LOCUS       sequence-446516-        8934 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for adding C-terminal SpyTag to the Plesiomonas ZOR0011 P2 
            phage major capsid protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8934)
  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 8934)
  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: pAX1_Z11 gpN:SpyTag
FEATURES             Location/Qualifiers
     source          1..8934
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             35..46
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     682..701
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(768..870)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             complement(984..1517)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(1706..1711,1712..1728,1729..1734))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     oriT            1998..2106
                     /note="incP origin of transfer"
     CDS             complement(2476..3426)
                     /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"
     rep_origin      3474..3696
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(4744..5535,5536..5604))
                     /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     5367..5386
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5605..5709)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5777..5795
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5833..5855)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5955..5974
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(7026..7064)
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     terminator      complement(7923..8009)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(8112..8206)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(8249..8934,1..28))
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(8289..8309)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     8904..8932
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
ORIGIN
        1 cagtgaaaag ttcttctcct ttacgcatgc ttaatttctc ctctttaatt ctaggtaccc
       61 gaacagtact atgtgattat accagccccc tcactacatg tcaagaataa actgccaaag
      121 actagttcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca
      181 caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa
      241 tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
      301 aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc aataacccct
      361 ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
      421 aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
      481 tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
      541 gccatacgta attccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
      601 taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
      661 gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc
      721 cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta
      781 gctcctgaaa atctcgacaa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg
      841 tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccaaaa gttggcccag
      901 ggcttcccgg tatcaacagg gacaccagga tttatttatt ctgcgaagtg atcttccgtc
      961 acaggtatcg atattgtcta gagttaggtg gcggtacttg ggtcgatatc aaagtgcatc
     1021 acttcttccc gtatgcccaa ctttgtatag agagccactg cgggatcgtc accgtaatct
     1081 gcttgcacgt agatcacata agcaccaagc gcgttggcct catgcttgag gagattgatg
     1141 agcgcggtgg caatgccctg cctccggtgc tcgccggaga ctgcgagatc atagatatag
     1201 atctcactac gcggctgctc aaacttgggc agaacgtaag ccgcgagagc gccaacaacc
     1261 gcttcttggt cgaaggcagc aagcgcgatg aatgtcttac tacggagcaa gttcccgagg
     1321 taatcggagt ccggctgatg ttgggagtag gtggctacgt ctccgaactc acgaccgaaa
     1381 agatcaagag cagcccgcat ggatttgact tggtcagggc cgagcctaca tgtgcgaatg
     1441 atgcccatac ttgagccacc taactttgtt ttagggcgac tgccctgctg cgtaacatcg
     1501 ttgctgctgc gtaacatcgt tgctgctcca taacatcaaa catcgaccca cggcgtaacg
     1561 cgcttgctgc ttggatgccc gaggcataga ctgtacaaaa aaacagtcat aacaagccat
     1621 gaaaaccgcc actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg
     1681 tgagcgcata cgctacttgc attacagttt acgaaccgaa caggcttatg tcaattcgat
     1741 ccattgctgt tgacaaaggg aatcagggga tcttggaatt ctcttccgct tcctcgctca
     1801 ctgacccgct gcgctcggtc gttcggctgc ggcgagcggt atcaggggat tccttaaggt
     1861 atactttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc ggtatatcca
     1921 tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt tccttggtgt
     1981 atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag cgggtgttcc
     2041 ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc tctgcgaggc
     2101 tggccgataa gctctgataa cgcaggaaag aacatgggca gttcaacctg ttgatagtac
     2161 gtactaagct ctcatgtttc acgtactaag ctctcaagtt taacgtacta agctctcatg
     2221 tttaacgaac taaaccctca tggctaacgt actaagctct catggctaac gtactaagct
     2281 ctcatgtttc acgtactaag ctctcatgtt tgaacaataa aattaatata aatcagcaac
     2341 ttaaatagcc tctaaggttt taagttttac atgggtatgg acagttttcc ctttgatatg
     2401 taacggtgaa cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca
     2461 agagccataa gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg
     2521 ttgtttttgc gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact
     2581 caaaaatttt gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt
     2641 ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc
     2701 attcattttt atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc
     2761 aacttggaaa atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt
     2821 gctgtaagtg tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa
     2881 ctcatggtag ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat
     2941 atttgccttg tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat
     3001 agagtatttg ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa
     3061 ctggaaaaga taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa
     3121 cttggcatag tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc
     3181 cacagttctc gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct
     3241 actgatgttc atcatctgaa cgtattggtt ataagtgaac gataccgtcc gttctttcct
     3301 tgtagggttt tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc
     3361 atgctccgtt aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc
     3421 agacatacat ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag
     3481 tcaatgataa ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac
     3541 ctttgctgga aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg
     3601 tgtttttttt gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa
     3661 gataaaaaga atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta
     3721 ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa
     3781 aggcttaagt agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata
     3841 ttccttttgt ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc
     3901 gctgcgctca cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg
     3961 attcatgcaa ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt
     4021 tttatggcgg gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc
     4081 tctgattttc cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa
     4141 tgcacccagt aaggcagcgg tatcatcaac ggggtctgac gctcagtgga acgaaaactc
     4201 acgttaaggg attttggtca tgagctttta aggtttaacg gttgtggaca acaagccagg
     4261 gatgtaacgc actgagaagc ccttagagcc tctcaaagca attttgagtg acacaggaac
     4321 acttaacggc tgacaaccgg ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4381 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4441 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4501 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4561 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4621 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4681 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4741 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4801 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4861 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4921 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4981 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5041 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5101 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5161 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5221 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5281 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5341 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5401 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5461 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5521 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5581 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5641 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5701 ggttccgcgt acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     5761 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga
     5821 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
     5881 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
     5941 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
     6001 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct
     6061 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctagagga
     6121 ccagccgcgt aacctggcaa aatcggttac ggttgagtaa taaatggatg ccctgcgtaa
     6181 gcgggctgca gtcgtcctag gggccggcct ccactcgagt ctggtcgacc ccgccatcag
     6241 gctgctcagg cgcaacaaac tccatcctgc attcctctga attggtaggc ggtggacggg
     6301 attttgatgc ggcatctgcc tgtcgccatc ccgtgccgcc tcgcgcgtgg gcacgttctg
     6361 ttatccgttg ttggcgttac gggcaattcg ctccagttgc tccatatctt ttttcacgcc
     6421 acagttctta tcgaactgga acgcctgact gatatgggca tacgccaaca ccggttgatt
     6481 gttatcgcgc aggccatacg ccaaaatttt atggagtttg gcccgtactt gatctggcat
     6541 atcgtgggag tcggtcagcg ccaatgtgcg ctggataagg tcaacatcga ccggctgttt
     6601 ggacgcatag gcatgaatgg tggcatcggc aatttcttcg gctacggcgc aggccgtctg
     6661 gcggttgtag cgctttggca tcaccagacc atgctttaat gcatactcgg caatatccaa
     6721 cgcccccgaa aaatcaccgg catccacacg ccaaatcatg atgttcatca gcacatcatc
     6781 ttgtgcccct ttccccgtcg ccaatgcccc agccacccac ggctggtaat acggtaagag
     6841 ttcgcgtttc agttcggcct tacgcaccgt ggactgcaca cgcttgagcc gacgctggtc
     6901 ttcttccagc ttgagcaaca tctggttgta cgccgatagg tttcgtaagg tagagccgcc
     6961 catttgggcg gactcctcag cctgaacacg cattaaatgg cgtcgtgcag gacttggcat
     7021 gatcatttag tgggtttgta ggcgtccacc atcacgatgt gggcgcctga accacctccc
     7081 gacgcctccg cagcagccgg ttgtgatgcc gccgcaacga atgagccgac tttgatgttt
     7141 tcgactaacg ccacacagcg gtaatcttcc accacgtagg cttcattcac ggactctagg
     7201 ttttcaatgc gatcacgttt tggattgtcc agaacggagc ggcggcgcgt gtcttcttgc
     7261 caataaatcg acaggttatc cagacgggtg atcatcagcg cattggctgg gaagtatggt
     7321 gcgcgtaccg cttgcaaacc gcccatgcgt ttctggctga taatcatctc agcggccatg
     7381 gcttcgctgt tgtcctgctc tttgttgacc agggggaaat acttgtcagc aagcagtgca
     7441 cggccacaaa tcaccaccag accgtcgtcg tcctgaaaca cggggtcaat gatttcattc
     7501 acggcatcca taaccagtgc gtccagattg gcgtaatcgc cccccttgcc cactttgata
     7561 accttggtca ccgagccatc atcggcggtg atgccatcca tcacatgcgt tggcgcatcg
     7621 ttacgcacct tctgcagcca accgatattc acatcttgca gcatggggtt tttcgcacgg
     7681 tcagaggttt tcgcgcggga tgtcccgttg aaaccaatca tgatgcggtc caaggcttgg
     7741 cgcttgataa tggcgttacg gatccgcagc tggaagtctt taaacttggc ccacaaatcc
     7801 agcttggcgt acggcagcgc cgtatcgaag ttggtctgtt cgcacttgta ttccacatca
     7861 ctaaatgcgg tagggtcggt cggctcacgc tcggggttta aactcgcgac tcctagcggc
     7921 ggatttgtcc tactcaggag agcgttcacc tacaaacaac agataaaacg aaaggcccag
     7981 tctttcgact gagcctttcg ttttatttga tgcctcaagc tagagagtca ttaccccagg
     8041 cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc actcatcgca
     8101 gtctagcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa
     8161 tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag ctcagctaat
     8221 taagctagcg tttaaacgag ctcgcttatt tgtatagttc atccatgcca tgtgtaatcc
     8281 cagcagctgt tacaaactca agaaggacca tgtggtctct cttttcgttg ggatctttcg
     8341 aaagggcaga ttgtgtggac aggtaatggt tgtctggtaa aaggacaggg ccatcgccaa
     8401 ttggagtatt ttgttgataa tggtctgcta gttgaacgct tccatcttca atgttgtgtc
     8461 taattttgaa gttaactttg attccattct tttgtttgtc tgccatgatg tatacattgt
     8521 gtgagttata gttgtattcc aatttgtgtc caagaatgtt tccatcttct ttaaaatcaa
     8581 taccttttaa ctcgattcta ttaacaaggg tatcaccttc aaacttgact tcagcacgtg
     8641 tcttgtagtt cccgtcatct ttgaaaaata tagttctttc ctgtacataa ccttcgggca
     8701 tggcactctt gaaaaagtca tgctgtttca tatgatctgg gtatctcgca aagcattgaa
     8761 caccataacc gaaagtagtg acaagtgttg gccatggaac aggtagtttt ccagtagtgc
     8821 aaataaattt aagggtaagt tttccgtatg ttgcatcacc ttcaccctct ccactgacag
     8881 aaaatttgtg cccattaaca tcaccatcta attcaacaag aattgggaca actc
//
