LOCUS       sequence-384007-        6834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Marker for yeast mitochondria. Integration of 2xGFP tag at OM14 C 
            terminus. Uses antibiotic resistance marker natMX6. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6834)
  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 6834)
  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: OM14-2EGFP-UG74-ter
FEATURES             Location/Qualifiers
     source          1..6834
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             466..1179
                     /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             1216..1932
                     /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             1216..1929
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      1953..2140
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2008..2032)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     gene            2215..3334
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        2215..2558
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             2559..3131
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      3137..3334
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    3397..3430
                     /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(4471..4490)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4472..4490)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4577..4594)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4748..5336)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4828..4847)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5507..6298,6299..6367))
                     /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     6130..6149
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6368..6472)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6540..6558
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6596..6618)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6718..6737
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(6818..6834,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(6818..6834,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttgctaaa cacgacagta acgctagccc aaattctgac
       61 tctgaagacg gtcaccacca caacaataag aaggaatgcg ctatcgaata tctgaaggca
      121 cggttaaata gtgcatcagc agtcgcttgc ggctacctcc aagcttttgt cagtaagacg
      181 caagactttg ccaaagtatg ctttttagaa cttcagaatc ccgttgtctt ggtcaacttg
      241 ttgctgcatt cttcagtggt atgttattta tgtaacgggt atgcgaacca caacgccaga
      301 ttcttgaagg ggaaacctaa ctctacagtc ttagcaacaa ccgccggcgc tctgggtctt
      361 ttgacgctgg acggtataat ttcaaagaaa tactactcca gatacgacaa gaaagcttcg
      421 tacgctgcag gtcgacggat cggtgacggt gctggtttaa ttaacatgtc taaaggtgaa
      481 gaattattca ctggtgttgt cccaattttg gttgaattag atggtgatgt taatggtcac
      541 aaattttctg tctccggtga aggtgaaggt gatgctactt acggtaaatt gaccttaaaa
      601 tttatttgta ctactggtaa attgccagtt ccatggccaa ccttagtcac tactttaact
      661 tatggtgttc aatgtttttc tagataccca gatcatatga aacaacatga ctttttcaag
      721 tctgccatgc cagaaggtta tgttcaagaa agaactattt ttttcaaaga tgacggtaac
      781 tacaagacca gagctgaagt caagtttgaa ggtgatacct tagttaatag aatcgaatta
      841 aaaggtattg attttaaaga agatggtaac attttaggtc acaaattgga atacaactat
      901 aactctcaca atgtttacat catggctgac aaacaaaaga atggtatcaa agttaacttc
      961 aaaattagac acaacattga agatggttct gttcaattag ctgaccatta tcaacaaaat
     1021 actccaattg gtgatggtcc agtcttgtta ccagacaacc attacttatc cactcaatct
     1081 gccttatcca aagatccaaa cgaaaagaga gaccacatgg tcttgttaga atttgttact
     1141 gctgctggta ttacccatgg tattgatgaa ttgtacaaaa ttccagttcc aacgcgtgat
     1201 ctagagaagc cattcatgtc taaaggtgaa gaattattca ctggtgttgt cccaattttg
     1261 gttgaattag atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt
     1321 gatgctactt acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt
     1381 ccatggccaa ccttagtcac tactttaact tatggtgttc aatgtttttc tagataccca
     1441 gatcatatga aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa
     1501 agaactattt ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa
     1561 ggtgatacct tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac
     1621 attttaggtc acaaattgga atacaactat aactctcaca atgtttacat catggctgac
     1681 aaacaaaaga atggtatcaa agttaacttc aaaattagac acaacattga agatggttct
     1741 gttcaattag ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta
     1801 ccagacaacc attacttatc cactcaatct gccttatcca aagatccaaa cgaaaagaga
     1861 gaccacatgg tcttgttaga atttgttact gctgctggta ttacccatgg tatggatgaa
     1921 ttatataaat aaggcgcgcc acttctaaat aagcgaattt cttatgattt atgattttta
     1981 ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt
     2041 taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg
     2101 tatagtatga ggtcgctctt attgaccaca cctctaccgg cagatccgct agggataaca
     2161 gggtaatata gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg
     2221 gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac
     2281 tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt
     2341 gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga
     2401 aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata
     2461 aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta
     2521 ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaccact cttgacgaca
     2581 cggcttaccg gtaccgcacc agtgtcccgg gggacgccga ggccatcgag gcactggatg
     2641 ggtccttcac caccgacacc gtcttccgcg tcaccgccac cggggacggc ttcaccctgc
     2701 gggaggtgcc ggtggacccg cccctgacca aggtgttccc cgacgacgaa tcggacgacg
     2761 aatcggacga cggggaggac ggcgacccgg actcccggac gttcgtcgcg tacggggacg
     2821 acggcgacct ggcgggcttc gtggtcgtct cgtactccgg ctggaaccgc cggctgaccg
     2881 tcgaggacat cgaggtcgcc ccggagcacc gggggcacgg ggtcgggcgc gcgttgatgg
     2941 ggctcgcgac ggagttcgcc cgcgagcggg gcgccgggca cctctggctg gaggtcacca
     3001 acgtcaacgc accggcgatc cacgcgtacc ggcggatggg gttcaccctc tgcggcctgg
     3061 acaccgccct gtacgacggc accgcctcgg acggcgagca ggcgctctac atgagcatgc
     3121 cctgccccta atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt
     3181 atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt
     3241 ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg
     3301 cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca
     3361 tccagtgtcg aaaacgagct ctcgagaacc cttaatataa cttcgtataa tgtatgctat
     3421 acgaagttat taggtgatat cagatccact agtggcctat gcggccgcgg catcctttca
     3481 cacactcaca cactcacaca ctttatatac atatattttt ataactatta ctgctgattt
     3541 atttgtaagg aaacgtgctt tcctcttcat cggtcaagtg ataagtttct ataatataat
     3601 agcttttctg tctactatca ttcttttttc atttcaagta cccttaattt gttttacccg
     3661 gaccacgaca ttttctcact acggcacttg agagctataa ctcaatgaac atgttgcttg
     3721 gagttatttg attgctctgc gtatcttaaa atagcggtct cgaatcaacc gtatgcaaca
     3781 tggtcctttt gaaatggctc gtatgccaat tggtcttctt taccgctttt tcgcatgcgt
     3841 ttaccgacta tctattaaag aagtgtgcgc aatctgggtt ttgccataga aacagggttt
     3901 atgcagaaaa tattgccaaa tctcatcact gctattacaa agtggacgcc gagtctattg
     3961 cacacgatcc tttagagaat gtgcttcatg ctaccataat taaaactata ccaagattgg
     4021 agggcgatga tatagccgtt cagttcccat tctctctctc ttttttacag gatcactcag
     4081 taaggttcac tataaatgag aaagagagaa tgccaaccaa cagcagcggt ttgttgatct
     4141 cttcacaacg gttcaatgag acctggaagt acgcattcga caagaaattt caagaggagg
     4201 cgaacaggac cagtattcca caattccact tccttaagca aaaacaaact gtgaactcat
     4261 tctggtcgaa aatatcttca tttttgtcac tttcaaactc cactgcagac acatttcatc
     4321 ttcgaaacgg tgatgtatcc gtagaaatct ttgctgaacc ttttcaattg aaagtttact
     4381 ggcaaaatgc gctgaaactt attgtaaacg agcaaaattt cctgaacatt gaacatcata
     4441 gaactaagca ggccgcggat ctgccggtct ccctatagtg agtcgtatta atttcgataa
     4501 gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4561 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4621 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4681 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4741 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4801 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4861 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4921 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4981 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5041 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5101 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5161 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5221 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5281 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5341 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5401 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5461 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5521 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5581 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5641 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5701 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5761 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5821 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5881 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5941 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6001 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6061 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6121 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6181 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6241 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6301 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6361 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6421 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6481 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     6541 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     6601 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     6661 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     6721 catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta gaacgcggct
     6781 acaattaata cataacctta tgtatcatac acatacgatt taggtgacac tata
//
