LOCUS       sequence-446758-        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"
     primer_bind     472..492
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      575..669
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      772..858
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             join(1477..1728,1729..2085)
                     /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     2020..2041
                     /label=LexA
                     /note="LexA DNA binding domain, forward primer"
     primer_bind     complement(2861..2880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2980..3002
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3040..3058)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3126..3230
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3231..3299,3300..4091)
                     /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(3449..3468)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5139..5361
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5409..6359
                     /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"
     oriT            6729..6837
                     /note="incP origin of transfer"
     promoter        join(7101..7106,7107..7123,7124..7129)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             7318..7851
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        7965..8067
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(8134..8153)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     RBS             8789..8800
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(8807..8988,1..532)
                     /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(8837..8865)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
ORIGIN
        1 cactactttc ggttatggtg ttcaatgctt tgcgagatac ccagatcata tgaaacagca
       61 tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa
      121 agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa
      181 tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt
      241 ggaatacaac tataactcac acaatgtata catcatggca gacaaacaaa agaatggaat
      301 caaagttaac ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca
      361 ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct
      421 gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca tggtccttct
      481 tgagtttgta acagctgctg ggattacaca tggcatggat gaactataca aataagcgag
      541 ctcgtttaaa cgctagctta attagctgag cttggactcc tgttgataga tccagtaatg
      601 acctcagaac tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt
      661 ggtgagaatc caagctagac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca
      721 gttattggtg cccttaaacg cctggggtaa tgactctcta gcttgaggca tcaaataaaa
      781 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
      841 ctcctgagta ggacaaatcc gccgctagga gtcgcgagtt taaaccccgg gaaattctgt
      901 ggtatccgct catgtttcgc gcggcgttac gcaaacccga atcatcggat ttaacggtac
      961 actgatattg acgctcataa tgtaaaaagg ttctttcaat ggccaataat accactggat
     1021 tcacccgaat tatcaaagct gctggctatt cctggaaagg tttacgcgct gcatggatca
     1081 acgaagcggc attccgtcag gaaggcgtag cggtattgtt ggcggtggtc atcgcctgct
     1141 ggctggatgt ggacgcaatt acccgcgtgc tgcttatcag ctccgtgatg ctggtgatga
     1201 ttgtggaaat tctcaatagc gccatcgaag cagtggttga ccgaattggc tctgaatacc
     1261 atgagctttc cggacgcgct aaagatatgg gatccgccgc ggtgctgatt gccattatcg
     1321 tcgccgtgat tacctggtgc attctgttat ggtcgcattt tggataaccc ttccagattt
     1381 cgataaatct ctggtttatt gtgcagttta tggttccaaa atcgcctttt gctgtatata
     1441 ctcacagcat aactgtatat acacccaggg ggcggaatga aagcgttaac ggccaggcaa
     1501 caagaggtgt ttgatctcat ccgtgatcac atcagccaga caggtatgcc gccgacgcgt
     1561 gcggaaatcg cgcagcgttt ggggttccgt tccccaaacg cggctgaaga acatctgaag
     1621 gcgctggcac gcaaaggcgt tattgaaatt gtttccggcg catcacgcgg gattcgtctg
     1681 ttgcaggaag aggaagaagg gttgccgctg gtaggtcgtg tggctgccga cgaaccgctt
     1741 ctggcgcaac agcatattga aggtcattat caggtcgatc cttccttgtt caagccgaat
     1801 gctgatttcc tgctgcgcgt cagcgggatg tcgatgaaag atatcggcat tatggatggt
     1861 gacttgctgg cagtgcataa aactcaggat gtacgtaacg gtcaggtcgt tgtcgcacgt
     1921 attgatgacg aagttaccgt taagcgcctg aaaaaacagg gcaataaagt cgaactgttg
     1981 ccagaaaata gcgagtttaa accaattgtc gttgaccttc gtcagcagag cttcaccatt
     2041 gaagggctgg cggttggggt tattcgcaac ggcgactggc tgtaacatat ctctgagacc
     2101 gcgatgccgc caggcgtcgc ggtttgtttt tcatctctct tcatcaggct tgtctgcatg
     2161 gcattcctca cttcatctga taaagcactc tggcatctcg ccttacccat gattttctcc
     2221 aatatcaccg ttccgttgct gggactggtc gatacggcgg taattggtca tcttgatagc
     2281 ccggtttatt tgggcggcgt ggcggttggt gcaacggcga ccagctttct ctttatgctg
     2341 ttgctgtttt tacgcatgag caccaccggg ctgactgcgc aggcttatgg tgccaaaaat
     2401 cctcaggcat tagcccgtgc gctggtgcaa ccgttgctgt tggcgttggg ggctggggcg
     2461 ttaattgcgc tgctgcgtac accgattatc gatctggcgc tgcatattgt tggcggtagt
     2521 gaagcggtcc tggaacaggc gcggcgcttt cttgaaatcc gctggttaag cgctccggcg
     2581 tcgctggcga atctggtatt acggggtcga ccagactcga gtggaggccg gcccctagga
     2641 cgactgcagc ccgcttacgc agggcatcca tttattactc aaccgtaacc gattttgcca
     2701 ggttacgcgg ctggtcctct agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     2761 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     2821 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
     2881 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     2941 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3001 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     3061 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     3121 atgtacgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3181 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3241 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     3301 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     3361 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     3421 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     3481 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     3541 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     3601 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     3661 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     3721 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     3781 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     3841 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     3901 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     3961 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4021 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4081 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4141 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4201 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4261 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4321 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4381 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     4441 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagccggtt
     4501 gtcagccgtt aagtgttcct gtgtcactca aaattgcttt gagaggctct aagggcttct
     4561 cagtgcgtta catccctggc ttgttgtcca caaccgttaa accttaaaag ctcatgacca
     4621 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgttgat gataccgctg
     4681 ccttactggg tgcattagcc agtctgaatg acctgtcacg ggataatccg aagtggtcag
     4741 actggaaaat cagagggcag gaactgctga acagcaaaaa gtcagatagc accacatagc
     4801 agacccgcca taaaacgccc tgagaagccc gtgacgggct tttcttgtat tatgggtagt
     4861 ttccttgcat gaatccataa aaggcgcctg tagtgccatt tacccccatt cactgccaga
     4921 gccgtgagcg cagcgaactg aatgtcacga aaaagacagc gactcaggtg cctgatggtc
     4981 ggagacaaaa ggaatattca gcgatttgcc cgattgcggc cgcaaccgag cttgcgaggg
     5041 tgctacttaa gcctttaggg ttttaaggtc tgttttgtag aggagcaaac agcgtttgcg
     5101 acatcctttt gtaatactgc ggaactgact aaagtagtga gttatacaca gggctgggat
     5161 ctattctttt tatctttttt tattctttct ttattctata aattataacc acttgaatat
     5221 aaacaaaaaa aacacacaaa ggtctagcgg aatttacaga gggtctagca gaatttacaa
     5281 gttttccagc aaaggtctag cagaatttac agatacccac aactcaaagg aaaaggacta
     5341 gtaattatca ttgactagcc catctcaatt ggtatagtga ttaaaatcac ctagaccaat
     5401 tgagatgtat gtctgaatta gttgttttca aagcaaatga actagcgatt agtcgctatg
     5461 acttaacgga gcatgaaacc aagctaattt tatgctgtgt ggcactactc aaccccacga
     5521 ttgaaaaccc tacaaggaaa gaacggacgg tatcgttcac ttataaccaa tacgttcaga
     5581 tgatgaacat cagtagggaa aatgcttatg gtgtattagc taaagcaacc agagagctga
     5641 tgacgagaac tgtggaaatc aggaatcctt tggttaaagg ctttgagatt ttccagtgga
     5701 caaactatgc caagttctca agcgaaaaat tagaattagt ttttagtgaa gagatattgc
     5761 cttatctttt ccagttaaaa aaattcataa aatataatct ggaacatgtt aagtcttttg
     5821 aaaacaaata ctctatgagg atttatgagt ggttattaaa agaactaaca caaaagaaaa
     5881 ctcacaaggc aaatatagag attagccttg atgaatttaa gttcatgtta atgcttgaaa
     5941 ataactacca tgagtttaaa aggcttaacc aatgggtttt gaaaccaata agtaaagatt
     6001 taaacactta cagcaatatg aaattggtgg ttgataagcg aggccgcccg actgatacgt
     6061 tgattttcca agttgaacta gatagacaaa tggatctcgt aaccgaactt gagaacaacc
     6121 agataaaaat gaatggtgac aaaataccaa caaccattac atcagattcc tacctacata
     6181 acggactaag aaaaacacta cacgatgctt taactgcaaa aattcagctc accagttttg
     6241 aggcaaaatt tttgagtgac atgcaaagta agtatgatct caatggttcg ttctcatggc
     6301 tcacgcaaaa acaacgaacc acactagaga acatactggc taaatacgga aggatctgag
     6361 gttcttatgg ctcttgtatc tatcagtgaa gcatcaagac taacaaacaa aagtagaaca
     6421 actgttcacc gttacatatc aaagggaaaa ctgtccatac ccatgtaaaa cttaaaacct
     6481 tagaggctat ttaagttgct gatttatatt aattttattg ttcaaacatg agagcttagt
     6541 acgtgaaaca tgagagctta gtacgttagc catgagagct tagtacgtta gccatgaggg
     6601 tttagttcgt taaacatgag agcttagtac gttaaacttg agagcttagt acgtgaaaca
     6661 tgagagctta gtacgtacta tcaacaggtt gaactgccca tgttctttcc tgcgttatca
     6721 gagcttatcg gccagcctcg cagagcagga ttcccgttga gcaccgccag gtgcgaataa
     6781 gggacagtga agaaggaaca cccgctcgcg ggtgggccta cttcacctat cctgcccggc
     6841 tgacgccgtt ggatacacca aggaaagtct acacgaaccc tttggcaaaa tcctgtatat
     6901 cgtgcgaaaa aggatggata taccgaaaaa atcgctataa tgaccccgaa gcagggttat
     6961 gcagcggaaa gtatacctta aggaatcccc tgataccgct cgccgcagcc gaacgaccga
     7021 gcgcagcggg tcagtgagcg aggaagcgga agagaattcc aagatcccct gattcccttt
     7081 gtcaacagca atggatcgaa ttgacataag cctgttcggt tcgtaaactg taatgcaagt
     7141 agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga acgcagcggt ggtaacggcg
     7201 cagtggcggt tttcatggct tgttatgact gtttttttgt acagtctatg cctcgggcat
     7261 ccaagcagca agcgcgttac gccgtgggtc gatgtttgat gttatggagc agcaacgatg
     7321 ttacgcagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaggtggc
     7381 tcaagtatgg gcatcattcg cacatgtagg ctcggccctg accaagtcaa atccatgcgg
     7441 gctgctcttg atcttttcgg tcgtgagttc ggagacgtag ccacctactc ccaacatcag
     7501 ccggactccg attacctcgg gaacttgctc cgtagtaaga cattcatcgc gcttgctgcc
     7561 ttcgaccaag aagcggttgt tggcgctctc gcggcttacg ttctgcccaa gtttgagcag
     7621 ccgcgtagtg agatctatat ctatgatctc gcagtctccg gcgagcaccg gaggcagggc
     7681 attgccaccg cgctcatcaa tctcctcaag catgaggcca acgcgcttgg tgcttatgtg
     7741 atctacgtgc aagcagatta cggtgacgat cccgcagtgg ctctctatac aaagttgggc
     7801 atacgggaag aagtgatgca ctttgatatc gacccaagta ccgccaccta actctagaca
     7861 atatcgatac ctgtgacgga agatcacttc gcagaataaa taaatcctgg tgtccctgtt
     7921 gataccggga agccctgggc caacttttgg cgaaaatgag acgttgatcg gcacgtaaga
     7981 ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttgtcg
     8041 agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt
     8101 gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt
     8161 acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat
     8221 aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg
     8281 gaattacgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt
     8341 tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac
     8401 gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg
     8461 gcctatttcc ctaaaggggt tattgagaat atgtttttcg tctcagccaa tccctgggtg
     8521 agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc
     8581 accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt
     8641 catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac
     8701 tgcgatgaac tagtctttgg cagtttattc ttgacatgta gtgagggggc tggtataatc
     8761 acatagtact gttcgggtac ctagaattaa agaggagaaa ttaagcatgc gtaaaggaga
     8821 agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg ttaatgggca
     8881 caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac ttacccttaa
     8941 atttatttgc actactggaa aactacctgt tccatggcca acacttgt
//
