LOCUS       sequence-472224-        5056 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Repression of gene VIII by Ptac promoter .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5056)
  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 5056)
  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: pHK201
FEATURES             Location/Qualifiers
     source          1..5056
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..624)
                     /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     532..551
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     781..798
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     782..798
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    818..836
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    843..861
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    917..931
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     RBS             932..943
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             944..1657
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     protein_bind    1658..1677
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     promoter        2010..2044
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     RBS             2173..2195
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2202..2423
                     /codon_start=1
                     /gene="VIII"
                     /product="pVIII"
                     /label=M13 gene VIII
                     /note="major coat protein"
                     /translation="MKKSLALKASVAVATLVPMLSFAAEGDDPAKAAFNSLQASATEYI
                     GYAWAMVVVIVGATIGIKLFKKFTSKAS"
     primer_bind     complement(2699..2717)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2713..2760
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      2769..2855
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2947..2974
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3103..3894)
                     /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(3895..3986)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ttaggaccca ctttcacatt taagttgttt ttctaatccg catataatca attcaaggcc
       61 gaataagaag gctggctctg caccttggtg atcaaataat tcgatagctt gtcgtaataa
      121 tggcggcata ctatcagtag taggtgtttc cctttcttct ttagcgactt gatgctcttg
      181 atcttccaat acgcaaccta aagtaaaatg ccccacagcg ctgagtgcat ataatgcatt
      241 ctctagtgaa aaaccttgtt ggcataaaaa ggctaattga ttttcgagag tttcatactg
      301 tttttctgta ggccgtgtac ctaaatgtac ttttgctcca tcgcgatgac ttagtaaagc
      361 acatctaaaa cttttagcgt tattacgtaa aaaatcttgc cagctttccc cttctaaagg
      421 gcaaaagtga gtatggtgcc tatctaacat ctcaatggct aaggcgtcga gcaaagcccg
      481 cttatttttt acatgccaat acaatgtagg ctgctctaca cctagcttct gggcgagttt
      541 acgggttgtt aaaccttcga ttccgacctc attaagcagc tctaatgcgc tgttaatcac
      601 tttactttta tctaatctgg acattttttt cctcctggat ccttaatttt taaagtatgg
      661 gcaatcaatt gctcctgtta aaattgcttt agaaatactt tggcagcggt ttgttgtatt
      721 gagtttcatt tgcgcattgg ttaaatggaa agtgacagta cgctcactgc gaattcgagt
      781 tgtaaaacga cggccagtac tccaccgttg gcttttttcc ctatcagtga tagagattga
      841 catccctatc agtgatagag ataatgagca caagagattt ctacacgatt gagcactgtc
      901 tcagcctgcg gtccggctgc cgtataggca gaaagaggag aaaatgagca caaaaaagaa
      961 accattaaca caagagcagc ttgaggacgc acgtcgcctt aaagcaattt atgaaaaaaa
     1021 gaaaaatgaa cttggcttat cccaggaatc tgtcgcagac aagatgggga tggggcagtc
     1081 aggcgttggt gctttattta atggcatcaa tgcattaaat gcttataacg ccgcattgct
     1141 tgcaaaaatt ctcaaagtta gcgttgaaga atttagccct tcaatcgcca gagaaatcta
     1201 cgagatgtat gaagcggtta gtatgcagcc gtcacttaga agtgagtatg agtaccctgt
     1261 tttttctcat gttcaggcag ggatgttctc acctgagctt agaaccttta ccaaaggtga
     1321 tgcggagaga tgggtaagca caaccaaaaa agccagtgat tctgcattct ggcttgaggt
     1381 tgaaggtaat tccatgaccg caccaacagg ctccaagcca agctttcctg acggaatgtt
     1441 aattctcgtt gaccctgagc aggctgttga gccaggtgat ttctgcatag ccagacttgg
     1501 gggtgatgag tttaccttca agaaactgat cagggatagc ggtcaggtgt ttttacaacc
     1561 actaaaccca cagtacccaa tgatcccatg caatgagagt tgttccgttg tggggaaagt
     1621 tatcgctagt cagtggcctg aagagacgtt tggctaagtt cactgccgta taggcagtcg
     1681 ctgggacgcc cgccatggtt cagccaaaaa acttaagacc gccggtcttg tccactacct
     1741 tgcagtaatg cggtggacag gatcggcggt tttcttttct cttctcaatt cttctgacct
     1801 gtaacgaata atagatagta aagtagtctc cgattgagtt ttctctgccg agtcccaccc
     1861 agttctgtga tttcagtaag ttggtaattg atacactgtt gcgagaactg ctgcctggta
     1921 gtagataggt tgttattgag taagaaggta aagtgaacga aatccctgaa actgagactg
     1981 tagaaaataa gcttcagcct gcggtccggt tgacagctag ctcagtccta ggtataatgc
     2041 tagctcgctg ggacgcccgg ggactacact tacgaaacta tggattgctc agcctgcggt
     2101 ccggtaacac cgtgcgtgtt gactatttta cctctggcgg tgataatggt tgctcgctgg
     2161 gacgcccgta attttgttta actttaagaa ggagatatac catgaaaaag tctttagccc
     2221 tcaaagcctc tgtagccgtt gctaccctcg ttccgatgct gtctttcgct gctgagggtg
     2281 acgatcccgc aaaagcggcc tttaactccc tgcaagcctc agcgaccgaa tatatcggtt
     2341 atgcgtgggc gatggttgtt gtcattgtcg gcgcaactat cggtatcaag ctgtttaaga
     2401 aattcacctc gaaagcaagc tgactcggta ccaaattcca gaaaagaggc ctcccgaaag
     2461 gggggccttt tttcgttttg gtcctaatag ataaaggata ggtctggtag tgttgttcgt
     2521 tctcgcaggt aaatcaataa tactcagcag ttccgtagac ttttcagtgg gacagggtag
     2581 cgataacaga tagattgtaa taagacacag taggtgctcg tagttgcgtg aagagaaccg
     2641 ctcaggaaat ccagtcagaa gtattggtaa tcgttgaaaa ctcagtcgac tgctgccacc
     2701 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     2761 ccaggcatca aataaaacga aaggctcagt cggaagactg ggcctttcgt tttatctgtt
     2821 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg
     2881 tgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa
     2941 ctaagcagaa ggccatcctg acggatggcc tttttgcgtt tcagatctac cggtaaacca
     3001 gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcatcgtg
     3061 gccggatctt gcggcccctc ggcttgaacg aattgttaga cattatttgc cgactacctt
     3121 ggtgatctcg cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa
     3181 gcgatcttct tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg
     3241 ggccggcagg cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt
     3301 tactgcgctg taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca
     3361 gtcgggcggc gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc
     3421 aggaaccgga tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct
     3481 tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc
     3541 aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca
     3601 cggaatgatg tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc
     3661 tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc
     3721 aagccttacg gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgtcatc
     3781 cactgcggag ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac
     3841 gccaactacc tctgatagtt gagtcgatac ttcggcgatc accgcttccc tcatactctt
     3901 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3961 tgaatgtatt tagaaaaata aacaaatagc tagctcactc ggtcgctacg ctccgggcgt
     4021 gagactgcgg cgggcgctgc ggacacatac aaagttaccc acagattccg tggataagca
     4081 ggggactaac atgtgaggca aaacagcagg gccgcgccgg tggcgttttt ccataggctc
     4141 cgccctcctg ccagagttca cataaacaga cgcttttccg gtgcatctgt gggagccgtg
     4201 aggctcaacc atgaatctga cagtacgggc gaaacccgac aggacttaaa gatccccacc
     4261 gtttccggcg ggtcgctccc tcttgcgctc tcctgttccg accctgccgt ttaccggata
     4321 cctgttccgc ctttctccct tacgggaagt gtggcgcttt ctcatagctc acacactggt
     4381 atctcggctc ggtgtaggtc gtttgctcca agctgggctg taagcaagaa ctccccgttc
     4441 agcccgactg ctgcgcctta tccggtaact gttcacttga gtccaacccg gaaaagcacg
     4501 gtaaaacgcc actggcagca gccattggta actgggagtt cgcagaggat ttgtttagct
     4561 aaacacgcgg ttgctcttga agtgtgcgcc aaagtccggc tacactggaa ggacagattt
     4621 ggttgctgtg ctctgcgaaa gccagttacc acggttaagc agttccccaa ctgacttaac
     4681 cttcgatcaa accacctccc caggtggttt tttcgaaggt taagtcagtt ggggaactgc
     4741 ttaaccttcg atcaaaccac ctccccaggt ggttttttcg tttacagggc aaaagattac
     4801 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctactgaac cgctctagat
     4861 ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt
     4921 gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgctccta
     4981 ggatgcagtg actcgcacat ttccccgaaa agtgccacct gaattcgaaa tccctgaaac
     5041 tgagactgta gaaaat
//
