LOCUS       sequence-382342-        7460 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Yeast expression of Aur1 under GAL promoter. Uses antibiotic 
            resistance marker hphMX6. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7460)
  AUTHORS   Li D, Yang SG, He CW, Zhang ZT, Liang Y, Li H, Zhu J, Su X, Gong Q, 
            Xie Z
  TITLE     Excess diacylglycerol at the endoplasmic reticulum disrupts 
            endomembrane homeostasis and autophagy.
  JOURNAL   BMC Biol. 2020 Aug 28;18(1):107. doi: 10.1186/s12915-020-00837-w.
  PUBMED    32859196
REFERENCE   2  (bases 1 to 7460)
  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: "BMC Biol.";
            date: "2020-08-28"; volume: "18(1):107. doi"; pages: " 
            10.1186/s12915-020-00837-w"
COMMENT     Sequence Label: GAL-Aur1-2GFP-Hygromycin
FEATURES             Location/Qualifiers
     source          1..7460
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    53..86
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            141..1716
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        141..484
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             485..1513
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             485..1513
                     /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      1519..1716
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1779..1812
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1852..2293
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    1852..1969
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     2264..2287
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             2300..3502
                     /codon_start=1
                     /gene="S. cerevisiae AUR1 with a dominant F158Y mutation"
                     /product="aureobasidin A-resistant mutant of S. cerevisiae 
                     inositol phosphorylceramide synthase (Aeed et al., 2009)"
                     /label=AurR
                     /note="confers fungal resistance to aureobasidin A"
                     /translation="MANPFSRWFLSERPPNCHVADLETSLDPHQTLLKVQKYKPALSDW
                     VHYIFLGSIMLFVFITNPAPWIFKILFYCFLGTLFIIPATSQFFFNALPILTWVALYFT
                     SSYFPDDRRPPITVKVLPAVETILYGDNLSDILATSTNSFLDILAWLPYGLFHFGAPFV
                     VAAILFVFGPPTVLQGYAFAFGYMNLFGVIMQNVFPAAPPWYKILYGLQSANYDMHGSP
                     GGLARIDKLLGINMYTTAFSNSSVIFGAFPSLHSGCATMEALFFCYCFPKLKPLFIAYV
                     CWLWWSTMYLTHHYFVDLMAGSVLSYVIFQYTKYTHLPIVDTSLFCRWSYTSIEKYDIS
                     KSDPLAADSNDIESVPLSNLELDFDLNMTDEPSVSPSLFDGSTSVSRSSATSITSLGVK
                     RA"
     CDS             3554..4267
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             4304..5017
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     protein_bind    5037..5070
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5097..5116)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5098..5116)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5203..5220)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5374..5962)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5454..5473)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6133..6924,6925..6993))
                     /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     6756..6775
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6994..7098)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7166..7184
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7222..7244)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7344..7363
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7444..7460,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7444..7460,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg acaaccctta atataacttc
       61 gtataatgta tgctatacga agttattagg tctagagatc tgtttagctt gcctcgtccc
      121 cgccgggtca cccggccagc gacatggagg cccagaatac cctccttgac agtcttgacg
      181 tgcgcagctc aggggcatga tgtgactgtc gcccgtacat ttagcccata catccccatg
      241 tataatcatt tgcatccata cattttgatg gccgcacggc gcgaagcaaa aattacggct
      301 cctcgctgca gacctgcgag cagggaaacg ctcccctcac agacgcgttg aattgtcccc
      361 acgccgcgcc cctgtagaga aatataaaag gttaggattt gccactgagg ttcttctttc
      421 atatacttcc ttttaaaatc ttgctaggat acagttctca catcacatcc gaacataaac
      481 aaccatgggt aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa
      541 gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag
      601 cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta
      661 caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct
      721 tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt
      781 cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc
      841 aatggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc
      901 gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca
      961 tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct
     1021 cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga
     1081 tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag
     1141 cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg
     1201 gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg
     1261 atcgccacgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt
     1321 ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg
     1381 atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac
     1441 cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag
     1501 ggcaaaggaa taatcagtac tgacaataaa aagattcttg ttttcaagaa cttgtcattt
     1561 gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt gatttatatt
     1621 ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca
     1681 tgcgtcaatc gtatgtgaat gctggtcgct atactgctgt cgattcgata ctaacgccgc
     1741 catccagtgt cgaaaacgag ctctcgagaa cccttaatat aacttcgtat aatgtatgct
     1801 atacgaagtt attaggtgat atcagatcca ctagtggcct atgcggccgc gcggattaga
     1861 agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt cctcgtcttc
     1921 accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga acaataaaga
     1981 ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac ctggccccac
     2041 aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga ttagtttttt
     2101 agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat taacagatat
     2161 ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc ggtttgtatt
     2221 acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac ctctatactt
     2281 taacgtcaag gagccgcgga tggcaaaccc tttttcgaga tggtttctat cagagagacc
     2341 tccaaactgc catgtagccg atttagaaac aagtttagat ccccatcaaa cgttgttgaa
     2401 ggtgcaaaaa tacaaacccg ctttaagcga ctgggtgcat tacatcttct tgggatccat
     2461 catgctgttt gtgttcatta ctaatcccgc accttggatc ttcaagatcc ttttttattg
     2521 tttcttgggc actttattca tcattccagc tacgtcacag tttttcttca atgccttgcc
     2581 catcctaaca tgggtggcgc tgtatttcac ttcatcgtac tttccagatg accgcaggcc
     2641 tcctattact gtcaaagtgt taccagcggt ggaaacaatt ttatacggcg acaatttaag
     2701 tgatattctt gcaacatcga cgaattcctt tttggacatt ttagcatggt taccgtacgg
     2761 actatttcat tttggggccc catttgtcgt tgctgccatc ttattcgtat ttggtccacc
     2821 aactgttttg caaggttatg cttttgcatt tggttatatg aacctgtttg gtgttatcat
     2881 gcaaaatgtc tttccagccg ctcccccatg gtataaaatt ctctatggat tgcaatcagc
     2941 caactatgat atgcatggct cgcctggtgg attagctaga attgataagc tactcggtat
     3001 taatatgtat actacagctt tttcaaattc ctccgtcatt ttcggtgctt ttccttcact
     3061 gcattccggg tgtgctacta tggaagccct gtttttctgt tattgttttc caaaattgaa
     3121 gcccttgttt attgcttatg tttgctggtt atggtggtca actatgtatc tgacacacca
     3181 ttattttgta gaccttatgg caggttctgt gctgtcatac gttattttcc agtacacaaa
     3241 gtacacacat ttaccaattg tagatacatc tcttttttgc agatggtcat acacttcaat
     3301 tgagaaatac gatatatcaa agagtgatcc attggctgca gattcaaacg atatcgaaag
     3361 tgtccctttg tccaacttgg aacttgactt tgatcttaat atgactgatg aacccagtgt
     3421 aagcccttcg ttatttgatg gatctacttc tgtttctcgt tcgtccgcca cgtctataac
     3481 gtcactaggt gtaaagaggg ctgcttcgta cgctgcaggt cgtgggattc ctgggttaat
     3541 taacatcgct agcatgagta aaggagaaga acttttcact ggagttgtcc caattcttgt
     3601 tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga
     3661 tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc
     3721 atggccaaca cttgtcacta ctttcactta tggtgttcaa tgcttttcaa gatacccaga
     3781 tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag
     3841 aactatattt ttcaaagatg acgggaacta caagacacgt gctgaagtca agtttgaagg
     3901 tgataccctt gttaatagaa tcgagttaaa aggtattgat tttaaagaag atggaaacat
     3961 tcttggacac aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa
     4021 acaaaagaat ggaatcaaag ttaacttcaa aattagacac aacattgaag atggaagcgt
     4081 tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc
     4141 agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga
     4201 ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact
     4261 atacaaacct ccagttccaa cgcgtgatct agagaagcca ttcatgagta aaggagaaga
     4321 acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa
     4381 attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta cccttaaatt
     4441 tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta ctttcactta
     4501 tggtgttcaa tgcttttcaa gatacccaga tcatatgaaa cggcatgact ttttcaagag
     4561 tgccatgccc gaaggttatg tacaggaaag aactatattt ttcaaagatg acgggaacta
     4621 caagacacgt gctgaagtca agtttgaagg tgataccctt gttaatagaa tcgagttaaa
     4681 aggtattgat tttaaagaag atggaaacat tcttggacac aaattggaat acaactataa
     4741 ctcacacaat gtatacatca tggcagacaa acaaaagaat ggaatcaaag ttaacttcaa
     4801 aattagacac aacattgaag atggaagcgt tcaactagca gaccattatc aacaaaatac
     4861 tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcca cacaatctgc
     4921 cctttcgaaa gatcccaacg aaaagagaga ccacatggtc cttcttgagt ttgtaacagc
     4981 tgctgggatt acacatggca tggatgaact atacaaacct aggtgaggcg cgccatataa
     5041 cttcgtataa tgtatgctat acgaagttat taggtctaga gcggatctgc cggtctccct
     5101 atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg
     5161 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5221 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5281 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5341 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5401 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5461 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5521 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5581 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5641 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5701 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5761 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5821 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5881 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5941 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     6001 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     6061 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     6121 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6181 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6241 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6301 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6361 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6421 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6481 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6541 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6601 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6661 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6721 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6781 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6841 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6901 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6961 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     7021 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     7081 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     7141 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     7201 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     7261 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     7321 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     7381 ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
     7441 acgatttagg tgacactata
//
