LOCUS       sequence-446744-        7432 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for inserting SpyCatcher003 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_SpyCatcher003:mNG
FEATURES             Location/Qualifiers
     source          1..7432
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      640..862
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             910..1860
                     /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(2519..3310,3311..3379))
                     /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     3142..3161
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3380..3484)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3552..3570
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3608..3630)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3730..3749
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4163..4179
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4164..4180
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      4208..4302
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      4405..4491
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4552..4599
                     /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(4729..5262)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(5451..5456,5457..5473,5474..5479))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    5520..5567
                     /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(5623..5628,5629..5644,5645..5651)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     RBS             5675..5697
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5709..6047
                     /codon_start=1
                     /product="improved SpyCatcher variant that reacts with 
                     SpyTag003 at a rate approaching the diffusion limit (Keeble
                     et al., 2019)"
                     /label=SpyCatcher003
                     /translation="VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATME
                     LRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDG
                     EATEGDAHT"
     CDS             6063..6773
                     /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      6318..6327
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     mobile_element  6915..7080
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     oriT            join(7384..7432,1..60)
                     /note="incP origin of transfer"
ORIGIN
        1 actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg aggctggccg
       61 ataagctctg ataacgcagg aaagaacatg ggcagttcaa cctgttgata gtacatgacc
      121 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgttga tgataccgct
      181 gccttactgg gtgcattagc cagtctgaat gacctgtcac gggataatcc gaagtggtca
      241 gactggaaaa tcagagggca ggaactgctg aacagcaaaa agtcagatag caccacatag
      301 cagacccgcc ataaaacgcc ctgagaagcc cgtgacgggc ttttcttgta ttatgggtag
      361 tttccttgca tgaatccata aaaggcgcct gtagtgccat ttacccccat tcactgccag
      421 agccgtgagc gcagcgaact gaatgtcacg aaaaagacag cgactcaggt gcctgatggt
      481 cggagacaaa aggaatattc agcgatttgc ccgattgcgg ccgcaaccga gcttgcgagg
      541 gtgctactta agcctttagg gttttaaggt ctgttttgta gaggagcaaa cagcgtttgc
      601 gacatccttt tgtaatactg cggaactgac taaagtagtg agttatacac agggctggga
      661 tctattcttt ttatcttttt ttattctttc tttattctat aaattataac cacttgaata
      721 taaacaaaaa aaacacacaa aggtctagcg gaatttacag agggtctagc agaatttaca
      781 agttttccag caaaggtcta gcagaattta cagataccca caactcaaag gaaaaggact
      841 agtaattatc attgactagc ccatctcaat tggtatagtg attaaaatca cctagaccaa
      901 ttgagatgta tgtctgaatt agttgttttc aaagcaaatg aactagcgat tagtcgctat
      961 gacttaacgg agcatgaaac caagctaatt ttatgctgtg tggcactact caaccccacg
     1021 attgaaaacc ctacaaggaa agaacggacg gtatcgttca cttataacca atacgttcag
     1081 atgatgaaca tcagtaggga aaatgcttat ggtgtattag ctaaagcaac cagagagctg
     1141 atgacgagaa ctgtggaaat caggaatcct ttggttaaag gctttgagat tttccagtgg
     1201 acaaactatg ccaagttctc aagcgaaaaa ttagaattag tttttagtga agagatattg
     1261 ccttatcttt tccagttaaa aaaattcata aaatataatc tggaacatgt taagtctttt
     1321 gaaaacaaat actctatgag gatttatgag tggttattaa aagaactaac acaaaagaaa
     1381 actcacaagg caaatataga gattagcctt gatgaattta agttcatgtt aatgcttgaa
     1441 aataactacc atgagtttaa aaggcttaac caatgggttt tgaaaccaat aagtaaagat
     1501 ttaaacactt acagcaatat gaaattggtg gttgataagc gaggccgccc gactgatacg
     1561 ttgattttcc aagttgaact agatagacaa atggatctcg taaccgaact tgagaacaac
     1621 cagataaaaa tgaatggtga caaaatacca acaaccatta catcagattc ctacctacat
     1681 aacggactaa gaaaaacact acacgatgct ttaactgcaa aaattcagct caccagtttt
     1741 gaggcaaaat ttttgagtga catgcaaagt aagtatgatc tcaatggttc gttctcatgg
     1801 ctcacgcaaa aacaacgaac cacactagag aacatactgg ctaaatacgg aaggatctga
     1861 ggttcttatg gctcttgtat ctatcagtga agcatcaaga ctaacaaaca aaagtagaac
     1921 aactgttcac cgttacatat caaagggaaa actgtccata cccatgtaag aaaaaaaaga
     1981 atatataagg cttttaaagc ttttaaggtt taacggttgt ggacaacaag ccagggatgt
     2041 aacgcactga gaagccctta gagcctctca aagcaatttt gagtgacaca ggaacactta
     2101 acggctgaca accggctggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2161 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2221 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2281 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2341 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2401 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2461 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2521 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2581 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2641 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2701 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2761 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     2821 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     2881 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     2941 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3001 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     3061 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3121 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3181 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3241 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3301 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3361 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3421 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3481 cgcgtacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     3541 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     3601 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     3661 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     3721 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
     3781 cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca
     3841 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagcta gaggaccagc
     3901 cgcgtaacct ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg
     3961 tgtaggcgga caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt
     4021 cttaaactag acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat
     4081 actggacttt tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc
     4141 gtattaaaga ggggcgtggg gttcgaggtc gacggtatcg ataagctagc ttaattagct
     4201 gagcttggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
     4261 agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaagcta gactgcgatg
     4321 agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa acgcctgggg
     4381 taatgactct ctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     4441 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccgcta
     4501 ggagcttgcg gcccggacga tatcatgcat gacgatcgaa ttggggatct tgaagtacct
     4561 attccgaagt tcctattctc tagaaagtat aggaacttca gagcgctttt gaagctgatg
     4621 tgcttaaaaa cttactcaat ggaattctag atcgaattgg ccgcggcgtt gtgacaattt
     4681 accgaacaac tccgcggccg ggaagccgat ctcggcttga acgaattgtt aggtggcggt
     4741 acttgggtcg atatcaaagt gcatcacttc ttcccgtatg cccaactttg tatagagagc
     4801 cactgcggga tcgtcaccgt aatctgcttg cacgtagatc acataagcac caagcgcgtt
     4861 ggcctcatgc ttgaggagat tgatgagcgc ggtggcaatg ccctgcctcc ggtgctcgcc
     4921 ggagactgcg agatcataga tatagatctc actacgcggc tgctcaaact tgggcagaac
     4981 gtaagccgcg agagcgccaa caaccgcttc ttggtcgaag gcagcaagcg cgatgaatgt
     5041 cttactacgg agcaagttcc cgaggtaatc ggagtccggc tgatgttggg agtaggtggc
     5101 tacgtctccg aactcacgac cgaaaagatc aagagcagcc cgcatggatt tgacttggtc
     5161 agggccgagc ctacatgtgc gaatgatgcc catacttgag ccacctaact ttgttttagg
     5221 gcgactgccc tgctgcgtaa catcgttgct gctgcgtaac atcgttgctg ctccataaca
     5281 tcaaacatcg acccacggcg taacgcgctt gctgcttgga tgcccgaggc atagactgta
     5341 caaaaaaaca gtcataacaa gccatgaaaa ccgccactgc gccgttacca ccgctgcgtt
     5401 cggtcaaggt tctggaccag ttgcgtgagc gcatacgcta cttgcattac agtttacgaa
     5461 ccgaacaggc ttatgtcaat tcgatccatt gctgttgaca aagggaatca ggggatcttg
     5521 aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcaga gcgcttttga
     5581 agctaattcg gagctcacta gtggatcccc caattcatcg atttgacaat taatcatcgg
     5641 ctcgtataat gtgtggagga tccctagaaa taattttgtt taactttaag aaggagatat
     5701 acataatggt aaccacctta tcaggtttat caggtgagca aggtccgtcc ggtgatatga
     5761 caactgaaga agatagtgct acccatatta aattctcaaa acgtgatgag gacggccgtg
     5821 agttagctgg tgcaactatg gagttgcgtg attcatctgg taaaactatt agtacatgga
     5881 tttcagatgg acatgtgaag gatttctacc tgtatccagg aaaatataca tttgtcgaaa
     5941 ccgcagcacc agacggttat gaggtagcaa ctccaattga atttacagtt aatgaggacg
     6001 gtcaggttac tgtagatggt gaagcaactg aaggtgacgc tcatactgga tccagtggta
     6061 gcatggtgag caagggcgag gaggataaca tggcctctct cccagcgaca catgagttac
     6121 acatctttgg ctccatcaac ggtgtggact ttgacatggt gggtcagggc accggcaatc
     6181 caaatgatgg ttatgaggag ttaaacctga agtccaccaa gggtgacctc cagttctccc
     6241 cctggattct ggtccctcat atcgggtatg gcttccatca gtacctgccc taccctgacg
     6301 ggatgtcgcc tttccaggcc gccatggtag atggctccgg ataccaagtc catcgcacaa
     6361 tgcagtttga agatggtgcc tcccttactg ttaactaccg ctacacctac gagggaagcc
     6421 acatcaaagg agaggcccag gtgaagggga ctggtttccc tgctgacggt cctgtgatga
     6481 ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa gacttacccc aacgacaaaa
     6541 ccatcatcag tacctttaag tggagttaca ccactggaaa tggcaagcgc taccggagca
     6601 ctgcgcggac cacctacacc tttgccaagc caatggcggc taactatctg aagaaccagc
     6661 cgatgtacgt gttccgtaag acggagctca agcactccaa gaccgagctc aacttcaagg
     6721 agtggcaaaa ggcctttacc gatgtgatgg gcatggacga gctgtacaag taagcggccg
     6781 cacacatggc atggatgagc tctacaaata atgagctagc ccgcctaatg agcgggcttt
     6841 tttttctcgg cctaggggct gcaggaattc ctcgagaagc ttgggcccgg tacctcgcga
     6901 aggccttgca ggccaaccag ataagtgaaa tctagttcca aactattttg tcatttttaa
     6961 ttttcgtatt agcttacgac gctacaccca gttcccatct attttgtcac tcttccctaa
     7021 ataatcctta aaaactccat ttccacccct cccagttccc aactattttg tccgcccaca
     7081 gcggggcatt tttcttcctg ttatgtttgg gcgctgcatt aatgaatcgg ccaacgcgcg
     7141 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga cccgctgcgc
     7201 tcggtcgttc ggctgcggcg agcggtatca ggggattcct taaggtatac tttccgctgc
     7261 ataaccctgc ttcggggtca ttatagcgat tttttcggta tatccatcct ttttcgcacg
     7321 atatacagga ttttgccaaa gggttcgtgt agactttcct tggtgtatcc aacggcgtca
     7381 gccgggcagg ataggtgaag taggcccacc cgcgagcggg tgttccttct tc
//
