LOCUS       sequence-382616-        7497 bp    DNA     circular SYN 15-DEC-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 7497)
  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 7497)
  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-DGK1-3HA
FEATURES             Location/Qualifiers
     source          1..7497
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      42..239
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    302..335
                     /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(1394..1413)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1395..1413)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1500..1517)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1671..2259)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1751..1770)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2430..3221,3222..3290))
                     /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     3053..3072
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3291..3395)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3463..3481
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3519..3541)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3641..3660
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3741..3759
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3741..3758
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             5706..5795
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     terminator      5834..6021
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(5889..5913)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    6073..6106
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     gene            join(6161..7497,1..239)
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        6161..6504
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             join(6505..7497,1..36)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             join(6505..7497,1..36)
                     /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"
ORIGIN
        1 cgacgcccca gcactcgtcc gagggcaaag gaataatcag tactgacaat aaaaagattc
       61 ttgttttcaa gaacttgtca tttgtatagt ttttttatat tgtagttgtt ctattttaat
      121 caaatgttag cgtgatttat attttttttc gcctcgacat catctgccca gatgcgaagt
      181 taagtgcgca gaaagtaata tcatgcgtca atcgtatgtg aatgctggtc gctatactgc
      241 tgtcgattcg atactaacgc cgccatccag tgtcgaaaac gagctctcga gaacccttaa
      301 tataacttcg tataatgtat gctatacgaa gttattaggt gatatcagat ccactagtgg
      361 cctatgcggc cgcggaagca tatataccaa aaacaggagt gttttatatt cttcggtaag
      421 tcttttttac agtaactgga gtgcgcgatt cagtttatct gtggttaacg atccagcacc
      481 tcttcgatct ttggcatcca caaattattt ttctaaatat cctacatatc cgagtataga
      541 gttagaacct gtgtaaatta ttcagcagta tttccgtatt tttcacgcca ttagaaacat
      601 tcgacacaag cagttttatg aaaacattta tgttactaat atatgtgcca acaagcgcat
      661 ccaaatgggg cgcaacatta ttatttaatc gtaaacctac accatatttt catcagtaaa
      721 aaaaggcttt ttcatgcccc aactttgtca caaagcaatc atatcagaga acaataaaaa
      781 agaacaatcc tgcgaaatac gattaccaag atcctgaacc agcgttcatt tatcttggca
      841 tctcaaggat atatggttta tctgcgatta ccgcccagtg gccttattga tattgatatc
      901 attgtgtacg ttagaacaga aaaaaagaaa tttggaaaat ttttcaacct catcgaaaat
      961 tttcagaaaa gcaagaatta tttatgcttt cgcaaaaatt tcgcatataa gttatttttg
     1021 aaatagcagt cgataccaaa ttagctgtag aacctcaacc tcctcataca atggcttacg
     1081 ttttaactga aacttcagct ggttatgctc ttttaaaagc ttccgataag aaaatctaca
     1141 aatcttcaag cttaattcaa gacttggata gctctgacaa agtcttgaag gaattcaaga
     1201 tcgccgcctt ttccaagttc aactctgcag ctaacgcttt agaggaagcc aactccatta
     1261 ttgaaggtaa agtttcttcc caattagaaa aacttctgga agaaattaaa aaggacaaga
     1321 agtcgacctt gattgttagt gaaactaaac tggcaaacgc tattaataag ttgggccgcg
     1381 gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat
     1441 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     1501 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     1561 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1621 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1681 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1741 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     1801 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     1861 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     1921 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1981 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     2041 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     2101 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2161 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2221 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2281 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2341 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2401 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     2461 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2521 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2581 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     2641 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     2701 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     2761 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     2821 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     2881 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     2941 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3001 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3061 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3121 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3181 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3241 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3301 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3361 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3421 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     3481 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     3541 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     3601 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     3661 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc
     3721 ttatgtatca tacacatacg atttaggtga cactatagaa cgcggccgcc agctgaagct
     3781 tctatagaac taccatccag gaattataga ggaaattaac tgaatcagag atctattttg
     3841 aaacattcat ttacatgtaa ttgtctgcaa taaagcaata tttttgaaat atgcaagttt
     3901 actaacaaga ataaattctt ttttgatttt tatctttaac agtggctcat ttcaaagaat
     3961 accaagtcat tggtcgtcgt ttaccaactg aatccgttcc agaaccaaag ttgttcagaa
     4021 tgagaatttt tgcttcaaat gaagtcatcg ccaagtctcg ttactggtat ttcttgcaaa
     4081 aattgcacaa ggttaagaag gcttctggtg aaattgtttc catcaaccaa atcaacgaag
     4141 ctcacccaac caaggtcaag aacttcggtg tttgggttag atacgattcc agatctggta
     4201 ctcacaacat gtacaaggaa atcagagacg tatccagagt tgctgccgtc gaaaccttat
     4261 accaagacat ggctgctaga cacagagcta gatttagatc tattcacatc ttgaaggttg
     4321 ctgaaattga aaagactgct gatgtcaaga gacaatacgt caagcaattc ttgaccaagg
     4381 atttaaaatt cccattacct cacagagtcc aaaaatctac caagactttc tcttacaaga
     4441 gaccatcaac cttctactaa gccaacaaga tatttctacg ttttttagta taataaaatt
     4501 ttgtataatt cattttgtag attcaaggaa attttaaaat atatcctgtt tcttctctta
     4561 cattatgcac taccagcgtg aaagcacaat gtcatgccat gtatctttac aaaatccagg
     4621 gtccatagcg ggtaacaaat tattggtttt tctgcgataa atataaaacg tagcgtaatt
     4681 ataaaaatcc tgagcggtgt tgtcgtagag cagttgaaca ttacgtaaac agatatctac
     4741 gcatttctgt catttggatt catacagggt ttacagacaa tatggggacc gaagatgcca
     4801 ttgcccttcc aaatagcacg ctagagccgc gtaccgaagc taagcaaaga ctatcatcta
     4861 agagtcatca agtctcggcg aaagtaacga ttccagcaaa agaagaaatt agtagtagcg
     4921 atgacgatgc acacgttcca gtgacagaaa tacatttgaa atctcatgaa tggttcggcg
     4981 attttataac taaacatgaa attccccgta aggtgttcca ttcttccatt ggctttatta
     5041 ctttgtacct gtatacgcag ggtattaatt ataaaaatgt tttatggcct ttgatatacg
     5101 ccttcatcat attgtttatt ttggatctga taagactaaa ctggcccttt ttcaatatgc
     5161 tttactgtag aactgtgggt gcgctaatga gaaaaaagga gattcataca tacaatgggg
     5221 tattgtggta cattcttggg ttaatctttt cttttaactt tttctctaaa gatgttacct
     5281 taatatcgtt atttttgcta agttggtccg atacagccgc cgcaactatt ggaagaaagt
     5341 atggtcattt aacacccaaa gtggcaagaa ataaatccct tgcaggttcg atagctgcgt
     5401 ttacagttgg tgttatcacc tgctgggtat tttatggcta ttttgttcct gcctacaact
     5461 acgtcaacaa acctggcgag atccaatgga gcccagaaac aagcagatta agtttgaata
     5521 tgctatcctt gttaggtggt gtggtagctg ctttgagtga aggtatagat ttgttcaact
     5581 gggatgataa tttcactatt cctgtcctgt catcactttt tatgaacgca gtaatcaaaa
     5641 cattcaagaa acaagcttcg tacgctgcag gtcgtgggat tcctgggtta attaacatcg
     5701 ctagctaccc atacgatgtt cctgactatg cgggctatcc ctatgacgtc ccggactatg
     5761 caggatccta tccatatgac gttccagatt acgctgctca gtgccctagg tgaggcgcgc
     5821 cacttctaaa taagcgaatt tcttatgatt tatgattttt attattaaat aagttataaa
     5881 aaaaataagt gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt
     5941 cttgagtaac tctttcctgt aggtcaggtt gctttctcag gtatagtatg aggtcgctct
     6001 tattgaccac acctctaccg gcagatccgc tagggataac agggtaatat ctgcaggtcg
     6061 acaaccctta atataacttc gtataatgta tgctatacga agttattagg tctagagatc
     6121 tgtttagctt gcctcgtccc cgccgggtca cccggccagc gacatggagg cccagaatac
     6181 cctccttgac agtcttgacg tgcgcagctc aggggcatga tgtgactgtc gcccgtacat
     6241 ttagcccata catccccatg tataatcatt tgcatccata cattttgatg gccgcacggc
     6301 gcgaagcaaa aattacggct cctcgctgca gacctgcgag cagggaaacg ctcccctcac
     6361 agacgcgttg aattgtcccc acgccgcgcc cctgtagaga aatataaaag gttaggattt
     6421 gccactgagg ttcttctttc atatacttcc ttttaaaatc ttgctaggat acagttctca
     6481 catcacatcc gaacataaac aaccatgggt aaaaagcctg aactcaccgc gacgtctgtc
     6541 gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc
     6601 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat
     6661 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg
     6721 ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc
     6781 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt
     6841 ctgcagccgg tcgcggaggc aatggatgcg atcgctgcgg ccgatcttag ccagacgagc
     6901 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata
     6961 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt
     7021 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc
     7081 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata
     7141 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac
     7201 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg
     7261 aggcatccgg agcttgcagg atcgccacgg ctccgggcgt atatgctccg cattggtctt
     7321 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt
     7381 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc
     7441 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaac
//
