LOCUS       sequence-446750-        7432 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for inserting SpyCatcher fused to mNeonGreen at the bacterial 
            Tn7 site .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7432)
  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 7432)
  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:mNG
FEATURES             Location/Qualifiers
     source          1..7432
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      649..871
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             919..1869
                     /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(2528..3319,3320..3388))
                     /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     3151..3170
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3389..3493)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3561..3579
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3617..3639)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3739..3758
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4172..4188
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4173..4189
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      4217..4311
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      4414..4500
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4561..4608
                     /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(4738..5271)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(5460..5465,5466..5482,5483..5488))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    5529..5576
                     /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(5632..5637,5638..5653,5654..5660)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     RBS             5684..5706
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5718..6056
                     /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             6072..6782
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      6327..6336
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     mobile_element  6924..7089
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     oriT            join(7393..7432,1..69)
                     /note="incP origin of transfer"
ORIGIN
        1 tccttcttca ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga
       61 ggctggccga taagctctga taacgcagga aagaacatgg gcagttcaac ctgttgatag
      121 tacatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgttgat
      181 gataccgctg ccttactggg tgcattagcc agtctgaatg acctgtcacg ggataatccg
      241 aagtggtcag actggaaaat cagagggcag gaactgctga acagcaaaaa gtcagatagc
      301 accacatagc agacccgcca taaaacgccc tgagaagccc gtgacgggct tttcttgtat
      361 tatgggtagt ttccttgcat gaatccataa aaggcgcctg tagtgccatt tacccccatt
      421 cactgccaga gccgtgagcg cagcgaactg aatgtcacga aaaagacagc gactcaggtg
      481 cctgatggtc ggagacaaaa ggaatattca gcgatttgcc cgattgcggc cgcaaccgag
      541 cttgcgaggg tgctacttaa gcctttaggg ttttaaggtc tgttttgtag aggagcaaac
      601 agcgtttgcg acatcctttt gtaatactgc ggaactgact aaagtagtga gttatacaca
      661 gggctgggat ctattctttt tatctttttt tattctttct ttattctata aattataacc
      721 acttgaatat aaacaaaaaa aacacacaaa ggtctagcgg aatttacaga gggtctagca
      781 gaatttacaa gttttccagc aaaggtctag cagaatttac agatacccac aactcaaagg
      841 aaaaggacta gtaattatca ttgactagcc catctcaatt ggtatagtga ttaaaatcac
      901 ctagaccaat tgagatgtat gtctgaatta gttgttttca aagcaaatga actagcgatt
      961 agtcgctatg acttaacgga gcatgaaacc aagctaattt tatgctgtgt ggcactactc
     1021 aaccccacga ttgaaaaccc tacaaggaaa gaacggacgg tatcgttcac ttataaccaa
     1081 tacgttcaga tgatgaacat cagtagggaa aatgcttatg gtgtattagc taaagcaacc
     1141 agagagctga tgacgagaac tgtggaaatc aggaatcctt tggttaaagg ctttgagatt
     1201 ttccagtgga caaactatgc caagttctca agcgaaaaat tagaattagt ttttagtgaa
     1261 gagatattgc cttatctttt ccagttaaaa aaattcataa aatataatct ggaacatgtt
     1321 aagtcttttg aaaacaaata ctctatgagg atttatgagt ggttattaaa agaactaaca
     1381 caaaagaaaa ctcacaaggc aaatatagag attagccttg atgaatttaa gttcatgtta
     1441 atgcttgaaa ataactacca tgagtttaaa aggcttaacc aatgggtttt gaaaccaata
     1501 agtaaagatt taaacactta cagcaatatg aaattggtgg ttgataagcg aggccgcccg
     1561 actgatacgt tgattttcca agttgaacta gatagacaaa tggatctcgt aaccgaactt
     1621 gagaacaacc agataaaaat gaatggtgac aaaataccaa caaccattac atcagattcc
     1681 tacctacata acggactaag aaaaacacta cacgatgctt taactgcaaa aattcagctc
     1741 accagttttg aggcaaaatt tttgagtgac atgcaaagta agtatgatct caatggttcg
     1801 ttctcatggc tcacgcaaaa acaacgaacc acactagaga acatactggc taaatacgga
     1861 aggatctgag gttcttatgg ctcttgtatc tatcagtgaa gcatcaagac taacaaacaa
     1921 aagtagaaca actgttcacc gttacatatc aaagggaaaa ctgtccatac ccatgtaaga
     1981 aaaaaaagaa tatataaggc ttttaaagct tttaaggttt aacggttgtg gacaacaagc
     2041 cagggatgta acgcactgag aagcccttag agcctctcaa agcaattttg agtgacacag
     2101 gaacacttaa cggctgacaa ccggctggta acaggattag cagagcgagg tatgtaggcg
     2161 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2221 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2281 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2341 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2401 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2461 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2521 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2581 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2641 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2701 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     2761 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     2821 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     2881 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     2941 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3001 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3061 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3121 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3181 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3241 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3301 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3361 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3421 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3481 taggggttcc gcgtacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     3541 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     3601 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3661 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3721 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     3781 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
     3841 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctag
     3901 aggaccagcc gcgtaacctg gcaaaatcgg ttacggttga gtaataaatg gatgccctgc
     3961 gtaagcgggt gtaggcggac aataaagtct taaactgaac aaaatagatc taaactatga
     4021 caataaagtc ttaaactaga cagaatagtt gtaaactgaa atcagtccag ttatgctgtg
     4081 aaaaagcata ctggactttt gttatggcta aagcaaactc ttcattttct gaagtgcaaa
     4141 ttgcccgtcg tattaaagag gggcgtgggg ttcgaggtcg acggtatcga taagctagct
     4201 taattagctg agcttggact cctgttgata gatccagtaa tgacctcaga actccatctg
     4261 gatttgttca gaacgctcgg ttgccgccgg gcgtttttta ttggtgagaa tccaagctag
     4321 actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaa
     4381 cgcctggggt aatgactctc tagcttgagg catcaaataa aacgaaaggc tcagtcgaaa
     4441 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     4501 ccgccgctag gagcttgcgg cccggacgat atcatgcatg acgatcgaat tggggatctt
     4561 gaagtaccta ttccgaagtt cctattctct agaaagtata ggaacttcag agcgcttttg
     4621 aagctgatgt gcttaaaaac ttactcaatg gaattctaga tcgaattggc cgcggcgttg
     4681 tgacaattta ccgaacaact ccgcggccgg gaagccgatc tcggcttgaa cgaattgtta
     4741 ggtggcggta cttgggtcga tatcaaagtg catcacttct tcccgtatgc ccaactttgt
     4801 atagagagcc actgcgggat cgtcaccgta atctgcttgc acgtagatca cataagcacc
     4861 aagcgcgttg gcctcatgct tgaggagatt gatgagcgcg gtggcaatgc cctgcctccg
     4921 gtgctcgccg gagactgcga gatcatagat atagatctca ctacgcggct gctcaaactt
     4981 gggcagaacg taagccgcga gagcgccaac aaccgcttct tggtcgaagg cagcaagcgc
     5041 gatgaatgtc ttactacgga gcaagttccc gaggtaatcg gagtccggct gatgttggga
     5101 gtaggtggct acgtctccga actcacgacc gaaaagatca agagcagccc gcatggattt
     5161 gacttggtca gggccgagcc tacatgtgcg aatgatgccc atacttgagc cacctaactt
     5221 tgttttaggg cgactgccct gctgcgtaac atcgttgctg ctgcgtaaca tcgttgctgc
     5281 tccataacat caaacatcga cccacggcgt aacgcgcttg ctgcttggat gcccgaggca
     5341 tagactgtac aaaaaaacag tcataacaag ccatgaaaac cgccactgcg ccgttaccac
     5401 cgctgcgttc ggtcaaggtt ctggaccagt tgcgtgagcg catacgctac ttgcattaca
     5461 gtttacgaac cgaacaggct tatgtcaatt cgatccattg ctgttgacaa agggaatcag
     5521 gggatcttga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttcagag
     5581 cgcttttgaa gctaattcgg agctcactag tggatccccc aattcatcga tttgacaatt
     5641 aatcatcggc tcgtataatg tgtggaggat ccctagaaat aattttgttt aactttaaga
     5701 aggagatata cataatggtt gataccttat caggtttatc aagtgagcaa ggtcagtccg
     5761 gtgatatgac aattgaagaa gatagtgcta cccatattaa attctcaaaa cgtgatgagg
     5821 acggcaaaga gttagctggt gcaactatgg agttgcgtga ttcatctggt aaaactatta
     5881 gtacatggat ttcagatgga caagtgaaag atttctacct gtatccagga aaatatacat
     5941 ttgtcgaaac cgcagcacca gacggttatg aggtagcaac tgctattacc tttacagtta
     6001 atgagcaagg tcaggttact gtaaatggca aagcaactaa aggtgacgct catattggat
     6061 ccagtggtag catggtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
     6121 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
     6181 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
     6241 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
     6301 accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccgga taccaagtcc
     6361 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
     6421 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
     6481 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
     6541 acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat ggcaagcgct
     6601 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
     6661 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     6721 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagt
     6781 aagcggccgc acacatggca tggatgagct ctacaaataa tgagctagcc cgcctaatga
     6841 gcgggctttt ttttctcggc ctaggggctg caggaattcc tcgagaagct tgggcccggt
     6901 acctcgcgaa ggccttgcag gccaaccaga taagtgaaat ctagttccaa actattttgt
     6961 catttttaat tttcgtatta gcttacgacg ctacacccag ttcccatcta ttttgtcact
     7021 cttccctaaa taatccttaa aaactccatt tccacccctc ccagttccca actattttgt
     7081 ccgcccacag cggggcattt ttcttcctgt tatgtttggg cgctgcatta atgaatcggc
     7141 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     7201 ccgctgcgct cggtcgttcg gctgcggcga gcggtatcag gggattcctt aaggtatact
     7261 ttccgctgca taaccctgct tcggggtcat tatagcgatt ttttcggtat atccatcctt
     7321 tttcgcacga tatacaggat tttgccaaag ggttcgtgta gactttcctt ggtgtatcca
     7381 acggcgtcag ccgggcagga taggtgaagt aggcccaccc gcgagcgggt gt
//
