LOCUS       sequence-329332-        8708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Peroxisome localized Vac8-RFP in yeasts. Made replacing 
            the first 21 base pairs of the VAC8 ORF with the first 390 base 
            pairs from the PEX14 ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8708)
  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 8708)
  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 PEX14-VAC8-RFP
FEATURES             Location/Qualifiers
     source          1..8708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     115..134
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(627..1721)
                     /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(1722..2129)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(2430..2885)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2567..2588)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2779..2798
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3011..3033
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3025..3042
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3026..3042
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3052..3071
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3052..3070
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5565..6236
                     /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(5692..5710)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5960..5979
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     6154..6177
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(6321..6345)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(6412..6432)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6412..6430)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6451..6467)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6451..6467)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6464..6486)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6475..6491
                     /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(6499..6505,6506..6523,6524..6529))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6544..6565
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6682..6699)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6853..7441)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6933..6952)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7612..8403,8404..8472))
                     /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     8235..8254
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8473..8577)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8645..8663
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(join(8701..8708,1..15))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
       61 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
      121 cagagcagat tgtactgaga gtgcaccata tcgactacgt cgttaaggcc gtttctgaca
      181 gagtaaaatt cttgagggaa ctttcaccat tatgggaaat ggttcaagaa ggtattgact
      241 taaactccat caaatggtca ggtcattgag tgttttttat ttgttgtatt tttttttttt
      301 agagaaaatc ctccaatata taaattagga atcatagttt catgattttc tgttacacct
      361 aactttttgt gtggtgccct cctccttgtc aatattaatg ttaaagtgca attctttttc
      421 cttatcacgt tgagccatta gtatcaattt gcttacctgt attcctttac atcctccttt
      481 ttctccttct tgataaatgt atgtagattg cgtatatagt ttcgtctacc ctatgaacat
      541 attccatttt gtaatttcgt gtcgtttcta ttatgaattt catttataaa gtttatgtac
      601 aaatatcata aaaaaagaga atctttttaa gcaaggattt tcttaacttc ttcggcgaca
      661 gcatcaccga cttcggtggt actgttggaa ccacctaaat caccagttct gatacctgca
      721 tccaaaacct ttttaactgc atcttcaatg gccttacctt cttcaggcaa gttcaatgac
      781 aatttcaaca tcattgcagc agacaagata gtggcgatag ggttgacctt attctttggc
      841 aaatctggag cagaaccgtg gcatggttcg tacaaaccaa atgcggtgtt cttgtctggc
      901 aaagaggcca aggacgcaga tggcaacaaa cccaaggaac ctgggataac ggaggcttca
      961 tcggagatga tatcaccaaa catgttgctg gtgattataa taccatttag gtgggttggg
     1021 ttcttaacta ggatcatggc ggcagaatca atcaattgat gttgaacctt caatgtaggg
     1081 aattcgttct tgatggtttc ctccacagtt tttctccata atcttgaaga ggccaaaaca
     1141 ttagctttat ccaaggacca aataggcaat ggtggctcat gttgtagggc catgaaagcg
     1201 gccattcttg tgattctttg cacttctgga acggtgtatt gttcactatc ccaagcgaca
     1261 ccatcaccat cgtcttcctt tctcttacca aagtaaatac ctcccactaa ttctctgaca
     1321 acaacgaagt cagtaccttt agcaaattgt ggcttgattg gagataagtc taaaagagag
     1381 tcggatgcaa agttacatgg tcttaagttg gcgtacaatt gaagttcttt acggattttt
     1441 agtaaacctt gttcaggtct aacactaccg gtaccccatt taggaccacc cacagcacct
     1501 aacaaaacgg catcagcctt cttggaggct tccagcgcct catctggaag tggaacacct
     1561 gtagcatcga tagcagcacc accaattaaa tgattttcga aatcgaactt gacattggaa
     1621 cgaacatcag aaatagcttt aagaacctta atggcttcgg ctgtgatttc ttgaccaacg
     1681 tggtcacctg gcaaaacgac gatcttctta ggggcagaca ttagaatggt atatccttga
     1741 aatatatata tatatattgc tgaaatgtaa aaggtaagaa aagttagaaa gtaagacgat
     1801 tgctaaccac ctattggaaa aaacaatagg tccttaaata atattgtcaa cttcaagtat
     1861 tgtgatgcaa gcatttagtc atgaacgctt ctctattcta tatgaaaagc cggttccggc
     1921 gctctcacct ttcctttttc tcccaatttt tcagttgaaa aaggtatatg cgtcaggcga
     1981 cctctgaaat taacaaaaaa tttccagtca tcgaatttga ttctgtgcga tagcgcccct
     2041 gtgtgttctc gttatgttga ggaaaaaaat aatggttgct aagagattcg aactcttgca
     2101 tcttacgata cctgagtatt cccacagtta actgcggtca agatatttct tgaatcaggc
     2161 gccttagacc gctcggccaa acaaccaatt acttgttgag aaatagagta taattatcct
     2221 ataaatataa cgtttttgaa cacacatgaa caaggaagta caggacaatt gattttgaag
     2281 agaatgtgga ttttgatgta attgttggga ttccattttt aataaggcaa taatattagg
     2341 tatgtagata tactagaagt tctcctcgac cggtcgatat gcggtgtgaa ataccgcaca
     2401 gatgcgtaag gagaaaatac cgcatcagga aattgtaagc gttaatattt tgttaaaatt
     2461 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     2521 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     2581 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     2641 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     2701 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     2761 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     2821 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     2881 cgcgtccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     2941 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     3001 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac
     3061 tcactatagg gcgaattggg taccgaggac tcaaaacgaa aaggaaaaaa atacgagtgg
     3121 aaagaaaaaa tgaacccttt ccagtaattg ataataatac aatcattcat tattagaaat
     3181 atattagttc tcaattcaag gcagaatagt gctgtcattg tgatcaaatt tttgtgtgag
     3241 tctgctttgc tttctgtttt tttttgttga ctcgctgttc aatctcctac ttttcctttt
     3301 gttgttaaaa taaggtatta tatatttata caaccctata aaaagcaatt agccaatttc
     3361 caatcctacg aagacaggaa ctgagcaaac tataagggtg ttctttcttc tgtactatat
     3421 atacatttgc aactatgagt gacgtggtca gtaaagatcg taaggcattg ttcgactcag
     3481 cagtatcctt tttaaaggat gagtccatta aagatgctcc acttttaaag aaaatcgaat
     3541 ttttaaaatc caaagggtta acagaaaagg agattgaaat agccatgaaa gagcccaaga
     3601 aagacggtat cgtaggcgat gaagtatcga aaaaaattgg tagtactgag aatagagcct
     3661 cacaggatat gtatctctat gaagcgatgc caccaacgct gccccacagg gattggaagg
     3721 actattttgt gatggctact gccacagctg ggctgttgta tggtgcatat gaagtaacta
     3781 gaaggtatgt gataccaaat attttaccag aagcaaaaag caagttgaaa gattcttcag
     3841 acgaggccag tgtctcacca attgccgata atgaaagaga agcggtgact ttattactag
     3901 ggtatttgga ggataaagac caattagact tttactctgg tggtccacta aaggccctaa
     3961 ctactttggt gtattcagat aatctaaatt tgcaaaggag cgccgctctg gcattcgcag
     4021 aaattactga aaaatacgtc cgtcaggtct ctagagaggt actggaaccc atcttaatat
     4081 tattacaaag ccaagatcca cagattcaag tggccgcttg tgcagccttg ggtaatttgg
     4141 ccgttaataa tgaaaataaa ttattaattg tggaaatggg aggcttagag ccattaataa
     4201 accaaatgat gggtgacaat gtagaagttc aatgtaatgc tgttggatgc attaccaatt
     4261 tagccacaag ggatgacaat aaacataaga ttgctacttc tggtgcttta attccattga
     4321 ctaagctagc taaatccaaa catataagag ttcagagaaa tgctacaggt gcattattaa
     4381 atatgacaca ttctgaggaa aatagaaaag aattggttaa tgcaggtgct gtgcctgtat
     4441 tggtatcttt actgtcgtcc accgatcctg atgttcagta ctattgtact actgctttat
     4501 caaatatagc agtggatgaa gccaatcgca agaagttggc tcaaactgag ccaagattgg
     4561 tttccaaatt ggttagtctg atggattctc catcttcaag ggtaaaatgt caagctactt
     4621 tggctttacg aaacctagcg tccgatacaa gctaccaatt agaaattgta agggctggtg
     4681 ggttgcctca tttggtaaag ttgattcaaa gtgattctat accactggtt ttggctagtg
     4741 tggcatgcat tagaaacatt tctattcacc ctctaaatga aggtttaatt gttgatgcag
     4801 gtttcttaaa accattagta agattgttgg actataagga ttcagaagaa attcaatgtc
     4861 acgccgtatc tactttgaga aatttggctg catcatctga aaaaaacaga aaagagttct
     4921 tcgaaagtgg tgctgtcgaa aaatgtaagg aactggctct agattcacct gttagtgttc
     4981 aaagtgaaat ttccgcatgt tttgctattt tagccctagc agatgtctcc aaattggatc
     5041 tactggaagc aaatatctta gatgctttaa tccctatgac tttttcccag aaccaagaag
     5101 tttctggtaa tgctgctgct gctttggcaa atctatgttc aagagtgaat aattatacaa
     5161 aaattattga agcatgggat cgtccgaatg aaggcattcg tggatttctg attagatttt
     5221 tgaaaagtga ctatgctacc tttgaacaca ttgcgctatg gacaatttta caattgctag
     5281 aaagtcataa tgataaagtg gaagatttgg ttaaaaatga tgacgacatc attaacggag
     5341 tgagaaaaat ggcagatgtg acctttgagc gtttacaaag atcaggaatt gatgttaaaa
     5401 atccggggag caacaacaat ccaagctcga atgataacaa cagtaataac aatgacacag
     5461 gatctgaaca tcaacctgta gaggatgcaa gtttggaatt gtataatatt actcaacaga
     5521 ttttacaatt tttacatcgg atccccgggt taattaacag atccgcctcc tccgaggacg
     5581 tcatcaagga gttcatgcgc ttcaaggtgc gcatggaggg ctccgtgaac ggccacgagt
     5641 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     5701 aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtcccct cagttccagt
     5761 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
     5821 tccccgaggg cttcaagtgg gggcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     5881 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     5941 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcca
     6001 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagatg aggctgaagc
     6061 tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacatggcc aagaagcccg
     6121 tgcagctgcc cggcgcctac aagaccgaca tcaagctgga catcacctcc cacaacgagg
     6181 actacaccat cgtggaacag tacgagcgcg ccgagggccg ccactccacc ggcgccggat
     6241 cttagggcgc gccacttcta aataagcgaa tttcttatga tttatgattt ttattattaa
     6301 ataagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg
     6361 aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc ccctttagtg
     6421 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     6481 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     6541 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6601 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6661 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     6721 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     6781 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     6841 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6901 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6961 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     7021 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     7081 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     7141 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     7201 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     7261 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     7321 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     7381 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     7441 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     7501 agggattttt gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     7561 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     7621 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     7681 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     7741 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     7801 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     7861 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7921 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7981 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     8041 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     8101 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     8161 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     8221 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     8281 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     8341 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     8401 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     8461 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     8521 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     8581 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     8641 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     8701 cctctgac
//
