LOCUS       sequence-426034-        7178 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Endogenous tagging of B. dendrobatidis Myo17D with a red fluorescent
            protein, hygromycin B selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7178)
  AUTHORS   Brody SM, Kalinka E, Prostak SM, Carvalho T, Man J, James TY, 
            Fritz-Laylin LK
  TITLE     Homology-mediated transformation of frog-killing fungus 
            Batrachochytrium dendrobatidis illuminates chytrid development and 
            pathogenesis.
  JOURNAL   Proc Natl Acad Sci U S A. 2025 Nov 4;122(44):e2507572122. doi: 
            10.1073/pnas.2507572122. Epub 2025 Oct 28.
  PUBMED    41150711
REFERENCE   2  (bases 1 to 7178)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2025-11-4"; volume: "122(44):e2507572122. 
            doi"; pages: " 10.1073/pnas.2507572122. Epub 2025 Oct 28"
COMMENT     Sequence Label: pEK48
FEATURES             Location/Qualifiers
     source          1..7178
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      2086..2273
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2141..2165)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             2573..3601
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2573..3601
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     primer_bind     complement(5014..5030)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5014..5030)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5027..5049)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(5202..5790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5282..5301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(5907..5931)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(6031..6822,6823..6891))
                     /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     6654..6673
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6892..6996)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7095..7117
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7109..7126
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7110..7126
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 aacgtgcctt tgcttgtttc accggctcta ttgacagaat actcggagaa cagtgtagac
       61 acattgcaca tgaagaattt actgcatttg ggagaagatc gatacctgac tacactgatg
      121 ttgaagcatt ttcctacgat gcgtaccaag tttgctggcg aggcgcgttg ctcaacagtt
      181 gtaccagaca aatggtctgt gctactttcg cagcgtcgtc gttggattaa ttctacagta
      241 cacaatctgt ttgaattggt gtttctggat caactgtgtg gactatgttg tttctctatg
      301 agatttgtag tgtttttgga tctatttgct acatttgtgc agccagcaat cgttatttat
      361 attgtatatc taatctactc tgtttccact tcaacagata cattccctct tttatcgatt
      421 attctgattg gatgcatcta tgggttccaa gtgattatat ttttgatcaa gcgagaatgg
      481 cagcatattg cgtggatgat tatctacctg cttgccattc cagtgttttc attctggatt
      541 ccatgctatt cattctggca tttcgatgac ttttcatggg gcaatacgcg tgtggtggtt
      601 ggagagggta agaaaacggt atatgtggcg gatgccgagc catttgatat cagatcgatt
      661 ccactgaagc gatggaagga ctttgaagga agtggtggag caggaagtgt agacagtcga
      721 ggtggtgtag atgcgtggga acatggatca caagcatcgg gatccatagt gcaagcagga
      781 gcacaccgta ataaggcagc tggatatgca ggatccgagt attcatcacc aggtggtggt
      841 gtctaccatc gcggttcagc agcatccaac tatgcagggt cggcgtatgg gggaccagcc
      901 ccatctattt caggtagcta ccatggcgga ttaccagtga ttgctcaatc tacagtgccg
      961 gcaagataca atacacacga tctgacacca ggacaagcgg cagatccaca acgattttca
     1021 atgatatcgt ttggagccta tccaacacaa actaatgaga tgcagaatgc acgcaattca
     1081 tcagcatcgc ttgttccagg acctttccca tcacaacaaa cgaatagata ttcaatgctt
     1141 tctaatacgt cgcaacaggt tcctgtacaa actggaccaa cggatgaaca gattttagca
     1201 gatattcgac aaatattatc atcaggcgat ttgatgacac tgaccaagaa acagatacga
     1261 gatgatttgg gacaacgatt tggagtagat ttgcgaccac gcaaagaaac aatcaatcga
     1321 atgattgatg aagtgcttca aggacggctt ggaggaggag gatccatggt gtctaaagga
     1381 gaggaactga ttaaggaaaa catgagaatg aaggtggtga tggaaggctc tgtgaatggc
     1441 caccagttca aatgtacagg ggagggggaa ggaaggcctt acgagggcgt gcagaccatg
     1501 agaatcaagg tgattgaggg gggcccactg ccctttgcct tcgacattct ggccacttct
     1561 ttcatgtatg gctccaggac ctttatcaag taccctgccg acatccctga ctttttcaaa
     1621 cagagctttc ccgagggatt cacatgggag cgggtgactc ggtatgagga tggaggggtg
     1681 gtgaccgtga ctcaggatac tagcctggag gacggcgaac tggtgtacaa cgtgaaggtg
     1741 agaggcgtga actttccatc taatggacct gtgatgcaga agaaaaccaa aggatgggag
     1801 cctaatactg agatgatgta tcccgccgat ggcggcctga gaggctacac cgatatcgcc
     1861 ctgaaggtgg atggaggagg ccacctgcac tgcaatttcg tgacaaccta tagaagcaaa
     1921 aagacagtgg ggaacattaa aatgcccggc gtgcatgccg tggatcacag gctggaaagg
     1981 atcgaagaat ccgacaatga aacatacgtg gtgcagaggg aagtggccgt ggccaaatac
     2041 agcaatctgg ggggcggaat ggacgagctg tacaaataag tcgacgcgaa tttcttatga
     2101 tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt
     2161 gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg
     2221 ttgctttctc aggtatagta tgaggtcgct cttattgacc acacctctac cggggcgcgc
     2281 cttttatgct ccaagcggag acgatgaggc cgatattaca gtggcttagt agcgtgtaca
     2341 atatttccat aaaaggctac atggtgagtt tttcacgtga ccgtaattag tcccaattag
     2401 gctttattgt atattgctac taagcatcgt taaatgacct gctcgaagct tcgtctataa
     2461 aaggcgggcg tgtgtgcgct tcccccgcta cgtctttttc acaaacactt tgcgacttga
     2521 ccaagacctt tcacccgcac cactctcatc catcacacac acaaattaat taatgggtaa
     2581 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     2641 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     2701 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     2761 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     2821 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     2881 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     2941 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     3001 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     3061 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     3121 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     3181 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     3241 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     3301 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     3361 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     3421 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     3481 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     3541 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     3601 actcgagaat cacggctggt gacaaaaagc atgagaatgg agagataaaa taaaaagtca
     3661 tcagtaacca ccaccatcag tgtgtcatgt gtttggggtg gtacttgcat aaattgattt
     3721 gacacgcttt tatgtggtcg agtttgttat tgtttggcag ccttttttgg agtgttttgc
     3781 aaatatcggt aaattgttcc atcagttgat gtgaatgacc agatcatcca ttgtgatttc
     3841 tgtaccaagg caacaactcg atgtttaaca caagagttta ctaaacgatc tactgatcaa
     3901 gttgatataa aaacatgtcg tttggttttg ttcaaacacg atccttggta ttctacttga
     3961 agtctaggtt aaccttgagc aatcgtgtag ttttaccaac gaagaatgca tgaaatacaa
     4021 catcagtaac ctgataaata ccatcagaat atgattaaaa ctggattcaa ctataaaatg
     4081 attttgtgca ttctcgtcag aattaaccaa tttaagagta aaacgaagct ttcaaaccaa
     4141 cagtggtggc gttgaaacga gtggctagaa acgatagcac caaattaccc ttaaaagtgg
     4201 caagagagtt gtctagactc ttgtagactc aagccagcaa tcactcatgc aactcattgc
     4261 tacattccta cttgttctgg ctgctgctct gtccaacgct cagagtctgt ctggatccac
     4321 ggctcctcca aaaggatgcg aagtttgtaa tacgtttatt ggacaaatgc agacttgcaa
     4381 gactactgct gctcctgctt ctgaagctca agtaagtttg aatgcgttgt tgctgcgatt
     4441 gctacgattg ctactggata ctaacttgca ttgtagtggg tcgatactgc tactaaacta
     4501 gctgcttgca tctgtcccgt ctttgcatct gccaactcgg acacttgttc catctgcatt
     4561 caaaccactg atcccacttc tcctgctacc aaattgtttt tcaaactcaa gcaggattgc
     4621 ttcactggtg ccatggcttc aactgctgtt gacattgctt ccatcttcaa tgtcaccgtc
     4681 aatgcaaaca gtattcctgt cagtccatcc tctactgcaa acccacctgc aaactcgggt
     4741 acaactgttc ccaaacaaac tcgttcttct gccgtgtcca tatataccgc aacattgagc
     4801 tccatcgtac tgtcttcgct gttttcttct cttctctttc tttagactgt ttagttattt
     4861 cattcatctc taattgtttt tgttggatcc atcgatttct ttctggatca acagtcttga
     4921 catagaccca acaccagtaa ttgaatcttg tttactaagg ctaggtggag gctcagtgat
     4981 gataagtctg cgatggtgga tgcatgtgtc atggtcatag ctgtttcctg tgtgaaattg
     5041 ttatccgctc agagggcaca atcctattcc gcgctatccg acaatctcca agacattagg
     5101 tggagttcag ttcggcgtat ggcatatgtc gctggaaaga acatgtgagc aaaaggccag
     5161 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5221 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5281 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5341 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5401 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct caaagctggg ctgtgtgcac
     5461 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5521 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5581 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5641 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5701 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5761 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5821 gacgctctat tcaacaaagc cgccgtcccg tcaagtcagc gtaaatgggt agggggcttc
     5881 aaatcgtcct cgtgatacca attcggagcc tgcttttttg tacaaacttg ttgataatgg
     5941 caattcaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6001 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6061 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6121 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagagccacg
     6181 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6241 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6301 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6361 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6421 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6481 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6541 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6601 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6661 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6721 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6781 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6841 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6901 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6961 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccaga
     7021 tacctgaaac aaaacccatc gtacggccaa ggaagtctcc aataactgtg atccaccaca
     7081 agcgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtcatg cataatccgc
     7141 acgcatctgg aataaggaag tgccattccg cctgacct
//
