LOCUS       sequence-422255-        9135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of 2xGFP tag at VPH1 C terminus, use auxotrophic marker 
            URA3(Kluyveromyces lactis) for selection. Marker for yeast vacuolar 
            membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9135)
  AUTHORS   He CW, Cui XF, Ma SJ, Xu Q, Ran YP, Chen WZ, Mu JX, Li H, Zhu J, 
            Gong Q, Xie Z
  TITLE     Membrane recruitment of Atg8 by Hfl1 facilitates turnover of 
            vacuolar membrane proteins in yeast cells approaching stationary 
            phase.
  JOURNAL   BMC Biol. 2021 Jun 4;19(1):117. doi: 10.1186/s12915-021-01048-7.
  PUBMED    34088313
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: "BMC Biol.";
            date: "2021-06-4"; volume: "19(1):117. doi"; pages: " 
            10.1186/s12915-021-01048-7"
COMMENT     Sequence Label: Vph1-2GFP-URA
FEATURES             Location/Qualifiers
     source          1..9135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2707..3420
                     /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             3457..4173
                     /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             3457..4170
                     /codon_start=1
                     /product="green fluorescent protein"
                     /label=GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     terminator      4194..4381
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(4249..4273)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    4664..4697
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     terminator      5652..5849
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    5903..5936
                     /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(6779..6798)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6780..6798)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6885..6902)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7056..7644)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7136..7155)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7815..8606,8607..8675))
                     /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     8438..8457
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8676..8780)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8848..8866
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(8904..8926)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     9026..9045
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(9126..9135,1..9)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(9126..9135,1..8)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 cactatagaa cgcggccgcc agctgaagct tgctagcttt tacctttctc tatacaacaa
       61 gtgagggttt gaacttttaa atccctttta atttgaatca aaaaaaaaac atttaaaggt
      121 tacacaagga aaataatggc agagaaggag gaagcgattt ttcgctctgc tgaaatggct
      181 ttagtccaat tctatattcc tcaagaaatt tcaagagact ctgcttacac tttaggtcaa
      241 ttgggtcttg ttcaattccg tgacttgaac tctaaggtgc gtgcgtttca aagaactttc
      301 gtgaacgaaa ttagaagact ggataatgta gaaagacagt atcgttactt ttattctctt
      361 ttgaagaaac acgatattaa gctctacgaa ggagacacgg acaaatattt ggacggctca
      421 ggtgaattgt acgttccacc aagcggttca gtgatagatg attatgtccg gaacgcttca
      481 tatttggaag aaagattgat tcaaatggag gatgcaaccg atcaaattga agtccagaaa
      541 aatgacttgg aacagtatcg ctttattttg cagtcaggtg atgaattttt cttgaagggt
      601 gataataccg acagcacttc ctatatggat gaagacatga tcgacgctaa tggggaaaac
      661 attgctgctg ctatcggtgc ttctgtaaac tatgtcactg gtgtcattgc tagagacaaa
      721 gttgccacct tagaacaaat tctttggaga gtattaagag gtaacctttt cttcaaaact
      781 gttgaaattg aacaacctgt ttatgatgtc aaaaccaggg agtataaaca taaaaatgct
      841 tttatcgtat tttctcacgg tgatctgatt attaaaagaa tcagaaagat tgcggaatca
      901 ttggatgcca atctttacga tgttgactct tccaacgagg gtagatcaca acaattggcc
      961 aaggtcaaca agaatttgag tgatttgtac acagttttga aaaccacttc taccacttta
     1021 gaaagtgaat tatatgccat tgccaaagaa ttggactctt ggttccaaga tgttacccgt
     1081 gaaaaggcga tttttgaaat tttgaacaag tctaactatg ataccaatag aaagattttg
     1141 attgctgaag gttggatacc aagagacgaa ttggctactt tgcaagctcg tcttggtgaa
     1201 atgatcgcaa gattgggtat tgatgtccca tccattatcc aagtcctgga tacaaaccac
     1261 actccaccta ccttccacag aactaacaag tttactgctg gtttccaaag tatctgtgac
     1321 tgttacggta ttgctcagta cagagaaatc aatgctggtt tacccacaat tgtcactttc
     1381 cctttcatgt ttgccatcat gtttggtgat atgggtcacg ggttcttaat gaccttagcc
     1441 gcattgtctc ttgtattgaa tgaaaagaaa atcaacaaaa tgaaaagagg cgaaattttc
     1501 gatatggcct tcactggtag atacattatt ttgttgatgg gtgtcttttc catgtacaca
     1561 ggtttccttt acaacgatat cttctctaaa actatgacta ttttcaagtc tggttggaaa
     1621 tggcctgatc attggaaaaa aggtgagagt attactgcta catcggtggg tacataccct
     1681 atcggtttag attgggcttg gcatggaact gaaaatgctt tgttattttc taattcttac
     1741 aaaatgaaac tatcaatttt aatggggttc atccacatga cctattctta tttcttttcg
     1801 ttggctaacc acctatactt taactctatg attgatatca tcggtaactt tattcctggt
     1861 ttgctattta tgcaaggtat ctttggttat ctttccgttt gtattgttta caaatgggct
     1921 gttgattggg ttaaggacgg aaagcctgct ccaggtttgt taaatatgtt gatcaacatg
     1981 tttttatcac caggaactat tgacgatgaa ttataccctc atcaagcaaa ggtccaagtg
     2041 tttttgttgt tgatggcctt ggtttgtatt ccttggttgc tattggtgaa gccattacat
     2101 ttcaaattca ctcataaaaa gaaatctcac gaaccactgc catcgactga agcagatgct
     2161 agttctgaag atttggaagc acaacaatta atttccgcga tggacgccga tgacgctgaa
     2221 gaagaagaag ttggttctgg atctcatggt gaagactttg gtgatattat gattcatcaa
     2281 gttattcata caattgaatt ctgtttgaat tgtgtttcgc acactgcatc ctatttacgt
     2341 ttatgggcct tatcattggc acatgctcaa ttgtctagtg ttttatggac aatgacaatt
     2401 caaattgcct ttggatttag aggatttgtg ggtgtgttta tgacggttgc actttttgcc
     2461 atgtggttcg cactaacatg tgcagttctt gttttgatgg aaggtacatc tgccatgctt
     2521 cattccttac gtttgcactg ggttgaatct atgtccaagt ttttcgtggg tgaaggttta
     2581 ccatacgaac cattcgcatt tgagtataaa gacatggaag tcgctgttgc tagtgcaagc
     2641 tcttccgctt caagcgcttc gtacgctgca ggtcgacgga tcggtgacgg tgctggttta
     2701 attaacatgt ctaaaggtga agaattattc actggtgttg tcccaatttt ggttgaatta
     2761 gatggtgatg ttaatggtca caaattttct gtctccggtg aaggtgaagg tgatgctact
     2821 tacggtaaat tgaccttaaa atttatttgt actactggta aattgccagt tccatggcca
     2881 accttagtca ctactttaac ttatggtgtt caatgttttt ctagataccc agatcatatg
     2941 aaacaacatg actttttcaa gtctgccatg ccagaaggtt atgttcaaga aagaactatt
     3001 tttttcaaag atgacggtaa ctacaagacc agagctgaag tcaagtttga aggtgatacc
     3061 ttagttaata gaatcgaatt aaaaggtatt gattttaaag aagatggtaa cattttaggt
     3121 cacaaattgg aatacaacta taactctcac aatgtttaca tcatggctga caaacaaaag
     3181 aatggtatca aagttaactt caaaattaga cacaacattg aagatggttc tgttcaatta
     3241 gctgaccatt atcaacaaaa tactccaatt ggtgatggtc cagtcttgtt accagacaac
     3301 cattacttat ccactcaatc tgccttatcc aaagatccaa acgaaaagag agaccacatg
     3361 gtcttgttag aatttgttac tgctgctggt attacccatg gtattgatga attgtacaaa
     3421 attccagttc caacgcgtga tctagagaag ccattcatgt ctaaaggtga agaattattc
     3481 actggtgttg tcccaatttt ggttgaatta gatggtgatg ttaatggtca caaattttct
     3541 gtctccggtg aaggtgaagg tgatgctact tacggtaaat tgaccttaaa atttatttgt
     3601 actactggta aattgccagt tccatggcca accttagtca ctactttaac ttatggtgtt
     3661 caatgttttt ctagataccc agatcatatg aaacaacatg actttttcaa gtctgccatg
     3721 ccagaaggtt atgttcaaga aagaactatt tttttcaaag atgacggtaa ctacaagacc
     3781 agagctgaag tcaagtttga aggtgatacc ttagttaata gaatcgaatt aaaaggtatt
     3841 gattttaaag aagatggtaa cattttaggt cacaaattgg aatacaacta taactctcac
     3901 aatgtttaca tcatggctga caaacaaaag aatggtatca aagttaactt caaaattaga
     3961 cacaacattg aagatggttc tgttcaatta gctgaccatt atcaacaaaa tactccaatt
     4021 ggtgatggtc cagtcttgtt accagacaac cattacttat ccactcaatc tgccttatcc
     4081 aaagatccaa acgaaaagag agaccacatg gtcttgttag aatttgttac tgctgctggt
     4141 attacccatg gtatggatga attatataaa taaggcgcgc cacttctaaa taagcgaatt
     4201 tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat
     4261 tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt
     4321 aggtcaggtt gctttctcag gtatagtatg aggtcgctct tattgaccac acctctaccg
     4381 gcagatccgc tagggataac agggtaatat agatctgttt agcttgcctc gtccccgccg
     4441 ggtcacccgg ccagcgacat ggaggcccag aataccctcc ttgacagtct tgacgtgcgc
     4501 agctcagggg catgatgtga ctgtcgcccg tacatttagc ccatacatcc ccatgtataa
     4561 tcatttgcat ccatacattt tgatggccgc acggcgcgaa gcaaaaatta cggctcctcg
     4621 ctgcggacct gcgagcaggg aaacgctccc ctcacagacg cgtataactt cgtataatgt
     4681 atgctatacg aagttattga attgtcccca cgccgcgccc ctgtagagaa atataaaagg
     4741 ttaggatttg ccactgaggt tcttctttca tatacttcct tttaaaatct tgctaggata
     4801 cagttctcac atcacatccg aacataaaca accatgacag tcaacactaa gacctatagt
     4861 gagagagcag aaactcatgc ctcaccagta gcacaacgat tatttcgatt aatggaactg
     4921 aagaaaacca atttatgtgc atcaattgat gttgatacca ctaaggaatt ccttgaatta
     4981 attgataaat tgggtcctta tgtatgctta atcaagactc atattgatat aatcaatgat
     5041 ttttcctatg aatccactat tgaaccatta ttagaacttt cacgtaaaca tcaatttatg
     5101 atttttgaag atagaaaatt tgctgatatt ggtaataccg tgaagaaaca atatattggt
     5161 ggagtttata aaattagtag ttgggcagat attactaatg ctcatggtgt cactgggaat
     5221 ggagtagttg aaggattaaa acagggagct aaagaaacca ccaccaacca agagccaaga
     5281 gggttattga tgttagctga attatcatca gtgggatcat tagcatatgg agaatattct
     5341 caaaaaactg ttgaaattgc taaatccgat aaggaatttg ttattggatt tattgcccaa
     5401 cgtgatatgg gtggacaaga agaaggattt gattggctta ttatgacacc tggagttgga
     5461 ttagatgata aaggtgatgg attaggacaa caatatagaa ctgttgatga agttgttagc
     5521 actggaactg atattatcat tgttggtaga ggattgtttg gtaaaggaag agatccagat
     5581 attgaaggta aaaggtatag agatgctggt tggaatgctt atttgaaaaa gactggccaa
     5641 ttataatcag tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt
     5701 ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc
     5761 gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca
     5821 atcgtatgtg aatgctggtc gctatactgc tgtcgattcg atactaacgc cgccatccag
     5881 tgtcgaaaac gagctcgaat tcataacttc gtataatgta tgctatacga agttatatcg
     5941 atgatatcag atccactagt gaggactttt aaaaaaagcg aaacatttaa gtacttcatt
     6001 aaataataaa tgatgctact atatagtgag taaaccttat tatatactat caatgctaat
     6061 tcaataaagt atttaaagta tcaaccaatg cttaaaaaga gtcaaaaaaa agggaaaaag
     6121 aaataggaaa gccaaactta tctcatgaaa acgttagact tgcaatccaa tccaccagtg
     6181 accatcattt gccttggggt aacgttagaa tcgaaatcca catccaggtc taaacagttg
     6241 acaaatcctg tatgcaactt ttcccaaacg tgtaagcact gccccgtgtt cagatcccaa
     6301 catctgacag atttatcatc agcacaagaa aacaagtatt ggcctcgaat actaatatct
     6361 ctaacccacg aaagatgccc cttcaacgta agaacacatc tgaaatttga atctgtagga
     6421 ttcggtatgg gtgctctatg ggccattaac gtaggtagag gtatctccca gatttttatc
     6481 gttctatctc ttgacgctga gacacaatat tgcatccccc aattcttata tcgatccgtt
     6541 gatggtgttc taaaccttat ctccggagag tcttcgatga aatgaataaa tttaactttc
     6601 tctatgggaa attcatgacc cgtgccaaca ctaagaccat tacctgatgg ccaatgtgtt
     6661 aaccttaatg tagtgtcatg tgaaccagag atgatgtaat cgcctagaac gtctatggaa
     6721 cgaacccatt gggaatgagg cctactagtg gcctatgcgg ccgcggatct gccggtctcc
     6781 ctatagtgag tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa
     6841 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     6901 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     6961 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     7021 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     7081 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     7141 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     7201 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     7261 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     7321 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     7381 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     7441 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     7501 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     7561 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     7621 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     7681 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     7741 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     7801 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     7861 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     7921 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     7981 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     8041 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     8101 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     8161 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     8221 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     8281 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     8341 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     8401 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     8461 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     8521 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     8581 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     8641 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     8701 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     8761 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     8821 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     8881 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     8941 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     9001 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     9061 atggacatat tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac
     9121 atacgattta ggtga
//
