LOCUS       sequence-382502-        9202 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 9202)
  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 9202)
  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-PAH1-3HA
FEATURES             Location/Qualifiers
     source          1..9202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     177..196
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     430..447
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     534..553
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        534..552
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(1601..1634)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            complement(1697..3272)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     terminator      complement(1697..1894)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(1900..2928)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             complement(1900..2928)
                     /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"
     promoter        complement(2929..3272)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     protein_bind    complement(3327..3360)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      complement(3412..3599)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     3520..3544
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(3638..3727)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     promoter        complement(7390..7408)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(7391..7408)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(7489..7508)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7608..7630
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7668..7686)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7754..7858
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7859..7927,7928..8719)
                     /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(8077..8096)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(8890..9202,1..276)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
       61 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
      121 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
      181 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
      241 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
      301 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
      361 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
      421 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
      481 ctccccgcgc gttggccgat tcattaatgc aggttaacct ggcttatcga aattaatacg
      541 actcactata gggagaccgg cagatccgcg gtagctggca aaattggtat tattctctct
      601 cttaaaggat tatgatcctg ttggtaccca gattctaggt ctccattgaa caatgattga
      661 aaaatttccc accattccag taaaaacgtg tcattagcgt ccatcagcat tttcgaaggt
      721 aatagcccat cttcactaac aatgtcaccc ttagatgcaa tatccattaa tccctgcttc
      781 aggctggcgt tcaagctagt tttcccaccc gaagcagatc cgtttattct agataaagga
      841 aggtcagctt cgcttaaaaa ttttgcggca gagttccagt aattgttttt gattaaatag
      901 ttataaatat gggcgtacaa taattgctta gagttcttag ccattacatc cgaaacgaaa
      961 actcgagaat ccgcatcaaa ctgcaactga gtatggctat tcaaaccttt gatattggga
     1021 gctgagctaa aatccgtgtt actagagcgc tcatttgtat taatattggt cgtgttatcc
     1081 atatctttat catgcacctt tttcttattt cactgcagtt taacgaaaaa ttgacaaaag
     1141 aaccgggaga taccaaaggg aaaaaccaaa agagtcacac aggattgcaa tagaaatctt
     1201 tctggtcttt tctactatgg tgattttatt atacccctcc tcttcctgtt atacacgcat
     1261 tgtatttcgg cattgcctct tggccgcttt tttcacctga cgtaatacct gttgcagcaa
     1321 gcgagtttcg tgtgcgtata gaaaagtatt taccttgaca tttcgaagaa gtatgtaatt
     1381 accaaatagc tcagaagaac acttggtacg taacaatcat cgtttacctt tttcttctat
     1441 caaaaaattt tcttcttttt tttttccttc tcttttctta aataaaattg aaaaattgtt
     1501 acttagtact tcataatttt acttaatatg cagtatggat cgttataaat aatattcggc
     1561 ccgcggccgc ataggccact agtggatctg atatcaccta ataacttcgt atagcataca
     1621 ttatacgaag ttatattaag ggttctcgag agctcgtttt cgacactgga tggcggcgtt
     1681 agtatcgaat cgacagcagt atagcgacca gcattcacat acgattgacg catgatatta
     1741 ctttctgcgc acttaacttc gcatctgggc agatgatgtc gaggcgaaaa aaaatataaa
     1801 tcacgctaac atttgattaa aatagaacaa ctacaatata aaaaaactat acaaatgaca
     1861 agttcttgaa aacaagaatc tttttattgt cagtactgat tattcctttg ccctcggacg
     1921 agtgctgggg cgtcggtttc cactatcggc gagtacttct acacagccat cggtccagac
     1981 ggccgcgctt ctgcgggcga tttgtgtacg cccgacagtc ccggctccgg atcggacgat
     2041 tgcgtcgcat cgaccctgcg cccaagctgc atcatcgaaa ttgccgtcaa ccaagctctg
     2101 atagagttgg tcaagaccaa tgcggagcat atacgcccgg agccgtggcg atcctgcaag
     2161 ctccggatgc ctccgctcga agtagcgcgt ctgctgctcc atacaagcca accacggcct
     2221 ccagaagaag atgttggcga cctcgtattg ggaatccccg aacatcgcct cgctccagtc
     2281 aatgaccgct gttatgcggc cattgtccgt caggacattg ttggagccga aatccgcgtg
     2341 cacgaggtgc cggacttcgg ggcagtcctc ggcccaaagc atcagctcat cgagagcctg
     2401 cgcgacggac gcactgacgg tgtcgtccat cacagtttgc cagtgataca catggggatc
     2461 agcaatcgcg catatgaaat cacgccatgt agtgtattga ccgattcctt gcggtccgaa
     2521 tgggccgaac ccgctcgtct ggctaagatc ggccgcagcg atcgcatcca ttgcctccgc
     2581 gaccggctgc agaacagcgg gcagttcggt ttcaggcagg tcttgcaacg tgacaccctg
     2641 tgcacggcgg gagatgcaat aggtcaggct ctcgctgaat tccccaatgt caagcacttc
     2701 cggaatcggg agcgcggccg atgcaaagtg ccgataaaca taacgatctt tgtagaaacc
     2761 atcggcgcag ctatttaccc gcaggacata tccacgccct cctacatcga agctgaaagc
     2821 acgagattct tcgccctccg agagctgcat caggtcggag acgctgtcga acttttcgat
     2881 cagaaacttc tcgacagacg tcgcggtgag ttcaggcttt ttacccatgg ttgtttatgt
     2941 tcggatgtga tgtgagaact gtatcctagc aagattttaa aaggaagtat atgaaagaag
     3001 aacctcagtg gcaaatccta accttttata tttctctaca ggggcgcggc gtggggacaa
     3061 ttcaacgcgt ctgtgagggg agcgtttccc tgctcgcagg tctgcagcga ggagccgtaa
     3121 tttttgcttc gcgccgtgcg gccatcaaaa tgtatggatg caaatgatta tacatgggga
     3181 tgtatgggct aaatgtacgg gcgacagtca catcatgccc ctgagctgcg cacgtcaaga
     3241 ctgtcaagga gggtattctg ggcctccatg tcgctggccg ggtgacccgg cggggacgag
     3301 gcaagctaaa cagatctcta gacctaataa cttcgtatag catacattat acgaagttat
     3361 attaagggtt gtcgacctgc agatattacc ctgttatccc tagcggatct gccggtagag
     3421 gtgtggtcaa taagagcgac ctcatactat acctgagaaa gcaacctgac ctacaggaaa
     3481 gagttactca agaataagaa ttttcgtttt aaaacctaag agtcacttta aaatttgtat
     3541 acacttattt tttttataac ttatttaata ataaaaatca taaatcataa gaaattcgct
     3601 tatttagaag tggcgcgcct cacctagggc actgagcagc gtaatctgga acgtcatatg
     3661 gataggatcc tgcatagtcc gggacgtcat agggatagcc cgcatagtca ggaacatcgt
     3721 atgggtagct agcgatgtta attaacccag gaatcccacg acctgcagcg tacgaagctt
     3781 gatcttcgaa ttcatcttcg tcgaattcat cgtcatcgaa ttcatcgctt actcttgaat
     3841 ccaaatcttt atcgtccatt gagttgtggg gtagctgatt gcgccctgtt tcattaacac
     3901 tttgctccct tcgtgattcg gtatctgaat tcccaagtga atgtacgtca tttgaaacgt
     3961 tgatctttga aacaattctt gttggagcgc cggcctttga cacagagagc tttttgaact
     4021 ctttatcgat actagtcgcg gctgcagaag atgctctcct tgatttagtt ctattgtaat
     4081 tggagtcttc atcatccata tcgtctaaat atcttagttt tggcgatgca agtggagaac
     4141 ctagctccaa atatatttgt tttccaatgt cccctttgga cattgtagat ttattgggcg
     4201 tggatcctga gctactatgg gaactcacca ggtcagacgc attttctaca tcagacgaag
     4261 cggctaaaac ttcgttgtta tttgtgctgc cgctcacatt tctttgcggg gctttagtga
     4321 ccttggcggt tttgactgaa tttgccctat ttctactaat gttgctttgt tgtgatacgt
     4381 cagtatcttc atcgatgtta tcagaatcat cattgctaat atccgataag ttgtcgatgt
     4441 cgactaacgg gtctctccaa aaatttacgt ctgtgaattt ctcttcctga ttgcctctaa
     4501 atagcgtttt gcgacctgaa gtaatttctg agtcaaagtt atccaacgtt ctattagggg
     4561 gggagccagg aaccatggaa gtattagttc ttagatcgac actatcaagg ctgactggtg
     4621 gaaagaaatg atcgacaagc tcattgatat gaatatagga gcttctgtaa cctgctaact
     4681 ccaataattc catatgaacc tcaccctctg tatttattgt gaaaattctt gaactaggta
     4741 tccccacagt tctgtaagat aaagcatcag taatcctatt accaaagccg gcaaaaaatg
     4801 gtgttgattt ttcctctgta tccacttcgt tatcactgtc ttcaaaatac aaggatctta
     4861 tgtcgtttag acacgcgatt ttaaagactt caggtttttt tagtattact tcccgcctta
     4921 acgcagccat cgttctatcg ggtgataaaa tcacaggccc atttggtagt ttgctgccat
     4981 tctgttcaat tgatcgcaaa taactcctcg tggaatcagc ttgtccagca cttcttgcag
     5041 ttagatagag tatattatag ccattcctgg agatctcgct aaataacttg gctacaccca
     5101 agtgcgtcca gtcttttcct atcattgcca gaacatggcc taaagcgtcc gattttgtga
     5161 tggtgccatc aatatcactg ataacaattg gaacatccca cctccaaacg aataattttg
     5221 acgtaacaat agcttttccg tgatctacgg aaaatttcag atcattttca ccataagtta
     5281 aatttaggca ctttaactgg tcattagtca atcttatcgt tcgtatgtat cttttctcgg
     5341 tctcaccgct ccctgctgtt cctttcttgc tagttttttc acttttactg ctttgttctt
     5401 tgctaaatga tgtttcgtcc tcagtatcgg aaccagaagg tgaagaagat aacgaattca
     5461 aagtacttcc tgattccatt gcatctaagc ctaaaactga tccgcttgtg gccattgttg
     5521 gaggcgtacc tggaggagat aaatcagtaa ggtgctcgta aggatttacg atcttgatgt
     5581 tgccattttt gtcctccttg ataaaactgg aaatatctga atcattaccg aattcgtcct
     5641 ttaacaactg cttcagttgt atgtctgtgt catgcatcat attcttgttt ggcttgtaac
     5701 cttcagtgtc tagtagtaag tcgccattgt tatcaagttt actgggtata tggatttcag
     5761 tgagtttttt gtttagtttt tgctgaaagt tctttgtacg ctgctcaaca agcttcctct
     5821 cttcaactga atctggtggt gtcgttgtag aggattcagt aggagaaagc gaccctgtag
     5881 tttcactatt tttactgcct gaatctccag tgtcattgat atctaaaaaa tctggttcct
     5941 ctagcacttt cttttccttc ttcttatttt cattttcacc ttcaccttca ccctcggttc
     6001 ctccttctaa gatggatgtt tcaggtgatt gaggggggct tgatgtggcg ctcatcacgg
     6061 gcgacacaag caattcgtca gggacatcag tgacctggtc acccatctcg aaaacgaaat
     6121 aggcttctcc agaatcactc agtttcattg gcatattact cagtttctca tttataaaca
     6181 cttggacttt cttttgagat ggctttagaa tttgaaattt gccgaacctc acatgaaagg
     6241 gagaacatga tagccttccg tctggatgct ccactacaat gacatctata gcacctgata
     6301 gcgtagccgg attgatagaa gaccatgttt tagacacaga cccaagagct ctgcctacgt
     6361 actgcataat cgaccgatgt gtctatcttc agtaatttct tccctgtatg tttcttagaa
     6421 ggtaaaactg tgattacgaa ggtgtagtgt ccaattggtc tgattaacac ttcttgaaca
     6481 cgttttacta aattgttgaa tgtattttct ttataaacac aagcccccac gatggcctac
     6541 tcttcacttg tccttgctct tccttctacc ctcaccgtgc gctgctttca acgctggccg
     6601 gaaaagccga gaaaaagaga tagataatag agaaaagaga gcccattgtc taaagggagg
     6661 gaaaccgaag gacgaaaagg gcccgcgaca gaccgtttag agaatgagca gcacgtgggc
     6721 acttcgattt tgtctcccag tgatgtagtg ttatgatata caagatgagt ttggaccctg
     6781 ttcaagcaaa tctgtcacat ttgttagact tttttttaaa cgagccctcc aaacaagaac
     6841 gctcagtgtt aggaactgcg aacgaagaga gaaatgcgta ttaaacttag atgagtattc
     6901 ttgccgtaaa tgactatata caaaacttat tcggaagaac ctatatctat gtatgtgaaa
     6961 tgataagtac aagacccact gtgatgtgca gtgatggcga atagggtgag gtcccttgta
     7021 ctgtctttat tgtagccgct tgtaggaaaa gttttatcac ataaaattat cctctagaga
     7081 gttagatgag ccagaaatca ggctcttagt ccgttggaaa tgcgttactt aatccgcgta
     7141 gggtcatttg atacagaagc aacattatac tgctctgaac acttgtccag acgaatctag
     7201 ggccaacgcc actaaagaaa cctaacacac cttcagattg gtagaccgtc cgaaggctga
     7261 gcgaaatcga gttggaaagt gctgccggtc tgccgtttgt tacatgtgga tgtgttactg
     7321 agtatgattt gttgctttgg ctgggcggct gttgagtttg gactctaaag cttcagctgg
     7381 cggccgcgtt ctatagtgtc acctaaatcg tatgtgtatg atacataagg ttatgtatta
     7441 attgtagccg cgttctaacg acaatatgtc catatggtgc actctcagta caatctgctc
     7501 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     7561 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     7621 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     7681 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     7741 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     7801 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     7861 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     7921 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     7981 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     8041 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     8101 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     8161 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     8221 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     8281 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     8341 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     8401 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     8461 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     8521 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     8581 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     8641 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     8701 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     8761 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     8821 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     8881 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     8941 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     9001 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     9061 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     9121 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     9181 accggataag gcgcagcggt cg
//
