LOCUS       sequence-496971-        5348 bp    DNA     circular SYN 12-JAN-2026
DEFINITION  Repression of gene VIII by Ptet promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5348)
  AUTHORS   Kusumawardhani H, Zoppi F, Avendano R, Schaerli Y
  TITLE     Engineering intercellular communication using M13 phagemid and 
            CRISPR-based gene regulation for multicellular computing in 
            Escherichia coli.
  JOURNAL   Nat Commun. 2025 Apr 15;16(1):3569. doi: 10.1038/s41467-025-58760-z.
  PUBMED    40234414
REFERENCE   2  (bases 1 to 5348)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 12, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2025-04-15"; volume: "16(1):3569. doi"; pages: " 
            10.1038/s41467-025-58760-z"
COMMENT     Sequence Label: pHK101
FEATURES             Location/Qualifiers
     source          1..5348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1..1083)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     1045..1064
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1204..1209,1210..1225,1226..1232)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1240..1256
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     protein_bind    1304..1318
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease 
                     (Haurwitz et al., 2010)"
     RBS             1319..1330
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1331..2044
                     /codon_start=1
                     /gene="cI"
                     /product="phage lambda repressor"
                     /label=lambda repressor
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLAKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     protein_bind    2045..2064
                     /label=Csy4 Site
                     /bound_moiety="Pseudomonas aeruginosa Csy4 (Cas6f)"
                     /note="cleavage site for the Csy4 endoribonuclease (Cermak 
                     et al. 2017)"
     RBS             2465..2487
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2494..2715
                     /codon_start=1
                     /gene="VIII"
                     /product="pVIII"
                     /label=M13 gene VIII
                     /note="major coat protein"
                     /translation="MKKSLALKASVAVATLVPMLSFAAEGDDPAKAAFNSLQASATEYI
                     GYAWAMVVVIVGATIGIKLFKKFTSKAS"
     primer_bind     complement(2991..3009)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3005..3052
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3061..3147
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3239..3266
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3395..4186)
                     /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(4187..4278)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
       61 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
      121 gactggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
      181 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
      241 acatgagcta tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
      301 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
      361 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
      421 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
      481 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
      541 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cctcatggga
      601 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
      661 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
      721 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
      781 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
      841 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
      901 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
      961 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     1021 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     1081 catattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     1141 tttgcgccat tcgatggcgc gccgccattg cggccgcgcc gtcactgcgt cttttacgag
     1201 ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaattaa
     1261 gagatttcta cacgattgag cactgtctca gcctgcggtc cggctgccgt ataggcagaa
     1321 agaggagaaa atgagcacaa aaaagaaacc attaacacaa gagcagcttg aggacgcacg
     1381 tcgccttaaa gcaatttatg aaaaaaagaa aaatgaactt ggcttatccc aggaatctgt
     1441 cgcagacaag atggggatgg ggcagtcagg cgttggtgct ttatttaatg gcatcaatgc
     1501 attaaatgct tataacgccg cattgcttgc aaaaattctc aaagttagcg ttgaagaatt
     1561 tagcccttca atcgccagag aaatctacga gatgtatgaa gcggttagta tgcagccgtc
     1621 acttagaagt gagtatgagt accctgtttt ttctcatgtt caggcaggga tgttctcacc
     1681 tgagcttaga acctttacca aaggtgatgc ggagagatgg gtaagcacaa ccaaaaaagc
     1741 cagtgattct gcattctggc ttgaggttga aggtaattcc atgaccgcac caacaggctc
     1801 caagccaagc tttcctgacg gaatgttaat tctcgttgac cctgagcagg ctgttgagcc
     1861 aggtgatttc tgcatagcca gacttggggg tgatgagttt accttcaaga aactgatcag
     1921 ggatagcggt caggtgtttt tacaaccact aaacccacag tacccaatga tcccatgcaa
     1981 tgagagttgt tccgttgtgg ggaaagttat cgctagtcag tggcctgaag agacgtttgg
     2041 ctaagttcac tgccgtatag gcagtcgctg ggacgcccgc catggttcag ccaaaaaact
     2101 taagaccgcc ggtcttgtcc actaccttgc agtaatgcgg tggacaggat cggcggtttt
     2161 cttttctctt ctcaattctt ctgacctgta acgaataata gatagtaaag tagtctccga
     2221 ttgagttttc tctgccgagt cccacccagt tctgtgattt cagtaagttg gtaattgata
     2281 cactgttgcg agaactgctg cctggtagta gataggttgt tattgagtaa gaaggtaaag
     2341 tgaacgaaat ccctgaaact gagactgtag aaaataagtt tcagcctgcg gtccggtaac
     2401 accgtgcgtg ttgactattt tacctctggc ggtgataatg gttgctcgct gggacgcccg
     2461 taattttgtt taactttaag aaggagatat accatgaaaa agtctttagc cctcaaagcc
     2521 tctgtagccg ttgctaccct cgttccgatg ctgtctttcg ctgctgaggg tgacgatccc
     2581 gcaaaagcgg cctttaactc cctgcaagcc tcagcgaccg aatatatcgg ttatgcgtgg
     2641 gcgatggttg ttgtcattgt cggcgcaact atcggtatca agctgtttaa gaaattcacc
     2701 tcgaaagcaa gctgactcgg taccaaattc cagaaaagag gcctcccgaa aggggggcct
     2761 tttttcgttt tggtcctaat agataaagga taggtctggt agtgttgttc gttctcgcag
     2821 gtaaatcaat aatactcagc agttccgtag acttttcagt gggacagggt agcgataaca
     2881 gatagattgt aataagacac agtaggtgct cgtagttgcg tgaagagaac cgctcaggaa
     2941 atccagtcag aagtattggt aatcgttgaa aactcagtcg actgctgcca ccgctgagca
     3001 ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgccaggcat
     3061 caaataaaac gaaaggctca gtcggaagac tgggcctttc gttttatctg ttgtttgtcg
     3121 gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgtgaagcaa
     3181 cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca aactaagcag
     3241 aaggccatcc tgacggatgg cctttttgcg tttcagatct accggtaaac cagcaataga
     3301 cataagcggc tatttaacga ccctgccctg aaccgacgac cgggtcatcg tggccggatc
     3361 ttgcggcccc tcggcttgaa cgaattgtta gacattattt gccgactacc ttggtgatct
     3421 cgcctttcac gtagtggaca aattcttcca actgatctgc gcgcgaggcc aagcgatctt
     3481 cttcttgtcc aagataagcc tgtctagctt caagtatgac gggctgatac tgggccggca
     3541 ggcgctccat tgcccagtcg gcagcgacat ccttcggcgc gattttgccg gttactgcgc
     3601 tgtaccaaat gcgggacaac gtaagcacta catttcgctc atcgccagcc cagtcgggcg
     3661 gcgagttcca tagcgttaag gtttcattta gcgcctcaaa tagatcctgt tcaggaaccg
     3721 gatcaaagag ttcctccgcc gctggaccta ccaaggcaac gctatgttct cttgcttttg
     3781 tcagcaagat agccagatca atgtcgatcg tggctggctc gaagatacct gcaagaatgt
     3841 cattgcgctg ccattctcca aattgcagtt cgcgcttagc tggataacgc cacggaatga
     3901 tgtcgtcgtg cacaacaatg gtgacttcta cagcgcggag aatctcgctc tctccagggg
     3961 aagccgaagt ttccaaaagg tcgttgatca aagctcgccg cgttgtttca tcaagcctta
     4021 cggtcaccgt aaccagcaaa tcaatatcac tgtgtggctt caggccgtca tccactgcgg
     4081 agccgtacaa atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg acgccaacta
     4141 cctctgatag ttgagtcgat acttcggcga tcaccgcttc cctcatactc ttcctttttc
     4201 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4261 tttagaaaaa taaacaaata gctagctcac tcggtcgcta cgctccgggc gtgagactgc
     4321 ggcgggcgct gcggacacat acaaagttac ccacagattc cgtggataag caggggacta
     4381 acatgtgagg caaaacagca gggccgcgcc ggtggcgttt ttccataggc tccgccctcc
     4441 tgccagagtt cacataaaca gacgcttttc cggtgcatct gtgggagccg tgaggctcaa
     4501 ccatgaatct gacagtacgg gcgaaacccg acaggactta aagatcccca ccgtttccgg
     4561 cgggtcgctc cctcttgcgc tctcctgttc cgaccctgcc gtttaccgga tacctgttcc
     4621 gcctttctcc cttacgggaa gtgtggcgct ttctcatagc tcacacactg gtatctcggc
     4681 tcggtgtagg tcgtttgctc caagctgggc tgtaagcaag aactccccgt tcagcccgac
     4741 tgctgcgcct tatccggtaa ctgttcactt gagtccaacc cggaaaagca cggtaaaacg
     4801 ccactggcag cagccattgg taactgggag ttcgcagagg atttgtttag ctaaacacgc
     4861 ggttgctctt gaagtgtgcg ccaaagtccg gctacactgg aaggacagat ttggttgctg
     4921 tgctctgcga aagccagtta ccacggttaa gcagttcccc aactgactta accttcgatc
     4981 aaaccacctc cccaggtggt tttttcgaag gttaagtcag ttggggaact gcttaacctt
     5041 cgatcaaacc acctccccag gtggtttttt cgtttacagg gcaaaagatt acgcgcagaa
     5101 aaaaaggatc tcaagaagat cctttgatct tttctactga accgctctag atttcagtgc
     5161 aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag ttgtaattct
     5221 catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgctcc taggatgcag
     5281 tgactcgcac atttccccga aaagtgccac ctgaattcga aatccctgaa actgagactg
     5341 tagaaaat
//
