LOCUS       sequence-329325-        8404 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a myristoylation mutant Vac8-GFP in yeasts, mutation is 
            Gly2Ala.
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-GFP
FEATURES             Location/Qualifiers
     source          1..8404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(95..550)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(232..253)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     444..463
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     676..698
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     690..707
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     691..707
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     717..736
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        717..735
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2912..2940)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3535..3555
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3680..3704)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3771..3791)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3771..3789)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3810..3826)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3810..3826)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3823..3845)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3834..3850
                     /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(3858..3864,3865..3882,3883..3888))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3903..3924
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4041..4058)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4212..4800)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4292..4311)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4971..5762,5763..5831))
                     /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     5594..5613
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5832..5936)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6004..6022
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6060..6082)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6182..6201
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(6696..7790)
                     /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(7791..8198)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 ttaggtatgt agatatacta gaagttctcc tcgaccggtc gatatgcggt gtgaaatacc
       61 gcacagatgc gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta
      121 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
      181 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
      241 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
      301 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
      361 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
      421 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
      481 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
      541 cagggcgcgt ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
      601 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
      661 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat
      721 acgactcact atagggcgaa ttgggtaccg aggactcaaa acgaaaagga aaaaaatacg
      781 agtggaaaga aaaaatgaac cctttccagt aattgataat aatacaatca ttcattatta
      841 gaaatatatt agttctcaat tcaaggcaga atagtgctgt cattgtgatc aaatttttgt
      901 gtgagtctgc tttgctttct gttttttttt gttgactcgc tgttcaatct cctacttttc
      961 cttttgttgt taaaataagg tattatatat ttatacaacc ctataaaaag caattagcca
     1021 atttccaatc ctacgaagac aggaactgag caaactataa gggtgttctt tcttctgtac
     1081 tatatataca tttgcaacta tggcttcatg ttgtagttgc ttgaaagatt cttcagacga
     1141 ggccagtgtc tcaccaattg ccgataatga aagagaagcg gtgactttat tactagggta
     1201 tttggaggat aaagaccaat tagactttta ctctggtggt ccactaaagg ccctaactac
     1261 tttggtgtat tcagataatc taaatttgca aaggagcgcc gctctggcat tcgcagaaat
     1321 tactgaaaaa tacgtccgtc aggtctctag agaggtactg gaacccatct taatattatt
     1381 acaaagccaa gatccacaga ttcaagtggc cgcttgtgca gccttgggta atttggccgt
     1441 taataatgaa aataaattat taattgtgga aatgggaggc ttagagccat taataaacca
     1501 aatgatgggt gacaatgtag aagttcaatg taatgctgtt ggatgcatta ccaatttagc
     1561 cacaagggat gacaataaac ataagattgc tacttctggt gctttaattc cattgactaa
     1621 gctagctaaa tccaaacata taagagttca gagaaatgct acaggtgcat tattaaatat
     1681 gacacattct gaggaaaata gaaaagaatt ggttaatgca ggtgctgtgc ctgtattggt
     1741 atctttactg tcgtccaccg atcctgatgt tcagtactat tgtactactg ctttatcaaa
     1801 tatagcagtg gatgaagcca atcgcaagaa gttggctcaa actgagccaa gattggtttc
     1861 caaattggtt agtctgatgg attctccatc ttcaagggta aaatgtcaag ctactttggc
     1921 tttacgaaac ctagcgtccg atacaagcta ccaattagaa attgtaaggg ctggtgggtt
     1981 gcctcatttg gtaaagttga ttcaaagtga ttctatacca ctggttttgg ctagtgtggc
     2041 atgcattaga aacatttcta ttcaccctct aaatgaaggt ttaattgttg atgcaggttt
     2101 cttaaaacca ttagtaagat tgttggacta taaggattca gaagaaattc aatgtcacgc
     2161 cgtatctact ttgagaaatt tggctgcatc atctgaaaaa aacagaaaag agttcttcga
     2221 aagtggtgct gtcgaaaaat gtaaggaact ggctctagat tcacctgtta gtgttcaaag
     2281 tgaaatttcc gcatgttttg ctattttagc cctagcagat gtctccaaat tggatctact
     2341 ggaagcaaat atcttagatg ctttaatccc tatgactttt tcccagaacc aagaagtttc
     2401 tggtaatgct gctgctgctt tggcaaatct atgttcaaga gtgaataatt atacaaaaat
     2461 tattgaagca tgggatcgtc cgaatgaagg cattcgtgga tttctgatta gatttttgaa
     2521 aagtgactat gctacctttg aacacattgc gctatggaca attttacaat tgctagaaag
     2581 tcataatgat aaagtggaag atttggttaa aaatgatgac gacatcatta acggagtgag
     2641 aaaaatggca gatgtgacct ttgagcgttt acaaagatca ggaattgatg ttaaaaatcc
     2701 ggggagcaac aacaatccaa gctcgaatga taacaacagt aataacaatg acacaggatc
     2761 tgaacatcaa cctgtagagg atgcaagttt ggaattgtat aatattactc aacagatttt
     2821 acaattttta catcggatcc ccgggttaat taattcgcga attcggggag gcggaggtgg
     2881 agctagcaaa ggagaagaac tcttcactgg agttgtccca attcttgttg aattagatgg
     2941 tgatgttaac ggccacaagt tctctgtcag tggagagggt gaaggtgatg caacatacgg
     3001 aaaacttacc ctgaagttca tctgcactac tggcaaactg cctgttccat ggccaacact
     3061 agtcactact ctgtgctatg gtgttcaatg cttttcaaga tacccggatc atatgaaacg
     3121 gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaaagga ccatcttctt
     3181 caaagatgac ggcaactaca agacacgtgc tgaagtcaag tttgaaggtg atacccttgt
     3241 taatagaatc gagttaaaag gtattgactt caaggaagat ggcaacattc tgggacacaa
     3301 attggaatac aactataact cacacaatgt atacatcatg gcagacaaac aaaagaatgg
     3361 aatcaaagtg aacttcaaga cccgccacaa cattgaagat ggaagcgttc aactagcaga
     3421 ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta
     3481 cctgtccaca caatctgccc tttcgaaaga tcccaacgaa aagagagacc acatggtcct
     3541 tcttgagttt gtaacagctg ctgggattac acatggcatg gatgaactgt acaacatcga
     3601 ttgaggcgcg ccacttctaa ataagcgaat ttcttatgat ttatgatttt tattattaaa
     3661 taagttataa aaaaaataag tgtatacaaa ttttaaagtg actcttaggt tttaaaacga
     3721 aaattcttat tcttgagtaa ctctttcctg taggtcaggt tgctttctcc cctttagtga
     3781 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3841 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3901 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3961 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4021 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     4081 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     4141 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     4201 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     4261 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     4321 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     4381 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4441 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4501 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4561 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4621 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4681 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4741 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4801 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4861 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4921 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4981 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5041 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5101 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5161 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     5221 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     5281 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     5341 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     5401 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5461 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     5521 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5581 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5641 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5701 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5761 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5821 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5881 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5941 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     6001 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     6061 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     6121 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     6181 gcggcatcag agcagattgt actgagagtg caccatatcg actacgtcgt taaggccgtt
     6241 tctgacagag taaaattctt gagggaactt tcaccattat gggaaatggt tcaagaaggt
     6301 attgacttaa actccatcaa atggtcaggt cattgagtgt tttttatttg ttgtattttt
     6361 ttttttttta gagaaaatcc tccaatatat aaattaggaa tcatagtttc atgattttct
     6421 gttacaccta actttttgtg tggtgccctc ctccttgtca atattaatgt taaagtgcaa
     6481 ttctttttcc ttatcacgtt gagccattag tatcaatttg cttacctgta ttcctttaca
     6541 tcctcctttt tctccttctt gataaatgta tgtagattgc gtatatagtt tcgtctaccc
     6601 tatgaacata ttccattttg taatttcgtg tcgtttctat tatgaatttc atttataaag
     6661 tttatgtaca aatatcataa aaaaagagaa tctttttaag caaggatttt cttaacttct
     6721 tcggcgacag catcaccgac ttcggtggta ctgttggaac cacctaaatc accagttctg
     6781 atacctgcat ccaaaacctt tttaactgca tcttcaatgg ccttaccttc ttcaggcaag
     6841 ttcaatgaca atttcaacat cattgcagca gacaagatag tggcgatagg gttgacctta
     6901 ttctttggca aatctggagc agaaccgtgg catggttcgt acaaaccaaa tgcggtgttc
     6961 ttgtctggca aagaggccaa ggacgcagat ggcaacaaac ccaaggaacc tgggataacg
     7021 gaggcttcat cggagatgat atcaccaaac atgttgctgg tgattataat accatttagg
     7081 tgggttgggt tcttaactag gatcatggcg gcagaatcaa tcaattgatg ttgaaccttc
     7141 aatgtaggga attcgttctt gatggtttcc tccacagttt ttctccataa tcttgaagag
     7201 gccaaaacat tagctttatc caaggaccaa ataggcaatg gtggctcatg ttgtagggcc
     7261 atgaaagcgg ccattcttgt gattctttgc acttctggaa cggtgtattg ttcactatcc
     7321 caagcgacac catcaccatc gtcttccttt ctcttaccaa agtaaatacc tcccactaat
     7381 tctctgacaa caacgaagtc agtaccttta gcaaattgtg gcttgattgg agataagtct
     7441 aaaagagagt cggatgcaaa gttacatggt cttaagttgg cgtacaattg aagttcttta
     7501 cggattttta gtaaaccttg ttcaggtcta acactaccgg taccccattt aggaccaccc
     7561 acagcaccta acaaaacggc atcagccttc ttggaggctt ccagcgcctc atctggaagt
     7621 ggaacacctg tagcatcgat agcagcacca ccaattaaat gattttcgaa atcgaacttg
     7681 acattggaac gaacatcaga aatagcttta agaaccttaa tggcttcggc tgtgatttct
     7741 tgaccaacgt ggtcacctgg caaaacgacg atcttcttag gggcagacat tagaatggta
     7801 tatccttgaa atatatatat atatattgct gaaatgtaaa aggtaagaaa agttagaaag
     7861 taagacgatt gctaaccacc tattggaaaa aacaataggt ccttaaataa tattgtcaac
     7921 ttcaagtatt gtgatgcaag catttagtca tgaacgcttc tctattctat atgaaaagcc
     7981 ggttccggcg ctctcacctt tcctttttct cccaattttt cagttgaaaa aggtatatgc
     8041 gtcaggcgac ctctgaaatt aacaaaaaat ttccagtcat cgaatttgat tctgtgcgat
     8101 agcgcccctg tgtgttctcg ttatgttgag gaaaaaaata atggttgcta agagattcga
     8161 actcttgcat cttacgatac ctgagtattc ccacagttaa ctgcggtcaa gatatttctt
     8221 gaatcaggcg ccttagaccg ctcggccaaa caaccaatta cttgttgaga aatagagtat
     8281 aattatccta taaatataac gtttttgaac acacatgaac aaggaagtac aggacaattg
     8341 attttgaaga gaatgtggat tttgatgtaa ttgttgggat tccattttta ataaggcaat
     8401 aata
//
