LOCUS       sequence-329327-        8404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a myristoylation and palmitoylation mutant Vac8-GFP in 
            yeasts, mutations are Gly2Ala, Cys4Ala, Cys5Ala, Cys7Ala.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8404)
  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 8404)
  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-G2A,C4A,C5A,C7A-GFP
FEATURES             Location/Qualifiers
     source          1..8404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     22..40
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(78..100)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     200..219
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(714..1808)
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     promoter        complement(1809..2216)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2517..2972)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2654..2675)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2866..2885
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3098..3120
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3112..3129
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3113..3129
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3139..3158
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3139..3157
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5334..5362)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5957..5977
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(6102..6126)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(6193..6213)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6193..6211)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6232..6248)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6232..6248)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6245..6267)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6256..6272
                     /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(6280..6286,6287..6304,6305..6310))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6325..6346
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6463..6480)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6634..7222)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6714..6733)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7393..8184,8185..8253))
                     /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     8016..8035
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8254..8358)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
       61 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
      121 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
      181 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatcgac
      241 tacgtcgtta aggccgtttc tgacagagta aaattcttga gggaactttc accattatgg
      301 gaaatggttc aagaaggtat tgacttaaac tccatcaaat ggtcaggtca ttgagtgttt
      361 tttatttgtt gtattttttt tttttttaga gaaaatcctc caatatataa attaggaatc
      421 atagtttcat gattttctgt tacacctaac tttttgtgtg gtgccctcct ccttgtcaat
      481 attaatgtta aagtgcaatt ctttttcctt atcacgttga gccattagta tcaatttgct
      541 tacctgtatt cctttacatc ctcctttttc tccttcttga taaatgtatg tagattgcgt
      601 atatagtttc gtctacccta tgaacatatt ccattttgta atttcgtgtc gtttctatta
      661 tgaatttcat ttataaagtt tatgtacaaa tatcataaaa aaagagaatc tttttaagca
      721 aggattttct taacttcttc ggcgacagca tcaccgactt cggtggtact gttggaacca
      781 cctaaatcac cagttctgat acctgcatcc aaaacctttt taactgcatc ttcaatggcc
      841 ttaccttctt caggcaagtt caatgacaat ttcaacatca ttgcagcaga caagatagtg
      901 gcgatagggt tgaccttatt ctttggcaaa tctggagcag aaccgtggca tggttcgtac
      961 aaaccaaatg cggtgttctt gtctggcaaa gaggccaagg acgcagatgg caacaaaccc
     1021 aaggaacctg ggataacgga ggcttcatcg gagatgatat caccaaacat gttgctggtg
     1081 attataatac catttaggtg ggttgggttc ttaactagga tcatggcggc agaatcaatc
     1141 aattgatgtt gaaccttcaa tgtagggaat tcgttcttga tggtttcctc cacagttttt
     1201 ctccataatc ttgaagaggc caaaacatta gctttatcca aggaccaaat aggcaatggt
     1261 ggctcatgtt gtagggccat gaaagcggcc attcttgtga ttctttgcac ttctggaacg
     1321 gtgtattgtt cactatccca agcgacacca tcaccatcgt cttcctttct cttaccaaag
     1381 taaatacctc ccactaattc tctgacaaca acgaagtcag tacctttagc aaattgtggc
     1441 ttgattggag ataagtctaa aagagagtcg gatgcaaagt tacatggtct taagttggcg
     1501 tacaattgaa gttctttacg gatttttagt aaaccttgtt caggtctaac actaccggta
     1561 ccccatttag gaccacccac agcacctaac aaaacggcat cagccttctt ggaggcttcc
     1621 agcgcctcat ctggaagtgg aacacctgta gcatcgatag cagcaccacc aattaaatga
     1681 ttttcgaaat cgaacttgac attggaacga acatcagaaa tagctttaag aaccttaatg
     1741 gcttcggctg tgatttcttg accaacgtgg tcacctggca aaacgacgat cttcttaggg
     1801 gcagacatta gaatggtata tccttgaaat atatatatat atattgctga aatgtaaaag
     1861 gtaagaaaag ttagaaagta agacgattgc taaccaccta ttggaaaaaa caataggtcc
     1921 ttaaataata ttgtcaactt caagtattgt gatgcaagca tttagtcatg aacgcttctc
     1981 tattctatat gaaaagccgg ttccggcgct ctcacctttc ctttttctcc caatttttca
     2041 gttgaaaaag gtatatgcgt caggcgacct ctgaaattaa caaaaaattt ccagtcatcg
     2101 aatttgattc tgtgcgatag cgcccctgtg tgttctcgtt atgttgagga aaaaaataat
     2161 ggttgctaag agattcgaac tcttgcatct tacgatacct gagtattccc acagttaact
     2221 gcggtcgaga tatttcttga atcaggcgcc ttagaccgct cggccaaaca accaattact
     2281 tgttgagaaa tagagtataa ttatcctata aatataacgt ttttgaacac acatgaacaa
     2341 ggaagtacag gacaattgat tttgaagaga atgtggattt tgatgtaatt gttgggattc
     2401 catttttaat aaggcaataa tattaggtat gtagatatac tagaagttct cctcgaccgg
     2461 tcgatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat
     2521 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     2581 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     2641 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     2701 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     2761 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     2821 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     2881 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     2941 cgccgcgctt aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt
     3001 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     3061 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     3121 acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac cgaggactca
     3181 aaacgaaaag gaaaaaaata cgagtggaaa gaaaaaatga accctttcca gtaattgata
     3241 ataatacaat cattcattat tagaaatata ttagttctca attcaaggca gaatagtgct
     3301 gtcattgtga tcaaattttt gtgtgagtct gctttgcttt ctgttttttt ttgttgactc
     3361 gctgttcaat ctcctacttt tccttttgtt gttaaaataa ggtattatat atttatacaa
     3421 ccctataaaa agcaattagc caatttccaa tcctacgaag acaggaactg agcaaactat
     3481 aagggtgttc tttcttctgt actatatata catttgcaac tatggcttca gctgctagtg
     3541 ccttgaaaga ttcttcagac gaggccagtg tctcaccaat tgccgataat gaaagagaag
     3601 cggtgacttt attactaggg tatttggagg ataaagacca attagacttt tactctggtg
     3661 gtccactaaa ggccctaact actttggtgt attcagataa tctaaatttg caaaggagcg
     3721 ccgctctggc attcgcagaa attactgaaa aatacgtccg tcaggtctct agagaggtac
     3781 tggaacccat cttaatatta ttacaaagcc aagatccaca gattcaagtg gccgcttgtg
     3841 cagccttggg taatttggcc gttaataatg aaaataaatt attaattgtg gaaatgggag
     3901 gcttagagcc attaataaac caaatgatgg gtgacaatgt agaagttcaa tgtaatgctg
     3961 ttggatgcat taccaattta gccacaaggg atgacaataa acataagatt gctacttctg
     4021 gtgctttaat tccattgact aagctagcta aatccaaaca tataagagtt cagagaaatg
     4081 ctacaggtgc attattaaat atgacacatt ctgaggaaaa tagaaaagaa ttggttaatg
     4141 caggtgctgt gcctgtattg gtatctttac tgtcgtccac cgatcctgat gttcagtact
     4201 attgtactac tgctttatca aatatagcag tggatgaagc caatcgcaag aagttggctc
     4261 aaactgagcc aagattggtt tccaaattgg ttagtctgat ggattctcca tcttcaaggg
     4321 taaaatgtca agctactttg gctttacgaa acctagcgtc cgatacaagc taccaattag
     4381 aaattgtaag ggctggtggg ttgcctcatt tggtaaagtt gattcaaagt gattctatac
     4441 cactggtttt ggctagtgtg gcatgcatta gaaacatttc tattcaccct ctaaatgaag
     4501 gtttaattgt tgatgcaggt ttcttaaaac cattagtaag attgttggac tataaggatt
     4561 cagaagaaat tcaatgtcac gccgtatcta ctttgagaaa tttggctgca tcatctgaaa
     4621 aaaacagaaa agagttcttc gaaagtggtg ctgtcgaaaa atgtaaggaa ctggctctag
     4681 attcacctgt tagtgttcaa agtgaaattt ccgcatgttt tgctatttta gccctagcag
     4741 atgtctccaa attggatcta ctggaagcaa atatcttaga tgctttaatc cctatgactt
     4801 tttcccagaa ccaagaagtt tctggtaatg ctgctgctgc tttggcaaat ctatgttcaa
     4861 gagtgaataa ttatacaaaa attattgaag catgggatcg tccgaatgaa ggcattcgtg
     4921 gatttctgat tagatttttg aaaagtgact atgctacctt tgaacacatt gcgctatgga
     4981 caattttaca attgctagaa agtcataatg ataaagtgga agatttggtt aaaaatgatg
     5041 acgacatcat taacggagtg agaaaaatgg cagatgtgac ctttgagcgt ttacaaagat
     5101 caggaattga tgttaaaaat ccggggagca acaacaatcc aagctcgaat gataacaaca
     5161 gtaataacaa tgacacagga tctgaacatc aacctgtaga ggatgcaagt ttggaattgt
     5221 ataatattac tcaacagatt ttacaatttt tacatcggat ccccgggtta attaattcgc
     5281 gaattcgggg aggcggaggt ggagctagca aaggagaaga actcttcact ggagttgtcc
     5341 caattcttgt tgaattagat ggtgatgtta acggccacaa gttctctgtc agtggagagg
     5401 gtgaaggtga tgcaacatac ggaaaactta ccctgaagtt catctgcact actggcaaac
     5461 tgcctgttcc atggccaaca ctagtcacta ctctgtgcta tggtgttcaa tgcttttcaa
     5521 gatacccgga tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg
     5581 tacaggaaag gaccatcttc ttcaaagatg acggcaacta caagacacgt gctgaagtca
     5641 agtttgaagg tgataccctt gttaatagaa tcgagttaaa aggtattgac ttcaaggaag
     5701 atggcaacat tctgggacac aaattggaat acaactataa ctcacacaat gtatacatca
     5761 tggcagacaa acaaaagaat ggaatcaaag tgaacttcaa gacccgccac aacattgaag
     5821 atggaagcgt tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg
     5881 tccttttacc agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg
     5941 aaaagagaga ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca
     6001 tggatgaact gtacaacatc gattgaggcg cgccacttct aaataagcga atttcttatg
     6061 atttatgatt tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag
     6121 tgactcttag gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag
     6181 gttgctttct cccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     6241 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     6301 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     6361 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6421 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     6481 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     6541 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     6601 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     6661 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     6721 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     6781 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     6841 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     6901 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     6961 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7021 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     7081 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     7141 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     7201 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     7261 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     7321 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     7381 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     7441 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     7501 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     7561 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     7621 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     7681 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     7741 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     7801 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     7861 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     7921 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     7981 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     8041 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     8101 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     8161 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     8221 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     8281 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     8341 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     8401 tatt
//
