LOCUS       sequence-384002-        8303 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Marker for yeast mitochondria. Integration of 2xGFP tag at ACH1 C 
            terminus. Uses auxotrophic marker URA3(Candida albicans). .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8303)
  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 8303)
  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: ACH1-2EGFP-ter-URA3
FEATURES             Location/Qualifiers
     source          1..8303
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        94..112
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     94..111
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1764..2477
                     /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"
     CDS             2514..3230
                     /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             2514..3227
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      3251..3438
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3306..3330)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3721..3754
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      4709..4906
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    4960..4993
                     /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(6050..6069)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6051..6069)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6156..6173)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6327..6915)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6407..6426)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7086..7877,7878..7946))
                     /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     7709..7728
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7947..8051)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8119..8137
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8175..8197)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     join(8297..8303,1..13)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa
       61 ttaatacata accttatgta tcatacacat acgatttagg tgacactata gaacgcggcc
      121 gccagctgaa gcttatgaca atttctaatt tgttaaagca gagagttagg tatgctccct
      181 atctgaaaaa agttaaggaa gctcacgagc ttattccatt gttcaagaat ggtcagtacc
      241 ttgggtggtc cggttttaca ggagtgggta ctcccaaggc agtgccggag gcactgatag
      301 atcacgtgga gaagaacaat ttacaaggga agttgagatt caaccttttt gttggagctt
      361 ctgctggtcc agaggaaaac cgttgggctg aacacgacat gatcattaag agagcccctc
      421 atcaagtagg gaaacccatt gcaaaggcaa ttaaccaggg tagaattgag ttctttgata
      481 aacatctgtc catgttccct caggatctga catacgggtt ctacaccagg gaaagaaaag
      541 acaacaaaat ccttgattat actataatcg aggcaacggc cattaaagag gacgggtcta
      601 tcgtcccagg tccctctgtc ggtggttctc cagaattcat tacagtcagt gataaagtga
      661 ttattgaggt taacacggct acgccttcgt tcgagggtat tcacgatata gacatgcccg
      721 tgaacccacc tttcaggaaa ccatacccat atctgaaagt ggacgacaag tgtggtgttg
      781 actccatccc ggttgatcct gaaaaggttg ttgcgattgt ggagtccacc atgagggacc
      841 aggtcccacc aaatacgccc tctgacgaca tgtccagggc tattgcaggt catttggtcg
      901 agtttttcag aaacgaggta aaacatggta ggctacctga aaacctgctg cctttacaaa
      961 gtggtatagg taacattgct aacgctgtca ttgaagggct tgctggcgcc caattcaagc
     1021 acttgactgt atggacggaa gtgctgcagg actcgttctt ggatcttttc gagaacggat
     1081 ctttggacta cgccactgct acttccgtga gattgactga aaagggtttc gacagagcct
     1141 ttgcaaactg ggaaaatttc aaacacagat tgtgtttgag atctcaagtt gtctcgaaca
     1201 atccggaaat gatccgtaga ttgggtgtca tcgccatgaa taccccagta gaagttgaca
     1261 tttacgcgca cgccaattct acaaatgtga atggttcccg tatgttgaac gggttgggtg
     1321 gatctgctga tttcttgaga aatgcaaagt tgtccatcat gcatgccccc tctgcaagac
     1381 caactaaagt agaccctacc ggtatctcta ccattgttcc tatggcctct catgtagatc
     1441 aaactgagca tgacctggac atcttggtca ctgaccaagg tttggcggat ctaagaggtc
     1501 tatcgcctaa ggaaagagcc cgtgaaatca tcaacaagtg tgctcatccc gattatcaag
     1561 ctttgttgac cgattacttg gacagagcag agcattacgc taaaaagcac aattgcttgc
     1621 atgaaccaca catgctaaag aatgctttca agttccacac caacttagct gaaaagggta
     1681 caatgaaggt cgacagctgg gaaccagttg acgcttcgta cgctgcaggt cgacggatcg
     1741 gtgacggtgc tggtttaatt aacatgtcta aaggtgaaga attattcact ggtgttgtcc
     1801 caattttggt tgaattagat ggtgatgtta atggtcacaa attttctgtc tccggtgaag
     1861 gtgaaggtga tgctacttac ggtaaattga ccttaaaatt tatttgtact actggtaaat
     1921 tgccagttcc atggccaacc ttagtcacta ctttaactta tggtgttcaa tgtttttcta
     1981 gatacccaga tcatatgaaa caacatgact ttttcaagtc tgccatgcca gaaggttatg
     2041 ttcaagaaag aactattttt ttcaaagatg acggtaacta caagaccaga gctgaagtca
     2101 agtttgaagg tgatacctta gttaatagaa tcgaattaaa aggtattgat tttaaagaag
     2161 atggtaacat tttaggtcac aaattggaat acaactataa ctctcacaat gtttacatca
     2221 tggctgacaa acaaaagaat ggtatcaaag ttaacttcaa aattagacac aacattgaag
     2281 atggttctgt tcaattagct gaccattatc aacaaaatac tccaattggt gatggtccag
     2341 tcttgttacc agacaaccat tacttatcca ctcaatctgc cttatccaaa gatccaaacg
     2401 aaaagagaga ccacatggtc ttgttagaat ttgttactgc tgctggtatt acccatggta
     2461 ttgatgaatt gtacaaaatt ccagttccaa cgcgtgatct agagaagcca ttcatgtcta
     2521 aaggtgaaga attattcact ggtgttgtcc caattttggt tgaattagat ggtgatgtta
     2581 atggtcacaa attttctgtc tccggtgaag gtgaaggtga tgctacttac ggtaaattga
     2641 ccttaaaatt tatttgtact actggtaaat tgccagttcc atggccaacc ttagtcacta
     2701 ctttaactta tggtgttcaa tgtttttcta gatacccaga tcatatgaaa caacatgact
     2761 ttttcaagtc tgccatgcca gaaggttatg ttcaagaaag aactattttt ttcaaagatg
     2821 acggtaacta caagaccaga gctgaagtca agtttgaagg tgatacctta gttaatagaa
     2881 tcgaattaaa aggtattgat tttaaagaag atggtaacat tttaggtcac aaattggaat
     2941 acaactataa ctctcacaat gtttacatca tggctgacaa acaaaagaat ggtatcaaag
     3001 ttaacttcaa aattagacac aacattgaag atggttctgt tcaattagct gaccattatc
     3061 aacaaaatac tccaattggt gatggtccag tcttgttacc agacaaccat tacttatcca
     3121 ctcaatctgc cttatccaaa gatccaaacg aaaagagaga ccacatggtc ttgttagaat
     3181 ttgttactgc tgctggtatt acccatggta tggatgaatt atataaataa ggcgcgccac
     3241 ttctaaataa gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa
     3301 aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt
     3361 gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagtatgagg tcgctcttat
     3421 tgaccacacc tctaccggca gatccgctag ggataacagg gtaatataga tctgtttagc
     3481 ttgcctcgtc cccgccgggt cacccggcca gcgacatgga ggcccagaat accctccttg
     3541 acagtcttga cgtgcgcagc tcaggggcat gatgtgactg tcgcccgtac atttagccca
     3601 tacatcccca tgtataatca tttgcatcca tacattttga tggccgcacg gcgcgaagca
     3661 aaaattacgg ctcctcgctg cggacctgcg agcagggaaa cgctcccctc acagacgcgt
     3721 ataacttcgt ataatgtatg ctatacgaag ttattgaatt gtccccacgc cgcgcccctg
     3781 tagagaaata taaaaggtta ggatttgcca ctgaggttct tctttcatat acttcctttt
     3841 aaaatcttgc taggatacag ttctcacatc acatccgaac ataaacaacc atgacagtca
     3901 acactaagac ctatagtgag agagcagaaa ctcatgcctc accagtagca caacgattat
     3961 ttcgattaat ggaactgaag aaaaccaatt tatgtgcatc aattgatgtt gataccacta
     4021 aggaattcct tgaattaatt gataaattgg gtccttatgt atgcttaatc aagactcata
     4081 ttgatataat caatgatttt tcctatgaat ccactattga accattatta gaactttcac
     4141 gtaaacatca atttatgatt tttgaagata gaaaatttgc tgatattggt aataccgtga
     4201 agaaacaata tattggtgga gtttataaaa ttagtagttg ggcagatatt actaatgctc
     4261 atggtgtcac tgggaatgga gtagttgaag gattaaaaca gggagctaaa gaaaccacca
     4321 ccaaccaaga gccaagaggg ttattgatgt tagctgaatt atcatcagtg ggatcattag
     4381 catatggaga atattctcaa aaaactgttg aaattgctaa atccgataag gaatttgtta
     4441 ttggatttat tgcccaacgt gatatgggtg gacaagaaga aggatttgat tggcttatta
     4501 tgacacctgg agttggatta gatgataaag gtgatggatt aggacaacaa tatagaactg
     4561 ttgatgaagt tgttagcact ggaactgata ttatcattgt tggtagagga ttgtttggta
     4621 aaggaagaga tccagatatt gaaggtaaaa ggtatagaga tgctggttgg aatgcttatt
     4681 tgaaaaagac tggccaatta taatcagtac tgacaataaa aagattcttg ttttcaagaa
     4741 cttgtcattt gtatagtttt tttatattgt agttgttcta ttttaatcaa atgttagcgt
     4801 gatttatatt ttttttcgcc tcgacatcat ctgcccagat gcgaagttaa gtgcgcagaa
     4861 agtaatatca tgcgtcaatc gtatgtgaat gctggtcgct atactgctgt cgattcgata
     4921 ctaacgccgc catccagtgt cgaaaacgag ctcgaattca taacttcgta taatgtatgc
     4981 tatacgaagt tatatcgatg atatcagatc cactagtggc ctatgcggcc gcgggtttgt
     5041 gcgcaaaccg agagatgagt atttaacaaa aaaaagaaag gaaatgatat gattatgatt
     5101 ttatgtttat aaagctttta tccaatgcgt tgttttttct tgcatattta tactttttgc
     5161 gctcatggag ggagttaatc aatacgcatg acgtctagtt aattcacagg tagtactgta
     5221 tatttatatg tttacacaat aattatgtat taagtagtga ttagtaaaaa aaactaagag
     5281 gttgaaagtc atcaaccctt atattaattt caacgaattt cgctgatgac gtcagggtag
     5341 ttctcttgct taggtcaatg taaaaatgcc agggtaatac aaaaccaagc tctgaaccgc
     5401 ccaacttatc tgtaatgatg catgtatacc ttacaatgat tttttttctc ctattatgga
     5461 attaatattg gaatccatgt ttgtgaacag ccttctccgc aatttagctt caagctaata
     5521 agtggttatt gtttgcgcgc gcttgaaggc aaggatcatc gcttttttct tctcaccatt
     5581 gaatttaagg gacgccgatt agcataatac acttatcaac aacaaccgga tgtgtgttat
     5641 ctatcagtgg tggaaggatt ggtaagcaat ttggtttgat ccagaggact aagaaaaaaa
     5701 aaacggctca tcaaagaaat tagttcaatt gagtggttta atagacaaag tttggaaaga
     5761 taacagaagt agaaaaagta aaaatctaca catgacactc aacacatgtg gacagcaaga
     5821 gggcagatag ataagtcgac actgcaaatt gttgtgtatg taatagccct gggtgcaggt
     5881 tggccaaaac tgttcaacag ctcgcccatt tgaaggttca acaggccgcc agaaatgttc
     5941 aacagattgt gccgctatga tacgtaactt atatatagca gcgtgtataa tattaggacc
     6001 ataataaaat gaatggcatg aaaataacga cccgcggatc tgccggtctc cctatagtga
     6061 gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca acgcgcgggg
     6121 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     6181 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     6241 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     6301 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     6361 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6421 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6481 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6541 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6601 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6661 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6721 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6781 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6841 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6901 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6961 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     7021 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     7081 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     7141 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     7201 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     7261 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     7321 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     7381 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     7441 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     7501 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7561 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7621 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7681 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     7741 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7801 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7861 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7921 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7981 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     8041 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     8101 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     8161 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     8221 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     8281 ggctggctta actatgcggc atc
//
