LOCUS       sequence-446747-        7441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for inserting SpyCatcher fused to mCitrine at the bacterial Tn7
            site .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7441)
  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 7441)
  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: pTn7xTS_SpyCatcher:mCitrine
FEATURES             Location/Qualifiers
     source          1..7441
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      472..694
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             742..1692
                     /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"
     CDS             complement(join(2351..3142,3143..3211))
                     /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     2974..2993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3212..3316)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3384..3402
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3440..3462)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3562..3581
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3995..4011
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3996..4012
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      4040..4134
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      4237..4323
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4384..4431
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(4561..5094)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(5283..5288,5289..5305,5306..5311))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    5352..5399
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        join(5455..5460,5461..5476,5477..5483)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     RBS             5507..5529
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5541..5879
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     CDS             join(5895..5897,5898..5900,5901..6614)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5940..5961)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6201..6220)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6548..6569
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     mobile_element  6756..6921
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     oriT            7225..7333
                     /note="incP origin of transfer"
ORIGIN
        1 gatgataccg ctgccttact gggtgcatta gccagtctga atgacctgtc acgggataat
       61 ccgaagtggt cagactggaa aatcagaggg caggaactgc tgaacagcaa aaagtcagat
      121 agcaccacat agcagacccg ccataaaacg ccctgagaag cccgtgacgg gcttttcttg
      181 tattatgggt agtttccttg catgaatcca taaaaggcgc ctgtagtgcc atttaccccc
      241 attcactgcc agagccgtga gcgcagcgaa ctgaatgtca cgaaaaagac agcgactcag
      301 gtgcctgatg gtcggagaca aaaggaatat tcagcgattt gcccgattgc ggccgcaacc
      361 gagcttgcga gggtgctact taagccttta gggttttaag gtctgttttg tagaggagca
      421 aacagcgttt gcgacatcct tttgtaatac tgcggaactg actaaagtag tgagttatac
      481 acagggctgg gatctattct ttttatcttt ttttattctt tctttattct ataaattata
      541 accacttgaa tataaacaaa aaaaacacac aaaggtctag cggaatttac agagggtcta
      601 gcagaattta caagttttcc agcaaaggtc tagcagaatt tacagatacc cacaactcaa
      661 aggaaaagga ctagtaatta tcattgacta gcccatctca attggtatag tgattaaaat
      721 cacctagacc aattgagatg tatgtctgaa ttagttgttt tcaaagcaaa tgaactagcg
      781 attagtcgct atgacttaac ggagcatgaa accaagctaa ttttatgctg tgtggcacta
      841 ctcaacccca cgattgaaaa ccctacaagg aaagaacgga cggtatcgtt cacttataac
      901 caatacgttc agatgatgaa catcagtagg gaaaatgctt atggtgtatt agctaaagca
      961 accagagagc tgatgacgag aactgtggaa atcaggaatc ctttggttaa aggctttgag
     1021 attttccagt ggacaaacta tgccaagttc tcaagcgaaa aattagaatt agtttttagt
     1081 gaagagatat tgccttatct tttccagtta aaaaaattca taaaatataa tctggaacat
     1141 gttaagtctt ttgaaaacaa atactctatg aggatttatg agtggttatt aaaagaacta
     1201 acacaaaaga aaactcacaa ggcaaatata gagattagcc ttgatgaatt taagttcatg
     1261 ttaatgcttg aaaataacta ccatgagttt aaaaggctta accaatgggt tttgaaacca
     1321 ataagtaaag atttaaacac ttacagcaat atgaaattgg tggttgataa gcgaggccgc
     1381 ccgactgata cgttgatttt ccaagttgaa ctagatagac aaatggatct cgtaaccgaa
     1441 cttgagaaca accagataaa aatgaatggt gacaaaatac caacaaccat tacatcagat
     1501 tcctacctac ataacggact aagaaaaaca ctacacgatg ctttaactgc aaaaattcag
     1561 ctcaccagtt ttgaggcaaa atttttgagt gacatgcaaa gtaagtatga tctcaatggt
     1621 tcgttctcat ggctcacgca aaaacaacga accacactag agaacatact ggctaaatac
     1681 ggaaggatct gaggttctta tggctcttgt atctatcagt gaagcatcaa gactaacaaa
     1741 caaaagtaga acaactgttc accgttacat atcaaaggga aaactgtcca tacccatgta
     1801 agaaaaaaaa gaatatataa ggcttttaaa gcttttaagg tttaacggtt gtggacaaca
     1861 agccagggat gtaacgcact gagaagccct tagagcctct caaagcaatt ttgagtgaca
     1921 caggaacact taacggctga caaccggctg gtaacaggat tagcagagcg aggtatgtag
     1981 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2041 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2101 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2161 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2221 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2281 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     2341 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2401 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     2461 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     2521 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     2581 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     2641 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     2701 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     2761 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     2821 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     2881 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     2941 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3001 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3061 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3121 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3181 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3241 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3301 aaataggggt tccgcgtaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     3361 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     3421 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     3481 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     3541 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     3601 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat
     3661 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     3721 tagaggacca gccgcgtaac ctggcaaaat cggttacggt tgagtaataa atggatgccc
     3781 tgcgtaagcg ggtgtaggcg gacaataaag tcttaaactg aacaaaatag atctaaacta
     3841 tgacaataaa gtcttaaact agacagaata gttgtaaact gaaatcagtc cagttatgct
     3901 gtgaaaaagc atactggact tttgttatgg ctaaagcaaa ctcttcattt tctgaagtgc
     3961 aaattgcccg tcgtattaaa gaggggcgtg gggttcgagg tcgacggtat cgataagcta
     4021 gcttaattag ctgagcttgg actcctgttg atagatccag taatgacctc agaactccat
     4081 ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc
     4141 tagactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt
     4201 aaacgcctgg ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg
     4261 aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca
     4321 aatccgccgc taggagcttg cggcccggac gatatcatgc atgacgatcg aattggggat
     4381 cttgaagtac ctattccgaa gttcctattc tctagaaagt ataggaactt cagagcgctt
     4441 ttgaagctga tgtgcttaaa aacttactca atggaattct agatcgaatt ggccgcggcg
     4501 ttgtgacaat ttaccgaaca actccgcggc cgggaagccg atctcggctt gaacgaattg
     4561 ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact tcttcccgta tgcccaactt
     4621 tgtatagaga gccactgcgg gatcgtcacc gtaatctgct tgcacgtaga tcacataagc
     4681 accaagcgcg ttggcctcat gcttgaggag attgatgagc gcggtggcaa tgccctgcct
     4741 ccggtgctcg ccggagactg cgagatcata gatatagatc tcactacgcg gctgctcaaa
     4801 cttgggcaga acgtaagccg cgagagcgcc aacaaccgct tcttggtcga aggcagcaag
     4861 cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa tcggagtccg gctgatgttg
     4921 ggagtaggtg gctacgtctc cgaactcacg accgaaaaga tcaagagcag cccgcatgga
     4981 tttgacttgg tcagggccga gcctacatgt gcgaatgatg cccatacttg agccacctaa
     5041 ctttgtttta gggcgactgc cctgctgcgt aacatcgttg ctgctgcgta acatcgttgc
     5101 tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg gatgcccgag
     5161 gcatagactg tacaaaaaaa cagtcataac aagccatgaa aaccgccact gcgccgttac
     5221 caccgctgcg ttcggtcaag gttctggacc agttgcgtga gcgcatacgc tacttgcatt
     5281 acagtttacg aaccgaacag gcttatgtca attcgatcca ttgctgttga caaagggaat
     5341 caggggatct tgaagttcct attccgaagt tcctattctc tagaaagtat aggaacttca
     5401 gagcgctttt gaagctaatt cggagctcac tagtggatcc cccaattcat cgatttgaca
     5461 attaatcatc ggctcgtata atgtgtggag gatccctaga aataattttg tttaacttta
     5521 agaaggagat atacataatg gttgatacct tatcaggttt atcaagtgag caaggtcagt
     5581 ccggtgatat gacaattgaa gaagatagtg ctacccatat taaattctca aaacgtgatg
     5641 aggacggcaa agagttagct ggtgcaacta tggagttgcg tgattcatct ggtaaaacta
     5701 ttagtacatg gatttcagat ggacaagtga aagatttcta cctgtatcca ggaaaatata
     5761 catttgtcga aaccgcagca ccagacggtt atgaggtagc aactgctatt acctttacag
     5821 ttaatgagca aggtcaggtt actgtaaatg gcaaagcaac taaaggtgac gctcatattg
     5881 gatccagtgg tagcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     5941 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     6001 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     6061 tgccctggcc caccctcgtg accaccttcg gctacggcct gatgtgcttc gcccgctacc
     6121 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     6181 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     6241 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     6301 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     6361 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     6421 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     6481 tgcccgacaa ccactacctg agctaccagt ccaagctgag caaagacccc aacgagaagc
     6541 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     6601 agctgtacaa gtaagcggcc gcacacatgg catggatgag ctctacaaat aatgagctag
     6661 cccgcctaat gagcgggctt ttttttctcg gcctaggggc tgcaggaatt cctcgagaag
     6721 cttgggcccg gtacctcgcg aaggccttgc aggccaacca gataagtgaa atctagttcc
     6781 aaactatttt gtcattttta attttcgtat tagcttacga cgctacaccc agttcccatc
     6841 tattttgtca ctcttcccta aataatcctt aaaaactcca tttccacccc tcccagttcc
     6901 caactatttt gtccgcccac agcggggcat ttttcttcct gttatgtttg ggcgctgcat
     6961 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     7021 tcgctcactg acccgctgcg ctcggtcgtt cggctgcggc gagcggtatc aggggattcc
     7081 ttaaggtata ctttccgctg cataaccctg cttcggggtc attatagcga ttttttcggt
     7141 atatccatcc tttttcgcac gatatacagg attttgccaa agggttcgtg tagactttcc
     7201 ttggtgtatc caacggcgtc agccgggcag gataggtgaa gtaggcccac ccgcgagcgg
     7261 gtgttccttc ttcactgtcc cttattcgca cctggcggtg ctcaacggga atcctgctct
     7321 gcgaggctgg ccgataagct ctgataacgc aggaaagaac atgggcagtt caacctgttg
     7381 atagtacatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     7441 t
//
