttta cggtagttat caaccagaga cggcacgatc agcggaatat
     3241 tcatgcatgc atgcaggaac aggctgtcaa tatagttatc tttttcagag tgagcgaagc
     3301 tcagcatacc aaagccattg tttgcggtat ggatcgggaa cgaaaaaccc gtaatcaggc
     3361 cgctggtttt ggcttctttg atcacgttcg gagatttttt gttgaccgca ttgttttcaa
     3421 agatattcca gttaatcggg gagtgattgg agttactgta gtccacgatc ggatcatatt
     3481 taatcagatt ggcgtcatcg taatactgac gccatttttt cgggtagttg tccaggatcg
     3541 aaatatctga tttgaccata ctgtgcggat agataatcgc cagcaggtaa tattcgcaat
     3601 gaaccatttt ggtcatatcg ctcaggcact ggttgatgtc gttgtttgag cgacaagctt
     3661 tgattttgtt gatgatgcgg tacgtatcgt cggcgttgat gtttttcata cccatctctt
     3721 tatccttacc tattgtttgt cgcaagtttt gcgtgttata tatcattaaa acggtaatgg
     3781 attgacattt gattctaata aattggattt ttgtcacact attgtatcgc tgggaataca
     3841 attacttaac ataagcacct gtaggatcgt acaggtcaat aaacagttga tagggcttct
     3901 ccgttacagc ctgcggtccg gacctgtagg atcgtacagg tttacgcaag aaaatggttt
     3961 gttatagtcg aatatcccta tcagtgatag agatcgctgg gacgcccgaa gagatttcta
     4021 cacgattgag cactgtctca gcctgcggtc cggctgccgt ataggcagaa agaggagaaa
     4081 atgagcacaa aaaagaaacc attaacacaa gagcagcttg aggacgcacg tcgccttaaa
     4141 gcaatttatg aaaaaaagaa aaatgaactt ggcttatccc aggaatctgt cgcagacaag
     4201 atggggatgg ggcagtcagg cgttggtgct ttatttaatg gcatcaatgc attaaatgct
     4261 tataacgccg cattgcttgc aaaaattctc aaagttagcg ttgaagaatt tagcccttca
     4321 atcgccagag aaatctacga gatgtatgaa gcggttagta tgcagccgtc acttagaagt
     4381 gagtatgagt accctgtttt ttctcatgtt caggcaggga tgttctcacc tgagcttaga
     4441 acctttacca aaggtgatgc ggagagatgg gtaagcacaa ccaaaaaagc cagtgattct
     4501 gcattctggc ttgaggttga aggtaattcc atgaccgcac caacaggctc caagccaagc
     4561 tttcctgacg gaatgttaat tctcgttgac cctgagcagg ctgttgagcc aggtgatttc
     4621 tgcatagcca gacttggggg tgatgagttt accttcaaga aactgatcag ggatagcggt
     4681 caggtgtttt tacaaccact aaacccacag tacccaatga tcccatgcaa tgagagttgt
     4741 tccgttgtgg ggaaagttat cgctagtcag tggcctgaag agacgtttgg ctaagttcac
     4801 tgccgtatag gcagtcgctg ggacgcccgc catggttcag ccaaaaaact taagaccgcc
     4861 ggtcttgtcc actaccttgc agtaatgcgg tggacaggat cggcggtttt cttttctctt
     4921 ctcaattctt ctgacctgta acgaataata gatagtaaag tagtctccga ttgagttttc
     4981 tctgccgagt cccacccagt tctgtgattt cagtaagttg gtaattgata cactgttgcg
     5041 agaactgctg cctggtagta gataggttgt tattgagtaa gaaggtaaag tgaacgaaat
     5101 ccctgaaact gagactgtag aaaataagct tcagcctgcg gtccggttga cagctagctc
     5161 agtcctaggt ataatgctag ctcgctggga cgcccgggga ctacacttac gaaactattg
     5221 attgctcagc ctgcggtccg gtaacaccgt gcgtgttgac tattttacct ctggcggtga
     5281 taatggttgc tcgctgggac gcccgtaatt ttgtttaact ttaagaagga gatataccat
     5341 gaaaaagtct ttagccctca aagcctctgt agccgttgct accctcgttc cgatgctgtc
     5401 tttcgctgct gagggtgacg atcccgcaaa agcggccttt aactccctgc aagcctcagc
     5461 gaccgaatat atcggttatg cgtgggcgat ggttgttgtc attgtcggcg caactatcgg
     5521 tatcaagctg tttaagaaat tcacctcgaa agcaagctga ctcggtacca aattccagaa
     5581 aagaggcctc ccgaaagggg ggcctttttt cgttttggtc ctaatagata aaggataggt
     5641 ctggtagtgt tgttcgttct cgcaggtaaa tcaataatac tcagcagttc cgtagacttt
     5701 tcagtgggac agggtagcga taacagatag attgtaataa gacacagtag gtgctcgtag
     5761 ttgcgtgaag agaaccgctc aggaaatcca gtcagaagta ttggtaatcg ttgaaaactc
     5821 agtcgactgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     5881 tcttgagggg ttttttgcca ggcatcaaat aaaacgaaag gctcagtcgg aagactgggc
     5941 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     6001 agcggatttg aacgttgtga agcaacggcc cggagggtgg cgggcaggac gcccgccata
     6061 aactgccagg catcaaacta agcagaaggc catcctgacg gatggccttt ttgcgtttca
     6121 gatctaccgg taaaccagca atagacataa gcggctattt a
//
