LOCUS       sequence-417145-        6192 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Reporter gene expression in B. dendrobatidis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6192)
  AUTHORS   Kalinka E, Brody SM, Swafford AJM, Medina EM, Fritz-Laylin LK
  TITLE     Genetic transformation of the frog-killing chytrid fungus 
            Batrachochytrium dendrobatidis.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Jan 23;121(4):e2317928121. doi: 
            10.1073/pnas.2317928121. Epub 2024 Jan 18.
  PUBMED    38236738
REFERENCE   2  (bases 1 to 6192)
  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: "2024-01-23"; volume: "121(4):e2317928121. 
            doi"; pages: " 10.1073/pnas.2317928121. Epub 2024 Jan 18"
COMMENT     Sequence Label: pEK36
FEATURES             Location/Qualifiers
     source          1..6192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(155..172)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(326..914)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(406..425)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1009..1824)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1009..1824)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1732..1751
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1816..1835
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     misc_feature    2117..2141
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    2318..2339
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2354..2359,2360..2377,2378..2384)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2392..2408
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2397..2419
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2416..2432
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2416..2432
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     terminator      complement(2447..2634)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     2555..2579
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(join(2655..3368,3369..3371,3372..3374))
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(2700..2721)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3308..3329
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(3390..4415)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(3390..4415)
                     /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(4714..4731)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4714..4730)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4723..4745)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_feature    4926..4950
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(5117..5920)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(5921..6136)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
ORIGIN
        1 gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat
       61 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
      121 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
      181 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      241 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      301 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      361 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      421 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      481 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
      541 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
      601 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
      661 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
      721 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
      781 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
      841 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
      901 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
      961 aaaactcacg ttaagggatt ttggtcatgc aggatcatga attaattctt agaaaaactc
     1021 atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg
     1081 aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag
     1141 atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc
     1201 ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga
     1261 gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc
     1321 gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag
     1381 acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg
     1441 caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac
     1501 ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg
     1561 gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat
     1621 ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc
     1681 atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc
     1741 ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga
     1801 cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag
     1861 ttttattgtt catgatctgg atcacaggca gcaacgctct gtcatcgtta caatcaacat
     1921 gctaccctcc gcgagatcat ccgtgtttca aacccggcag cttagttgcc gttcttccga
     1981 atagcatcgg taacatgagc aaagtctgcc gccttacaac ggctctcccg ctgacgccgt
     2041 cccggactga tgggctgcct gtatcgagtg gtgattttgt gccgagctgc cggtcgggga
     2101 gctgttggct ggctggtggc aggatatatt gtggtgtaaa caaattgacg cttagacaac
     2161 ttaataacac attgcggacg tttttaatgt actgaattaa cgccgaattg ctctagccaa
     2221 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     2281 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
     2341 aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg
     2401 gataacaatt tcacacagga aacagctatg acatgattac gaattcccgg tagaggtgtg
     2461 gtcaataaga gcgacctcat actatacctg agaaagcaac ctgacctaca ggaaagagtt
     2521 actcaagaat aagaattttc gttttaaaac ctaagagtca ctttaaaatt tgtatacact
     2581 tatttttttt ataacttatt taataataaa aatcataaat cataagaaat tcgcttattt
     2641 agaagtggcg cgccttactt gtacagctcg tccatgccat gtgtaatccc ggcggcggtc
     2701 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagcttggac
     2761 tgatggctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc
     2821 tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcaa cgttgtggcg gatcttgaag
     2881 ttagccttga tgcagttctt ctgcttgtcg gccgtgatat agacgtagtg gctgttgaag
     2941 ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc
     3001 tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtaggta
     3061 ccgtcgtcct tgaaagagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg
     3121 aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcaggccac gccgtagccg
     3181 aaggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc
     3241 agggtcagct tgccgttggt ggcatcgccc tcgccctcgc cgcggacgct gaacttgtgg
     3301 ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg
     3361 cccttgctca ccatggatcc tcctcctcct tcctttgccc tcggacgagt gctggggcgt
     3421 cggtttccac tatcggcgag tacttctaca cagccatcgg tccagacggc cgcgcttctg
     3481 cgggcgattt gtgtacgccc gacagtcccg gctccggatc ggacgattgc gtcgcatcga
     3541 ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca agctctgata gagttggtca
     3601 agaccaatgc ggagcatata cgcccggagc cgcggcgatc ctgcaagctc cggatgcctc
     3661 cgctcgaagt agcgcgtctg ctgctccata caagccaacc acggcctcca gaagaagatg
     3721 ttggcgacct cgtattggga atccccgaac atcgcctcgc tccagtcaat gaccgctgtt
     3781 atgcggccat tgtccgtcag gacattgttg gagccgaaat ccgcgtgcac gaggtgccgg
     3841 acttcggggc agtcctcggc ccaaagcatc agctcatcga gagcctgcgc gacggacgca
     3901 ctgacggtgt cgtccatcac agtttgccag tgatacacat ggggatcagc aatcgcgcat
     3961 atgaaatcac gccatgtagt gtattgaccg attccttgcg gtccgaatgg gccgaacccg
     4021 ctcgtctggc taagatcggc cgcagcgatc gcatccatgg cctccgcgac cggctgcaga
     4081 acagcgggca gttcggtttc aggcaggtct tgcaacgtga caccctgtgc acggcgggag
     4141 atgcaatagg tcaggctctc gctgaattcc ccaatgtcaa gcacttccgg aatcgggagc
     4201 gcggccgatg caaagtgccg ataaacataa cgatctttgt agaaaccatc ggcgcagcta
     4261 tttacccgca ggacatatcc acgccctcct acatcgaagc tgaaagcacg agattcttcg
     4321 ccctccgaga gctgcatcag gtcggagacg ctgtcgaact tttcgatcag aaacttctcg
     4381 acagacgtcg cggtgagttc aggcttttta cccattaatt aatttgtgtg tgtgatggat
     4441 gagagtggtg cgggtgaaag gtcttggtca agtcgcaaag tgtttgtgaa aaagacgtag
     4501 cgggggaagc gcacacacgc ccgcctttta tagacgaagc ttcgagcagg tcatttaacg
     4561 atgcttagta gcaatataca ataaagccta attgggacta attacggtca cgtgaaaaac
     4621 tcaccatgta gccttttatg gaaatattgt acacgctact aagccactgt aatatcggcc
     4681 tcatcgtctc cgcttggagc ataaaagctt ggcactggcc gtcgttttac aacgtcgtga
     4741 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4801 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4861 tggcgaatga gcttgagctt ggatcagatt gtcgtttccc gccttcagtt taaactatca
     4921 gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataat
     4981 cggatattta aaagggcgtg aaaaggttta tccgttcgtc catttgtatg tgcatgccag
     5041 gtaataactg atataattaa attgaagctc taatttgtga gtttagtata catgcattta
     5101 cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct tcccagcctg
     5161 cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat agtcctcttc
     5221 caacaataat aatgtcagat cctgtagaga ccacatcatc cacggttcta tactgttgac
     5281 ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac caatcgtaac
     5341 cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa tctttgtcgc
     5401 tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag cccttgcatg
     5461 acaattctgc tagcatcaaa aggcctctag gttcctttgt tacttcttct gccgcctgct
     5521 tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg tctgcccatt
     5581 ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca atgtcagcaa
     5641 attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt agcggcttaa
     5701 ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt aaacaaattt
     5761 tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca tccaatgaag
     5821 cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca acaggactag
     5881 gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt cgtttcctgc
     5941 aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt tcttcaacac
     6001 tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct tccttctgtt
     6061 cggagattac cgaatcaaaa aaatttcaag gaaaccgaaa tcaaaaaaaa gaataaaaaa
     6121 aaaatgatga attgaaaagg tggtatggtg cactctcagt acaatctgct ctgatgccgc
     6181 atagttaagc ca
//
