LOCUS       sequence-446513-        9014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for adding C-terminal SpyTag to the E. coli HS P2 phage major 
            capsid protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9014)
  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 9014)
  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_HS_gpN:SpyTag
FEATURES             Location/Qualifiers
     source          1..9014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     459..479
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      562..656
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      759..845
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             1740..1778
                     /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"
     primer_bind     complement(2874..2893)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2993..3015
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3053..3071)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3139..3243
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3244..3312,3313..4104)
                     /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     complement(3462..3481)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5152..5374
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             5422..6372
                     /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
                     ELDRQMDLVTELENNQIKMNGDKIPTTITLDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     oriT            6742..6850
                     /note="incP origin of transfer"
     promoter        join(7114..7119,7120..7136,7137..7142)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             7331..7864
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        7978..8080
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(8147..8166)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     RBS             8802..8813
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(8820..9014,1..519)
                     /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(8850..8878)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
ORIGIN
        1 tatggtgttc aatgctttgc gagataccca gatcatatga aacagcatga ctttttcaag
       61 agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac
      121 tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta
      181 aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga atacaactat
      241 aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa agttaacttc
      301 aaaattagac acaacattga agatggaagc gttcaactag cagaccatta tcaacaaaat
      361 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct
      421 gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca
      481 gctgctggga ttacacatgg catggatgaa ctatacaaat aagcgagctc gtttaaacgc
      541 tagcttaatt agctgagctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc
      601 atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa
      661 gctagactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc
      721 ttaaacgcct ggggtaatga ctctctagct tgaggcatca aataaaacga aaggctcagt
      781 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
      841 caaatccgcc gctaggagtc gcgagtttaa accccggtgt tggcagcacc attgccggaa
      901 ccactgatac caccgcgaaa gagcgtgaac ctgtcgatcc gacgctgatg gtcgatgtgg
      961 aatataaatg cgagcagacc aactttgaca cggtgctgac ctacgcgaag ctggacctgt
     1021 gggcgaagtt tcaggatttc caggtgcgta tccgtgacgc catcgtgaaa cgtcaggcac
     1081 tggaccgcat catgatcggc tttaacggcg tgaagcgtgc gaaaacctcc aaccgtagcg
     1141 aaaacccgct gctgcaggat gtgaataaag gctggctgca gaaaatccgt gaggatgcac
     1201 cggatcacgt catgggcagc accaccacgg gcggtgaaac tacaccgggt gcggtgaaag
     1261 tcgggaaagg tggcgaatat gccaacctgg acgccgtagt gatggatgcc gtcaatgagc
     1321 ttatcgacgt ggtttaccag gacgatgacg atctggtggt tatttgcggg cgtgaactgc
     1381 tgtctgacaa gtatttcccg ctggtcaaca aagagcagga aaacagtgaa aaactggctg
     1441 ccgatatgat catcagtcag aaacgcatgg gtggcctgca ggccgtgcgt gcgccgttct
     1501 tcccgccgaa tgcgctgcta atcacccgtc tggataacct gtccatctac tggcaggaag
     1561 acacccgccg ccgttcagtt atcgacaacc cgaaacgtga ccggattgaa aactttgaat
     1621 ccgttaacga agcctatgtg gttgaggact accgctgcgc cgcactggtg gaaaacatcc
     1681 agattggcga tttcagcgcc gccgtagcag aaaccggaga gtcgggaggt ggttcaggcg
     1741 cccacatcgt gatggtggac gcctacaaac ccactaaata atccatgagc ctgagtcccg
     1801 cacggcagca tcgcctgcgc gttcaggctg aacaggccgc ccgcgagggc ggcagtgttc
     1861 gccacgcatc gggctatgac ctgatgctgc tgcaactggc ggaagaccgc cgccgtctca
     1921 agggcgttca gtccacggtc aaaaaagcgg aaatcaaggt ggagctgctg ccgaagtacg
     1981 ccgcctgggc agagggtgtc ctggctgccg gaggcgctca acaggatgac gtgctgatgt
     2041 acgtgatgct gtggcgcatt gatgccggag attatgccgg ggcgctggag atcgggcgtc
     2101 atgccctgcg tcatggctgg gtgatgccgc tgggtaaccg caacgtgcag accgtgctgg
     2161 cagaggaaat ggcagacgcc gcgcagagcg caatgcttgc cgccaccggc tttgatgccg
     2221 atctgttgct gcagacgctg gagctgacag acggtctgga tatgccggac cagtcacggg
     2281 cgcgtctgca taaagcgatt ggcgctgtcc tgagtgaaaa caatccggct tccgccctta
     2341 atcatctcaa ccatgcgtta cagctcgatc cccgctgtgg cgtgaaaaaa gacaaacagc
     2401 agctggagcg cagactgcgc aatgacagcc gctgacagaa cgtgcccccg cgcacgggcg
     2461 gcacggggtg gcgaaaggca ctgccacatc aaaaccccgt ccaccgccct ctatttcagg
     2521 agaaagcagc atgaagtttg ttgcgctaga acaggcaccg gaacaggcgg aaatcatcag
     2581 aaatacgccg ttctggcctg atgtggacct gtcggggggt cgaccagact cgagtggagg
     2641 ccggccccta ggacgactgc agcccgctta cgcagggcat ccatttatta ctcaaccgta
     2701 accgattttg ccaggttacg cggctggtcc tctagctggc gtaatagcga agaggcccgc
     2761 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat
     2821 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     2881 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     2941 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3001 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     3061 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     3121 acttttcggg gaaatgtacg cggaacccct atttgtttat ttttctaaat acattcaaat
     3181 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3241 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3301 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3361 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     3421 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     3481 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3541 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3601 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3661 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3721 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3781 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3841 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3901 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3961 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4021 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4081 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4141 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4201 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4261 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4321 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4381 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4441 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4501 ttaccagccg gttgtcagcc gttaagtgtt cctgtgtcac tcaaaattgc tttgagaggc
     4561 tctaagggct tctcagtgcg ttacatccct ggcttgttgt ccacaaccgt taaaccttaa
     4621 aagctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgtt
     4681 gatgataccg ctgccttact gggtgcatta gccagtctga atgacctgtc acgggataat
     4741 ccgaagtggt cagactggaa aatcagaggg caggaactgc tgaacagcaa aaagtcagat
     4801 agcaccacat agcagacccg ccataaaacg ccctgagaag cccgtgacgg gcttttcttg
     4861 tattatgggt agtttccttg catgaatcca taaaaggcgc ctgtagtgcc atttaccccc
     4921 attcactgcc agagccgtga gcgcagcgaa ctgaatgtca cgaaaaagac agcgactcag
     4981 gtgcctgatg gtcggagaca aaaggaatat tcagcgattt gcccgattgc ggccgcaacc
     5041 gagcttgcga gggtgctact taagccttta gggttttaag gtctgttttg tagaggagca
     5101 aacagcgttt gcgacatcct tttgtaatac tgcggaactg actaaagtag tgagttatac
     5161 acagggctgg gatctattct ttttatcttt ttttattctt tctttattct ataaattata
     5221 accacttgaa tataaacaaa aaaaacacac aaaggtctag cggaatttac agagggtcta
     5281 gcagaattta caagttttcc agcaaaggtc tagcagaatt tacagatacc cacaactcaa
     5341 aggaaaagga ctagtaatta tcattgacta gcccatctca attggtatag tgattaaaat
     5401 cacctagacc aattgagatg tatgtctgaa ttagttgttt tcaaagcaaa tgaactagcg
     5461 attagtcgct atgacttaac ggagcatgaa accaagctaa ttttatgctg tgtggcacta
     5521 ctcaacccca cgattgaaaa ccctacaagg aaagaacgga cggtatcgtt cacttataac
     5581 caatacgttc agatgatgaa catcagtagg gaaaatgctt atggtgtatt agctaaagca
     5641 accagagagc tgatgacgag aactgtggaa atcaggaatc ctttggttaa aggctttgag
     5701 attttccagt ggacaaacta tgccaagttc tcaagcgaaa aattagaatt agtttttagt
     5761 gaagagatat tgccttatct tttccagtta aaaaaattca taaaatataa tctggaacat
     5821 gttaagtctt ttgaaaacaa atactctatg aggatttatg agtggttatt aaaagaacta
     5881 acacaaaaga aaactcacaa ggcaaatata gagattagcc ttgatgaatt taagttcatg
     5941 ttaatgcttg aaaataacta ccatgagttt aaaaggctta accaatgggt tttgaaacca
     6001 ataagtaaag atttaaacac ttacagcaat atgaaattgg tggttgataa gcgaggccgc
     6061 ccgactgata cgttgatttt ccaagttgaa ctagatagac aaatggatct cgtaaccgaa
     6121 cttgagaaca accagataaa aatgaatggt gacaaaatac caacaaccat tacattagat
     6181 tcctacctac ataacggact aagaaaaaca ctacacgatg ctttaactgc aaaaattcag
     6241 ctcaccagtt ttgaggcaaa atttttgagt gacatgcaaa gtaagtatga tctcaatggt
     6301 tcgttctcat ggctcacgca aaaacaacga accacactag agaacatact ggctaaatac
     6361 ggaaggatct gaggttctta tggctcttgt atctatcagt gaagcatcaa gactaacaaa
     6421 caaaagtaga acaactgttc accgttacat atcaaaggga aaactgtcca tacccatgta
     6481 aaacttaaaa ccttagaggc tatttaagtt gctgatttat attaatttta ttgttcaaac
     6541 atgagagctt agtacgtgaa acatgagagc ttagtacgtt agccatgaga gcttagtacg
     6601 ttagccatga gggtttagtt cgttaaacat gagagcttag tacgttaaac ttgagagctt
     6661 agtacgtgaa acatgagagc ttagtacgta ctatcaacag gttgaactgc ccatgttctt
     6721 tcctgcgtta tcagagctta tcggccagcc tcgcagagca ggattcccgt tgagcaccgc
     6781 caggtgcgaa taagggacag tgaagaagga acacccgctc gcgggtgggc ctacttcacc
     6841 tatcctgccc ggctgacgcc gttggataca ccaaggaaag tctacacgaa ccctttggca
     6901 aaatcctgta tatcgtgcga aaaaggatgg atataccgaa aaaatcgcta taatgacccc
     6961 gaagcagggt tatgcagcgg aaagtatacc ttaaggaatc ccctgatacc gctcgccgca
     7021 gccgaacgac cgagcgcagc gggtcagtga gcgaggaagc ggaagagaat tccaagatcc
     7081 cctgattccc tttgtcaaca gcaatggatc gaattgacat aagcctgttc ggttcgtaaa
     7141 ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc
     7201 ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt tgtacagtct
     7261 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg
     7321 agcagcaacg atgttacgca gcagcaacga tgttacgcag cagggcagtc gccctaaaac
     7381 aaagttaggt ggctcaagta tgggcatcat tcgcacatgt aggctcggcc ctgaccaagt
     7441 caaatccatg cgggctgctc ttgatctttt cggtcgtgag ttcggagacg tagccaccta
     7501 ctcccaacat cagccggact ccgattacct cgggaacttg ctccgtagta agacattcat
     7561 cgcgcttgct gccttcgacc aagaagcggt tgttggcgct ctcgcggctt acgttctgcc
     7621 caagtttgag cagccgcgta gtgagatcta tatctatgat ctcgcagtct ccggcgagca
     7681 ccggaggcag ggcattgcca ccgcgctcat caatctcctc aagcatgagg ccaacgcgct
     7741 tggtgcttat gtgatctacg tgcaagcaga ttacggtgac gatcccgcag tggctctcta
     7801 tacaaagttg ggcatacggg aagaagtgat gcactttgat atcgacccaa gtaccgccac
     7861 ctaactctag acaatatcga tacctgtgac ggaagatcac ttcgcagaat aaataaatcc
     7921 tggtgtccct gttgataccg ggaagccctg ggccaacttt tggcgaaaat gagacgttga
     7981 tcggcacgta agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt
     8041 ttttgagttg tcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg
     8101 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc
     8161 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac
     8221 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat
     8281 gaatgctcat ccggaattac gtatggcaat gaaagacggt gagctggtga tatgggatag
     8341 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag
     8401 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta
     8461 cggtgaaaac ctggcctatt tccctaaagg ggttattgag aatatgtttt tcgtctcagc
     8521 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt
     8581 cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct
     8641 ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga
     8701 attacaacag tactgcgatg aactagtctt tggcagttta ttcttgacat gtagtgaggg
     8761 ggctggtata atcacatagt actgttcggg tacctagaat taaagaggag aaattaagca
     8821 tgcgtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg
     8881 atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca acatacggaa
     8941 aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg ccaacacttg
     9001 tcactacttt cggt
//
