LOCUS       sequence-389064-        6203 bp    DNA     circular SYN 15-APR-2025
DEFINITION  DGK1 overexpression. Promotes the formation of phosphatidate from 
            diacylglycerol. Uses antibiotic resistance marker hphMX6. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6203)
  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 6203)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-DGK1-3HA
FEATURES             Location/Qualifiers
     source          1..6203
                     /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             3506..3595
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      3634..3821
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3689..3713)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3840..3859)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3841..3859)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3946..3963)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4117..4705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4197..4216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4876..5667,5668..5736))
                     /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     5499..5518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5737..5841)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5909..5927
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5965..5987)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6087..6106
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6187..6203,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6187..6203,1)
                     /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 gggaccgaag atgccattgc ccttccaaat agcacgctag agccgcgtac
     2641 cgaagctaag caaagactat catctaagag tcatcaagtc tcggcgaaag taacgattcc
     2701 agcaaaagaa gaaattagta gtagcgatga cgatgcacac gttccagtga cagaaataca
     2761 tttgaaatct catgaatggt tcggcgattt tataactaaa catgaaattc cccgtaaggt
     2821 gttccattct tccattggct ttattacttt gtacctgtat acgcagggta ttaattataa
     2881 aaatgtttta tggcctttga tatacgcctt catcatattg tttattttgg atctgataag
     2941 actaaactgg ccctttttca atatgcttta ctgtagaact gtgggtgcgc taatgagaaa
     3001 aaaggagatt catacataca atggggtatt gtggtacatt cttgggttaa tcttttcttt
     3061 taactttttc tctaaagatg ttaccttaat atcgttattt ttgctaagtt ggtccgatac
     3121 agccgccgca actattggaa gaaagtatgg tcatttaaca cccaaagtgg caagaaataa
     3181 atcccttgca ggttcgatag ctgcgtttac agttggtgtt atcacctgct gggtatttta
     3241 tggctatttt gttcctgcct acagctacgt caacaaacct ggcgagatcc aatggagccc
     3301 agaaacaagc agattaagtt tgaatatgct atccttgtta ggtggtgtgg tagctgcttt
     3361 gagtgaaggt atagatttgt tcaactggga tgataatttc actattcctg tcctgtcatc
     3421 actttttatg aacgcagtaa tcaaaacatt caagaaatcg tacgctgcag gtcgtgggat
     3481 tcctgggtta attaacatcg ctagctaccc atacgatgtt cctgactatg cgggctatcc
     3541 ctatgacgtc ccggactatg caggatccta tccatatgac gttccagatt acgctgctca
     3601 gtgccctagg tgaggcgcgc cacttctaaa taagcgaatt tcttatgatt tatgattttt
     3661 attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga ctcttaggtt
     3721 ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt gctttctcag
     3781 gtatagtatg aggtcgctct tattgaccac acctctaccg gccgcggatc tgccggtctc
     3841 cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca
     3901 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3961 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4021 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4081 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4141 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4201 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4261 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4321 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4381 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4441 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4501 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4561 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4621 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4681 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4741 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4801 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4861 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4921 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4981 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5041 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5101 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5161 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5221 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5281 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5341 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5401 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5461 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5521 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5581 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5641 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5701 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5761 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5821 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     5881 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     5941 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     6001 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     6061 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     6121 tatggacata ttgtcgttag aacgcggcta caattaatac ataaccttat gtatcataca
     6181 catacgattt aggtgacact ata
//
