LOCUS       sequence-441197-        8988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to create a non-inducible lexA by introducing a G85D mutation 
            in E. coli HS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8988)
  AUTHORS   Ortiz de Ora L, Wiles ET, Zund M, Banuelos MS, Haro-Ramirez N, Suder
            DS, Ujagar N, Ayala-Angulo J, Trinh C, Knitter C, Gonen S, Nicholas 
            DA, Wiles TJ
  TITLE     Phollow reveals in situ phage transmission dynamics in the zebrafish
            gut microbiome at single-virion resolution.
  JOURNAL   Nat Microbiol. 2025 Apr 18. doi: 10.1038/s41564-025-01981-1.
  PUBMED    40251446
REFERENCE   2  (bases 1 to 8988)
  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 
            Microbiol. 2025 Apr 18. doi: 10.1038/s41564-025-01981-1."
COMMENT     Sequence Label: pAX1_HS_lexA(Ind-)
FEATURES             Location/Qualifiers
     source          1..8988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            370..478
                     /note="incP origin of transfer"
     promoter        join(742..747,748..764,765..770)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             959..1492
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        1606..1708
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1775..1794)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     RBS             2430..2441
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2448..3161
                     /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(2478..2506)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3101..3121
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3204..3298
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      3401..3487
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             join(4106..4357,4358..4714)
                     /codon_start=1
                     /gene="lexA"
                     /product="E. coli DNA-binding protein that represses genes 
                     involved in the SOS response to DNA damage"
                     /label=LexA
                     /note="The N-terminal DNA-binding domain recognizes the 
                     16-bp palindromic sequence 5'-CTGTATATATATACAG-3'."
                     /translation="MKALTARQQEVFDLIRDHISQTGMPPTRAEIAQRLGFRSPNAAEE
                     HLKALARKGVIEIVSGASRGIRLLQEEEEGLPLVGRVAADEPLLAQQHIEGHYQVDPSL
                     FKPNADFLLRVSGMSMKDIGIMDGDLLAVHKTQDVRNGQVVVARIDDEVTVKRLKKQGN
                     KVELLPENSEFKPIVVDLRQQSFTIEGLAVGVIRNGDWL"
     primer_bind     4649..4670
                     /label=LexA
                     /note="LexA DNA binding domain, forward primer"
     primer_bind     complement(5490..5509)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5609..5631
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5669..5687)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5755..5859
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5860..5928,5929..6720)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6078..6097)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7768..7990
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             8038..8988
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 ggttcttatg gctcttgtat ctatcagtga agcatcaaga ctaacaaaca aaagtagaac
       61 aactgttcac cgttacatat caaagggaaa actgtccata cccatgtaaa acttaaaacc
      121 ttagaggcta tttaagttgc tgatttatat taattttatt gttcaaacat gagagcttag
      181 tacgtgaaac atgagagctt agtacgttag ccatgagagc ttagtacgtt agccatgagg
      241 gtttagttcg ttaaacatga gagcttagta cgttaaactt gagagcttag tacgtgaaac
      301 atgagagctt agtacgtact atcaacaggt tgaactgccc atgttctttc ctgcgttatc
      361 agagcttatc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
      421 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
      481 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
      541 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
      601 tgcagcggaa agtatacctt aaggaatccc ctgataccgc tcgccgcagc cgaacgaccg
      661 agcgcagcgg gtcagtgagc gaggaagcgg aagagaattc caagatcccc tgattccctt
      721 tgtcaacagc aatggatcga attgacataa gcctgttcgg ttcgtaaact gtaatgcaag
      781 tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg tggtaacggc
      841 gcagtggcgg ttttcatggc ttgttatgac tgtttttttg tacagtctat gcctcgggca
      901 tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat
      961 gttacgcagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaggtgg
     1021 ctcaagtatg ggcatcattc gcacatgtag gctcggccct gaccaagtca aatccatgcg
     1081 ggctgctctt gatcttttcg gtcgtgagtt cggagacgta gccacctact cccaacatca
     1141 gccggactcc gattacctcg ggaacttgct ccgtagtaag acattcatcg cgcttgctgc
     1201 cttcgaccaa gaagcggttg ttggcgctct cgcggcttac gttctgccca agtttgagca
     1261 gccgcgtagt gagatctata tctatgatct cgcagtctcc ggcgagcacc ggaggcaggg
     1321 cattgccacc gcgctcatca atctcctcaa gcatgaggcc aacgcgcttg gtgcttatgt
     1381 gatctacgtg caagcagatt acggtgacga tcccgcagtg gctctctata caaagttggg
     1441 catacgggaa gaagtgatgc actttgatat cgacccaagt accgccacct aactctagac
     1501 aatatcgata cctgtgacgg aagatcactt cgcagaataa ataaatcctg gtgtccctgt
     1561 tgataccggg aagccctggg ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag
     1621 aggttccaac tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttgtc
     1681 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt
     1741 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg
     1801 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa
     1861 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc
     1921 ggaattacgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg
     1981 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga
     2041 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct
     2101 ggcctatttc cctaaagggg ttattgagaa tatgtttttc gtctcagcca atccctgggt
     2161 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt
     2221 caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
     2281 tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta
     2341 ctgcgatgaa ctagtctttg gcagtttatt cttgacatgt agtgaggggg ctggtataat
     2401 cacatagtac tgttcgggta cctagaatta aagaggagaa attaagcatg cgtaaaggag
     2461 aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat gttaatgggc
     2521 acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa cttaccctta
     2581 aatttatttg cactactgga aaactacctg ttccatggcc aacacttgtc actactttcg
     2641 gttatggtgt tcaatgcttt gcgagatacc cagatcatat gaaacagcat gactttttca
     2701 agagtgccat gcccgaaggt tatgtacagg aaagaactat atttttcaaa gatgacggga
     2761 actacaagac acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat agaatcgagt
     2821 taaaaggtat tgattttaaa gaagatggaa acattcttgg acacaaattg gaatacaact
     2881 ataactcaca caatgtatac atcatggcag acaaacaaaa gaatggaatc aaagttaact
     2941 tcaaaattag acacaacatt gaagatggaa gcgttcaact agcagaccat tatcaacaaa
     3001 atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg tccacacaat
     3061 ctgccctttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt gagtttgtaa
     3121 cagctgctgg gattacacat ggcatggatg aactatacaa ataagcgagc tcgtttaaac
     3181 gctagcttaa ttagctgagc ttggactcct gttgatagat ccagtaatga cctcagaact
     3241 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     3301 aagctagact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc
     3361 ccttaaacgc ctggggtaat gactctctag cttgaggcat caaataaaac gaaaggctca
     3421 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
     3481 gacaaatccg ccgctaggag tcgcgagttt aaaccccggg aaattctgtg gtatccgctc
     3541 atgtttcgcg cggcgttacg caaacccgaa tcatcggatt taacggtaca ctgatattga
     3601 cgctcataat gtaaaaaggt tctttcaatg gccaataata ccactggatt cacccgaatt
     3661 atcaaagctg ctggctattc ctggaaaggt ttacgcgctg catggatcaa cgaagcggca
     3721 ttccgtcagg aaggcgtagc ggtattgttg gcggtggtca tcgcctgctg gctggatgtg
     3781 gacgcaatta cccgcgtgct gcttatcagc tccgtgatgc tggtgatgat tgtggaaatt
     3841 ctcaatagcg ccatcgaagc agtggttgac cgaattggct ctgaatacca tgagctttcc
     3901 ggacgcgcta aagatatggg atccgccgcg gtgctgattg ccattatcgt cgccgtgatt
     3961 acctggtgca ttctgttatg gtcgcatttt ggataaccct tccagatttc gataaatctc
     4021 tggtttattg tgcagtttat ggttccaaaa tcgccttttg ctgtatatac tcacagcata
     4081 actgtatata cacccagggg gcggaatgaa agcgttaacg gccaggcaac aagaggtgtt
     4141 tgatctcatc cgtgatcaca tcagccagac aggtatgccg ccgacgcgtg cggaaatcgc
     4201 gcagcgtttg gggttccgtt ccccaaacgc ggctgaagaa catctgaagg cgctggcacg
     4261 caaaggcgtt attgaaattg tttccggcgc atcacgcggg attcgtctgt tgcaggaaga
     4321 ggaagaaggg ttgccgctgg taggtcgtgt ggctgccgac gaaccgcttc tggcgcaaca
     4381 gcatattgaa ggtcattatc aggtcgatcc ttccttgttc aagccgaatg ctgatttcct
     4441 gctgcgcgtc agcgggatgt cgatgaaaga tatcggcatt atggatggtg acttgctggc
     4501 agtgcataaa actcaggatg tacgtaacgg tcaggtcgtt gtcgcacgta ttgatgacga
     4561 agttaccgtt aagcgcctga aaaaacaggg caataaagtc gaactgttgc cagaaaatag
     4621 cgagtttaaa ccaattgtcg ttgaccttcg tcagcagagc ttcaccattg aagggctggc
     4681 ggttggggtt attcgcaacg gcgactggct gtaacatatc tctgagaccg cgatgccgcc
     4741 aggcgtcgcg gtttgttttt catctctctt catcaggctt gtctgcatgg cattcctcac
     4801 ttcatctgat aaagcactct ggcatctcgc cttacccatg attttctcca atatcaccgt
     4861 tccgttgctg ggactggtcg atacggcggt aattggtcat cttgatagcc cggtttattt
     4921 gggcggcgtg gcggttggtg caacggcgac cagctttctc tttatgctgt tgctgttttt
     4981 acgcatgagc accaccgggc tgactgcgca ggcttatggt gccaaaaatc ctcaggcatt
     5041 agcccgtgcg ctggtgcaac cgttgctgtt ggcgttgggg gctggggcgt taattgcgct
     5101 gctgcgtaca ccgattatcg atctggcgct gcatattgtt ggcggtagtg aagcggtcct
     5161 ggaacaggcg cggcgctttc ttgaaatccg ctggttaagc gctccggcgt cgctggcgaa
     5221 tctggtatta cggggtcgac cagactcgag tggaggccgg cccctaggac gactgcagcc
     5281 cgcttacgca gggcatccat ttattactca accgtaaccg attttgccag gttacgcggc
     5341 tggtcctcta gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     5401 cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     5461 atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     5521 cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     5581 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     5641 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     5701 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtacgcgga
     5761 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     5821 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     5881 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     5941 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     6001 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     6061 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     6121 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     6181 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     6241 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     6301 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     6361 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     6421 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     6481 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     6541 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     6601 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     6661 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     6721 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     6781 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6841 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6901 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6961 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     7021 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     7081 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagccggttg tcagccgtta
     7141 agtgttcctg tgtcactcaa aattgctttg agaggctcta agggcttctc agtgcgttac
     7201 atccctggct tgttgtccac aaccgttaaa ccttaaaagc tcatgaccaa aatcccttaa
     7261 cgtgagtttt cgttccactg agcgtcagac cccgttgatg ataccgctgc cttactgggt
     7321 gcattagcca gtctgaatga cctgtcacgg gataatccga agtggtcaga ctggaaaatc
     7381 agagggcagg aactgctgaa cagcaaaaag tcagatagca ccacatagca gacccgccat
     7441 aaaacgccct gagaagcccg tgacgggctt ttcttgtatt atgggtagtt tccttgcatg
     7501 aatccataaa aggcgcctgt agtgccattt acccccattc actgccagag ccgtgagcgc
     7561 agcgaactga atgtcacgaa aaagacagcg actcaggtgc ctgatggtcg gagacaaaag
     7621 gaatattcag cgatttgccc gattgcggcc gcaaccgagc ttgcgagggt gctacttaag
     7681 cctttagggt tttaaggtct gttttgtaga ggagcaaaca gcgtttgcga catccttttg
     7741 taatactgcg gaactgacta aagtagtgag ttatacacag ggctgggatc tattcttttt
     7801 atcttttttt attctttctt tattctataa attataacca cttgaatata aacaaaaaaa
     7861 acacacaaag gtctagcgga atttacagag ggtctagcag aatttacaag ttttccagca
     7921 aaggtctagc agaatttaca gatacccaca actcaaagga aaaggactag taattatcat
     7981 tgactagccc atctcaattg gtatagtgat taaaatcacc tagaccaatt gagatgtatg
     8041 tctgaattag ttgttttcaa agcaaatgaa ctagcgatta gtcgctatga cttaacggag
     8101 catgaaacca agctaatttt atgctgtgtg gcactactca accccacgat tgaaaaccct
     8161 acaaggaaag aacggacggt atcgttcact tataaccaat acgttcagat gatgaacatc
     8221 agtagggaaa atgcttatgg tgtattagct aaagcaacca gagagctgat gacgagaact
     8281 gtggaaatca ggaatccttt ggttaaaggc tttgagattt tccagtggac aaactatgcc
     8341 aagttctcaa gcgaaaaatt agaattagtt tttagtgaag agatattgcc ttatcttttc
     8401 cagttaaaaa aattcataaa atataatctg gaacatgtta agtcttttga aaacaaatac
     8461 tctatgagga tttatgagtg gttattaaaa gaactaacac aaaagaaaac tcacaaggca
     8521 aatatagaga ttagccttga tgaatttaag ttcatgttaa tgcttgaaaa taactaccat
     8581 gagtttaaaa ggcttaacca atgggttttg aaaccaataa gtaaagattt aaacacttac
     8641 agcaatatga aattggtggt tgataagcga ggccgcccga ctgatacgtt gattttccaa
     8701 gttgaactag atagacaaat ggatctcgta accgaacttg agaacaacca gataaaaatg
     8761 aatggtgaca aaataccaac aaccattaca tcagattcct acctacataa cggactaaga
     8821 aaaacactac acgatgcttt aactgcaaaa attcagctca ccagttttga ggcaaaattt
     8881 ttgagtgaca tgcaaagtaa gtatgatctc aatggttcgt tctcatggct cacgcaaaaa
     8941 caacgaacca cactagagaa catactggct aaatacggaa ggatctga
//
