LOCUS       sequence-446752-        9216 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used to create a markerless deletion of the E. coli HS P2 phage.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9216)
  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 9216)
  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∆P2
FEATURES             Location/Qualifiers
     source          1..9216
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     358..378
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      461..555
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      658..744
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2975..2994)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3094..3116
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3154..3172)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3240..3344
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3345..3413,3414..4205)
                     /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(3563..3582)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5253..5475
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5523..6473
                     /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            6843..6951
                     /note="incP origin of transfer"
     promoter        join(7215..7220,7221..7237,7238..7243)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             7432..7965
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        8079..8181
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(8248..8267)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     RBS             8903..8914
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(8921..9216,1..418)
                     /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(8951..8979)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
ORIGIN
        1 tttcaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct
       61 tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca
      121 caaattggaa tacaactata actcacacaa tgtatacatc atggcagaca aacaaaagaa
      181 tggaatcaaa gttaacttca aaattagaca caacattgaa gatggaagcg ttcaactagc
      241 agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca
      301 ttacctgtcc acacaatctg ccctttcgaa agatcccaac gaaaagagag accacatggt
      361 ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac tatacaaata
      421 agcgagctcg tttaaacgct agcttaatta gctgagcttg gactcctgtt gatagatcca
      481 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
      541 tttattggtg agaatccaag ctagactgcg atgagtggca gggcggggcg taattttttt
      601 aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt gaggcatcaa
      661 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
      721 aacgctctcc tgagtaggac aaatccgccg ctaggagtcg cgagtttaaa ccccgatgtc
      781 agccagagtg ccagacttaa tatcatcatc ccgatcaatg cgcaggacat cgtgaccagg
      841 atgacattac gtgtcccaaa gcgtttcact aaccacgccg agcagagaat accgctcatc
      901 gaaccgatcg acagaccaaa gagaacaccg cccatttcag cgatcgagac agagagaata
      961 tcgcggatag caggcgtacg ggttgcccag gacgccatta acaggcctgg caaaaagaag
     1021 aacataaaca gcgcccaggt tcggcgtttc aatgcattac gtgaagaatt tacggtcata
     1081 gatcacgtca aaataagaag agggaagaca acattagcaa ggttgtgtac atttgtacac
     1141 aattgcagac agaggaaatg acatgcgtcg cgctaacgat ccgcaacggc gagaaaaaat
     1201 tatccaggcc acgctggagg cggtgaaact ttacggaata catgctgtta cgcaccgcaa
     1261 aattgctacc cttgccgggg taccgttggg atcgatgacc tactattttt caggaattga
     1321 tgagttgttg ctggaggcgt tcagcagttt tactgagatt atgtcccggc aatatcaggc
     1381 attttttagc gatgttagtg atgctcaagg cgcatgccag gctatcaccg atatgatcta
     1441 cagctcacag gttgcaaagc cggataatat ggagctgatg taccagctct acgcgctggc
     1501 tagccgaaaa ccgctattaa aaacggtaat gcaaaactgg atgcagcgca gtcagcaaac
     1561 gctcgaacaa tggtttgaac ccggaaccgc ccgcgcgctt gatgcgttta ttgaggggat
     1621 gacgctgcat tttgtcaccg accgtaagcc gctatcgcgc gaggagattt tgaggatggt
     1681 tgagagggtt gcagggtagt agataaattt tagataacaa aaaacccatc aaccttgaac
     1741 cgaaatggcg gggttgatgg gctccacaaa atggggacat caaagaaaag cagtggcact
     1801 aattaagact gatgccctgc ggaaaagttc tgcggttgtg caaaaaaatt tcattttcag
     1861 ggcaacttca gttttatcct aatcctggcc ataccatgac gatgattgtc cctgccagcg
     1921 tcagcaggac gttggcgatt gcataggtgc ccgcatagcc cagcgccggg atgttactgc
     1981 gagctgtatc actgatgatc tccattgccg gcgcgcaggt acgtgcgccc atcattgcgc
     2041 cgaacaacag cgcgcggttc attcgcaata cataagcacc gaacaagaaa cagataacca
     2101 cgggcaccag actgacaatc aatccggcaa tcaacatctg accgccaatc gcgcccaggc
     2161 cgttattaat accgctaccg gcgctcagac caacgcctgc cataaacacc atcaagccga
     2221 actctttcac catgcttaat gcaccctgcg gaatgtaacc gaaggtcggg tggttagcac
     2281 gcataaagcc cagcataatt ccggcgaata acaacccggc agcgttcccc atgccgaaac
     2341 tgaatgtgct gaactggaag gtgatcatcc cgatcatcag cccaataaca aagaaggcgc
     2401 aaaatgccag caggtcagtg acctggctgt gaatcgagat aaagccgatg cgatcggcga
     2461 tggtttttac gcgacgggca tcaccgctga cttgtaaaac gtcacctttg ttaagcacga
     2521 cgttgtcatc tatcggcatc tcaatctggc tacgaatgac gcggttaagg aagcaaccgt
     2581 gatcggtcaa cttcagttgt gcgagacgtt tacctacagc gttatggttt ttaacgacca
     2641 cttcttcagt gacgatacgc atgtcgagaa ggtcacgatc gaaaacttct ttaccgttac
     2701 ggaagctggg atcgaggggg tcgaccagac tcgagtggag gccggcccct aggacgactg
     2761 cagcccgctt acgcagggca tccatttatt actcaaccgt aaccgatttt gccaggttac
     2821 gcggctggtc ctctagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2881 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     2941 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3001 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3061 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3121 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     3181 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtac
     3241 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3301 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3361 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3421 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3481 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3541 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3601 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3661 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3721 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3781 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3841 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3901 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3961 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     4021 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     4081 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4141 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4201 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4261 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4321 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4381 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4441 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4501 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4561 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagcc ggttgtcagc
     4621 cgttaagtgt tcctgtgtca ctcaaaattg ctttgagagg ctctaagggc ttctcagtgc
     4681 gttacatccc tggcttgttg tccacaaccg ttaaacctta aaagctcatg accaaaatcc
     4741 cttaacgtga gttttcgttc cactgagcgt cagaccccgt tgatgatacc gctgccttac
     4801 tgggtgcatt agccagtctg aatgacctgt cacgggataa tccgaagtgg tcagactgga
     4861 aaatcagagg gcaggaactg ctgaacagca aaaagtcaga tagcaccaca tagcagaccc
     4921 gccataaaac gccctgagaa gcccgtgacg ggcttttctt gtattatggg tagtttcctt
     4981 gcatgaatcc ataaaaggcg cctgtagtgc catttacccc cattcactgc cagagccgtg
     5041 agcgcagcga actgaatgtc acgaaaaaga cagcgactca ggtgcctgat ggtcggagac
     5101 aaaaggaata ttcagcgatt tgcccgattg cggccgcaac cgagcttgcg agggtgctac
     5161 ttaagccttt agggttttaa ggtctgtttt gtagaggagc aaacagcgtt tgcgacatcc
     5221 ttttgtaata ctgcggaact gactaaagta gtgagttata cacagggctg ggatctattc
     5281 tttttatctt tttttattct ttctttattc tataaattat aaccacttga atataaacaa
     5341 aaaaaacaca caaaggtcta gcggaattta cagagggtct agcagaattt acaagttttc
     5401 cagcaaaggt ctagcagaat ttacagatac ccacaactca aaggaaaagg actagtaatt
     5461 atcattgact agcccatctc aattggtata gtgattaaaa tcacctagac caattgagat
     5521 gtatgtctga attagttgtt ttcaaagcaa atgaactagc gattagtcgc tatgacttaa
     5581 cggagcatga aaccaagcta attttatgct gtgtggcact actcaacccc acgattgaaa
     5641 accctacaag gaaagaacgg acggtatcgt tcacttataa ccaatacgtt cagatgatga
     5701 acatcagtag ggaaaatgct tatggtgtat tagctaaagc aaccagagag ctgatgacga
     5761 gaactgtgga aatcaggaat cctttggtta aaggctttga gattttccag tggacaaact
     5821 atgccaagtt ctcaagcgaa aaattagaat tagtttttag tgaagagata ttgccttatc
     5881 ttttccagtt aaaaaaattc ataaaatata atctggaaca tgttaagtct tttgaaaaca
     5941 aatactctat gaggatttat gagtggttat taaaagaact aacacaaaag aaaactcaca
     6001 aggcaaatat agagattagc cttgatgaat ttaagttcat gttaatgctt gaaaataact
     6061 accatgagtt taaaaggctt aaccaatggg ttttgaaacc aataagtaaa gatttaaaca
     6121 cttacagcaa tatgaaattg gtggttgata agcgaggccg cccgactgat acgttgattt
     6181 tccaagttga actagataga caaatggatc tcgtaaccga acttgagaac aaccagataa
     6241 aaatgaatgg tgacaaaata ccaacaacca ttacatcaga ttcctaccta cataacggac
     6301 taagaaaaac actacacgat gctttaactg caaaaattca gctcaccagt tttgaggcaa
     6361 aatttttgag tgacatgcaa agtaagtatg atctcaatgg ttcgttctca tggctcacgc
     6421 aaaaacaacg aaccacacta gagaacatac tggctaaata cggaaggatc tgaggttctt
     6481 atggctcttg tatctatcag tgaagcatca agactaacaa acaaaagtag aacaactgtt
     6541 caccgttaca tatcaaaggg aaaactgtcc atacccatgt aaaacttaaa accttagagg
     6601 ctatttaagt tgctgattta tattaatttt attgttcaaa catgagagct tagtacgtga
     6661 aacatgagag cttagtacgt tagccatgag agcttagtac gttagccatg agggtttagt
     6721 tcgttaaaca tgagagctta gtacgttaaa cttgagagct tagtacgtga aacatgagag
     6781 cttagtacgt actatcaaca ggttgaactg cccatgttct ttcctgcgtt atcagagctt
     6841 atcggccagc ctcgcagagc aggattcccg ttgagcaccg ccaggtgcga ataagggaca
     6901 gtgaagaagg aacacccgct cgcgggtggg cctacttcac ctatcctgcc cggctgacgc
     6961 cgttggatac accaaggaaa gtctacacga accctttggc aaaatcctgt atatcgtgcg
     7021 aaaaaggatg gatataccga aaaaatcgct ataatgaccc cgaagcaggg ttatgcagcg
     7081 gaaagtatac cttaaggaat cccctgatac cgctcgccgc agccgaacga ccgagcgcag
     7141 cgggtcagtg agcgaggaag cggaagagaa ttccaagatc ccctgattcc ctttgtcaac
     7201 agcaatggat cgaattgaca taagcctgtt cggttcgtaa actgtaatgc aagtagcgta
     7261 tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg
     7321 cggttttcat ggcttgttat gactgttttt ttgtacagtc tatgcctcgg gcatccaagc
     7381 agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc
     7441 agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttagg tggctcaagt
     7501 atgggcatca ttcgcacatg taggctcggc cctgaccaag tcaaatccat gcgggctgct
     7561 cttgatcttt tcggtcgtga gttcggagac gtagccacct actcccaaca tcagccggac
     7621 tccgattacc tcgggaactt gctccgtagt aagacattca tcgcgcttgc tgccttcgac
     7681 caagaagcgg ttgttggcgc tctcgcggct tacgttctgc ccaagtttga gcagccgcgt
     7741 agtgagatct atatctatga tctcgcagtc tccggcgagc accggaggca gggcattgcc
     7801 accgcgctca tcaatctcct caagcatgag gccaacgcgc ttggtgctta tgtgatctac
     7861 gtgcaagcag attacggtga cgatcccgca gtggctctct atacaaagtt gggcatacgg
     7921 gaagaagtga tgcactttga tatcgaccca agtaccgcca cctaactcta gacaatatcg
     7981 atacctgtga cggaagatca cttcgcagaa taaataaatc ctggtgtccc tgttgatacc
     8041 gggaagccct gggccaactt ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc
     8101 aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt gtcgagattt
     8161 tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata
     8221 tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat
     8281 aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac
     8341 aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaatta
     8401 cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc
     8461 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc
     8521 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
     8581 ttccctaaag gggttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc
     8641 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg
     8701 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat
     8761 gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat
     8821 gaactagtct ttggcagttt attcttgaca tgtagtgagg gggctggtat aatcacatag
     8881 tactgttcgg gtacctagaa ttaaagagga gaaattaagc atgcgtaaag gagaagaact
     8941 tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt
     9001 ttctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc ttaaatttat
     9061 ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt tcggttatgg
     9121 tgttcaatgc tttgcgagat acccagatca tatgaaacag catgactttt tcaagagtgc
     9181 catgcccgaa ggttatgtac aggaaagaac tatatt
//
