LOCUS       sequence-389062-        7919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PAH1 overexpression. Promotes the formation of diacylglycerol from 
            phosphatidate. Uses antibiotic resistance marker hphMX6. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7919)
  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 7919)
  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: UG75-ADH1-PAH1-3HA
FEATURES             Location/Qualifiers
     source          1..7919
                     /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        1853..2557
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 promoter
                     /note="promoter for alcohol dehydrogenase 1"
     CDS             5222..5311
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      5350..5537
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5405..5429)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(5556..5575)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5557..5575)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5662..5679)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5833..6421)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5913..5932)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6592..7383,7384..7452))
                     /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     7215..7234
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7453..7557)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7625..7643
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7681..7703)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7803..7822
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7903..7919,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7903..7919,1)
                     /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 ggatcctttt
     1861 gttgtttccg ggtgtacaat atggacttcc tcttttctgg caaccaaacc catacatcgg
     1921 gattcctata ataccttcgt tggtctccct aacatgtagg tggcggaggg gagatataca
     1981 atagaacaga taccagacaa gacataatgg gctaaacaag actacaccaa ttacactgcc
     2041 tcattgatgg tggtacataa cgaactaata ctgtagccct agacttgata gccatcatca
     2101 tatcgaagtt tcactaccct ttttccattt gccatctatt gaagtaataa taggcgcatg
     2161 caacttcttt tctttttttt tcttttctct ctcccccgtt gttgtctcac catatccgca
     2221 atgacaaaaa aatgatggaa gacactaaag gaaaaaatta acgacaaaga cagcaccaac
     2281 agatgtcgtt gttccagagc tgatgagggg tatctcgaag cacacgaaac tttttccttc
     2341 cttcattcac gcacactact ctctaatgag caacggtata cggccttcct tccagttact
     2401 tgaatttgaa ataaaaaaaa gtttgctgtc ttgctatcaa gtataaatag acctgcaatt
     2461 attaatcttt tgtttcctcg tcattgttct cgttcccttt cttccttgtt tctttttctg
     2521 cacaatattt caagctatac caagcataca atcaactatc tcatatacaa tgtctatccc
     2581 aatcgatatg cagtacgtag gcagagctct tgggtctgtg tctaaaacat ggtcttctat
     2641 caatccggct acgctatcag gtgctataga tgtcattgta gtggagcatc cagacggaag
     2701 gctatcatgt tctccctttc atgtgaggtt cggcaaattt caaattctaa agccatctca
     2761 aaagaaagtc caagtgttta taaatgagaa actgagtaat atgccaatga aactgagtga
     2821 ttctggagaa gcctatttcg ttttcgagat gggtgaccag gtcactgatg tccctgacga
     2881 attgcttgtg tcgcccgtga tgagcgccac atcaagcccc cctcaatcac ctgaaacatc
     2941 catcttagaa ggaggaaccg agggtgaagg tgaaggtgaa aatgaaaata agaagaagga
     3001 aaagaaagtg ctagaggaac cagatttttt agatatcaat gacactggag attcaggcag
     3061 taaaaatagt gaaactacag ggtcgctttc tcctactgaa tcctctacaa cgacaccacc
     3121 agattcagtt gaagagagga agcttgttga gcagcgtaca aagaactttc agcaaaaact
     3181 aaacaaaaaa ctcactgaaa tccatatacc cagtaaactt gataacaatg gcgacttact
     3241 actagacact gaaggttaca agccaaacaa gaatatgatg catgacacag acatacaact
     3301 gaagcagttg ttaaaggacg aattcggtaa tgattcagat atttccagtt ttatcaagga
     3361 ggacaaaaat ggcaacatca agatcgtaaa tccttacgag caccttactg atttatctcc
     3421 tccaggtacg cctccaacaa tggccacaag cggatcagtt ttaggcttag atgcaatgga
     3481 atcaggaagt actttgaatt cgttatcttc ttcaccttct ggttccgata ctgaggacga
     3541 aacatcattt agcaaagaac aaagcagtaa aagtgaaaaa actagcaaga aaggaacagc
     3601 agggagcggt gagaccgaga aaagatacat acgaacgata agattgacta atgaccagtt
     3661 aaagtgccta aatttaactt atggtgaaaa tgatctgaaa ttttccgtag atcacggaaa
     3721 agctattgtt acgtcaaaat tattcgtttg gaggtgggat gttccaattg ttatcagtga
     3781 tattgatggc accatcacaa aatcggacgc tttaggccat gttctggcaa tgataggaaa
     3841 agactggacg cacttgggtg tagccaagtt atttagcgag atctccagga atggctataa
     3901 tatactctat ctaactgcaa gaagtgctgg acaagctgat tccacgagga gttatttgcg
     3961 atcaattgaa cagaatggca gcaaactacc aaatgggcct gtgattttat cacccgatag
     4021 aacgatggct gcgttaaggc gggaagtaat actaaaaaaa cctgaagtct ttaaaatcgc
     4081 gtgtctaaac gacataagat ccttgtattt tgaagacagt gataacgaag tggatacaga
     4141 ggaaaaatca acaccatttt ttgccggctt tggtaatagg attactgatg ctttatctta
     4201 cagaactgtg gggataccta gttcaagaat tttcacaata aatacagagg gtgaggttca
     4261 tatggaatta ttggagttag caggttacag aagctcctat attcatatca atgagcttgt
     4321 cgatcatttc tttccaccag tcagccttga tagtgtcgat ctaagaacta atacttccat
     4381 ggttcctggc tcccccccta atagaacgtt ggataacttt gactcagaaa ttacttcagg
     4441 tcgcaaaacg ctatttagag gcaatcagga agagaaattc acagacgtaa atttttggag
     4501 agacccgtta gtcgacatcg acaacttatc ggatattagc aatgatgatt ctgataacat
     4561 cgatgaagat actgacgtat cacaacaaag caacattagt agaaataggg caaattcagt
     4621 caaaaccgcc aaggtcacta aagccccgca aagaaatgtg agcggcagca caaataacaa
     4681 cgaagtttta gccgcttcgt ctgatgtaga aaatgcgtct gacctggtga gttcccatag
     4741 tagctcagga tccacgccca ataaatctac aatgtccaaa ggggacattg gaaaacaaat
     4801 atatttggag ctaggttctc cacttgcatc gccaaaacta agatatttag acgatatgga
     4861 tgatgaagac tccaattaca atagaactaa atcaaggaga gcatcttctg cagccgcgac
     4921 tagtatcgat aaagagttca aaaagctctc tgtgtcaaag gccggcgctc caacaagaat
     4981 tgtttcaaag atcaacgttt caaatgacgt acattcactt gggaattcag ataccgaatc
     5041 acgaagggag caaagtgtta atgaaacagg gcgcaatcag ctaccccaca actcaatgga
     5101 cgataaagat ttggattcaa gagtaagcga tgaattcgat gacgatgaat tcgacgaaga
     5161 tgaattcgaa gattcgtacg ctgcaggtcg tgggattcct gggttaatta acatcgctag
     5221 ctacccatac gatgttcctg actatgcggg ctatccctat gacgtcccgg actatgcagg
     5281 atcctatcca tatgacgttc cagattacgc tgctcagtgc cctaggtgag gcgcgccact
     5341 tctaaataag cgaatttctt atgatttatg atttttatta ttaaataagt tataaaaaaa
     5401 ataagtgtat acaaatttta aagtgactct taggttttaa aacgaaaatt cttattcttg
     5461 agtaactctt tcctgtaggt caggttgctt tctcaggtat agtatgaggt cgctcttatt
     5521 gaccacacct ctaccggccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc
     5581 gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     5641 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5701 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5761 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5821 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5881 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5941 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6001 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6061 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6121 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6181 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6241 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6301 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6361 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6421 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6481 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6541 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6601 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6661 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6721 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6781 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6841 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6901 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6961 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7021 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7081 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7141 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7201 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7261 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7321 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7381 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7441 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7501 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7561 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     7621 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     7681 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     7741 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     7801 tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg
     7861 cggctacaat taatacataa ccttatgtat catacacata cgatttaggt gacactata
//
