LOCUS       sequence-329330-        8340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a myristoylation and palmitoylation mutant Vac8-RFP in 
            yeasts, mutations are Gly2Ala, Cys4Ala, Cys5Ala, Cys7Ala.
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-G2A,C4A,C5A,C7A-RFP
FEATURES             Location/Qualifiers
     source          1..8340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(65..520)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(202..223)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     414..433
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     646..668
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     660..677
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     661..677
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     687..706
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        687..705
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2831..3502
                     /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(2958..2976)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3226..3245
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3420..3443
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3587..3611)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3678..3698)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3678..3696)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3717..3733)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3717..3733)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3730..3752)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3741..3757
                     /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(3765..3771,3772..3789,3790..3795))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3810..3831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3948..3965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4119..4707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4199..4218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4878..5669,5670..5738))
                     /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     5501..5520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5739..5843)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5911..5929
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5967..5989)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6089..6108
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(6602..7696)
                     /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(7697..8104)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 tcgaccggtc gatatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
       61 caggaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
      121 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
      181 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
      241 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
      301 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
      361 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
      421 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
      481 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt ccattcgcca ttcaggctgc
      541 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
      601 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
      661 gtaaaacgac ggccagtgag cgcgcgtaat acgactcact atagggcgaa ttgggtaccg
      721 aggactcaaa acgaaaagga aaaaaatacg agtggaaaga aaaaatgaac cctttccagt
      781 aattgataat aatacaatca ttcattatta gaaatatatt agttctcaat tcaaggcaga
      841 atagtgctgt cattgtgatc aaatttttgt gtgagtctgc tttgctttct gttttttttt
      901 gttgactcgc tgttcaatct cctacttttc cttttgttgt taaaataagg tattatatat
      961 ttatacaacc ctataaaaag caattagcca atttccaatc ctacgaagac aggaactgag
     1021 caaactataa gggtgttctt tcttctgtac tatatataca tttgcaacta tggcttcagc
     1081 tgctagtgcc ttgaaagatt cttcagacga ggccagtgtc tcaccaattg ccgataatga
     1141 aagagaagcg gtgactttat tactagggta tttggaggat aaagaccaat tagactttta
     1201 ctctggtggt ccactaaagg ccctaactac tttggtgtat tcagataatc taaatttgca
     1261 aaggagcgcc gctctggcat tcgcagaaat tactgaaaaa tacgtccgtc aggtctctag
     1321 agaggtactg gaacccatct taatattatt acaaagccaa gatccacaga ttcaagtggc
     1381 cgcttgtgca gccttgggta atttggccgt taataatgaa aataaattat taattgtgga
     1441 aatgggaggc ttagagccat taataaacca aatgatgggt gacaatgtag aagttcaatg
     1501 taatgctgtt ggatgcatta ccaatttagc cacaagggat gacaataaac ataagattgc
     1561 tacttctggt gctttaattc cattgactaa gctagctaaa tccaaacata taagagttca
     1621 gagaaatgct acaggtgcat tattaaatat gacacattct gaggaaaata gaaaagaatt
     1681 ggttaatgca ggtgctgtgc ctgtattggt atctttactg tcgtccaccg atcctgatgt
     1741 tcagtactat tgtactactg ctttatcaaa tatagcagtg gatgaagcca atcgcaagaa
     1801 gttggctcaa actgagccaa gattggtttc caaattggtt agtctgatgg attctccatc
     1861 ttcaagggta aaatgtcaag ctactttggc tttacgaaac ctagcgtccg atacaagcta
     1921 ccaattagaa attgtaaggg ctggtgggtt gcctcatttg gtaaagttga ttcaaagtga
     1981 ttctatacca ctggttttgg ctagtgtggc atgcattaga aacatttcta ttcaccctct
     2041 aaatgaaggt ttaattgttg atgcaggttt cttaaaacca ttagtaagat tgttggacta
     2101 taaggattca gaagaaattc aatgtcacgc cgtatctact ttgagaaatt tggctgcatc
     2161 atctgaaaaa aacagaaaag agttcttcga aagtggtgct gtcgaaaaat gtaaggaact
     2221 ggctctagat tcacctgtta gtgttcaaag tgaaatttcc gcatgttttg ctattttagc
     2281 cctagcagat gtctccaaat tggatctact ggaagcaaat atcttagatg ctttaatccc
     2341 tatgactttt tcccagaacc aagaagtttc tggtaatgct gctgctgctt tggcaaatct
     2401 atgttcaaga gtgaataatt atacaaaaat tattgaagca tgggatcgtc cgaatgaagg
     2461 cattcgtgga tttctgatta gatttttgaa aagtgactat gctacctttg aacacattgc
     2521 gctatggaca attttacaat tgctagaaag tcataatgat aaagtggaag atttggttaa
     2581 aaatgatgac gacatcatta acggagtgag aaaaatggca gatgtgacct ttgagcgttt
     2641 acaaagatca ggaattgatg ttaaaaatcc ggggagcaac aacaatccaa gctcgaatga
     2701 taacaacagt aataacaatg acacaggatc tgaacatcaa cctgtagagg atgcaagttt
     2761 ggaattgtat aatattactc aacagatttt acaattttta catcggatcc ccgggttaat
     2821 taacagatcc gcctcctccg aggacgtcat caaggagttc atgcgcttca aggtgcgcat
     2881 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     2941 cgagggcacc cagaccgcca agctgaaggt gaccaagggc ggccccctgc ccttcgcctg
     3001 ggacatcctg tcccctcagt tccagtacgg ctccaaggcc tacgtgaagc accccgccga
     3061 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggggc gcgtgatgaa
     3121 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     3181 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     3241 gaagaccatg ggctgggagg cctccaccga gcggatgtac cccgaggacg gcgccctgaa
     3301 gggcgagatc aagatgaggc tgaagctgaa ggacggcggc cactacgacg ccgaggtcaa
     3361 gaccacctac atggccaaga agcccgtgca gctgcccggc gcctacaaga ccgacatcaa
     3421 gctggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg agcgcgccga
     3481 gggccgccac tccaccggcg ccggatctta gggcgcgcca cttctaaata agcgaatttc
     3541 ttatgattta tgatttttat tattaaataa gttataaaaa aaataagtgt atacaaattt
     3601 taaagtgact cttaggtttt aaaacgaaaa ttcttattct tgagtaactc tttcctgtag
     3661 gtcaggttgc tttctcccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca
     3721 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     3781 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     3841 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     3901 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     3961 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4021 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4081 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4141 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4201 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     4261 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     4321 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     4381 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     4441 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     4501 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     4561 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     4621 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     4681 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     4741 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     4801 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     4861 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     4921 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     4981 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5041 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5101 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5161 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     5221 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     5281 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     5341 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     5401 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     5461 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     5521 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     5581 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     5641 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     5701 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     5761 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     5821 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     5881 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     5941 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     6001 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     6061 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     6121 catatcgact acgtcgttaa ggccgtttct gacagagtaa aattcttgag ggaactttca
     6181 ccattatggg aaatggttca agaaggtatt gacttaaact ccatcaaatg gtcaggtcat
     6241 tgagtgtttt ttatttgttg tatttttttt tttttagaga aaatcctcca atatataaat
     6301 taggaatcat agtttcatga ttttctgtta cacctaactt tttgtgtggt gccctcctcc
     6361 ttgtcaatat taatgttaaa gtgcaattct ttttccttat cacgttgagc cattagtatc
     6421 aatttgctta cctgtattcc tttacatcct cctttttctc cttcttgata aatgtatgta
     6481 gattgcgtat atagtttcgt ctaccctatg aacatattcc attttgtaat ttcgtgtcgt
     6541 ttctattatg aatttcattt ataaagttta tgtacaaata tcataaaaaa agagaatctt
     6601 tttaagcaag gattttctta acttcttcgg cgacagcatc accgacttcg gtggtactgt
     6661 tggaaccacc taaatcacca gttctgatac ctgcatccaa aaccttttta actgcatctt
     6721 caatggcctt accttcttca ggcaagttca atgacaattt caacatcatt gcagcagaca
     6781 agatagtggc gatagggttg accttattct ttggcaaatc tggagcagaa ccgtggcatg
     6841 gttcgtacaa accaaatgcg gtgttcttgt ctggcaaaga ggccaaggac gcagatggca
     6901 acaaacccaa ggaacctggg ataacggagg cttcatcgga gatgatatca ccaaacatgt
     6961 tgctggtgat tataatacca tttaggtggg ttgggttctt aactaggatc atggcggcag
     7021 aatcaatcaa ttgatgttga accttcaatg tagggaattc gttcttgatg gtttcctcca
     7081 cagtttttct ccataatctt gaagaggcca aaacattagc tttatccaag gaccaaatag
     7141 gcaatggtgg ctcatgttgt agggccatga aagcggccat tcttgtgatt ctttgcactt
     7201 ctggaacggt gtattgttca ctatcccaag cgacaccatc accatcgtct tcctttctct
     7261 taccaaagta aatacctccc actaattctc tgacaacaac gaagtcagta cctttagcaa
     7321 attgtggctt gattggagat aagtctaaaa gagagtcgga tgcaaagtta catggtctta
     7381 agttggcgta caattgaagt tctttacgga tttttagtaa accttgttca ggtctaacac
     7441 taccggtacc ccatttagga ccacccacag cacctaacaa aacggcatca gccttcttgg
     7501 aggcttccag cgcctcatct ggaagtggaa cacctgtagc atcgatagca gcaccaccaa
     7561 ttaaatgatt ttcgaaatcg aacttgacat tggaacgaac atcagaaata gctttaagaa
     7621 ccttaatggc ttcggctgtg atttcttgac caacgtggtc acctggcaaa acgacgatct
     7681 tcttaggggc agacattaga atggtatatc cttgaaatat atatatatat attgctgaaa
     7741 tgtaaaaggt aagaaaagtt agaaagtaag acgattgcta accacctatt ggaaaaaaca
     7801 ataggtcctt aaataatatt gtcaacttca agtattgtga tgcaagcatt tagtcatgaa
     7861 cgcttctcta ttctatatga aaagccggtt ccggcgctct cacctttcct ttttctccca
     7921 atttttcagt tgaaaaaggt atatgcgtca ggcgacctct gaaattaaca aaaaatttcc
     7981 agtcatcgaa tttgattctg tgcgatagcg cccctgtgtg ttctcgttat gttgaggaaa
     8041 aaaataatgg ttgctaagag attcgaactc ttgcatctta caatacctga gtattcccac
     8101 agttaactgc ggtcaagata tttcttgaat caggcgcctt agaccgctcg gccaaacaac
     8161 caattacttg ttgagaaata gagtataatt atcctataaa tataacgttt ttgaacacac
     8221 atgaacaagg aagtacagga caattgattt tgaagagaat gtggattttg atgtaattgt
     8281 tgggattcca tttttaataa ggcaataata ttaggtatgt agatatacta gaagttctcc
//
