LOCUS       sequence-329326-        8403 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses a palmitoylation mutant Vac8-GFP in yeasts, mutations are 
            Cys4Ala, Cys5Ala, Cys7Ala.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8403)
  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 8403)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-C4A,C5A,C7A-GFP
FEATURES             Location/Qualifiers
     source          1..8403
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(107..562)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(244..265)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     456..475
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     688..710
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     702..719
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     703..719
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     729..748
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        729..747
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2924..2952)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3547..3567
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3692..3716)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3783..3803)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3783..3801)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3822..3838)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3822..3838)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3835..3857)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3846..3862
                     /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(3870..3876,3877..3894,3895..3900))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3915..3936
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4053..4070)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4224..4812)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4304..4323)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4983..5774,5775..5843))
                     /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     5606..5625
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5844..5948)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6016..6034
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6072..6094)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6194..6213
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(6707..7801)
                     /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(7802..8209)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 aaggcaataa tattaggtat gtagatatac tagaagttct cctcgaccgg tcgatatgcg
       61 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt
      121 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
      181 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
      241 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
      301 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
      361 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
      421 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
      481 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
      541 aatgcgccgc tacagggcgc gtccattcgc cattcaggct gcgcaactgt tgggaagggc
      601 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
      661 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
      721 agcgcgcgta atacgactca ctatagggcg aattgggtac cgaggactca aaacgaaaag
      781 gaaaaaaata cgagtggaaa gaaaaaatga accctttcca gtaattgata ataatacaat
      841 cattcattat tagaaatata ttagttctca attcaaggca gaatagtgct gtcattgtga
      901 tcaaattttt gtgtgagtct gctttgcttt ctgttttttt ttgttgactc gctgttcaat
      961 ctcctacttt tccttttgtt gttaaaataa ggtattatat atttatacaa ccctataaaa
     1021 agcaattagc caatttccaa tcctacgaag acaggaactg agcaaactat aagggtgttc
     1081 tttcttctgt actatatata catttgcaac tatgggttca gctgctagtg ccttgaaaga
     1141 ttcttcagac gaggccagtg tctcaccaat tgccgataat gaaagagaag cggtgacttt
     1201 attactaggg tatttggagg ataaagacca attagacttt tactctggtg gtccactaaa
     1261 ggccctaact actttggtgt attcagataa tctaaatttg caaaggagcg ccgctctggc
     1321 attcgcagaa attactgaaa aatacgtccg tcaggtctct agagaggtac tggaacccat
     1381 cttaatatta ttacaaagcc aagatccaca gattcaagtg gccgcttgtg cagccttggg
     1441 taatttggcc gttaataatg aaaataaatt attaattgtg gaaatgggag gcttagagcc
     1501 attaataaac caaatgatgg gtgacaatgt agaagttcaa tgtaatgctg ttggatgcat
     1561 taccaattta gccacaaggg atgacaataa acataagatt gctacttctg gtgctttaat
     1621 tccattgact aagctagcta aatccaaaca tataagagtt cagagaaatg ctacaggtgc
     1681 attattaaat atgacacatt ctgaggaaaa tagaaaagaa ttggttaatg caggtgctgt
     1741 gcctgtattg gtatctttac tgtcgtccac cgatcctgat gttcagtact attgtactac
     1801 tgctttatca aatatagcag tggatgaagc caatcgcaag aagttggctc aaactgagcc
     1861 aagattggtt tccaaattgg ttagtctgat ggattctcca tcttcaaggg taaaatgtca
     1921 agctactttg gctttacgaa acctagcgtc cgatacaagc taccaattag aaattgtaag
     1981 ggctggtggg ttgcctcatt tggtaaagtt gattcaaagt gattctatac cactggtttt
     2041 ggctagtgtg gcatgcatta gaaacatttc tattcaccct ctaaatgaag gtttaattgt
     2101 tgatgcaggt ttcttaaaac cattagtaag attgttggac tataaggatt cagaagaaat
     2161 tcaatgtcac gccgtatcta ctttgagaaa tttggctgca tcatctgaaa aaaacagaaa
     2221 agagttcttc gaaagtggtg ctgtcgaaaa atgtaaggaa ctggctctag attcacctgt
     2281 tagtgttcaa agtgaaattt ccgcatgttt tgctatttta gccctagcag atgtctccaa
     2341 attggatcta ctggaagcaa atatcttaga tgctttaatc cctatgactt tttcccagaa
     2401 ccaagaagtt tctggtaatg ctgctgctgc tttggcaaat ctatgttcaa gagtgaataa
     2461 ttatacaaaa attattgaag catgggatcg tccgaatgaa ggcattcgtg gatttctgat
     2521 tagatttttg aaaagtgact atgctacctt tgaacacatt gcgctatgga caattttaca
     2581 attgctagaa agtcataatg ataaagtgga agatttggtt aaaaatgatg acgacatcat
     2641 taacggagtg agaaaaatgg cagatgtgac ctttgagcgt ttacaaagat caggaattga
     2701 tgttaaaaat ccggggagca acaacaatcc aagctcgaat gataacaaca gtaataacaa
     2761 tgacacagga tctgaacatc aacctgtaga ggatgcaagt ttggaattgt ataatattac
     2821 tcaacagatt ttacaatttt tacatcggat ccccgggtta attaattcgc gaattcgggg
     2881 aggcggaggt ggagctagca aaggagaaga actcttcact ggagttgtcc caattcttgt
     2941 tgaattagat ggtgatgtta acggccacaa gttctctgtc agtggagagg gtgaaggtga
     3001 tgcaacatac ggaaaactta ccctgaagtt catctgcact actggcaaac tgcctgttcc
     3061 atggccaaca ctagtcacta ctctgtgcta tggtgttcaa tgcttttcaa gatacccgga
     3121 tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag
     3181 gaccatcttc ttcaaagatg acggcaacta caagacacgt gctgaagtca agtttgaagg
     3241 tgataccctt gttaatagaa tcgagttaaa aggtattgac ttcaaggaag atggcaacat
     3301 tctgggacac aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa
     3361 acaaaagaat ggaatcaaag tgaacttcaa gacccgccac aacattgaag atggaagcgt
     3421 tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc
     3481 agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga
     3541 ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact
     3601 gtacaacatc gattgaggcg cgccacttct aaataagcga atttcttatg atttatgatt
     3661 tttattatta aataagttat aaaaaaaata agtgtataca aattttaaag tgactcttag
     3721 gttttaaaac gaaaattctt attcttgagt aactctttcc tgtaggtcag gttgctttct
     3781 cccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg
     3841 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     3901 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     3961 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     4021 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4081 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4141 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4201 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4261 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4321 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4381 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4441 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4501 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4561 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4621 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     4681 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4741 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     4801 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     4861 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4921 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     4981 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     5041 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     5101 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     5161 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     5221 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     5281 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     5341 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     5401 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     5461 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     5521 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     5581 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     5641 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     5701 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     5761 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5821 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5881 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5941 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     6001 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     6061 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     6121 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     6181 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat cgactacgtc
     6241 gttaaggccg tttctgacag agtaaaattc ttgagggaac tttcaccatt atgggaaatg
     6301 gttcaagaag gtattgactt aaactccatc aaatggtcag gtcattgagt gttttttatt
     6361 tgttgtattt tttttttttt agagaaaatc ctccaatata taaattagga atcatagttt
     6421 catgattttc tgttacacct aactttttgt gtggtgccct cctccttgtc aatattaatg
     6481 ttaaagtgca attctttttc cttatcacgt tgagccatta gtatcaattt gcttacctgt
     6541 attcctttac atcctccttt ttctccttct tgataaatgt atgtagattg cgtatatagt
     6601 ttcgtctacc ctatgaacat attccatttt gtaatttcgt gtcgtttcta ttatgaattt
     6661 catttataaa gtttatgtac aaatatcata aaaaaagaga atctttttaa gcaaggattt
     6721 tcttaacttc ttcggcgaca gcatcaccga cttcggtggt actgttggaa ccacctaaat
     6781 caccagttct gatacctgca tccaaaacct ttttaactgc atcttcaatg gccttacctt
     6841 cttcaggcaa gttcaatgac aatttcaaca tcattgcagc agacaagata gtggcgatag
     6901 ggttgacctt attctttggc aaatctggag cagaaccgtg gcatggttcg tacaaaccaa
     6961 atgcggtgtt cttgtctggc aaagaggcca aggacgcaga tggcaacaaa cccaaggaac
     7021 ctgggataac ggaggcttca tcggagatga tatcaccaaa catgttgctg gtgattataa
     7081 taccatttag gtgggttggg ttcttaacta ggatcatggc ggcagaatca atcaattgat
     7141 gttgaacctt caatgtaggg aattcgttct tgatggtttc ctccacagtt tttctccata
     7201 atcttgaaga ggccaaaaca ttagctttat ccaaggacca aataggcaat ggtggctcat
     7261 gttgtagggc catgaaagcg gccattcttg tgattctttg cacttctgga acggtgtatt
     7321 gttcactatc ccaagcgaca ccatcaccat cgtcttcctt tctcttacca aagtaaatac
     7381 ctcccactaa ttctctgaca acaacgaagt cagtaccttt agcaaattgt ggcttgattg
     7441 gagataagtc taaaagagag tcggatgcaa agttacatgg tcttaagttg gcgtacaatt
     7501 gaagttcttt acggattttt agtaaacctt gttcaggtct aacactaccg gtaccccatt
     7561 taggaccacc cacagcacct aacaaaacgg catcagcctt cttggaggct tccagcgcct
     7621 catctggaag tggaacacct gtagcatcga tagcagcacc accaattaaa tgattttcga
     7681 aatcgaactt gacattggaa cgaacatcag aaatagcttt aagaacctta atggcttcgg
     7741 ctgtgatttc ttgaccaacg tggtcacctg gcaaaacgac gatcttctta ggggcagaca
     7801 ttagaatggt atatccttga aatatatata tatatattgc tgaaatgtaa aaggtaagaa
     7861 aagttagaaa gtaagacgat tgctaaccac ctattggaaa aaacaatagg tccttaaata
     7921 atattgtcaa cttcaagtat tgtgatgcaa gcatttagtc atgaacgctt ctctattcta
     7981 tatgaaaagc cggttccggc gctctcacct ttcctttttc tcccaatttt tcagttgaaa
     8041 aaggtatatg cgtcaggcga cctctgaaat taacaaaaaa tttccagtca tcgaatttga
     8101 ttctgtgcga tagcgcccct gtgtgttctc gttatgttga ggaaaaaaat aatggttgct
     8161 aagagattcg aactcttgca tcttacgata cctgagtatt cccacagtta actgcggtca
     8221 agatatttct tgaatcaggc gccttagacc gctcggccaa acaaccaatt acttgttgag
     8281 aaatagagta taattatcct ataaatataa cgtttttgaa cacacatgaa caaggaagta
     8341 caggacaatt gattttgaag agaatgtgga ttttgatgta attgttggga ttccattttt
     8401 aat
//
