LOCUS       sequence-422251-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of GFP-Pho8 as an additional copy in genome, expressed 
            under CUP1 promoter. Uses auxotrophic marker URA3 (Saccharomyces 
            cerevisiae) for selection. Marker for yeast vacuolar membrane..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  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 7302)
  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: PCUP1-GFP-Pho8-406
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..51
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(89..107)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        175..279
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(280..348,349..1140)
                     /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(498..517)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1311..1899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1800..1819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2053..2070
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2187..2208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2223..2228,2229..2246,2247..2253)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2261..2277
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2266..2288
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2285..2301
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2285..2301
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2320..2340
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2322..2340
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2359..2593
                     /gene="S. cerevisiae CUP1"
                     /label=CUP1 promoter
                     /note="inducible metallothionein promoter, regulated by 
                     Cu2+"
     CDS             2604..3317
                     /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"
     primer_bind     complement(5397..5416)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5398..5416)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5426..5443)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5426..5442)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5435..5457)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5583..6038
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5670..5689)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5880..5901
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6169..6972)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6973..7188)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(7212..7231)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
       61 tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat
      121 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
      181 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
      241 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
      301 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
      361 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
      421 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
      481 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
      541 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
      601 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
      661 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
      721 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
      781 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
      841 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
      901 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
      961 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     1021 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     1081 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     1141 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     1201 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     1261 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     1321 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1381 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1441 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     1501 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1561 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1621 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1681 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1741 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1801 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1861 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1921 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     1981 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     2041 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     2101 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     2161 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     2221 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
     2281 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
     2341 gaacaaaagc tggagctcct agttagaaaa agacattttt gctgtcagtc actgtcaaga
     2401 gattcttttg ctggcatttc ttctagaagc aaaaagagcg atgcgtcttt tccgctgaac
     2461 cgttccagca aaaaagacta ccaacgcaat atggattgtc agaatcatat aaaagagaag
     2521 caaataactc cttgtcttgt atcaattgca ttataatatc ttcttgttag tgcaatatca
     2581 tatagaagtc atcgactagt aacatgtcta aaggtgaaga attattcact ggtgttgtcc
     2641 caattttggt tgaattagat ggtgatgtta atggtcacaa attttctgtc tccggtgaag
     2701 gtgaaggtga tgctacttac ggtaaattga ccttaaaatt tatttgtact actggtaaat
     2761 tgccagttcc atggccaacc ttagtcacta ctttaactta tggtgttcaa tgtttttcta
     2821 gatacccaga tcatatgaaa caacatgact ttttcaagtc tgccatgcca gaaggttatg
     2881 ttcaagaaag aactattttt ttcaaagatg acggtaacta caagaccaga gctgaagtca
     2941 agtttgaagg tgatacctta gttaatagaa tcgaattaaa aggtattgat tttaaagaag
     3001 atggtaacat tttaggtcac aaattggaat acaactataa ctctcacaat gtttacatca
     3061 tggctgacaa acaaaagaat ggtatcaaag ttaacttcaa aattagacac aacattgaag
     3121 atggttctgt tcaattagct gaccattatc aacaaaatac tccaattggt gatggtccag
     3181 tcttgttacc agacaaccat tacttatcca ctcaatctgc cttatccaaa gatccaaacg
     3241 aaaagagaga ccacatggtc ttgttagaat ttgttactgc tgctggtatt acccatggta
     3301 ttgatgaatt gtacaaagga gccggaacaa gtgacatgat gactcacaca ttaccaagcg
     3361 aacagacacg tcttgttcct ggatctgact cgagctctcg tcctaaaaag agacggatct
     3421 cgaagagatc aaagataata gtatccactg tggtctgtat tggtttgttg ctggttttag
     3481 tgcaactggc atttccaagc agttttgcat tacgttctgc atcacacaag aagaagaatg
     3541 tcatattctt cgtgacggat gggatgggac ctgcgtcttt gtccatggca agatcattca
     3601 accagcatgt taatgattta cctatagacg acatcctgac acttgacgag cattttatcg
     3661 ggtcttcgag aacaaggtcc tcggactcac tagtcactga ctcagctgcg ggcgccaccg
     3721 catttgcatg tgcattaaag agctataatg gggccatcgg tgttgatccg catcacagac
     3781 cttgtggtac agtgctcgag gcggctaagt tagcaggtta tctcactggg cttgttgtta
     3841 cgacaagaat cacagatgcc actccggctt cattcagttc acatgttgat tacagatggc
     3901 aggaagatct tatcgctacg caccagttag gcgaatatcc tctggggaga gtagtagact
     3961 tactcatggg tggcggcagg agccatttct accctcaagg cgaaaaggct tctccatatg
     4021 gtcaccacgg tgctagaaaa gatggaagag atttaatcga tgaggctcaa tctaatggct
     4081 ggcagtacgt gggtgaccgt aaaaattttg attctttgtt gaaaagccac ggtgaaaacg
     4141 ttactttacc tttcttgggt ctctttgcag ataacgacat tccttttgaa attgacagag
     4201 atgaaaagga gtatccttcc cttaaggaac aagtcaaggt ggctttgggt gccttggaaa
     4261 aagcttccaa tgaggacaaa gattctaatg ggtttttctt aatggtggaa ggctccagaa
     4321 ttgatcatgc tggtcaccaa aacgatccag cttcgcaggt aagggaggtg ttggctttcg
     4381 atgaagcatt ccaatacgtt ttggagtttg ctgaaaattc cgatacggag actgttttgg
     4441 tctctacgtc ggatcatgaa acgggtggtt tagtcacttc aagacaagtc actgctagct
     4501 atcctcagta tgtctggtat ccacaggtgc tcgccaacgc cacccattca ggtgagtttt
     4561 tgaaaagaaa actagtagat tttgttcatg agcataaggg cgcaagcagc aaaatagaaa
     4621 atttcatcaa acatgagatc ctggaaaaag atttgggtat ttacgattac acggatagcg
     4681 atttagaaac attaattcat ttggatgaca acgcaaatgc tatccaagac aaactgaatg
     4741 acatggtatc atttagggcc caaattggct ggaccacgca tggtcacagc gcagtcgacg
     4801 tcaacatata tgcatacgcc aataagaaag ctacatggtc atatgtcttg aacaatttac
     4861 aaggcaatca cgaaaatacg gaagttggcc agttcttaga aaacttttta gaattgaacc
     4921 tgaacgaagt cactgatttg attagggaca ccaaacacac atcagatttt gatgctacag
     4981 aaatagctag tgaagtacaa cattatgacg aatactacca tgagttgacc aactgatcct
     5041 atctaactct ccctcttttt tcacatatta tttaatacgt tataatttat ttgaatatat
     5101 atatatatat atatgtatat atactaaccg catgacacgt actgtatcgc atttcgtttc
     5161 ggagtgcagg cgagttttag gtgcattcta tagcgtgaag aagtgaaatc tcgtcatttc
     5221 ttcaagttat gccattcagt ccagattgtt tcacatgctt tcatatagtc atttttgtcg
     5281 tcgcgaactt caaatttgaa aacatcatca tacagagaga tttctccaat aatgatcctt
     5341 tctttgtcgt tgtcatcatc ggcatcgtcg ttaagtgaag cgcggtaccc aattcgccct
     5401 atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     5461 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     5521 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     5581 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     5641 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     5701 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5761 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5821 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5881 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     5941 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     6001 taacgcgaat tttaacaaaa tattaacgct tacaatttcc tgatgcggta ttttctcctt
     6061 acgcatctgt gcggtatttc acaccgcata gggtaataac tgatataatt aaattgaagc
     6121 tctaatttgt gagtttagta tacatgcatt tacttataat acagtttttt agttttgctg
     6181 gccgcatctt ctcaaatatg cttcccagcc tgcttttctg taacgttcac cctctacctt
     6241 agcatccctt ccctttgcaa atagtcctct tccaacaata ataatgtcag atcctgtaga
     6301 gaccacatca tccacggttc tatactgttg acccaatgcg tctcccttgt catctaaacc
     6361 cacaccgggt gtcataatca accaatcgta accttcatct cttccaccca tgtctctttg
     6421 agcaataaag ccgataacaa aatctttgtc gctcttcgca atgtcaacag tacccttagt
     6481 atattctcca gtagataggg agcccttgca tgacaattct gctaacatca aaaggcctct
     6541 aggttccttt gttacttctt ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac
     6601 cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt ctgtatacac ccgcagagta
     6661 ctgcaatttg actgtattac caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt
     6721 gtacttggcg gataatgcct ttagcggctt aactgtgccc tccatggaaa aatcagtcaa
     6781 gatatccaca tgtgttttta gtaaacaaat tttgggacct aatgcttcaa ctaactccag
     6841 taattccttg gtggtacgaa catccaatga agcacacaag tttgtttgct tttcgtgcat
     6901 gatattaaat agcttggcag caacaggact aggatgagta gcagcacgtt ccttatatgt
     6961 agctttcgac atgatttatc ttcgtttcct gcaggttttt gttctgtgca gttgggttaa
     7021 gaatactggg caatttcatg tttcttcaac actacatatg cgtatatata ccaatctaag
     7081 tctgtgctcc ttccttcgtt cttccttctg ttcggagatt accgaatcaa aaaaatttca
     7141 aggaaaccga aatcaaaaaa aagaataaaa aaaaaatgat gaattgaaaa ggtggtatgg
     7201 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     7261 acacccgctg acgcgccctg acgggcttgt ctgctcccgg ca
//
