LOCUS       sequence-472232-        5367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible gene VIII by Pbad/lac hybrid promoter .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5367)
  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 5367)
  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: pHK328
FEATURES             Location/Qualifiers
     source          1..5367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(220..1302)
                     /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     1264..1283
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      1424..1451
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(1548..2426)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        2453..2737
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     2660..2679
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     protein_bind    2745..2761
                     /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)."
     RBS             2768..2790
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2797..3018
                     /codon_start=1
                     /gene="VIII"
                     /product="pVIII"
                     /label=M13 gene VIII
                     /note="major coat protein"
                     /translation="MKKSLALKASVAVATLVPMLSFAAEGDDPAKAAFNSLQASATEYI
                     GYAWAMVVVIVGATIGIKLFKKFTSKAS"
     primer_bind     complement(3294..3312)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3308..3355
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      3364..3450
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3542..3569
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             complement(3698..4489)
                     /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(4490..4581)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4629..5367)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
ORIGIN
        1 ttttctactg aaccgctcta gatttcagtg caatttatct cttcaaatgt agcacctgaa
       61 gtcagcccca tacgatataa gttgtaattc tcatgttagt catgccccgc gcccaccgga
      121 aggagctgac tgggttgctc ctaggatgca gtgactcgca catttccccg aaaagtgcca
      181 cctgaattcg aaatccctga aactgagact gtagaaaatt cactgcccgc tttccagtcg
      241 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
      301 cgtattgggc gccagggtgg tttttctttt caccagtgag actggcaaca gctgattgcc
      361 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
      421 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctat cttcggtatc
      481 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
      541 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
      601 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
      661 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
      721 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
      781 cagatgctcc acgcccagtc gcgtaccgtc ctcatgggag aaaataatac tgttgatggg
      841 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
      901 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
      961 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     1021 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     1081 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     1141 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     1201 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     1261 accggcatac tctgcgacat cgtataacgt tactggtttc atattcacca ccctgaattg
     1321 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggcgcg
     1381 ccgccattgc ggccgcgccg tcactgcgtc ttttactcac actggctcac cttcgggtgg
     1441 gcctttctgc gtttatatac tagagagaga atataaaaag ccagattatt aatccggctt
     1501 ttttattatt tgaattcgaa atccctgaaa ctgagactgt agaaaattta tgacaacttg
     1561 acggctacat cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg
     1621 gtgcattttt taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg
     1681 acggtggcga taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg
     1741 tcctcgcgcc agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc
     1801 gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg
     1861 tgatcgctga tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac
     1921 tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc
     1981 tccgaatagc gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa
     2041 tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag
     2101 ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg
     2161 cgagcctccg gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca
     2221 cccggtcggc aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg
     2281 agattgagaa tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg
     2341 ttggcctcaa tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc
     2401 aggggatcat tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc
     2461 aattgtccat attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta
     2521 accaaaccgg taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg
     2581 acaaaaacgc gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg
     2641 cacggcgtca cactttgcta tgccatagca tttttatcca taagattagc ggttcctacc
     2701 tgacgctttt tatcgcaact ctctactgtt tctccatacc ggaattgtga gcggataaca
     2761 atttaatttt gtttaacttt aagaaggaga tataccatga aaaagtcttt agccctcaaa
     2821 gcctctgtag ccgttgctac cctcgttccg atgctgtctt tcgctgctga gggtgacgat
     2881 cccgcaaaag cggcctttaa ctccctgcaa gcctcagcga ccgaatatat cggttatgcg
     2941 tgggcgatgg ttgttgtcat tgtcggcgca actatcggta tcaagctgtt taagaaattc
     3001 acctcgaaag caagctgact cggtaccaaa ttccagaaaa gaggcctccc gaaagggggg
     3061 ccttttttcg ttttggtcct aatagataaa ggataggtct ggtagtgttg ttcgttctcg
     3121 caggtaaatc aataatactc agcagttccg tagacttttc agtgggacag ggtagcgata
     3181 acagatagat tgtaataaga cacagtaggt gctcgtagtt gcgtgaagag aaccgctcag
     3241 gaaatccagt cagaagtatt ggtaatcgtt gaaaactcag tcgactgctg ccaccgctga
     3301 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgccagg
     3361 catcaaataa aacgaaaggc tcagtcggaa gactgggcct ttcgttttat ctgttgtttg
     3421 tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgtgaag
     3481 caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaactaag
     3541 cagaaggcca tcctgacgga tggccttttt gcgtttcaga tctaccggta aaccagcaat
     3601 agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtca tcgtggccgg
     3661 atcttgcggc ccctcggctt gaacgaattg ttagacatta tttgccgact accttggtga
     3721 tctcgccttt cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag gccaagcgat
     3781 cttcttcttg tccaagataa gcctgtctag cttcaagtat gacgggctga tactgggccg
     3841 gcaggcgctc cattgcccag tcggcagcga catccttcgg cgcgattttg ccggttactg
     3901 cgctgtacca aatgcgggac aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg
     3961 gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa
     4021 ccggatcaaa gagttcctcc gccgctggac ctaccaaggc aacgctatgt tctcttgctt
     4081 ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa
     4141 tgtcattgcg ctgccattct ccaaattgca gttcgcgctt agctggataa cgccacggaa
     4201 tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg gagaatctcg ctctctccag
     4261 gggaagccga agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc
     4321 ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg tcatccactg
     4381 cggagccgta caaatgtacg gccagcaacg tcggttcgag atggcgctcg atgacgccaa
     4441 ctacctctga tagttgagtc gatacttcgg cgatcaccgc ttccctcata ctcttccttt
     4501 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4561 gtatttagaa aaataaacaa atagctagct cactcggtcg ctacgctccg ggcgtgagac
     4621 tgcggcgggc gctgcggaca catacaaagt tacccacaga ttccgtggat aagcagggga
     4681 ctaacatgtg aggcaaaaca gcagggccgc gccggtggcg tttttccata ggctccgccc
     4741 tcctgccaga gttcacataa acagacgctt ttccggtgca tctgtgggag ccgtgaggct
     4801 caaccatgaa tctgacagta cgggcgaaac ccgacaggac ttaaagatcc ccaccgtttc
     4861 cggcgggtcg ctccctcttg cgctctcctg ttccgaccct gccgtttacc ggatacctgt
     4921 tccgcctttc tcccttacgg gaagtgtggc gctttctcat agctcacaca ctggtatctc
     4981 ggctcggtgt aggtcgtttg ctccaagctg ggctgtaagc aagaactccc cgttcagccc
     5041 gactgctgcg ccttatccgg taactgttca cttgagtcca acccggaaaa gcacggtaaa
     5101 acgccactgg cagcagccat tggtaactgg gagttcgcag aggatttgtt tagctaaaca
     5161 cgcggttgct cttgaagtgt gcgccaaagt ccggctacac tggaaggaca gatttggttg
     5221 ctgtgctctg cgaaagccag ttaccacggt taagcagttc cccaactgac ttaaccttcg
     5281 atcaaaccac ctccccaggt ggttttttcg tttacagggc aaaagattac gcgcagaaaa
     5341 aaaggatctc aagaagatcc tttgatc
//
