LOCUS       sequence-382612-        8087 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Marker for yeast mitochondria. Integration of 2xGFP tag at AFG3 C 
            terminus. Uses auxotrophic marker URA3(Candida albicans). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8087)
  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 8087)
  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: AFG3-2EGFP-ter-URA3
FEATURES             Location/Qualifiers
     source          1..8087
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(332..365)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      complement(419..616)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    complement(1571..1604)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      complement(1887..2074)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     1995..2019
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     CDS             complement(2095..2811)
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             complement(2098..2811)
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             complement(2848..3561)
                     /codon_start=1
                     /product="yeast-enhanced green fluorescent protein"
                     /label=yeGFP
                     /note="codons optimized for translation in S. cerevisiae 
                     and C. albicans"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGIDELYK"
     promoter        complement(4998..5016)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4999..5016)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5097..5116)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5216..5238
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5276..5294)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5362..5466
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5467..5535,5536..6327)
                     /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(5685..5704)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6498..7086
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6987..7006
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7240..7257
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7344..7363
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7344..7362
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggtcaatggt aacgaaggta gtaaccatga tgtgataatg cagaacgacg aaggtgaggt
       61 tggcgtgagc aacaacttga tcatcgacga ggaaactttt taagttgaga actaaaaagc
      121 agtattgttt tcgaatatgt attatttttt tttatttttt ttattactcg ttatcaatat
      181 atatatatac atgtatatta ttactaaaag aatgagaaaa taagatagga tcctggaagg
      241 gggatcaggt aaagatataa agtgagatga agaagccacc gttttatttc ccgcggccgc
      301 ataggccact agtggatctg atatcatcga tataacttcg tatagcatac attatacgaa
      361 gttatgaatt cgagctcgtt ttcgacactg gatggcggcg ttagtatcga atcgacagca
      421 gtatagcgac cagcattcac atacgattga cgcatgatat tactttctgc gcacttaact
      481 tcgcatctgg gcagatgatg tcgaggcgaa aaaaaatata aatcacgcta acatttgatt
      541 aaaatagaac aactacaata taaaaaaact atacaaatga caagttcttg aaaacaagaa
      601 tctttttatt gtcagtactg attataattg gccagtcttt ttcaaataag cattccaacc
      661 agcatctcta taccttttac cttcaatatc tggatctctt cctttaccaa acaatcctct
      721 accaacaatg ataatatcag ttccagtgct aacaacttca tcaacagttc tatattgttg
      781 tcctaatcca tcacctttat catctaatcc aactccaggt gtcataataa gccaatcaaa
      841 tccttcttct tgtccaccca tatcacgttg ggcaataaat ccaataacaa attccttatc
      901 ggatttagca atttcaacag ttttttgaga atattctcca tatgctaatg atcccactga
      961 tgataattca gctaacatca ataaccctct tggctcttgg ttggtggtgg tttctttagc
     1021 tccctgtttt aatccttcaa ctactccatt cccagtgaca ccatgagcat tagtaatatc
     1081 tgcccaacta ctaattttat aaactccacc aatatattgt ttcttcacgg tattaccaat
     1141 atcagcaaat tttctatctt caaaaatcat aaattgatgt ttacgtgaaa gttctaataa
     1201 tggttcaata gtggattcat aggaaaaatc attgattata tcaatatgag tcttgattaa
     1261 gcatacataa ggacccaatt tatcaattaa ttcaaggaat tccttagtgg tatcaacatc
     1321 aattgatgca cataaattgg ttttcttcag ttccattaat cgaaataatc gttgtgctac
     1381 tggtgaggca tgagtttctg ctctctcact ataggtctta gtgttgactg tcatggttgt
     1441 ttatgttcgg atgtgatgtg agaactgtat cctagcaaga ttttaaaagg aagtatatga
     1501 aagaagaacc tcagtggcaa atcctaacct tttatatttc tctacagggg cgcggcgtgg
     1561 ggacaattca ataacttcgt atagcataca ttatacgaag ttatacgcgt ctgtgagggg
     1621 agcgtttccc tgctcgcagg tccgcagcga ggagccgtaa tttttgcttc gcgccgtgcg
     1681 gccatcaaaa tgtatggatg caaatgatta tacatgggga tgtatgggct aaatgtacgg
     1741 gcgacagtca catcatgccc ctgagctgcg cacgtcaaga ctgtcaagga gggtattctg
     1801 ggcctccatg tcgctggccg ggtgacccgg cggggacgag gcaagctaaa cagatctata
     1861 ttaccctgtt atccctagcg gatctgccgg tagaggtgtg gtcaataaga gcgacctcat
     1921 actatacctg agaaagcaac ctgacctaca ggaaagagtt actcaagaat aagaattttc
     1981 gttttaaaac ctaagagtca ctttaaaatt tgtatacact tatttttttt ataacttatt
     2041 taataataaa aatcataaat cataagaaat tcgcttattt agaagtggcg cgccttattt
     2101 atataattca tccataccat gggtaatacc agcagcagta acaaattcta acaagaccat
     2161 gtggtctctc ttttcgtttg gatctttgga taaggcagat tgagtggata agtaatggtt
     2221 gtctggtaac aagactggac catcaccaat tggagtattt tgttgataat ggtcagctaa
     2281 ttgaacagaa ccatcttcaa tgttgtgtct aattttgaag ttaactttga taccattctt
     2341 ttgtttgtca gccatgatgt aaacattgtg agagttatag ttgtattcca atttgtgacc
     2401 taaaatgtta ccatcttctt taaaatcaat accttttaat tcgattctat taactaaggt
     2461 atcaccttca aacttgactt cagctctggt cttgtagtta ccgtcatctt tgaaaaaaat
     2521 agttctttct tgaacataac cttctggcat ggcagacttg aaaaagtcat gttgtttcat
     2581 atgatctggg tatctagaaa aacattgaac accataagtt aaagtagtga ctaaggttgg
     2641 ccatggaact ggcaatttac cagtagtaca aataaatttt aaggtcaatt taccgtaagt
     2701 agcatcacct tcaccttcac cggagacaga aaatttgtga ccattaacat caccatctaa
     2761 ttcaaccaaa attgggacaa caccagtgaa taattcttca cctttagaca tgaatggctt
     2821 ctctagatca cgcgttggaa ctggaatttt gtacaattca tcaataccat gggtaatacc
     2881 agcagcagta acaaattcta acaagaccat gtggtctctc ttttcgtttg gatctttgga
     2941 taaggcagat tgagtggata agtaatggtt gtctggtaac aagactggac catcaccaat
     3001 tggagtattt tgttgataat ggtcagctaa ttgaacagaa ccatcttcaa tgttgtgtct
     3061 aattttgaag ttaactttga taccattctt ttgtttgtca gccatgatgt aaacattgtg
     3121 agagttatag ttgtattcca atttgtgacc taaaatgtta ccatcttctt taaaatcaat
     3181 accttttaat tcgattctat taactaaggt atcaccttca aacttgactt cagctctggt
     3241 cttgtagtta ccgtcatctt tgaaaaaaat agttctttct tgaacataac cttctggcat
     3301 ggcagacttg aaaaagtcat gttgtttcat atgatctggg tatctagaaa aacattgaac
     3361 accataagtt aaagtagtga ctaaggttgg ccatggaact ggcaatttac cagtagtaca
     3421 aataaatttt aaggtcaatt taccgtaagt agcatcacct tcaccttcac cggagacaga
     3481 aaatttgtga ccattaacat caccatctaa ttcaaccaaa attgggacaa caccagtgaa
     3541 taattcttca cctttagaca tgttaattaa accagcaccg tcaccgatcc gtcgacctgc
     3601 agcgtacgaa gcatttgttg ctgcaggtgc ctcaggcggt tcggtattgc tctttggatc
     3661 caaatatttt tcaaaagcct cgttcctttc cttgaatggc cttggaccca ataatctaat
     3721 catgtcttct ctagtaattg cttccttacg tagcaattct tttgcgacaa gatcgacttt
     3781 gtccaaattt ttggttagca attctgtaca agctcggtgt gcatcatcta ctatagattt
     3841 aacttctaaa tcaatggttc ttgccgtttt attactgaag ggtttgttaa ctttgaagtt
     3901 tccatcattc tgatcaaacg acaggtagcc aatcttgggt gacattccta gggatgtaac
     3961 catggcattt gccatctgtg taaccttttt gaaatcatca tgagcaccgc tagtcaccga
     4021 tggaaaatgt agctcctcag aaacacggcc accaagagcc atgatcattc tatgtctgaa
     4081 ttgctcctca gatatcaaat attgatccgg tggtaggtac tgggcatagc ctaaagcacc
     4141 ttgtccacgc gggatgatgc ttactttcag aagtggatcc gcatatttta gaaaccaacc
     4201 acaaacggca tgtcctgcct catgataggc cactgacctt ttttcttcct tagaaaggac
     4261 tcttgttttc ttctctagtc ccgcaatgac tctttcaatg gcttgctcaa agtgatggat
     4321 agtgatatat gggtcattat gtctggcagc gattaatgca gcctcattac aagcattagc
     4381 gatatctgca ccagtaaaac ctggagtaag cgtagccaat ttcccagaaa gattattcat
     4441 atcatctgta agaaggggat ccagattcag tcttttcaag tgaaccaggt atatttgctg
     4501 cctaccattg acatcaggag aatcaatttg gatatgtcta tcgaaccttc ccggtctcat
     4561 caaagcatta tccaacacat cgggcctatt tgtacccgca aggactacga cttggtcgga
     4621 agtagtgaat ccgtccattt ccaccaataa ttgattcagc gtagcttctc tttcatcgtt
     4681 agcgccaccg agagcgccgc ctttgcctct ttctttaccg atagcgtcaa tttcatctat
     4741 aaagataatc gagggggcca tagaccttgc ttgagtaaac aagtctctta cacgtgaagc
     4801 acccacccca acgaacattt cgacgaactc agaacctgat actgacaaga agggcacgtt
     4861 cgcctcacct gcggtggcct tggccaagag agtcttaccg gtacctgggg gtccagaaag
     4921 aatagcgcct cgtggaatct tggcacccaa tttagtgtac ttacctgggt tcttcaagct
     4981 tcagctggcg gccgcgttct atagtgtcac ctaaatcgta tgtgtatgat acataaggtt
     5041 atgtattaat tgtagccgcg ttctaacgac aatatgtcca tatggtgcac tctcagtaca
     5101 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5161 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5221 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5281 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5341 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5401 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5461 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5521 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5581 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5641 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5701 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5761 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5821 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     5881 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     5941 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6001 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6061 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6121 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6181 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6241 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6301 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6361 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6421 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6481 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6541 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6601 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6661 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6721 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6781 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6841 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     6901 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     6961 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7021 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7081 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7141 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     7201 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     7261 gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc
     7321 aggttaacct ggcttatcga aattaatacg actcactata gggagaccgg cagatccgcg
     7381 gatggaattg agaaaacagt gtacgttgat aagttaatca gatgtaaatt tcaagataat
     7441 ttagagttct tgcagtggtt aaagaaacat tggattcgac ataaagatga aagcgtgtat
     7501 gatccagatg caaggcgcaa gtatcgtccc attataacga ataatagtgc aaccaaaccc
     7561 cgtacagtat ccaaccctac cactgcaaag aggtcatcaa gcacaggcac tgggagtgcc
     7621 atgtcaggag gattagcaac aagacattct tcgctgggaa taaacggtag cagaaaaacc
     7681 agtgttaccc aaggtcagtt ggtagcgata caagcggaat taactaaatc gcaggaaacg
     7741 atcggttcat tgaatgaaga gatagaacaa tacaaaggaa cagtaagtac tttagagatt
     7801 gaaagagaat tttacttcaa caaactacgc gacatagaaa tcctggtcca taccactcaa
     7861 gatttaataa atgaaggtgt ttataaattt aatgatgaga ccatcaccgg ccatggtaat
     7921 ggaaatgggg gagcattatt acggttcgtc aagaaggtgg aaagtatttt atatgctact
     7981 gcagaaggat ttgaaatgaa tgatggtgag gatgaattga atgataaaaa tctgggtgaa
     8041 catggaactg tgccgaatca gggcggctat gcgaacagca atggcga
//
