LOCUS       sequence-426055-        5436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  B. dendrobatidis plasmid for integration of target sequence at URA3 
            locus, hygromycin B selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5436)
  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 5436)
  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: pEK33
FEATURES             Location/Qualifiers
     source          1..5436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1146..2171
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1146..2171
                     /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"
     CDS             2187..2216
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     rep_origin      complement(3479..4067)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3559..3578)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(4184..4208)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(4308..5099,5100..5168))
                     /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     4931..4950
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5169..5273)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5372..5394
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5386..5403
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5387..5403
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gtgccattcc gcctgaccta ccggtaagtg gttgtattta ttgcattagt acagtctgca
       61 aatgtttgtt gatttctggg tttatttatt tcaatatatg cactatatat ggatacatag
      121 agcagtgatt atggttggaa ttccatttgc caatattgct tcaatagaac tgcaagaacg
      181 gatgcgcttt tttgatgtgc aggtaaatgt tttgtacaaa gtgtagtttg tattgctttg
      241 attggagata ttattgattt gcatgattta acacgcatag gcaaaagaaa ctggatcggg
      301 cattactggt aaagagtact acgaaaacat ttgtatgcgt gcagtcaatc agtctgtagg
      361 caagtttata tgatgtggta ctatacttga ctatttaata acattgcagg cgtattgtgc
      421 tttgtaacag gtcgcgcaat caggcacaag aatgactatg cagtcattat atttctagac
      481 atgcggttca agcgagaccg tattgtagct aaattgccgg aatggatccg taattgtggt
      541 ttgacattcc cgaatcagtt tgggccatct attgccatga ttagcaaggt atgagccata
      601 tttttgactg cgtttatcag atgtgtttgg tattctgact attttattct ccatcactag
      661 tttttcaatt cacatagaaa gcccaccaac aaaaaagaat agccatttga atttaccatt
      721 gtattcatgt aaaacgaaat aattggtttg gaatggccga gtattcgttg tactttcaat
      781 tctttcaaac aacgactatt ttataatgca aactataagt ttaaactgga ttgtttggta
      841 caattctaca ctgcacaatc acccaattca actacaatcc aaatgttggg ttcaactgcc
      901 atcaaactcc tgtattctaa tatcagatac gttatagttc aagaaaacaa agaatgcaac
      961 tgttggtttg atatcaatca ctaaaagaac agaacttgga ttagtaaatt tggtaaaaat
     1021 tatcatttcg tgagaccgat ttattctatg attttttttc cactttacat gaatacgaca
     1081 acacattagc gccaattgag ccgattgaag gtcttgaaca cttgctgata atttaaacta
     1141 aacatatggg taaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
     1201 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
     1261 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
     1321 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
     1381 ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
     1441 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
     1501 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
     1561 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
     1621 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
     1681 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     1741 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     1801 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     1861 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     1921 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     1981 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
     2041 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
     2101 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
     2161 gggcaaagga aggaggagga ggatccgagc agaagctgat cagcgaagag gatctgtaaa
     2221 agcttgcaga gcatgagttg gctcatccac tacacgtaca atctaaataa atagctattt
     2281 gctaatctgt gtatttccaa gactttattc tcacatcagt actctaccaa tgtgctatta
     2341 gagttaatta tttgctaaaa gcgatcctag ctctgcgctg cgttcggtag cacgctggac
     2401 ggcttcagca aagatggctc gaatcttgtt cccttcgagt gagttgagtg ctgcctcggt
     2461 ggtaccattt ggagatgtga ctttgcgtct taattcagct ggatcctcgc ctgtatctgt
     2521 caacatgcga cccgctccta aacaagtctg tgctgcaagt ccacgagcaa catctgcagg
     2581 aagccccaat ttcactcctg cggcttcgag gcactcgacc aacaggaaaa aataagctgg
     2641 accagatcct aacatgggtc aaaaaagcat cagttgatca atcaccaaat atattttaca
     2701 caaaatcagt gttcaataca ttgaaatgaa cttaccagat atgcctgtta cgacatccaa
     2761 cagagattcc tccttgaccc agtagttttg cttcgataca gacttgagca cagcagaaac
     2821 aatttccttt tgattggtgg tgactgaagg agctgcatac atacccgttg ctccttcgac
     2881 aaccaacgca ggagtgtttg gcatgcatcg cacaattgct ggtgactcgg tcatcccttt
     2941 cttggctgaa catgtcaacc acttcaacaa gtttgaaatg gtaattccag cagcaattga
     3001 cacaattaat ggcttgtgag tcaagacggc gtccgaaatg ccttcagcaa cggatctcat
     3061 gacttgaggc ttgacagcaa gaataagaac atcgacagga gtcgtttggc tttgattggt
     3121 aaacgcaatt gcctgagtgt ttgaggtggt gacctgaact ttaaaccgtg aaactagacg
     3181 agcacgagct tcgtcccagg gttctgacac gataatattg gatgcggaaa gaccggattt
     3241 caacagtccg ccaataattg caccagccat attgcctcca ccaataaagc caagacgcaa
     3301 cccagtaaag tctgtcatgt tgtaatgcca agactattgc gagaattgtt gagcatcaaa
     3361 ctaaaaataa aaagaagaat caagccaacc ctcaggcgcg ccgtgtcgct ggaaagaaca
     3421 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3481 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3541 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3601 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3661 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3721 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3781 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3841 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3901 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3961 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4021 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4081 tcttttctac ggggtctgac gctctattca acaaagccgc cgtcccgtca agtcagcgta
     4141 aatgggtagg gggcttcaaa tcgtcctcgt gataccaatt cggagcctgc ttttttgtac
     4201 aaacttgttg ataatggcaa ttcaaggatc ttcacctaga tccttttaaa ttaaaaatga
     4261 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     4321 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     4381 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     4441 ataccgcgag agccacgctc accggctcca gatttatcag caataaacca gccagccgga
     4501 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     4561 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     4621 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4681 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4741 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4801 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4861 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4921 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4981 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     5041 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     5101 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     5161 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     5221 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     5281 ccccgaaaag tgccagatac ctgaaacaaa acccatcgta cggccaagga agtctccaat
     5341 aactgtgatc caccacaagc gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     5401 agtcatgcat aatccgcacg catctggaat aaggaa
//
