LOCUS       sequence-268960-        9135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of 2xGFP tag at VPH1 C terminus, use auxotrophic marker 
            URA3(Candida albicans). Marker for yeast vacuole..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9135)
  AUTHORS   Zhu J, Zhang ZT, Tang SW, Zhao BS, Li H, Song JZ, Li D, Xie Z
  TITLE     A Validated Set of Fluorescent-Protein-Based Markers for Major 
            Organelles in Yeast (Saccharomyces cerevisiae).
  JOURNAL   MBio. 2019 Sep 3;10(5). pii: mBio.01691-19. doi: 
            10.1128/mBio.01691-19.
  PUBMED    31481383
REFERENCE   2  (bases 1 to 9135)
  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: "MBio."; 
            date: "2019-09-3"; volume: "10(5). pii: mBio.01691-19. doi"; pages: 
            " 10.1128/mBio.01691-19"
COMMENT     Sequence Label: ZJOM153
FEATURES             Location/Qualifiers
     source          1..9135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             2717..3430
                     /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             3467..4183
                     /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             3467..4180
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      4204..4391
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4259..4283)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    4674..4707
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      5662..5859
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    5913..5946
                     /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(6789..6808)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6790..6808)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6895..6912)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7066..7654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7146..7165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7825..8616,8617..8685))
                     /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     8448..8467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8686..8790)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8858..8876
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8914..8936)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     9036..9055
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tgctagcttt tacctttctc
       61 tatacaacaa gtgagggttt gaacttttaa atccctttta atttgaatca aaaaaaaaac
      121 atttaaaggt tacacaagga aaataatggc agagaaggag gaagcgattt ttcgctctgc
      181 tgaaatggct ttagtccaat tctatattcc tcaagaaatt tcaagagact ctgcttacac
      241 tttaggtcaa ttgggtcttg ttcaattccg tgacttgaac tctaaggtgc gtgcgtttca
      301 aagaactttc gtgaacgaaa ttagaagact ggataatgta gaaagacagt atcgttactt
      361 ttattctctt ttgaagaaac acgatattaa gctctacgaa ggagacacgg acaaatattt
      421 ggacggctca ggtgaattgt acgttccacc aagcggttca gtgatagatg attatgtccg
      481 gaacgcttca tatttggaag aaagattgat tcaaatggag gatgcaaccg atcaaattga
      541 agtccagaaa aatgacttgg aacagtatcg ctttattttg cagtcaggtg atgaattttt
      601 cttgaagggt gataataccg acagcacttc ctatatggat gaagacatga tcgacgctaa
      661 tggggaaaac attgctgctg ctatcggtgc ttctgtaaac tatgtcactg gtgtcattgc
      721 tagagacaaa gttgccacct tagaacaaat tctttggaga gtattaagag gtaacctttt
      781 cttcaaaact gttgaaattg aacaacctgt ttatgatgtc aaaaccaggg agtataaaca
      841 taaaaatgct tttatcgtat tttctcacgg tgatctgatt attaaaagaa tcagaaagat
      901 tgcggaatca ttggatgcca atctttacga tgttgactct tccaacgagg gtagatcaca
      961 acaattggcc aaggtcaaca agaatttgag tgatttgtac acagttttga aaaccacttc
     1021 taccacttta gaaagtgaat tatatgccat tgccaaagaa ttggactctt ggttccaaga
     1081 tgttacccgt gaaaaggcga tttttgaaat tttgaacaag tctaactatg ataccaatag
     1141 aaagattttg attgctgaag gttggatacc aagagacgaa ttggctactt tgcaagctcg
     1201 tcttggtgaa atgatcgcaa gattgggtat tgatgtccca tccattatcc aagtcctgga
     1261 tacaaaccac actccaccta ccttccacag aactaacaag tttactgctg gtttccaaag
     1321 tatctgtgac tgttacggta ttgctcagta cagagaaatc aatgctggtt tacccacaat
     1381 tgtcactttc cctttcatgt ttgccatcat gtttggtgat atgggtcacg ggttcttaat
     1441 gaccttagcc gcattgtctc ttgtattgaa tgaaaagaaa atcaacaaaa tgaaaagagg
     1501 cgaaattttc gatatggcct tcactggtag atacattatt ttgttgatgg gtgtcttttc
     1561 catgtacaca ggtttccttt acaacgatat cttctctaaa actatgacta ttttcaagtc
     1621 tggttggaaa tggcctgatc attggaaaaa aggtgagagt attactgcta catcggtggg
     1681 tacataccct atcggtttag attgggcttg gcatggaact gaaaatgctt tgttattttc
     1741 taattcttac aaaatgaaac tatcaatttt aatggggttc atccacatga cctattctta
     1801 tttcttttcg ttggctaacc acctatactt taactctatg attgatatca tcggtaactt
     1861 tattcctggt ttgctattta tgcaaggtat ctttggttat ctttccgttt gtattgttta
     1921 caaatgggct gttgattggg ttaaggacgg aaagcctgct ccaggtttgt taaatatgtt
     1981 gatcaacatg tttttatcac caggaactat tgacgatgaa ttataccctc atcaagcaaa
     2041 ggtccaagtg tttttgttgt tgatggcctt ggtttgtatt ccttggttgc tattggtgaa
     2101 gccattacat ttcaaattca ctcataaaaa gaaatctcac gaaccactgc catcgactga
     2161 agcagatgct agttctgaag atttggaagc acaacaatta atttccgcga tggacgccga
     2221 tgacgctgaa gaagaagaag ttggttctgg atctcatggt gaagactttg gtgatattat
     2281 gattcatcaa gttattcata caattgaatt ctgtttgaat tgtgtttcgc acactgcatc
     2341 ctatttacgt ttatgggcct tatcattggc acatgctcaa ttgtctagtg ttttatggac
     2401 aatgacaatt caaattgcct ttggatttag aggatttgtg ggtgtgttta tgacggttgc
     2461 actttttgcc atgtggttcg cactaacatg tgcagttctt gttttgatgg aaggtacatc
     2521 tgccatgctt cattccttac gtttgcactg ggttgaatct atgtccaagt ttttcgtggg
     2581 tgaaggttta ccatacgaac cattcgcatt tgagtataaa gacatggaag tcgctgttgc
     2641 tagtgcaagc tcttccgctt caagcgcttc gtacgctgca ggtcgacgga tcggtgacgg
     2701 tgctggttta attaacatgt ctaaaggtga agaattattc actggtgttg tcccaatttt
     2761 ggttgaatta gatggtgatg ttaatggtca caaattttct gtctccggtg aaggtgaagg
     2821 tgatgctact tacggtaaat tgaccttaaa atttatttgt actactggta aattgccagt
     2881 tccatggcca accttagtca ctactttaac ttatggtgtt caatgttttt ctagataccc
     2941 agatcatatg aaacaacatg actttttcaa gtctgccatg ccagaaggtt atgttcaaga
     3001 aagaactatt tttttcaaag atgacggtaa ctacaagacc agagctgaag tcaagtttga
     3061 aggtgatacc ttagttaata gaatcgaatt aaaaggtatt gattttaaag aagatggtaa
     3121 cattttaggt cacaaattgg aatacaacta taactctcac aatgtttaca tcatggctga
     3181 caaacaaaag aatggtatca aagttaactt caaaattaga cacaacattg aagatggttc
     3241 tgttcaatta gctgaccatt atcaacaaaa tactccaatt ggtgatggtc cagtcttgtt
     3301 accagacaac cattacttat ccactcaatc tgccttatcc aaagatccaa acgaaaagag
     3361 agaccacatg gtcttgttag aatttgttac tgctgctggt attacccatg gtattgatga
     3421 attgtacaaa attccagttc caacgcgtga tctagagaag ccattcatgt ctaaaggtga
     3481 agaattattc actggtgttg tcccaatttt ggttgaatta gatggtgatg ttaatggtca
     3541 caaattttct gtctccggtg aaggtgaagg tgatgctact tacggtaaat tgaccttaaa
     3601 atttatttgt actactggta aattgccagt tccatggcca accttagtca ctactttaac
     3661 ttatggtgtt caatgttttt ctagataccc agatcatatg aaacaacatg actttttcaa
     3721 gtctgccatg ccagaaggtt atgttcaaga aagaactatt tttttcaaag atgacggtaa
     3781 ctacaagacc agagctgaag tcaagtttga aggtgatacc ttagttaata gaatcgaatt
     3841 aaaaggtatt gattttaaag aagatggtaa cattttaggt cacaaattgg aatacaacta
     3901 taactctcac aatgtttaca tcatggctga caaacaaaag aatggtatca aagttaactt
     3961 caaaattaga cacaacattg aagatggttc tgttcaatta gctgaccatt atcaacaaaa
     4021 tactccaatt ggtgatggtc cagtcttgtt accagacaac cattacttat ccactcaatc
     4081 tgccttatcc aaagatccaa acgaaaagag agaccacatg gtcttgttag aatttgttac
     4141 tgctgctggt attacccatg gtatggatga attatataaa taaggcgcgc cacttctaaa
     4201 taagcgaatt tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt
     4261 gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac
     4321 tctttcctgt aggtcaggtt gctttctcag gtatagtatg aggtcgctct tattgaccac
     4381 acctctaccg gcagatccgc tagggataac agggtaatat agatctgttt agcttgcctc
     4441 gtccccgccg ggtcacccgg ccagcgacat ggaggcccag aataccctcc ttgacagtct
     4501 tgacgtgcgc agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc
     4561 ccatgtataa tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta
     4621 cggctcctcg ctgcggacct gcgagcaggg aaacgctccc ctcacagacg cgtataactt
     4681 cgtataatgt atgctatacg aagttattga attgtcccca cgccgcgccc ctgtagagaa
     4741 atataaaagg ttaggatttg ccactgaggt tcttctttca tatacttcct tttaaaatct
     4801 tgctaggata cagttctcac atcacatccg aacataaaca accatgacag tcaacactaa
     4861 gacctatagt gagagagcag aaactcatgc ctcaccagta gcacaacgat tatttcgatt
     4921 aatggaactg aagaaaacca atttatgtgc atcaattgat gttgatacca ctaaggaatt
     4981 ccttgaatta attgataaat tgggtcctta tgtatgctta atcaagactc atattgatat
     5041 aatcaatgat ttttcctatg aatccactat tgaaccatta ttagaacttt cacgtaaaca
     5101 tcaatttatg atttttgaag atagaaaatt tgctgatatt ggtaataccg tgaagaaaca
     5161 atatattggt ggagtttata aaattagtag ttgggcagat attactaatg ctcatggtgt
     5221 cactgggaat ggagtagttg aaggattaaa acagggagct aaagaaacca ccaccaacca
     5281 agagccaaga gggttattga tgttagctga attatcatca gtgggatcat tagcatatgg
     5341 agaatattct caaaaaactg ttgaaattgc taaatccgat aaggaatttg ttattggatt
     5401 tattgcccaa cgtgatatgg gtggacaaga agaaggattt gattggctta ttatgacacc
     5461 tggagttgga ttagatgata aaggtgatgg attaggacaa caatatagaa ctgttgatga
     5521 agttgttagc actggaactg atattatcat tgttggtaga ggattgtttg gtaaaggaag
     5581 agatccagat attgaaggta aaaggtatag agatgctggt tggaatgctt atttgaaaaa
     5641 gactggccaa ttataatcag tactgacaat aaaaagattc ttgttttcaa gaacttgtca
     5701 tttgtatagt ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat
     5761 attttttttc gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata
     5821 tcatgcgtca atcgtatgtg aatgctggtc gctatactgc tgtcgattcg atactaacgc
     5881 cgccatccag tgtcgaaaac gagctcgaat tcataacttc gtataatgta tgctatacga
     5941 agttatatcg atgatatcag atccactagt gaggactttt aaaaaaagcg aaacatttaa
     6001 gtacttcatt aaataataaa tgatgctact atatagtgag taaaccttat tatatactat
     6061 caatgctaat tcaataaagt atttaaagta tcaaccaatg cttaaaaaga gtcaaaaaaa
     6121 agggaaaaag aaataggaaa gccaaactta tctcatgaaa acgttagact tgcaatccaa
     6181 tccaccagtg accatcattt gccttggggt aacgttagaa tcgaaatcca catccaggtc
     6241 taaacagttg acaaatcctg tatgcaactt ttcccaaacg tgtaagcact gccccgtgtt
     6301 cagatcccaa catctgacag atttatcatc agcacaagaa aacaagtatt ggcctcgaat
     6361 actaatatct ctaacccacg aaagatgccc cttcaacgta agaacacatc tgaaatttga
     6421 atctgtagga ttcggtatgg gtgctctatg ggccattaac gtaggtagag gtatctccca
     6481 gatttttatc gttctatctc ttgacgctga gacacaatat tgcatccccc aattcttata
     6541 tcgatccgtt gatggtgttc taaaccttat ctccggagag tcttcgatga aatgaataaa
     6601 tttaactttc tctatgggaa attcatgacc cgtgccaaca ctaagaccat tacctgatgg
     6661 ccaatgtgtt aaccttaatg tagtgtcatg tgaaccagag atgatgtaat cgcctagaac
     6721 gtctatggaa cgaacccatt gggaatgagg cctactagtg gcctatgcgg ccgcggatct
     6781 gccggtctcc ctatagtgag tcgtattaat ttcgataagc caggttaacc tgcattaatg
     6841 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     6901 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     6961 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     7021 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     7081 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     7141 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     7201 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     7261 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     7321 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     7381 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     7441 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     7501 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     7561 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     7621 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     7681 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     7741 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     7801 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     7861 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     7921 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     7981 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     8041 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     8101 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     8161 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     8221 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     8281 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     8341 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     8401 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     8461 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     8521 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     8581 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     8641 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     8701 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     8761 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     8821 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     8881 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     8941 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     9001 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     9061 agtgcaccat atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg
     9121 tatcatacac atacg
//
