LOCUS       sequence-329321-        8404 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 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-GFP
FEATURES             Location/Qualifiers
     source          1..8404
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..57)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     157..176
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(671..1765)
                     /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(1766..2173)
                     /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     3055..3077
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3069..3086
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3070..3086
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3096..3115
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3096..3114
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5291..5319)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5914..5934
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(6059..6083)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(6150..6170)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6150..6168)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6189..6205)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6189..6205)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6202..6224)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6213..6229
                     /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(6237..6243,6244..6261,6262..6267))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6282..6303
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6420..6437)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6591..7179)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6671..6690)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7350..8141,8142..8210))
                     /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     7973..7992
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8211..8315)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8383..8401
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
       61 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
      121 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
      181 gagtgcacca tatcgactac gtcgttaagg ccgtttctga cagagtaaaa ttcttgaggg
      241 aactttcacc attatgggaa atggttcaag aaggtattga cttaaactcc atcaaatggt
      301 caggtcattg agtgtttttt atttgttgta tttttttttt ttttagagaa aatcctccaa
      361 tatataaatt aggaatcata gtttcatgat tttctgttac acctaacttt ttgtgtggtg
      421 ccctcctcct tgtcaatatt aatgttaaag tgcaattctt tttccttatc acgttgagcc
      481 attagtatca atttgcttac ctgtattcct ttacatcctc ctttttctcc ttcttgataa
      541 atgtatgtag attgcgtata tagtttcgtc taccctatga acatattcca ttttgtaatt
      601 tcgtgtcgtt tctattatga atttcattta taaagtttat gtacaaatat cataaaaaaa
      661 gagaatcttt ttaagcaagg attttcttaa cttcttcggc gacagcatca ccgacttcgg
      721 tggtactgtt ggaaccacct aaatcaccag ttctgatacc tgcatccaaa acctttttaa
      781 ctgcatcttc aatggcctta ccttcttcag gcaagttcaa tgacaatttc aacatcattg
      841 cagcagacaa gatagtggcg atagggttga ccttattctt tggcaaatct ggagcagaac
      901 cgtggcatgg ttcgtacaaa ccaaatgcgg tgttcttgtc tggcaaagag gccaaggacg
      961 cagatggcaa caaacccaag gaacctggga taacggaggc ttcatcggag atgatatcac
     1021 caaacatgtt gctggtgatt ataataccat ttaggtgggt tgggttctta actaggatca
     1081 tggcggcaga atcaatcaat tgatgttgaa ccttcaatgt agggaattcg ttcttgatgg
     1141 tttcctccac agtttttctc cataatcttg aagaggccaa aacattagct ttatccaagg
     1201 accaaatagg caatggtggc tcatgttgta gggccatgaa agcggccatt cttgtgattc
     1261 tttgcacttc tggaacggtg tattgttcac tatcccaagc gacaccatca ccatcgtctt
     1321 cctttctctt accaaagtaa atacctccca ctaattctct gacaacaacg aagtcagtac
     1381 ctttagcaaa ttgtggcttg attggagata agtctaaaag agagtcggat gcaaagttac
     1441 atggtcttaa gttggcgtac aattgaagtt ctttacggat ttttagtaaa ccttgttcag
     1501 gtctaacact accggtaccc catttaggac cacccacagc acctaacaaa acggcatcag
     1561 ccttcttgga ggcttccagc gcctcatctg gaagtggaac acctgtagca tcgatagcag
     1621 caccaccaat taaatgattt tcgaaatcga acttgacatt ggaacgaaca tcagaaatag
     1681 ctttaagaac cttaatggct tcggctgtga tttcttgacc aacgtggtca cctggcaaaa
     1741 cgacgatctt cttaggggca gacattagaa tggtatatcc ttgaaatata tatatatata
     1801 ttgctgaaat gtaaaaggta agaaaagtta gaaagtaaga cgattgctaa ccacctattg
     1861 gaaaaaacaa taggtcctta aataatattg tcaacttcaa gtattgtgat gcaagcattt
     1921 agtcatgaac gcttctctat tctatatgaa aagccggttc cggcgctctc acctttcctt
     1981 tttctcccaa tttttcagtt gaaaaaggta tatgcgtcag gcgacctctg aaattaacaa
     2041 aaaatttcca gtcatcgaat ttgattctgt gcgatagcgc ccctgtgtgt tctcgttatg
     2101 ttgaggaaaa aaataatggt tgctaagaga ttcgaactct tgcatcttac gatacctgag
     2161 tattcccaca gttaactgcg gtcaagatat ttcttgaatc aggcgcctta gaccgctcgg
     2221 ccaaacaacc aattacttgt tgagaaatag agtataatta tcctataaat ataacgtttt
     2281 tgaacacaca tgaacaagga agtacaggac aattgatttt gaagagaatg tggattttga
     2341 tgtaattgtt gggattccat ttttaataag gcaataatat taggtatgta gatatactag
     2401 aagttctcct cgaccggtcg atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2461 ataccgcatc aggaaattgt aagcgttaat 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 cattcgccat
     2941 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     3001 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     3061 cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat
     3121 tgggtaccga ggactcaaaa cgaaaaggaa aaaaatacga gtggaaagaa aaaatgaacc
     3181 ctttccagta attgataata atacaatcat tcattattag aaatatatta gttctcaatt
     3241 caaggcagaa tagtgctgtc attgtgatca aatttttgtg tgagtctgct ttgctttctg
     3301 tttttttttg ttgactcgct gttcaatctc ctacttttcc ttttgttgtt aaaataaggt
     3361 attatatatt tatacaaccc tataaaaagc aattagccaa tttccaatcc tacgaagaca
     3421 ggaactgagc aaactataag ggtgttcttt cttctgtact atatatacat ttgcaactat
     3481 gggttcatgt tgtagttgct tgaaagattc ttcagacgag gccagtgtct caccaattgc
     3541 cgataatgaa agagaagcgg tgactttatt actagggtat ttggaggata aagaccaatt
     3601 agacttttac tctggtggtc cactaaaggc cctaactact ttggtgtatt cagataatct
     3661 aaatttgcaa aggagcgccg ctctggcatt cgcagaaatt actgaaaaat acgtccgtca
     3721 ggtctctaga gaggtactgg aacccatctt aatattatta caaagccaag atccacagat
     3781 tcaagtggcc gcttgtgcag ccttgggtaa tttggccgtt aataatgaaa ataaattatt
     3841 aattgtggaa atgggaggct tagagccatt aataaaccaa atgatgggtg acaatgtaga
     3901 agttcaatgt aatgctgttg gatgcattac caatttagcc acaagggatg acaataaaca
     3961 taagattgct acttctggtg ctttaattcc attgactaag ctagctaaat ccaaacatat
     4021 aagagttcag agaaatgcta caggtgcatt attaaatatg acacattctg aggaaaatag
     4081 aaaagaattg gttaatgcag gtgctgtgcc tgtattggta tctttactgt cgtccaccga
     4141 tcctgatgtt cagtactatt gtactactgc tttatcaaat atagcagtgg atgaagccaa
     4201 tcgcaagaag ttggctcaaa ctgagccaag attggtttcc aaattggtta gtctgatgga
     4261 ttctccatct tcaagggtaa aatgtcaagc tactttggct ttacgaaacc tagcgtccga
     4321 tacaagctac caattagaaa ttgtaagggc tggtgggttg cctcatttgg taaagttgat
     4381 tcaaagtgat tctataccac tggttttggc tagtgtggca tgcattagaa acatttctat
     4441 tcaccctcta aatgaaggtt taattgttga tgcaggtttc ttaaaaccat tagtaagatt
     4501 gttggactat aaggattcag aagaaattca atgtcacgcc gtatctactt tgagaaattt
     4561 ggctgcatca tctgaaaaaa acagaaaaga gttcttcgaa agtggtgctg tcgaaaaatg
     4621 taaggaactg gctctagatt cacctgttag tgttcaaagt gaaatttccg catgttttgc
     4681 tattttagcc ctagcagatg tctccaaatt ggatctactg gaagcaaata tcttagatgc
     4741 tttaatccct atgacttttt cccagaacca agaagtttct ggtaatgctg ctgctgcttt
     4801 ggcaaatcta tgttcaagag tgaataatta tacaaaaatt attgaagcat gggatcgtcc
     4861 gaatgaaggc attcgtggat ttctgattag atttttgaaa agtgactatg ctacctttga
     4921 acacattgcg ctatggacaa ttttacaatt gctagaaagt cataatgata aagtggaaga
     4981 tttggttaaa aatgatgacg acatcattaa cggagtgaga aaaatggcag atgtgacctt
     5041 tgagcgttta caaagatcag gaattgatgt taaaaatccg gggagcaaca acaatccaag
     5101 ctcgaatgat aacaacagta ataacaatga cacaggatct gaacatcaac ctgtagagga
     5161 tgcaagtttg gaattgtata atattactca acagatttta caatttttac atcggatccc
     5221 cgggttaatt aattcgcgaa ttcggggagg cggaggtgga gctagcaaag gagaagaact
     5281 cttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaacg gccacaagtt
     5341 ctctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc tgaagttcat
     5401 ctgcactact ggcaaactgc ctgttccatg gccaacacta gtcactactc tgtgctatgg
     5461 tgttcaatgc ttttcaagat acccggatca tatgaaacgg catgactttt tcaagagtgc
     5521 catgcccgaa ggttatgtac aggaaaggac catcttcttc aaagatgacg gcaactacaa
     5581 gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt aatagaatcg agttaaaagg
     5641 tattgacttc aaggaagatg gcaacattct gggacacaaa ttggaataca actataactc
     5701 acacaatgta tacatcatgg cagacaaaca aaagaatgga atcaaagtga acttcaagac
     5761 ccgccacaac attgaagatg gaagcgttca actagcagac cattatcaac aaaatactcc
     5821 aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtccacac aatctgccct
     5881 ttcgaaagat cccaacgaaa agagagacca catggtcctt cttgagtttg taacagctgc
     5941 tgggattaca catggcatgg atgaactgta caacatcgat tgaggcgcgc cacttctaaa
     6001 taagcgaatt tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt
     6061 gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac
     6121 tctttcctgt aggtcaggtt gctttctccc ctttagtgag ggttaattgc gcgcttggcg
     6181 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     6241 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     6301 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6361 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6421 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6481 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6541 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6601 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6661 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6721 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6781 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6841 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6901 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6961 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     7021 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     7081 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     7141 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     7201 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     7261 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     7321 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7381 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7441 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7501 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7561 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7621 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7681 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7741 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7801 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7861 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7921 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7981 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     8041 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     8101 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     8161 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     8221 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     8281 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     8341 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     8401 ccct
//
