LOCUS       sequence-329329-        8340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Vac8-RFP in yeasts.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8340)
  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 8340)
  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-RFP
FEATURES             Location/Qualifiers
     source          1..8340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(90..545)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(227..248)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     439..458
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     671..693
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     685..702
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     686..702
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     712..731
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        712..730
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2856..3527
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWGRVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(2983..3001)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3251..3270
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3445..3468
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3612..3636)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3703..3723)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3703..3721)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3742..3758)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3742..3758)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3755..3777)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3766..3782
                     /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(3790..3796,3797..3814,3815..3820))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3835..3856
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3973..3990)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4144..4732)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4224..4243)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4903..5694,5695..5763))
                     /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     5526..5545
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5764..5868)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5936..5954
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5992..6014)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6114..6133
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(6627..7721)
                     /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(7722..8129)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 tatgtagata tactagaagt tctcctcgac cggtcgatat gcggtgtgaa ataccgcaca
       61 gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt
      121 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
      181 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
      241 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
      301 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
      361 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
      421 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
      481 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
      541 cgcgtccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
      601 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
      661 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac
      721 tcactatagg gcgaattggg taccgaggac tcaaaacgaa aaggaaaaaa atacgagtgg
      781 aaagaaaaaa tgaacccttt ccagtaattg ataataatac aatcattcat tattagaaat
      841 atattagttc tcaattcaag gcagaatagt gctgtcattg tgatcaaatt tttgtgtgag
      901 tctgctttgc tttctgtttt tttttgttga ctcgctgttc aatctcctac ttttcctttt
      961 gttgttaaaa taaggtatta tatatttata caaccctata aaaagcaatt agccaatttc
     1021 caatcctacg aagacaggaa ctgagcaaac tataagggtg ttctttcttc tgtactatat
     1081 atacatttgc aactatgggt tcatgttgta gttgcttgaa agattcttca gacgaggcca
     1141 gtgtctcacc aattgccgat aatgaaagag aagcggtgac tttattacta gggtatttgg
     1201 aggataaaga ccaattagac ttttactctg gtggtccact aaaggcccta actactttgg
     1261 tgtattcaga taatctaaat ttgcaaagga gcgccgctct ggcattcgca gaaattactg
     1321 aaaaatacgt ccgtcaggtc tctagagagg tactggaacc catcttaata ttattacaaa
     1381 gccaagatcc acagattcaa gtggccgctt gtgcagcctt gggtaatttg gccgttaata
     1441 atgaaaataa attattaatt gtggaaatgg gaggcttaga gccattaata aaccaaatga
     1501 tgggtgacaa tgtagaagtt caatgtaatg ctgttggatg cattaccaat ttagccacaa
     1561 gggatgacaa taaacataag attgctactt ctggtgcttt aattccattg actaagctag
     1621 ctaaatccaa acatataaga gttcagagaa atgctacagg tgcattatta aatatgacac
     1681 attctgagga aaatagaaaa gaattggtta atgcaggtgc tgtgcctgta ttggtatctt
     1741 tactgtcgtc caccgatcct gatgttcagt actattgtac tactgcttta tcaaatatag
     1801 cagtggatga agccaatcgc aagaagttgg ctcaaactga gccaagattg gtttccaaat
     1861 tggttagtct gatggattct ccatcttcaa gggtaaaatg tcaagctact ttggctttac
     1921 gaaacctagc gtccgataca agctaccaat tagaaattgt aagggctggt gggttgcctc
     1981 atttggtaaa gttgattcaa agtgattcta taccactggt tttggctagt gtggcatgca
     2041 ttagaaacat ttctattcac cctctaaatg aaggtttaat tgttgatgca ggtttcttaa
     2101 aaccattagt aagattgttg gactataagg attcagaaga aattcaatgt cacgccgtat
     2161 ctactttgag aaatttggct gcatcatctg aaaaaaacag aaaagagttc ttcgaaagtg
     2221 gtgctgtcga aaaatgtaag gaactggctc tagattcacc tgttagtgtt caaagtgaaa
     2281 tttccgcatg ttttgctatt ttagccctag cagatgtctc caaattggat ctactggaag
     2341 caaatatctt agatgcttta atccctatga ctttttccca gaaccaagaa gtttctggta
     2401 atgctgctgc tgctttggca aatctatgtt caagagtgaa taattataca aaaattattg
     2461 aagcatggga tcgtccgaat gaaggcattc gtggatttct gattagattt ttgaaaagtg
     2521 actatgctac ctttgaacac attgcgctat ggacaatttt acaattgcta gaaagtcata
     2581 atgataaagt ggaagatttg gttaaaaatg atgacgacat cattaacgga gtgagaaaaa
     2641 tggcagatgt gacctttgag cgtttacaaa gatcaggaat tgatgttaaa aatccgggga
     2701 gcaacaacaa tccaagctcg aatgataaca acagtaataa caatgacaca ggatctgaac
     2761 atcaacctgt agaggatgca agtttggaat tgtataatat tactcaacag attttacaat
     2821 ttttacatcg gatccccggg ttaattaaca gatccgcctc ctccgaggac gtcatcaagg
     2881 agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag ttcgagatcg
     2941 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca
     3001 agggcggccc cctgcccttc gcctgggaca tcctgtcccc tcagttccag tacggctcca
     3061 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg
     3121 gcttcaagtg ggggcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg
     3181 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc
     3241 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagcgga
     3301 tgtaccccga ggacggcgcc ctgaagggcg agatcaagat gaggctgaag ctgaaggacg
     3361 gcggccacta cgacgccgag gtcaagacca cctacatggc caagaagccc gtgcagctgc
     3421 ccggcgccta caagaccgac atcaagctgg acatcacctc ccacaacgag gactacacca
     3481 tcgtggaaca gtacgagcgc gccgagggcc gccactccac cggcgccgga tcttagggcg
     3541 cgccacttct aaataagcga atttcttatg atttatgatt tttattatta aataagttat
     3601 aaaaaaaata agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt
     3661 attcttgagt aactctttcc tgtaggtcag gttgctttct cccctttagt gagggttaat
     3721 tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3781 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3841 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3901 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3961 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4021 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4081 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4141 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4201 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4261 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4321 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4381 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4441 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4501 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4561 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4621 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4681 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4741 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4801 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4861 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4921 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4981 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5041 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5101 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5161 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5221 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5281 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5341 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5401 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5461 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5521 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5581 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5641 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5701 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5761 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5821 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5881 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     5941 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     6001 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     6061 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     6121 agagcagatt gtactgagag tgcaccatat cgactacgtc gttaaggccg tttctgacag
     6181 agtaaaattc ttgagggaac tttcaccatt atgggaaatg gttcaagaag gtattgactt
     6241 aaactccatc aaatggtcag gtcattgagt gttttttatt tgttgtattt tttttttttt
     6301 agagaaaatc ctccaatata taaattagga atcatagttt catgattttc tgttacacct
     6361 aactttttgt gtggtgccct cctccttgtc aatattaatg ttaaagtgca attctttttc
     6421 cttatcacgt tgagccatta gtatcaattt gcttacctgt attcctttac atcctccttt
     6481 ttctccttct tgataaatgt atgtagattg cgtatatagt ttcgtctacc ctatgaacat
     6541 attccatttt gtaatttcgt gtcgtttcta ttatgaattt catttataaa gtttatgtac
     6601 aaatatcata aaaaaagaga atctttttaa gcaaggattt tcttaacttc ttcggcgaca
     6661 gcatcaccga cttcggtggt actgttggaa ccacctaaat caccagttct gatacctgca
     6721 tccaaaacct ttttaactgc atcttcaatg gccttacctt cttcaggcaa gttcaatgac
     6781 aatttcaaca tcattgcagc agacaagata gtggcgatag ggttgacctt attctttggc
     6841 aaatctggag cagaaccgtg gcatggttcg tacaaaccaa atgcggtgtt cttgtctggc
     6901 aaagaggcca aggacgcaga tggcaacaaa cccaaggaac ctgggataac ggaggcttca
     6961 tcggagatga tatcaccaaa catgttgctg gtgattataa taccatttag gtgggttggg
     7021 ttcttaacta ggatcatggc ggcagaatca atcaattgat gttgaacctt caatgtaggg
     7081 aattcgttct tgatggtttc ctccacagtt tttctccata atcttgaaga ggccaaaaca
     7141 ttagctttat ccaaggacca aataggcaat ggtggctcat gttgtagggc catgaaagcg
     7201 gccattcttg tgattctttg cacttctgga acggtgtatt gttcactatc ccaagcgaca
     7261 ccatcaccat cgtcttcctt tctcttacca aagtaaatac ctcccactaa ttctctgaca
     7321 acaacgaagt cagtaccttt agcaaattgt ggcttgattg gagataagtc taaaagagag
     7381 tcggatgcaa agttacatgg tcttaagttg gcgtacaatt gaagttcttt acggattttt
     7441 agtaaacctt gttcaggtct aacactaccg gtaccccatt taggaccacc cacagcacct
     7501 aacaaaacgg catcagcctt cttggaggct tccagcgcct catctggaag tggaacacct
     7561 gtagcatcga tagcagcacc accaattaaa tgattttcga aatcgaactt gacattggaa
     7621 cgaacatcag aaatagcttt aagaacctta atggcttcgg ctgtgatttc ttgaccaacg
     7681 tggtcacctg gcaaaacgac gatcttctta ggggcagaca ttagaatggt atatccttga
     7741 aatatatata tatatattgc tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat
     7801 tgctaaccac ctattggaaa aaacaatagg tccttaaata atattgtcaa cttcaagtat
     7861 tgtgatgcaa gcatttagtc atgaacgctt ctctattcta tatgaaaagc cggttccggc
     7921 gctctcacct ttcctttttc tcccaatttt tcagttgaaa aaggtatatg cgtcaggcga
     7981 cctctgaaat taacaaaaaa tttccagtca tcgaatttga ttctgtgcga tagcgcccct
     8041 gtgtgttctc gttatgttga ggaaaaaaat aatggttgct aagagattcg aactcttgca
     8101 tcttacgata cctgagtatt cccacagtta actgcggtca agatatttct tgaatcaggc
     8161 gccttagacc gctcggccaa acaaccaatt acttgttgag aaatagagta taattatcct
     8221 ataaatataa cgtttttgaa cacacatgaa caaggaagta caggacaatt gattttgaag
     8281 agaatgtgga ttttgatgta attgttggga ttccattttt aataaggcaa taatattagg
//
