LOCUS       sequence-446637-        7420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for inserting SpyCatcher fused to mKate2 at the bacterial Tn7 
            site .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7420)
  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 7420)
  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:mKate2
FEATURES             Location/Qualifiers
     source          1..7420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      634..856
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             904..1854
                     /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(2513..3304,3305..3373))
                     /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     3136..3155
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3374..3478)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3546..3564
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3602..3624)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3724..3743
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4157..4173
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4158..4174
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      4202..4296
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      4399..4485
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4546..4593
                     /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(4723..5256)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(5445..5450,5451..5467,5468..5473))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    5514..5561
                     /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(5617..5622,5623..5638,5639..5645)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     RBS             5669..5691
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5703..6041
                     /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             6057..6755
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     mobile_element  6897..7062
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     oriT            join(7366..7420,1..54)
                     /note="incP origin of transfer"
ORIGIN
        1 ccttattcgc acctggcggt gctcaacggg aatcctgctc tgcgaggctg gccgataagc
       61 tctgataacg caggaaagaa catgggcagt tcaacctgtt gatagtacat gaccaaaatc
      121 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg ttgatgatac cgctgcctta
      181 ctgggtgcat tagccagtct gaatgacctg tcacgggata atccgaagtg gtcagactgg
      241 aaaatcagag ggcaggaact gctgaacagc aaaaagtcag atagcaccac atagcagacc
      301 cgccataaaa cgccctgaga agcccgtgac gggcttttct tgtattatgg gtagtttcct
      361 tgcatgaatc cataaaaggc gcctgtagtg ccatttaccc ccattcactg ccagagccgt
      421 gagcgcagcg aactgaatgt cacgaaaaag acagcgactc aggtgcctga tggtcggaga
      481 caaaaggaat attcagcgat ttgcccgatt gcggccgcaa ccgagcttgc gagggtgcta
      541 cttaagcctt tagggtttta aggtctgttt tgtagaggag caaacagcgt ttgcgacatc
      601 cttttgtaat actgcggaac tgactaaagt agtgagttat acacagggct gggatctatt
      661 ctttttatct ttttttattc tttctttatt ctataaatta taaccacttg aatataaaca
      721 aaaaaaacac acaaaggtct agcggaattt acagagggtc tagcagaatt tacaagtttt
      781 ccagcaaagg tctagcagaa tttacagata cccacaactc aaaggaaaag gactagtaat
      841 tatcattgac tagcccatct caattggtat agtgattaaa atcacctaga ccaattgaga
      901 tgtatgtctg aattagttgt tttcaaagca aatgaactag cgattagtcg ctatgactta
      961 acggagcatg aaaccaagct aattttatgc tgtgtggcac tactcaaccc cacgattgaa
     1021 aaccctacaa ggaaagaacg gacggtatcg ttcacttata accaatacgt tcagatgatg
     1081 aacatcagta gggaaaatgc ttatggtgta ttagctaaag caaccagaga gctgatgacg
     1141 agaactgtgg aaatcaggaa tcctttggtt aaaggctttg agattttcca gtggacaaac
     1201 tatgccaagt tctcaagcga aaaattagaa ttagttttta gtgaagagat attgccttat
     1261 cttttccagt taaaaaaatt cataaaatat aatctggaac atgttaagtc ttttgaaaac
     1321 aaatactcta tgaggattta tgagtggtta ttaaaagaac taacacaaaa gaaaactcac
     1381 aaggcaaata tagagattag ccttgatgaa tttaagttca tgttaatgct tgaaaataac
     1441 taccatgagt ttaaaaggct taaccaatgg gttttgaaac caataagtaa agatttaaac
     1501 acttacagca atatgaaatt ggtggttgat aagcgaggcc gcccgactga tacgttgatt
     1561 ttccaagttg aactagatag acaaatggat ctcgtaaccg aacttgagaa caaccagata
     1621 aaaatgaatg gtgacaaaat accaacaacc attacatcag attcctacct acataacgga
     1681 ctaagaaaaa cactacacga tgctttaact gcaaaaattc agctcaccag ttttgaggca
     1741 aaatttttga gtgacatgca aagtaagtat gatctcaatg gttcgttctc atggctcacg
     1801 caaaaacaac gaaccacact agagaacata ctggctaaat acggaaggat ctgaggttct
     1861 tatggctctt gtatctatca gtgaagcatc aagactaaca aacaaaagta gaacaactgt
     1921 tcaccgttac atatcaaagg gaaaactgtc catacccatg taagaaaaaa aagaatatat
     1981 aaggctttta aagcttttaa ggtttaacgg ttgtggacaa caagccaggg atgtaacgca
     2041 ctgagaagcc cttagagcct ctcaaagcaa ttttgagtga cacaggaaca cttaacggct
     2101 gacaaccggc tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2161 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2221 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2281 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2341 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2401 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2461 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     2521 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     2581 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     2641 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     2701 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     2761 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     2821 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     2881 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     2941 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3001 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3061 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3121 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3181 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3241 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3301 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3361 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     3421 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgta
     3481 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     3541 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     3601 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     3661 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     3721 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     3781 gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt
     3841 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctagaggac cagccgcgta
     3901 acctggcaaa atcggttacg gttgagtaat aaatggatgc cctgcgtaag cgggtgtagg
     3961 cggacaataa agtcttaaac tgaacaaaat agatctaaac tatgacaata aagtcttaaa
     4021 ctagacagaa tagttgtaaa ctgaaatcag tccagttatg ctgtgaaaaa gcatactgga
     4081 cttttgttat ggctaaagca aactcttcat tttctgaagt gcaaattgcc cgtcgtatta
     4141 aagaggggcg tggggttcga ggtcgacggt atcgataagc tagcttaatt agctgagctt
     4201 ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg
     4261 ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gctagactgc gatgagtggc
     4321 agggcggggc gtaatttttt taaggcagtt attggtgccc ttaaacgcct ggggtaatga
     4381 ctctctagct tgaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     4441 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gctaggagct
     4501 tgcggcccgg acgatatcat gcatgacgat cgaattgggg atcttgaagt acctattccg
     4561 aagttcctat tctctagaaa gtataggaac ttcagagcgc ttttgaagct gatgtgctta
     4621 aaaacttact caatggaatt ctagatcgaa ttggccgcgg cgttgtgaca atttaccgaa
     4681 caactccgcg gccgggaagc cgatctcggc ttgaacgaat tgttaggtgg cggtacttgg
     4741 gtcgatatca aagtgcatca cttcttcccg tatgcccaac tttgtataga gagccactgc
     4801 gggatcgtca ccgtaatctg cttgcacgta gatcacataa gcaccaagcg cgttggcctc
     4861 atgcttgagg agattgatga gcgcggtggc aatgccctgc ctccggtgct cgccggagac
     4921 tgcgagatca tagatataga tctcactacg cggctgctca aacttgggca gaacgtaagc
     4981 cgcgagagcg ccaacaaccg cttcttggtc gaaggcagca agcgcgatga atgtcttact
     5041 acggagcaag ttcccgaggt aatcggagtc cggctgatgt tgggagtagg tggctacgtc
     5101 tccgaactca cgaccgaaaa gatcaagagc agcccgcatg gatttgactt ggtcagggcc
     5161 gagcctacat gtgcgaatga tgcccatact tgagccacct aactttgttt tagggcgact
     5221 gccctgctgc gtaacatcgt tgctgctgcg taacatcgtt gctgctccat aacatcaaac
     5281 atcgacccac ggcgtaacgc gcttgctgct tggatgcccg aggcatagac tgtacaaaaa
     5341 aacagtcata acaagccatg aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca
     5401 aggttctgga ccagttgcgt gagcgcatac gctacttgca ttacagttta cgaaccgaac
     5461 aggcttatgt caattcgatc cattgctgtt gacaaaggga atcaggggat cttgaagttc
     5521 ctattccgaa gttcctattc tctagaaagt ataggaactt cagagcgctt ttgaagctaa
     5581 ttcggagctc actagtggat cccccaattc atcgatttga caattaatca tcggctcgta
     5641 taatgtgtgg aggatcccta gaaataattt tgtttaactt taagaaggag atatacataa
     5701 tggttgatac cttatcaggt ttatcaagtg agcaaggtca gtccggtgat atgacaattg
     5761 aagaagatag tgctacccat attaaattct caaaacgtga tgaggacggc aaagagttag
     5821 ctggtgcaac tatggagttg cgtgattcat ctggtaaaac tattagtaca tggatttcag
     5881 atggacaagt gaaagatttc tacctgtatc caggaaaata tacatttgtc gaaaccgcag
     5941 caccagacgg ttatgaggta gcaactgcta ttacctttac agttaatgag caaggtcagg
     6001 ttactgtaaa tggcaaagca actaaaggtg acgctcatat tggatccagt ggtagcatgg
     6061 tgagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc accgtgaaca
     6121 accaccactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca
     6181 tgagaatcaa ggcggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacca
     6241 gcttcatgta cggcagcaaa accttcatca accacaccca gggcatcccc gacttcttta
     6301 agcagtcctt ccccgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg
     6361 tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga
     6421 tcagaggggt gaacttccca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg
     6481 aggcctccac cgagaccctg taccccgctg acggcggcct ggaaggcaga gccgacatgg
     6541 ccctgaagct cgtgggcggg ggccacctga tctgcaactt gaagaccaca tacagatcca
     6601 agaaacccgc taagaacctc aagatgcccg gcgtctacta tgtggacaga agactggaaa
     6661 gaatcaagga ggccgacaaa gagacctacg tcgagcagca cgaggtggct gtggccagat
     6721 actgcgacct ccctagcaaa ctggggcaca gatgagcggc cgcacacatg gcatggatga
     6781 gctctacaaa taatgagcta gcccgcctaa tgagcgggct tttttttctc ggcctagggg
     6841 ctgcaggaat tcctcgagaa gcttgggccc ggtacctcgc gaaggccttg caggccaacc
     6901 agataagtga aatctagttc caaactattt tgtcattttt aattttcgta ttagcttacg
     6961 acgctacacc cagttcccat ctattttgtc actcttccct aaataatcct taaaaactcc
     7021 atttccaccc ctcccagttc ccaactattt tgtccgccca cagcggggca tttttcttcc
     7081 tgttatgttt gggcgctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     7141 attgggcgct cttccgcttc ctcgctcact gacccgctgc gctcggtcgt tcggctgcgg
     7201 cgagcggtat caggggattc cttaaggtat actttccgct gcataaccct gcttcggggt
     7261 cattatagcg attttttcgg tatatccatc ctttttcgca cgatatacag gattttgcca
     7321 aagggttcgt gtagactttc cttggtgtat ccaacggcgt cagccgggca ggataggtga
     7381 agtaggccca cccgcgagcg ggtgttcctt cttcactgtc
//
