LOCUS       sequence-323868-        8357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Vac8-GFP in yeasts..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8357)
  AUTHORS   Gatica D, Wen X, Cheong H, Klionsky DJ
  TITLE     Vac8 determines phagophore assembly site vacuolar localization 
            during nitrogen starvation-induced autophagy.
  JOURNAL   Autophagy. 2020 Jun 8. doi: 10.1080/15548627.2020.1776474.
  PUBMED    32508216
REFERENCE   2  (bases 1 to 8357)
  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: "Autophagy. 
            2020 Jun 8. doi: 10.1080/15548627.2020.1776474."
COMMENT     Sequence Label: pRS405 VAC8-GFP
FEATURES             Location/Qualifiers
     source          1..8357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(663..1757)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVAGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANLLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1770..2174)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2474..2929)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2611..2632)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2823..2842
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3059..3081
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3073..3090
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3074..3090
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3100..3119
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3100..3118
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5217..5927
                     /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(5244..5272)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5867..5887
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(6006..6030)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(6097..6117)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6097..6115)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6136..6152)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6136..6152)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6149..6171)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6160..6176
                     /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)."
     promoter        complement(join(6184..6190,6191..6208,6209..6214))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(6367..6384)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6538..7126)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6618..6637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7297..8088,8089..8157))
                     /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     7920..7939
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8158..8262)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8330..8348
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatcga ctacgtcgta aggccgtttc tgacagagta aaattcttga gggaactttc
      241 accattatgg gaaatgcttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca
      301 ttgagtgttt tttatttgtt gtattttttt ttttttagag aaaatcctcc aatatcaaat
      361 taggaatcgt agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
      421 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
      481 aatttgctta cctgtattcc tttactatcc tcctttttct ccttcttgat aaatgtatgt
      541 agattgcgta tatagtttcg tctaccctat gaacatattc cattttgtaa tttcgtgtcg
      601 tttctattat gaatttcatt tataaagttt atgtacaaat atcataaaaa aagagaatct
      661 ttttaagcaa ggattttctt aacttcttcg gcgacagcat caccgacttc ggtggtactg
      721 ttggaaccac ctaaatcacc agttctgata cctgcatcca aaaccttttt aactgcatct
      781 tcaatggcct taccttcttc aggcaagttc aatgacaatt tcaacatcat tgcagcagac
      841 aagatagtgg cgatagggtc aaccttattc tttggcaaat ctggagcaga accgtggcat
      901 ggttcgtaca aaccaaatgc ggtgttcttg tctggcaaag aggccaagga cgcagatggc
      961 aacaaaccca aggaacctgg gataacggag gcttcatcgg agatgatatc accaaacatg
     1021 ttgctggtga ttataatacc atttaggtgg gttgggttct taactaggat catggcggca
     1081 gaatcaatca attgatgttg aaccttcaat gtagggaatt cgttcttgat ggtttcctcc
     1141 acagtttttc tccataatct tgaagaggcc aaaagattag ctttatccaa ggaccaaata
     1201 ggcaatggtg gctcatgttg tagggccatg aaagcggcca ttcttgtgat tctttgcact
     1261 tctggaacgg tgtattgttc actatcccaa gcgacaccat caccatcgtc ttcctttctc
     1321 ttaccaaagt aaatacctcc cactaattct ctgacaacaa cgaagtcagt acctttagca
     1381 aattgtggct tgattggaga taagtctaaa agagagtcgg atgcaaagtt acatggtctt
     1441 aagttggcgt acaattgaag ttctttacgg atttttagta aaccttgttc aggtctaaca
     1501 ctaccggtac cccatttagg accagccaca gcacctaaca aaacggcatc aaccttcttg
     1561 gaggcttcca gcgcctcatc tggaagtggg acacctgtag catcgatagc agcaccacca
     1621 attaaatgat tttcgaaatc gaacttgaca ttggaacgaa catcagaaat agctttaaga
     1681 accttaatgg cttcggctgt gatttcttga ccaacgtggt cacctggcaa aacgacgatc
     1741 ttcttagggg cagacatagg ggcagacatt agaatggtat atccttgaaa tatatatata
     1801 tattgctgaa atgtaaaagg taagaaaagt tagaaagtaa gacgattgct aaccacctat
     1861 tggaaaaaac aataggtcct taaataatat tgtcaacttc aagtattgtg atgcaagcat
     1921 ttagtcatga acgcttctct attctatatg aaaagccggt tccggcctct cacctttcct
     1981 ttttctccca atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca
     2041 aaaaatttcc agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat
     2101 gttgaggaaa aaaataatgg ttgctaagag attcgaactc ttgcatctta cgatacctga
     2161 gtattcccac agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg
     2221 gccaaacaac caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt
     2281 ttgaacacac atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg
     2341 atgtaattgt tgggattcca tttttaataa ggcaataata ttaggtatgt ggatatacta
     2401 gaagttctcc tcgaccgtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2461 ataccgcatc aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt
     2521 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2581 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2641 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2701 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2761 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2821 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2881 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg
     2941 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     3001 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     3061 cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc
     3121 gaattgggta ccgaggactc aaaacgaaaa ggaaaaaaat acgagtggaa agaaaaaatg
     3181 aaccctttcc agtaattgat aataatacaa tcattcatta ttagaaatat attagttctc
     3241 aattcaaggc agaatagtgc tgtcattgtg atcaaatttt tgtgtgagtc tgctttgctt
     3301 tctgtttttt tttgttgact cgctgttcaa tctcctactt ttccttttgt tgttaaaata
     3361 aggtattata tatttataca accctataaa aagcaattag ccaatttcca atcctacgaa
     3421 gacaggaact gagcaaacta taagggtgtt ctttcttctg tactatatat acatttgcaa
     3481 ctatgggttc atgttgtagt tgcttgaaag attcttcaga cgaggccagt gtctcaccaa
     3541 ttgccgataa tgaaagagaa gcggtgactt tattactagg gtatttggag gataaagacc
     3601 aattagactt ttactctggt ggtccactaa aggccctaac tactttggtg tattcagata
     3661 atctaaattt gcaaaggagc gccgctctgg cattcgcaga aattactgaa aaatacgtcc
     3721 gtcaggtctc tagagaggta ctggaaccca tcttaatatt attacaaagc caagatccac
     3781 agattcaagt ggccgcttgt gcagccttgg gtaatttggc cgttaataat gaaaataaat
     3841 tattaattgt ggaaatggga ggcttagagc cattaataaa ccaaatgatg ggtgacaatg
     3901 tagaagttca atgtaatgct gttggatgca ttaccaattt agccacaagg gatgacaata
     3961 aacataagat tgctacttct ggtgctttaa ttccattgac taagctagct aaatccaaac
     4021 atataagagt tcagagaaat gctacaggtg cattattaaa tatgacacat tctgaggaaa
     4081 atagaaaaga attggttaat gcaggtgctg tgcctgtatt ggtatcttta ctgtcgtcca
     4141 ccgatcctga tgttcagtac tattgtacta ctgctttatc aaatatagca gtggatgaag
     4201 ccaatcgcaa gaagttggct caaactgagc caagattggt ttccaaattg gttagtctga
     4261 tggattctcc atcttcaagg gtaaaatgtc aagctacttt ggctttacga aacctagcgt
     4321 ccgatacaag ctaccaatta gaaattgtaa gggctggtgg gttgcctcat ttggtaaagt
     4381 tgattcaaag tgattctata ccactggttt tggctagtgt ggcatgcatt agaaacattt
     4441 ctattcaccc tctaaatgaa ggtttaattg ttgatgcagg tttcttaaaa ccattagtaa
     4501 gattgttgga ctataaggat tcagaagaaa ttcaatgtca cgccgtatct actttgagaa
     4561 atttggctgc atcatctgaa aaaaacagaa aagagttctt cgaaagtggt gctgtcgaaa
     4621 aatgtaagga actggctcta gattcacctg ttagtgttca aagtgaaatt tccgcatgtt
     4681 ttgctatttt agccctagca gatgtctcca aattggatct actggaagca aatatcttag
     4741 atgctttaat ccctatgact ttttcccaga accaagaagt ttctggtaat gctgctgctg
     4801 ctttggcaaa tctatgttca agagtgaata attatacaaa aattattgaa gcatgggatc
     4861 gtccgaatga aggcattcgt ggatttctga ttagattttt gaaaagtgac tatgctacct
     4921 ttgaacacat tgcgctatgg acaattttac aattgctaga aagtcataat gataaagtgg
     4981 aagatttggt taaaaatgat gacgacatca ttaacggagt gagaaaaatg gcagatgtga
     5041 cctttgagcg tttacaaaga tcaggaattg atgttaaaaa tccggggagc aacaacaatc
     5101 caagctcgaa tgataacaac agtaataaca atgacacagg atctgaacat caacctgtag
     5161 aggatgcaag tttggaattg tataatatta ctcaacagat tttacaattt ttacatagta
     5221 aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta
     5281 atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta
     5341 cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta
     5401 ctttcactta tggtgttcaa tgcttttcaa gatacccaga tcatatgaaa cggcatgact
     5461 ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt ttcaaagatg
     5521 acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt gttaatagaa
     5581 tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttggacac aaattggaat
     5641 acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat ggaatcaaag
     5701 ttaacttcaa aattagacac aacattgaag atggaagcgt tcaactagca gaccattatc
     5761 aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcca
     5821 cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc cttcttgagt
     5881 ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaatag ggcgcgccac
     5941 ttctaaataa gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa
     6001 aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt
     6061 gagtaactct ttcctgtagg tcaggttgct ttctcccctt tagtgagggt taattgcgcg
     6121 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     6181 acacaacata ggagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgaggta
     6241 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6301 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     6361 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     6421 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     6481 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     6541 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     6601 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     6661 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     6721 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     6781 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     6841 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     6901 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     6961 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     7021 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     7081 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     7141 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     7201 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     7261 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     7321 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7381 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     7441 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     7501 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     7561 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     7621 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     7681 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     7741 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     7801 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     7861 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     7921 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     7981 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8041 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8101 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     8161 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     8221 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     8281 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     8341 cacgaggccc tttcgtc
//
