LOCUS       sequence-423102-        7649 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Vac8-GFP under CPU1 promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7649)
  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 7649)
  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-Vac8-GFP-Ura
FEATURES             Location/Qualifiers
     source          1..7649
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2041..2751
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(2068..2096)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2691..2711
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3476..3663
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(3531..3555)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    3715..3748
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5200..5233
                     /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(5286..5305)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5287..5305)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5392..5409)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5563..6151)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5643..5662)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6322..7113,7114..7182))
                     /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     6945..6964
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7183..7287)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7355..7373
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7411..7433)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7533..7552
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(7633..7649,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(7633..7649,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg ctgcaggtcg tgggattcct gggttaatta
       61 gaaaaagaca tttttgctgt cagtcactgt caagagattc ttttgctggc atttcttcta
      121 gaagcaaaaa gagcgatgcg tcttttccgc tgaaccgttc cagcaaaaaa gactaccaac
      181 gcaatatgga ttgtcagaat catataaaag agaagcaaat aactccttgt cttgtatcaa
      241 ttgcattata atatcttctt gttagtgcaa tatcatatag aagtcatcat gggttcatgt
      301 tgtagttgct tgaaagattc ttcagacgag gccagtgtct caccaattgc cgataatgaa
      361 agagaagcgg tgactttatt actagggtat ttggaggata aagaccaatt agacttttac
      421 tctggtggtc cactaaaggc cctaactact ttggtgtatt cagataatct aaatttgcaa
      481 aggagcgccg ctctggcatt cgcagaaatt actgaaaaat acgtccgtca ggtctctaga
      541 gaggtactgg aacccatctt aatattatta caaagccaag atccacagat tcaagtggcc
      601 gcttgtgcag ccttgggtaa tttggccgtt aataatgaaa ataaattatt aattgtggaa
      661 atgggaggct tagagccatt aataaaccaa atgatgggtg acaatgtaga agttcaatgt
      721 aatgctgttg gatgcattac caatttagcc acaagggatg acaataaaca taagattgct
      781 acttctggtg ctttaattcc attgactaag ctagctaaat ccaaacatat aagagttcag
      841 agaaatgcta caggtgcatt attaaatatg acacattctg aggaaaatag aaaagaattg
      901 gttaatgcag gtgctgtgcc tgtattggta tctttactgt cgtccaccga tcctgatgtt
      961 cagtactatt gtactactgc tttatcaaat atagcagtgg atgaagccaa tcgcaagaag
     1021 ttggctcaaa ctgagccaag attggtttcc aaattggtta gtctgatgga ttctccatct
     1081 tcaagggtaa aatgtcaagc tactttggct ttacgaaacc tagcgtccga tacaagctac
     1141 caattagaaa ttgtaagggc tggtgggttg cctcatttgg taaagttgat tcaaagtgat
     1201 tctataccac tggttttggc tagtgtggca tgcattagaa acatttctat tcaccctcta
     1261 aatgaaggtt taattgttga tgcaggtttc ttaaaaccat tagtaagatt gttggactat
     1321 aaggattcag aagaaattca atgtcacgcc gtatctactt tgagaaattt ggctgcatca
     1381 tctgaaaaaa acagaaaaga gttcttcgaa agtggtgctg tcgaaaaatg taaggaactg
     1441 gctctagatt cacctgttag tgttcaaagt gaaatttccg catgttttgc tattttagcc
     1501 ctagcagatg tctccaaatt ggatctactg gaagcaaata tcttagatgc tttaatccct
     1561 atgacttttt cccagaacca agaagtttct ggtaatgctg ctgctgcttt ggcaaatcta
     1621 tgttcaagag tgaataatta tacaaaaatt attgaagcat gggatcgtcc gaatgaaggc
     1681 attcgtggat ttctgattag atttttgaaa agtgactatg ctacctttga acacattgcg
     1741 ctatggacaa ttttacaatt gctagaaagt cataatgata aagtggaaga tttggttaaa
     1801 aatgatgacg acatcattaa cggagtgaga aaaatggcag atgtgacctt tgagcgttta
     1861 caaagatcag gaattgatgt taaaaatccg gggagcaaca acaatccaag ctcgaatgat
     1921 aacaacagta ataacaatga cacaggatct gaacatcaac ctgtagagga tgcaagtttg
     1981 gaattgtata atattactca acagatttta caatttttac atttaattaa catcgctagc
     2041 agtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat
     2101 gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa
     2161 cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc aacacttgtc
     2221 actactttca cttatggtgt tcaatgcttt tcaagatacc cagatcatat gaaacggcat
     2281 gactttttca agagtgccat gcccgaaggt tatgtacagg aaagaactat atttttcaaa
     2341 gatgacggga actacaagac acgtgctgaa gtcaagtttg aaggtgatac ccttgttaat
     2401 agaatcgagt taaaaggtat tgattttaaa gaagatggaa acattcttgg acacaaattg
     2461 gaatacaact ataactcaca caatgtatac atcatggcag acaaacaaaa gaatggaatc
     2521 aaagttaact tcaaaattag acacaacatt gaagatggaa gcgttcaact agcagaccat
     2581 tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg
     2641 tccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt
     2701 gagtttgtaa cagctgctgg gattacacat ggcatggatg aactatacaa acctaggtga
     2761 ggcgcgcctt ttcttcaaaa gcgggactag gcattataat ttttatcaaa attttcttct
     2821 cagaagtttt attttctttc tctccatttc ttccatcttt ttaatctttg tttccttttt
     2881 ttacatcata ttacttctac ctactctact ctattctaat ttcaattgat tttttttatt
     2941 ttattttttt tttgttaata aaagaagaaa aaacttaaaa acgaataaga ggctgtcttt
     3001 gttccgtagt atatgcctat ttccttctag aagtttgaag tgtcgtaaat tttacctacg
     3061 gataatgttc gttacattgt tgaatggcaa ctgtagtgag gtaatgttca aaaagtgaga
     3121 aagcgggaga gaagggctac atacaaaaac caattctaga taacatctat atcttcgttt
     3181 ttagaatata ttgtaataaa acacaattga taatacagtt ctctcttcgt cctctgatgg
     3241 tgggagatct attaaaaata caagttagct gagcggggga aaaaaatagg tcttacccgg
     3301 attcgaaccg gggttgttcg gatcaaaacc gaaagtgata accactacac tataaaacct
     3361 cttaattatt gatttccaca ataattttat catatatcaa cactattctt cacatagttt
     3421 ttttaatccc ggtgtctctc atctgctaat gaaaatacgt tcaacttcta aataagcgaa
     3481 tttcttatga tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa
     3541 attttaaagt gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct
     3601 gtaggtcagg ttgctttctc aggtatagta tgaggtcgct cttattgacc acacctctac
     3661 cggcagatcc gctagggata acagggtaat atctgcaggt cgacaaccct taatataact
     3721 tcgtataatg tatgctatac gaagttatta ggtctagaga tcccaataca acagatcacg
     3781 tgatcttttt gtaagatgaa gttgaagtag tgttgcaccg tgccaatgca ggtggctatt
     3841 agattaaata tgtgatttgt tctattaagt ttcctgtata attaatgggg agcgctgatt
     3901 ctcttttggt acgcttccca tccagcattt ctgtatcttt caccttcaac cttaggatct
     3961 ctacccttgg cgaaaagtcc tctgccaaca atgatgatat ctgatccacc acttacaact
     4021 tcgtcgacgg ttctgtactg ctgacccaat gcatcgcctt tgtcgtctaa acctacacct
     4081 ggggtcatga ttagccaatc aaacccttct tctcttcctc ccatatcgtt ctgagcaatg
     4141 aacccaataa cgaaatcttt atcactcttt gcaatatcaa cggtaccctt agtatattca
     4201 ccgtgtgcta gagaaccctt ggaagacaat tcagcaagca tcaataatcc ccttggttct
     4261 ttggtgacct cttgcgcacc ttgtttcaag ccagcaacaa taccagcacc agtaaccccg
     4321 tgggcgttgg tgatatcaga ccattctgcg atacggtaaa cgcccgatgt atattgtaat
     4381 ttgactgtgt taccgatatc ggcgaatttt ctgtcctcaa atatcaagaa cttgtatttc
     4441 tctgccaatg ctttcaatgg aacgacagta ccctcataac tgaaatcatc caagatatca
     4501 acgtgtgttt tcaaaaggca aatgtatgga cccaacgttt caacaagttt caatagctca
     4561 tcagtcgaac gaacgtcaag agaagcacac aaattggtct tcttttcatc cattaaacgt
     4621 aaaagtttcg atgcaaccgg acttgcatga gtctcagctc tactggtata tgattttgtg
     4681 gacatggtgc aactaattga cgggagtgta ttgacgctgg cgtactggct ttcacaaaat
     4741 ggcccaatca caaccacatc ttagatagtt gaaatgactt tagataacat caattgagat
     4801 gagcttaatc atgtcaaagc taaaagtgtc accatgaacg acaattctta agcaaatcac
     4861 gtgatataga tccacgaata accaccattt gatgctcgag gcaagtaatg tgtgtaaaaa
     4921 aatgcgttac caccatccaa tgcagaccga tcttctaccc agaatcacat atatttatgt
     4981 accgagtacc ttttttctat cttccaattg cttctcccat atgattgtct ccgtaagctc
     5041 gaaatttcta agttggattt taatcttcac gcaggatgac agttcgatga gcttctgagg
     5101 agtgtttaga acataatcat ttatccatgg tctatctctt cttgtcgctt tttctcctcg
     5161 atagaaccta aataaaacga gctctcgaga acccttaata taacttcgta taatgtatgc
     5221 tatacgaagt tattaggtga tatcagatcc actagtggcc tatgcggccg cggatctgcc
     5281 ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat
     5341 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5401 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5461 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5521 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5581 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5641 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5701 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5761 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5821 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5881 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5941 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6001 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6061 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6121 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6181 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6241 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6301 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6361 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6421 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6481 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6541 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6601 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6661 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6721 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6781 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6841 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6901 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6961 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     7021 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7081 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7141 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7201 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7261 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     7321 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     7381 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     7441 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     7501 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     7561 gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat
     7621 catacacata cgatttaggt gacactata
//
