LOCUS       sequence-273666-        6950 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Hygromycin resistance from Spizellomyces Hsp70 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6950)
  AUTHORS   Medina EM, Robinson KA, Bellingham-Johnstun K, Ianiri G, Laplante C,
            Fritz-Laylin LK, Buchler NE
  TITLE     Genetic transformation of Spizellomyces punctatus, a resource for 
            studying chytrid biology and evolutionary cell biology.
  JOURNAL   Elife. 2020 May 11;9. pii: 52741. doi: 10.7554/eLife.52741.
  PUBMED    32392127
REFERENCE   2  (bases 1 to 6950)
  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: "Elife."; 
            date: "2020-05-11"; volume: "9. pii: 52741. doi"; pages: " 
            10.7554/eLife.52741"
COMMENT     Sequence Label: pEM03
FEATURES             Location/Qualifiers
     source          1..6950
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(246..461)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(485..504)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     604..626
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(664..682)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        750..854
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(855..923,924..1715)
                     /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(1073..1092)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1886..2474
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2375..2394
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2628..2645
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2762..2783
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2798..2803,2804..2821,2822..2828)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2836..2852
                     /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     2841..2863
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2860..2876
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2860..2876
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2895..2915
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2897..2915
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             4018..5046
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             4018..5046
                     /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"
     terminator      5068..5255
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5123..5147)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5620..5639)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5621..5639)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5649..5666)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5649..5665)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5658..5680)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5806..6261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5893..5912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6103..6124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(6392..6950,1..245))
                     /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"
ORIGIN
        1 gcggataatg cctttagcgg cttaactgtg ccctccatgg aaaaatcagt caagatatcc
       61 acatgtgttt ttagtaaaca aattttggga cctaatgctt caactaactc cagtaattcc
      121 ttggtggtac gaacatccaa tgaagcacac aagtttgttt gcttttcgtg catgatatta
      181 aatagcttgg cagcaacagg actaggatga gtagcagcac gttccttata tgtagctttc
      241 gacatgattt atcttcgttt cctgcaggtt tttgttctgt gcagttgggt taagaatact
      301 gggcaatttc atgtttcttc aacactacat atgcgtatat ataccaatct aagtctgtgc
      361 tccttccttc gttcttcctt ctgttcggag attaccgaat caaaaaaatt tcaaggaaac
      421 cgaaatcaaa aaaaagaata aaaaaaaaat gatgaattga aaaggtggta tggtgcactc
      481 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
      541 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
      601 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
      661 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
      721 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
      781 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
      841 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
      901 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
      961 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1021 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1081 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1141 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1201 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1261 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1321 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     1381 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     1441 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     1501 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     1561 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     1621 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     1681 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     1741 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     1801 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     1861 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     1921 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     1981 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2041 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2101 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2161 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2221 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2281 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     2341 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     2401 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     2461 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     2521 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     2581 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     2641 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     2701 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     2761 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     2821 gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg
     2881 attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag ctgagctctt
     2941 ttaaaatctt gtctttgtgc actcttctct ccctgtcata ttgctttacg tcgagagact
     3001 ggtcaaagga tccggtgaga tcattgagcg cattgttacc caaaatcagc atcgagaaat
     3061 taacaagatc gcgacgctgg gcgacgggaa catgtatgcg agcacattgg ataaggttgc
     3121 caaaggcggt cgatagagct ctagtatttg aatagtgatt cctgctgctc attctcacaa
     3181 atgtctatga atgtcgtctg tccatcgctt gaaatcaaag aatgcgatac atggcgtgtc
     3241 ctgatgcagc gattccaatt gtaaatgcgt gcatgtgttc catgggcctg cgttaaaatg
     3301 gattctgcat gtgcagcaat aaatcaataa aaggatgaga catgcagcaa ggaattcact
     3361 gaaagctatg gaaggatatc gaataaagaa caatttggaa gctcatagaa attggatgtg
     3421 atggtccaaa atggatttcc gttttgcttt ccgcgcgacg ctctttttct ttttacttgt
     3481 cttctttcaa caaacccccc cttcttctac tttctcgttg aaaacgatac aaggaagaaa
     3541 gcgagagttc aaaaggtaat aaaagccctt gcttttgcag tttgctttta cgtagagttt
     3601 gttcgcgtca tcagccagag cgccctttta cctggcaacg cgacgctttc aatttatttc
     3661 tcgctgtgtg aaatactttt catgctgact atcccttgtc catccccatt ccttccgtca
     3721 tttgcttgct agctcgctcg cgaagattcc ctctcaatcc ttctccctat ttggcaccct
     3781 cctcccgaca ccgggcacta acctcgccgc agatattctc atcggcgtcg tcgctattgg
     3841 agttgtaggc ctacttgcaa acttcctctt tcgcagaaca tcaagcagca ccaagcgtcg
     3901 gcgtaataaa aaacgcacga agcaagaagc tactcatcgt caacaggagc cgcttttaga
     3961 cgagcgacat aaagacacta ccagtaacgg accaaagatc agcacaattt aattaacatg
     4021 ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     4081 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     4141 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     4201 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     4261 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     4321 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     4381 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     4441 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     4501 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     4561 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     4621 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     4681 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     4741 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     4801 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     4861 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     4921 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     4981 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     5041 gaataatggc gcgccacttc taaataagcg aatttcttat gatttatgat ttttattatt
     5101 aaataagtta taaaaaaaat aagtgtatac aaattttaaa gtgactctta ggttttaaaa
     5161 cgaaaattct tattcttgag taactctttc ctgtaggtca ggttgctttc tcaggtatag
     5221 tatgaggtcg ctcttattga ccacacctct accggcagat ccgctaggga taacagggta
     5281 atattgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg gaggcccaga
     5341 ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt
     5401 acatttagcc catacatccc catgtataat catttgcatc catacatttt gatggccgca
     5461 cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga aacgctcccc
     5521 tcacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg
     5581 acgctcgaag gctttaattt gcggccggta cccaattcgc cctatagtga gtcgtattac
     5641 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     5701 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     5761 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg
     5821 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     5881 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     5941 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     6001 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     6061 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     6121 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     6181 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6241 aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     6301 ttcacaccgc atagggtaat aactgatata attaaattga agctctaatt tgtgagttta
     6361 gtatacatgc atttacttat aatacagttt tttagttttg ctggccgcat cttctcaaat
     6421 atgcttccca gcctgctttt ctgtaacgtt caccctctac cttagcatcc cttccctttg
     6481 caaatagtcc tcttccaaca ataataatgt cagatcctgt agagaccaca tcatccacgg
     6541 ttctatactg ttgacccaat gcgtctccct tgtcatctaa acccacaccg ggtgtcataa
     6601 tcaaccaatc gtaaccttca tctcttccac ccatgtctct ttgagcaata aagccgataa
     6661 caaaatcttt gtcgctcttc gcaatgtcaa cagtaccctt agtatattct ccagtagata
     6721 gggagccctt gcatgacaat tctgctaaca tcaaaaggcc tctaggttcc tttgttactt
     6781 cttctgccgc ctgcttcaaa ccgctaacaa tacctgggcc caccacaccg tgtgcattcg
     6841 taatgtctgc ccattctgct attctgtata cacccgcaga gtactgcaat ttgactgtat
     6901 taccaatgtc agcaaatttt ctgtcttcga agagtaaaaa attgtacttg
//
