LOCUS       sequence-446517-        9101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to create a markerless deletion of the E. coli HS tum gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9101)
  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 9101)
  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∆tum
FEATURES             Location/Qualifiers
     source          1..9101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     54..82
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     RBS             119..130
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     766..785
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(852..954)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(1068..1601)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1790..1795,1796..1812,1813..1818))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     oriT            2082..2190
                     /note="incP origin of transfer"
     CDS             complement(2560..3510)
                     /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"
     rep_origin      3558..3780
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(4828..5619,5620..5688))
                     /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     5451..5470
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5689..5793)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5861..5879
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5917..5939)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6039..6058
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     terminator      complement(8174..8260)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(8363..8457)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(8500..9101,1..112))
                     /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(8540..8560)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
ORIGIN
        1 ttgcatcacc ttcaccctct ccactgacag aaaatttgtg cccattaaca tcaccatcta
       61 attcaacaag aattgggaca actccagtga aaagttcttc tcctttacgc atgcttaatt
      121 tctcctcttt aattctaggt acccgaacag tactatgtga ttataccagc cccctcacta
      181 catgtcaaga ataaactgcc aaagactagt tcatcgcagt actgttgtaa ttcattaagc
      241 attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc
      301 agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg
      361 tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg
      421 aaaaacatat tctcaataac ccctttaggg aaataggcca ggttttcacc gtaacacgcc
      481 acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc
      541 gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat
      601 atcaccagct caccgtcttt cattgccata cgtaattccg gatgagcatt catcaggcgg
      661 gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa
      721 aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat
      781 gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt
      841 tttttctcca ttttagcttc cttagctcct gaaaatctcg acaactcaaa aaatacgccc
      901 ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg atcaacgtct
      961 cattttcgcc aaaagttggc ccagggcttc ccggtatcaa cagggacacc aggatttatt
     1021 tattctgcga agtgatcttc cgtcacaggt atcgatattg tctagagtta ggtggcggta
     1081 cttgggtcga tatcaaagtg catcacttct tcccgtatgc ccaactttgt atagagagcc
     1141 actgcgggat cgtcaccgta atctgcttgc acgtagatca cataagcacc aagcgcgttg
     1201 gcctcatgct tgaggagatt gatgagcgcg gtggcaatgc cctgcctccg gtgctcgccg
     1261 gagactgcga gatcatagat atagatctca ctacgcggct gctcaaactt gggcagaacg
     1321 taagccgcga gagcgccaac aaccgcttct tggtcgaagg cagcaagcgc gatgaatgtc
     1381 ttactacgga gcaagttccc gaggtaatcg gagtccggct gatgttggga gtaggtggct
     1441 acgtctccga actcacgacc gaaaagatca agagcagccc gcatggattt gacttggtca
     1501 gggccgagcc tacatgtgcg aatgatgccc atacttgagc cacctaactt tgttttaggg
     1561 cgactgccct gctgcgtaac atcgttgctg ctgcgtaaca tcgttgctgc tccataacat
     1621 caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca tagactgtac
     1681 aaaaaaacag tcataacaag ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc
     1741 ggtcaaggtt ctggaccagt tgcgtgagcg catacgctac ttgcattaca gtttacgaac
     1801 cgaacaggct tatgtcaatt cgatccattg ctgttgacaa agggaatcag gggatcttgg
     1861 aattctcttc cgcttcctcg ctcactgacc cgctgcgctc ggtcgttcgg ctgcggcgag
     1921 cggtatcagg ggattcctta aggtatactt tccgctgcat aaccctgctt cggggtcatt
     1981 atagcgattt tttcggtata tccatccttt ttcgcacgat atacaggatt ttgccaaagg
     2041 gttcgtgtag actttccttg gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta
     2101 ggcccacccg cgagcgggtg ttccttcttc actgtccctt attcgcacct ggcggtgctc
     2161 aacgggaatc ctgctctgcg aggctggccg ataagctctg ataacgcagg aaagaacatg
     2221 ggcagttcaa cctgttgata gtacgtacta agctctcatg tttcacgtac taagctctca
     2281 agtttaacgt actaagctct catgtttaac gaactaaacc ctcatggcta acgtactaag
     2341 ctctcatggc taacgtacta agctctcatg tttcacgtac taagctctca tgtttgaaca
     2401 ataaaattaa tataaatcag caacttaaat agcctctaag gttttaagtt ttacatgggt
     2461 atggacagtt ttccctttga tatgtaacgg tgaacagttg ttctactttt gtttgttagt
     2521 cttgatgctt cactgataga tacaagagcc ataagaacct cagatccttc cgtatttagc
     2581 cagtatgttc tctagtgtgg ttcgttgttt ttgcgtgagc catgagaacg aaccattgag
     2641 atcatactta ctttgcatgt cactcaaaaa ttttgcctca aaactggtga gctgaatttt
     2701 tgcagttaaa gcatcgtgta gtgtttttct tagtccgtta tgtaggtagg aatctgatgt
     2761 aatggttgtt ggtattttgt caccattcat ttttatctgg ttgttctcaa gttcggttac
     2821 gagatccatt tgtctatcta gttcaacttg gaaaatcaac gtatcagtcg ggcggcctcg
     2881 cttatcaacc accaatttca tattgctgta agtgtttaaa tctttactta ttggtttcaa
     2941 aacccattgg ttaagccttt taaactcatg gtagttattt tcaagcatta acatgaactt
     3001 aaattcatca aggctaatct ctatatttgc cttgtgagtt ttcttttgtg ttagttcttt
     3061 taataaccac tcataaatcc tcatagagta tttgttttca aaagacttaa catgttccag
     3121 attatatttt atgaattttt ttaactggaa aagataaggc aatatctctt cactaaaaac
     3181 taattctaat ttttcgcttg agaacttggc atagtttgtc cactggaaaa tctcaaagcc
     3241 tttaaccaaa ggattcctga tttccacagt tctcgtcatc agctctctgg ttgctttagc
     3301 taatacacca taagcatttt ccctactgat gttcatcatc tgaacgtatt ggttataagt
     3361 gaacgatacc gtccgttctt tccttgtagg gttttcaatc gtggggttga gtagtgccac
     3421 acagcataaa attagcttgg tttcatgctc cgttaagtca tagcgactaa tcgctagttc
     3481 atttgctttg aaaacaacta attcagacat acatctcaat tggtctaggt gattttaatc
     3541 actataccaa ttgagatggg ctagtcaatg ataattacta gtccttttcc tttgagttgt
     3601 gggtatctgt aaattctgct agacctttgc tggaaaactt gtaaattctg ctagaccctc
     3661 tgtaaattcc gctagacctt tgtgtgtttt ttttgtttat attcaagtgg ttataattta
     3721 tagaataaag aaagaataaa aaaagataaa aagaatagat cccagccctg tgtataactc
     3781 actactttag tcagttccgc agtattacaa aaggatgtcg caaacgctgt ttgctcctct
     3841 acaaaacaga ccttaaaacc ctaaaggctt aagtagcacc ctcgcaagct cggttgcggc
     3901 cgcaatcggg caaatcgctg aatattcctt ttgtctccga ccatcaggca cctgagtcgc
     3961 tgtctttttc gtgacattca gttcgctgcg ctcacggctc tggcagtgaa tgggggtaaa
     4021 tggcactaca ggcgcctttt atggattcat gcaaggaaac tacccataat acaagaaaag
     4081 cccgtcacgg gcttctcagg gcgttttatg gcgggtctgc tatgtggtgc tatctgactt
     4141 tttgctgttc agcagttcct gccctctgat tttccagtct gaccacttcg gattatcccg
     4201 tgacaggtca ttcagactgg ctaatgcacc cagtaaggca gcggtatcat caacggggtc
     4261 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagct tttaaggttt
     4321 aacggttgtg gacaacaagc cagggatgta acgcactgag aagcccttag agcctctcaa
     4381 agcaattttg agtgacacag gaacacttaa cggctgacaa ccggctggta acaggattag
     4441 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4501 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4561 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4621 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4681 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4741 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4801 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4861 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4921 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4981 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5041 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5101 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5161 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5221 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5281 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5341 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5401 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5461 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5521 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5581 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5641 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5701 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5761 tatttagaaa aataaacaaa taggggttcc gcgtacattt ccccgaaaag tgccacctga
     5821 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     5881 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     5941 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     6001 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     6061 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     6121 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     6181 ttcgctatta cgccagctag aggaccagcc gcgtaacctg gcaaaatcgg ttacggttga
     6241 gtaataaatg gatgccctgc gtaagcgggc tgcagtcgtc ctaggggccg gcctccactc
     6301 gagtctggtc gacccctgaa cttcattaca ccgcctgcat ttccattgta ttcgatagtc
     6361 ttaaccaatg agttactaac tcgacacagc ccatcaggaa cacgtttgat agaaatttct
     6421 gatgcagtat aagaagtacc atttggcggt gatatctcat tttttgcagc atcgtaacta
     6481 acataatcaa catagttacc gatttgccca tagagatttt ttaacgcaac agcttgaggg
     6541 ttatgataat tgcggtaaat tccattcccc tcactgcaat atgtaccagc agcgatagaa
     6601 gacaatgcac cattagccgc accaagttct agtacgtccg ttttaaatcc agtagcagat
     6661 gtgataatgg gatcgcccat gtaggcggta gcactttgcc caatagcagg cttcaccact
     6721 tcaatagcag tgatatttcg gttagaagca tgtggcacgc aaccagttag gattacagca
     6781 agagatattg gtaacgctac attattaatt ttcattttta gcctattatt cttttcttga
     6841 caaaaaacaa ggcgatatcc gattgacatc gcctctcact catatgtaac cctttttgat
     6901 tagtaaaaac aagcgtctat tgacaaaatc aatgcagcca gctgtcgtct tcccacacct
     6961 tctgcataat tttcatcact tgttttcttt cttcgtccag ttgcagtccg gtcagttcca
     7021 caccgttaga gctaccttta cggatacgga ttaccgtttt gggatacagg gggcgcagat
     7081 tgcggtaaag ctcggattca agggcgtcca gggtagactg gctaatcttc tgctctttat
     7141 cgatcattat ttcaatgcgc ataaaagtca cctcacatct ttcctcctat gacaccacaa
     7201 gaaatactgt atgtataacc actgtttata tttacagtat ataataatct tactgatgta
     7261 aaacgttttt tttacgctta tcagcttgat atgcctggta ttattaagag cacgaattgt
     7321 taaccagcgt aattaataca ggttccgcca cttatcatct tcctgcaaac gctggttccg
     7381 atagaagata cgcaggcctg ctcctgacgg aatactgccg ccgcgaagga gtaaatcgac
     7441 ctctttctcg ctgccatcaa atcctctgga tttcagttca tagacgagct gctgtcgttg
     7501 atggtctgta attcgctgtt tgtagtctct acgccgtttc ggtttcacca gacgtaacct
     7561 tgcagccagt tcccggcgct cctttttgtt catactgtgc aggtaatcgt gcaactcctt
     7621 gtcatccatg cgggtaaagt ccgttctggg gtccccatca gctgatttat ctttctcctg
     7681 ttggttcaaa ttttcagcaa ggggacagtt attgccacga gtccaagggg cgcaagcgcc
     7741 ctggtcggct gccgcctcct gaacgtcaac ggccttacga accattttcc acttcactgc
     7801 atgagtacaa atcttgccct ctgcaatggg tgaccagatg ccataaatac gaataccgtg
     7861 atcgccatag gcggtcggct cttcgttaat ttcataagct gttctgataa ggtgatattt
     7921 gcggggaacc agtacgccgc cctgcttcat aatgtaggtg gcaaaacaac cagcatcagc
     7981 agcagccaga atggcatcaa ggcgcgggtt atccagtacc ggcgcacctg cttttttgtc
     8041 accctgttgc cttgccgcct gaccagccag caatcgcaat tcacggtacg cctgacgccc
     8101 cggaatacca aagaagcgaa attgctgaac acgatgcaga gacggggttt aaactcgcga
     8161 ctcctagcgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac
     8221 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctcaag ctagagagtc
     8281 attaccccag gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc
     8341 cactcatcgc agtctagctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg
     8401 ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa
     8461 gctcagctaa ttaagctagc gtttaaacga gctcgcttat ttgtatagtt catccatgcc
     8521 atgtgtaatc ccagcagctg ttacaaactc aagaaggacc atgtggtctc tcttttcgtt
     8581 gggatctttc gaaagggcag attgtgtgga caggtaatgg ttgtctggta aaaggacagg
     8641 gccatcgcca attggagtat tttgttgata atggtctgct agttgaacgc ttccatcttc
     8701 aatgttgtgt ctaattttga agttaacttt gattccattc ttttgtttgt ctgccatgat
     8761 gtatacattg tgtgagttat agttgtattc caatttgtgt ccaagaatgt ttccatcttc
     8821 tttaaaatca atacctttta actcgattct attaacaagg gtatcacctt caaacttgac
     8881 ttcagcacgt gtcttgtagt tcccgtcatc tttgaaaaat atagttcttt cctgtacata
     8941 accttcgggc atggcactct tgaaaaagtc atgctgtttc atatgatctg ggtatctcgc
     9001 aaagcattga acaccataac cgaaagtagt gacaagtgtt ggccatggaa caggtagttt
     9061 tccagtagtg caaataaatt taagggtaag ttttccgtat g
//
