LOCUS Exported 8019 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Spizellomyces Hsp70 promoter driving expression of Hygromycin resistance fused to GFP. ACCESSION . VERSION . KEYWORDS pEM09 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8019) 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 8019) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8019 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 746..1334 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1235..1254 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1488..1505 /label=L4440 /note="L4440 vector, forward primer" promoter 1917..1947 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1955..1971 /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 1960..1982 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 1979..1995 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 1979..1995 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 2014..2034 /label=T3 /note="T3 promoter, forward primer" promoter 2016..2034 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" CDS 3135..4160 /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 4179..4892 /codon_start=1 /product="Aequoria victoria green fluorescent protein" /label=GFP /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITHGMDELYK" primer_bind complement(4206..4234) /label=GFP-R /note="GFP, reverse primer. Does NOT anneal to EGFP" primer_bind 4829..4849 /label=GFP-F /note="GFP, forward primer. Does NOT anneal to EGFP" terminator 4912..5099 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind complement(4967..4991) /label=pHybLex-R /note="ADH terminator, reverse primer" primer_bind complement(5549..5568) /label=T7 /note="T7 promoter, forward primer" promoter complement(5550..5568) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5578..5595) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(5578..5594) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5587..5609) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 5735..6190 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5822..5841) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6032..6053 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(6321..7124) /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(7125..7340) /gene="S. cerevisiae URA3" /label=URA3 promoter primer_bind complement(7364..7383) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7483..7505 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(7543..7561) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 7629..7733 /gene="bla" /label=AmpR promoter CDS 7734..575 /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(7952..7971) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 61 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 121 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 181 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 241 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 301 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 361 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 421 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 481 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 541 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 601 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 661 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 721 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 781 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 841 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 901 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 961 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 1021 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 1081 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 1141 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 1201 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 1261 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 1321 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 1381 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 1441 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 1501 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 1561 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 1621 ttaatgtgag ttacctcact cattaggccg ttgatatagg ccagtaacta atgcagctac 1681 tgcatagaat cgatcaagag actcgagttt cggcaatgga actgtgcgcg gagcatctcc 1741 aagtgtggat ttcgtagaga aggttgtgac tcctgccatt gcaaaagtag caaaaggctc 1801 ccagggatct tctctcacct cattgaaaaa agcctttgaa cttgcggaac aggtatgttg 1861 acaactgtac gcatagacaa catgagtcgg tctaatgcat actttgacac ctttcattta 1921 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca 1981 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa 2041 aagctggagc tgagctcttt taaaatcttg tctttgtgca ctcttctctc cctgtcatat 2101 tgctttacgt cgagagactg gtcaaaggat ccggtgagat cattgagcgc attgttaccc 2161 aaaatcagca tcgagaaatt aacaagatcg cgacgctggg cgacgggaac atgtatgcga 2221 gcacattgga taaggttgcc aaaggcggtc gatagagctc tagtatttga atagtgattc 2281 ctgctgctca ttctcacaaa tgtctatgaa tgtcgtctgt ccatcgcttg aaatcaaaga 2341 atgcgataca tggcgtgtcc tgatgcagcg attccaattg taaatgcgtg catgtgttcc 2401 atgggcctgc gttaaaatgg attctgcatg tgcagcaata aatcaataaa aggatgagac 2461 atgcagcaag gaattcactg aaagctatgg aaggatatcg aataaagaac aatttggaag 2521 ctcatagaaa ttggatgtga tggtccaaaa tggatttccg ttttgctttc cgcgcgacgc 2581 tctttttctt tttacttgtc ttctttcaac aaaccccccc ttcttctact ttctcgttga 2641 aaacgataca aggaagaaag cgagagttca aaaggtaata aaagcccttg cttttgcagt 2701 ttgcttttac gtagagtttg ttcgcgtcat cagccagagc gcccttttac ctggcaacgc 2761 gacgctttca atttatttct cgctgtgtga aatacttttc atgctgacta tcccttgtcc 2821 atccccattc cttccgtcat ttgcttgcta gctcgctcgc gaagattccc tctcaatcct 2881 tctccctatt tggcaccctc ctcccgacac cgggcactaa cctcgccgca gatattctca 2941 tcggcgtcgt cgctattgga gttgtaggcc tacttgcaaa cttcctcttt cgcagaacat 3001 caagcagcac caagcgtcgg cgtaataaaa aacgcacgaa gcaagaagct actcatcgtc 3061 aacaggagcc gcttttagac gagcgacata aagacactac cagtaacgga ccaaagatca 3121 gcacaattta attaatgggt aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc 3181 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc 3241 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg 3301 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc 3361 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg 3421 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg 3481 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc 3541 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg 3601 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg 3661 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg 3721 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca 3781 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct 3841 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg 3901 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct 3961 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg 4021 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg 4081 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca 4141 ctcgtccgag ggcaaaggaa ggaggaggag gaggaggaag taaaggagaa gaacttttca 4201 ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg 4261 tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca 4321 ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttcact tatggtgttc 4381 aatgcttttc aagataccca gatcatatga aacggcatga ctttttcaag agtgccatgc 4441 ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac tacaagacac 4501 gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta aaaggtattg 4561 attttaaaga agatggaaac attcttggac acaaattgga atacaactat aactcacaca 4621 atgtatacat catggcagac aaacaaaaga atggaatcaa agttaacttc aaaattagac 4681 acaacattga agatggaagc gttcaactag cagaccatta tcaacaaaat actccaattg 4741 gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct gccctttcga 4801 aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga 4861 ttacacatgg catggatgaa ctatacaaat aggcgcgcca cttctaaata agcgaatttc 4921 ttatgattta tgatttttat tattaaataa gttataaaaa aaataagtgt atacaaattt 4981 taaagtgact cttaggtttt aaaacgaaaa ttcttattct tgagtaactc tttcctgtag 5041 gtcaggttgc tttctcaggt atagtatgag gtcgctctta ttgaccacac ctctaccggc 5101 agatccgcta gggataacag ggtaatattg tttagcttgc ctcgtccccg ccgggtcacc 5161 cggccagcga caggaacacc cagggatagt gcagcaattg ttggtggaaa tgatggacaa 5221 attggaaata cgataagtgg ggattcattt tgggaaaaga caaaagaaag atgctttagg 5281 gacctatggg gggtgcgcag ctcaggggca tgatgtgact gtcgcccgta catttagccc 5341 atacatcccc atgtataatc atttgcatcc atacattttg atggccgcac ggcgcgaagc 5401 aaaaattacg gctcctcgct gcagacctgc gagcagggaa acgctcccct cacagacgcg 5461 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg 5521 ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact 5581 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 5641 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 5701 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc 5761 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 5821 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 5881 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 5941 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 6001 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 6061 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 6121 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 6181 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 6241 tagggtaata actgatataa ttaaattgaa gctctaattt gtgagtttag tatacatgca 6301 tttacttata atacagtttt ttagttttgc tggccgcatc ttctcaaata tgcttcccag 6361 cctgcttttc tgtaacgttc accctctacc ttagcatccc ttccctttgc aaatagtcct 6421 cttccaacaa taataatgtc agatcctgta gagaccacat catccacggt tctatactgt 6481 tgacccaatg cgtctccctt gtcatctaaa cccacaccgg gtgtcataat caaccaatcg 6541 taaccttcat ctcttccacc catgtctctt tgagcaataa agccgataac aaaatctttg 6601 tcgctcttcg caatgtcaac agtaccctta gtatattctc cagtagatag ggagcccttg 6661 catgacaatt ctgctaacat caaaaggcct ctaggttcct ttgttacttc ttctgccgcc 6721 tgcttcaaac cgctaacaat acctgggccc accacaccgt gtgcattcgt aatgtctgcc 6781 cattctgcta ttctgtatac acccgcagag tactgcaatt tgactgtatt accaatgtca 6841 gcaaattttc tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc ctttagcggc 6901 ttaactgtgc cctccatgga aaaatcagtc aagatatcca catgtgtttt tagtaaacaa 6961 attttgggac ctaatgcttc aactaactcc agtaattcct tggtggtacg aacatccaat 7021 gaagcacaca agtttgtttg cttttcgtgc atgatattaa atagcttggc agcaacagga 7081 ctaggatgag tagcagcacg ttccttatat gtagctttcg acatgattta tcttcgtttc 7141 ctgcaggttt ttgttctgtg cagttgggtt aagaatactg ggcaatttca tgtttcttca 7201 acactacata tgcgtatata taccaatcta agtctgtgct ccttccttcg ttcttccttc 7261 tgttcggaga ttaccgaatc aaaaaaattt caaggaaacc gaaatcaaaa aaaagaataa 7321 aaaaaaaatg atgaattgaa aaggtggtat ggtgcactct cagtacaatc tgctctgatg 7381 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 7441 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 7501 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat 7561 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 7621 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 7681 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 7741 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 7801 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 7861 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 7921 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 7981 acgccgggca agagcaactc ggtcgccgca tacactatt //