LOCUS       sequence-273591-        6464 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Hygromycin resistance from CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6464)
  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 6464)
  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: pEM01
FEATURES             Location/Qualifiers
     source          1..6464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(37..55)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        123..227
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(228..296,297..1088)
                     /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(446..465)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1259..1847
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1748..1767
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2001..2018
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2135..2156
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2171..2176,2177..2194,2195..2201)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2209..2225
                     /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     2214..2236
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2233..2249
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2233..2249
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2268..2288
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2270..2288
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        2312..2691
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2692..2895
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2846..2866
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             2905..3933
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2905..3933
                     /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      3955..4142
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4010..4034)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(4507..4526)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4508..4526)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4536..4553)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4536..4552)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4545..4567)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4693..5148
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4780..4799)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4990..5011
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5279..6082)
                     /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(6083..6298)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6322..6341)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6441..6463
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
       61 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
      121 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
      181 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
      241 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
      301 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
      361 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
      421 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
      481 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
      541 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
      601 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
      661 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
      721 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
      781 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
      841 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
      901 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
      961 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     1021 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     1081 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     1141 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     1201 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     1261 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     1321 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     1381 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     1441 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     1501 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     1561 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     1621 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     1681 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     1741 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     1801 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     1861 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     1921 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     1981 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
     2041 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     2101 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
     2161 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
     2221 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
     2281 actaaaggga acaaaagctg gagctgagct cgacattgat tattgactag ttattaatag
     2341 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     2401 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     2461 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     2521 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
     2581 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
     2641 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg
     2701 ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     2761 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     2821 tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     2881 tatataagca gagctttaat taacatgggt aaaaagcctg aactcaccgc gacgtctgtc
     2941 gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc
     3001 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     3061 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     3121 ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc
     3181 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt
     3241 ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc
     3301 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     3361 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     3421 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     3481 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     3541 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac
     3601 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     3661 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt
     3721 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     3781 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     3841 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga
     3901 cgccccagca ctcgtccgag ggcaaaggaa taatggcgcg ccacttctaa ataagcgaat
     3961 ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa
     4021 ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg
     4081 taggtcaggt tgctttctca ggtatagtat gaggtcgctc ttattgacca cacctctacc
     4141 ggcagatccg ctagggataa cagggtaata ttgtttagct tgcctcgtcc ccgccgggtc
     4201 acccggccag cgacatggag gcccagaata ccctccttga cagtcttgac gtgcgcagct
     4261 caggggcatg atgtgactgt cgcccgtaca tttagcccat acatccccat gtataatcat
     4321 ttgcatccat acattttgat ggccgcacgg cgcgaagcaa aaattacggc tcctcgctgc
     4381 agacctgcga gcagggaaac gctcccctca cagacgcgtg tacgcatgta acattatact
     4441 gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgcg gccggtaccc
     4501 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     4561 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     4621 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     4681 aatggcgaat ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4741 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4801 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4861 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4921 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4981 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     5041 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5101 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta
     5161 ttttctcctt acgcatctgt gcggtatttc acaccgcata gggtaataac tgatataatt
     5221 aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat acagtttttt
     5281 agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac
     5341 cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag
     5401 atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt
     5461 catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct cttccaccca
     5521 tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag
     5581 tacccttagt atattctcca gtagataggg agcccttgca tgacaattct gctaacatca
     5641 aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg ctaacaatac
     5701 ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtatacac
     5761 ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga
     5821 gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa
     5881 aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa
     5941 ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct
     6001 tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt
     6061 ccttatatgt agctttcgac atgatttatc ttcgtttcct gcaggttttt gttctgtgca
     6121 gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata
     6181 ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa
     6241 aaaaatttca aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa
     6301 ggtggtatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     6361 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     6421 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggt
//
