LOCUS       sequence-446512-        7139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for inserting PrecA upstream of mNeonGreen at the bacterial Tn7
            site for use as a RecA reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7139)
  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 7139)
  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_PrecA:mNeonGreen
FEATURES             Location/Qualifiers
     source          1..7139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      503..725
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             773..1723
                     /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(2382..3173,3174..3242))
                     /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     3005..3024
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3243..3347)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3415..3433
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3471..3493)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3593..3612
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4026..4042
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4027..4043
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     terminator      4071..4165
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      4268..4354
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4415..4462
                     /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(4592..5125)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(5314..5319,5320..5336,5337..5342))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    5383..5430
                     /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)."
     RBS             5603..5625
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5633..6343
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDKMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      5888..5897
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     mobile_element  6485..6650
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     oriT            6954..7062
                     /note="incP origin of transfer"
ORIGIN
        1 gttttcgttc cactgagcgt cagaccccgt tgatgatacc gctgccttac tgggtgcatt
       61 agccagtctg aatgacctgt cacgggataa tccgaagtgg tcagactgga aaatcagagg
      121 gcaggaactg ctgaacagca aaaagtcaga tagcaccaca tagcagaccc gccataaaac
      181 gccctgagaa gcccgtgacg ggcttttctt gtattatggg tagtttcctt gcatgaatcc
      241 ataaaaggcg cctgtagtgc catttacccc cattcactgc cagagccgtg agcgcagcga
      301 actgaatgtc acgaaaaaga cagcgactca ggtgcctgat ggtcggagac aaaaggaata
      361 ttcagcgatt tgcccgattg cggccgcaac cgagcttgcg agggtgctac ttaagccttt
      421 agggttttaa ggtctgtttt gtagaggagc aaacagcgtt tgcgacatcc ttttgtaata
      481 ctgcggaact gactaaagta gtgagttata cacagggctg ggatctattc tttttatctt
      541 tttttattct ttctttattc tataaattat aaccacttga atataaacaa aaaaaacaca
      601 caaaggtcta gcggaattta cagagggtct agcagaattt acaagttttc cagcaaaggt
      661 ctagcagaat ttacagatac ccacaactca aaggaaaagg actagtaatt atcattgact
      721 agcccatctc aattggtata gtgattaaaa tcacctagac caattgagat gtatgtctga
      781 attagttgtt ttcaaagcaa atgaactagc gattagtcgc tatgacttaa cggagcatga
      841 aaccaagcta attttatgct gtgtggcact actcaacccc acgattgaaa accctacaag
      901 gaaagaacgg acggtatcgt tcacttataa ccaatacgtt cagatgatga acatcagtag
      961 ggaaaatgct tatggtgtat tagctaaagc aaccagagag ctgatgacga gaactgtgga
     1021 aatcaggaat cctttggtta aaggctttga gattttccag tggacaaact atgccaagtt
     1081 ctcaagcgaa aaattagaat tagtttttag tgaagagata ttgccttatc ttttccagtt
     1141 aaaaaaattc ataaaatata atctggaaca tgttaagtct tttgaaaaca aatactctat
     1201 gaggatttat gagtggttat taaaagaact aacacaaaag aaaactcaca aggcaaatat
     1261 agagattagc cttgatgaat ttaagttcat gttaatgctt gaaaataact accatgagtt
     1321 taaaaggctt aaccaatggg ttttgaaacc aataagtaaa gatttaaaca cttacagcaa
     1381 tatgaaattg gtggttgata agcgaggccg cccgactgat acgttgattt tccaagttga
     1441 actagataga caaatggatc tcgtaaccga acttgagaac aaccagataa aaatgaatgg
     1501 tgacaaaata ccaacaacca ttacatcaga ttcctaccta cataacggac taagaaaaac
     1561 actacacgat gctttaactg caaaaattca gctcaccagt tttgaggcaa aatttttgag
     1621 tgacatgcaa agtaagtatg atctcaatgg ttcgttctca tggctcacgc aaaaacaacg
     1681 aaccacacta gagaacatac tggctaaata cggaaggatc tgaggttctt atggctcttg
     1741 tatctatcag tgaagcatca agactaacaa acaaaagtag aacaactgtt caccgttaca
     1801 tatcaaaggg aaaactgtcc atacccatgt aagaaaaaaa agaatatata aggcttttaa
     1861 agcttttaag gtttaacggt tgtggacaac aagccaggga tgtaacgcac tgagaagccc
     1921 ttagagcctc tcaaagcaat tttgagtgac acaggaacac ttaacggctg acaaccggct
     1981 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     2041 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     2101 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     2161 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     2221 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     2281 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     2341 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     2401 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     2461 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     2521 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     2581 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     2641 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     2701 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     2761 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     2821 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     2881 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     2941 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3001 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     3061 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     3121 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     3181 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     3241 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     3301 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgtac atttccccga
     3361 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     3421 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     3481 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     3541 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     3601 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
     3661 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     3721 tcggtgcggg cctcttcgct attacgccag ctagaggacc agccgcgtaa cctggcaaaa
     3781 tcggttacgg ttgagtaata aatggatgcc ctgcgtaagc gggtgtaggc ggacaataaa
     3841 gtcttaaact gaacaaaata gatctaaact atgacaataa agtcttaaac tagacagaat
     3901 agttgtaaac tgaaatcagt ccagttatgc tgtgaaaaag catactggac ttttgttatg
     3961 gctaaagcaa actcttcatt ttctgaagtg caaattgccc gtcgtattaa agaggggcgt
     4021 ggggttcgag gtcgacggta tcgataagct agcttaatta gctgagcttg gactcctgtt
     4081 gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg
     4141 ccgggcgttt tttattggtg agaatccaag ctagactgcg atgagtggca gggcggggcg
     4201 taattttttt aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt
     4261 gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     4321 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ctaggagctt gcggcccgga
     4381 cgatatcatg catgacgatc gaattgggga tcttgaagta cctattccga agttcctatt
     4441 ctctagaaag tataggaact tcagagcgct tttgaagctg atgtgcttaa aaacttactc
     4501 aatggaattc tagatcgaat tggccgcggc gttgtgacaa tttaccgaac aactccgcgg
     4561 ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg tcgatatcaa
     4621 agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg ggatcgtcac
     4681 cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca tgcttgagga
     4741 gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact gcgagatcat
     4801 agatatagat ctcactacgc ggctgctcaa acttgggcag aacgtaagcc gcgagagcgc
     4861 caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta cggagcaagt
     4921 tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct ccgaactcac
     4981 gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg agcctacatg
     5041 tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg ccctgctgcg
     5101 taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca tcgacccacg
     5161 gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa acagtcataa
     5221 caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac
     5281 cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca ggcttatgtc
     5341 aattcgatcc attgctgttg acaaagggaa tcaggggatc ttgaagttcc tattccgaag
     5401 ttcctattct ctagaaagta taggaacttc agagcgcttt tgaagctaat tcggagctca
     5461 ctagtggatc gcatgcattg cagaccttgt ggcaacaatt tctacaaaac acttgatact
     5521 gtatgagcat acagtataat tgcttcaaca gaacatattg actatccggt attacccggc
     5581 atgacggatc cctagaaata attttgttta actttaagaa ggagatatac atatggtgag
     5641 caagggcgag gaggataaga tggcgtcgct gccagcgaca catgagttac acatctttgg
     5701 ctccatcaac ggtgtggact ttgacatggt gggtcagggc accggcaatc caaatgatgg
     5761 ttatgaggag ttaaacctga agtccaccaa gggtgacctc cagttctccc cctggattct
     5821 ggtccctcat atcgggtatg gcttccatca gtacctgccc taccctgacg ggatgtcgcc
     5881 tttccaggcc gccatggtag atggctccgg ataccaagtc catcgcacaa tgcagtttga
     5941 agatggtgcc tcccttactg ttaactaccg ctacacctac gagggaagcc acatcaaagg
     6001 agaggcccag gtgaagggga ctggtttccc tgctgacggt cctgtgatga ccaactcgct
     6061 gaccgctgcg gactggtgca ggtcgaagaa gacttacccc aacgacaaaa ccatcatcag
     6121 tacctttaag tggagttaca ccactggaaa tggcaagcgc taccggagca ctgcgcggac
     6181 cacctacacc tttgccaagc caatggcggc taactatctg aagaaccagc cgatgtacgt
     6241 gttccgtaag acggagctca agcactccaa gaccgagctc aacttcaagg agtggcaaaa
     6301 ggcctttacc gatgtgatgg gcatggacga gctgtacaag taagcggccg cacacatggc
     6361 atggatgagc tctacaaata atgagctagc ccgcctaatg agcgggcttt tttttctcgg
     6421 cctaggggct gcaggaattc ctcgagaagc ttgggcccgg tacctcgcga aggccttgca
     6481 ggccaaccag ataagtgaaa tctagttcca aactattttg tcatttttaa ttttcgtatt
     6541 agcttacgac gctacaccca gttcccatct attttgtcac tcttccctaa ataatcctta
     6601 aaaactccat ttccacccct cccagttccc aactattttg tccgcccaca gcggggcatt
     6661 tttcttcctg ttatgtttgg gcgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6721 gtttgcgtat tgggcgctct tccgcttcct cgctcactga cccgctgcgc tcggtcgttc
     6781 ggctgcggcg agcggtatca ggggattcct taaggtatac tttccgctgc ataaccctgc
     6841 ttcggggtca ttatagcgat tttttcggta tatccatcct ttttcgcacg atatacagga
     6901 ttttgccaaa gggttcgtgt agactttcct tggtgtatcc aacggcgtca gccgggcagg
     6961 ataggtgaag taggcccacc cgcgagcggg tgttccttct tcactgtccc ttattcgcac
     7021 ctggcggtgc tcaacgggaa tcctgctctg cgaggctggc cgataagctc tgataacgca
     7081 ggaaagaaca tgggcagttc aacctgttga tagtacatga ccaaaatccc ttaacgtga
//
