LOCUS       sequence-329331-        8481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ER localized Vac8-RFP in yeasts. Made replacing the first 
            21 base pairs of the VAC8 ORF with the first 162 base pairs from the
            SEC66 ORF, which encode the Sec66 transmembrane domain..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8481)
  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 8481)
  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 SEC66-VAC8-RFP
FEATURES             Location/Qualifiers
     source          1..8481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(85..540)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(222..243)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     434..453
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     666..688
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     680..697
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     681..697
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     707..726
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        707..725
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2992..3663
                     /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(3119..3137)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3387..3406
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3581..3604
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3748..3772)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3839..3859)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3839..3857)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3878..3894)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3878..3894)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3891..3913)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3902..3918
                     /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(3926..3932,3933..3950,3951..3956))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3971..3992
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4109..4126)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4280..4868)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4360..4379)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5039..5830,5831..5899))
                     /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     5662..5681
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5900..6004)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6072..6090
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6128..6150)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6250..6269
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(6763..7857)
                     /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(7858..8265)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
ORIGIN
        1 agatatacta gaagttctcc tcgaccggtc gatatgcggt gtgaaatacc gcacagatgc
       61 gtaaggagaa aataccgcat caggaaattg taagcgttaa tattttgtta aaattcgcgt
      121 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
      181 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
      241 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
      301 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
      361 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
      421 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
      481 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
      541 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
      601 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      661 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
      721 atagggcgaa ttgggtaccg aggactcaaa acgaaaagga aaaaaatacg agtggaaaga
      781 aaaaatgaac cctttccagt aattgataat aatacaatca ttcattatta gaaatatatt
      841 agttctcaat tcaaggcaga atagtgctgt cattgtgatc aaatttttgt gtgagtctgc
      901 tttgctttct gttttttttt gttgactcgc tgttcaatct cctacttttc cttttgttgt
      961 taaaataagg tattatatat ttatacaacc ctataaaaag caattagcca atttccaatc
     1021 ctacgaagac aggaactgag caaactataa gggtgttctt tcttctgtac tatatataca
     1081 tttgcaacta tgtccgaatt taatgaaaca aaattctcca acaacgggac gttttttgaa
     1141 acggaagagc caattgtgga gacgaaatca atctccgttt ataccccact catatatgtc
     1201 tttattctgg tggtgtccct tgtgatgttt gcttcaagct acagaaagaa gttgaaagat
     1261 tcttcagacg aggccagtgt ctcaccaatt gccgataatg aaagagaagc ggtgacttta
     1321 ttactagggt atttggagga taaagaccaa ttagactttt actctggtgg tccactaaag
     1381 gccctaacta ctttggtgta ttcagataat ctaaatttgc aaaggagcgc cgctctggca
     1441 ttcgcagaaa ttactgaaaa atacgtccgt caggtctcta gagaggtact ggaacccatc
     1501 ttaatattat tacaaagcca agatccacag attcaagtgg ccgcttgtgc agccttgggt
     1561 aatttggccg ttaataatga aaataaatta ttaattgtgg aaatgggagg cttagagcca
     1621 ttaataaacc aaatgatggg tgacaatgta gaagttcaat gtaatgctgt tggatgcatt
     1681 accaatttag ccacaaggga tgacaataaa cataagattg ctacttctgg tgctttaatt
     1741 ccattgacta agctagctaa atccaaacat ataagagttc agagaaatgc tacaggtgca
     1801 ttattaaata tgacacattc tgaggaaaat agaaaagaat tggttaatgc aggtgctgtg
     1861 cctgtattgg tatctttact gtcgtccacc gatcctgatg ttcagtacta ttgtactact
     1921 gctttatcaa atatagcagt ggatgaagcc aatcgcaaga agttggctca aactgagcca
     1981 agattggttt ccaaattggt tagtctgatg gattctccat cttcaagggt aaaatgtcaa
     2041 gctactttgg ctttacgaaa cctagcgtcc gatacaagct accaattaga aattgtaagg
     2101 gctggtgggt tgcctcattt ggtaaagttg attcaaagtg attctatacc actggttttg
     2161 gctagtgtgg catgcattag aaacatttct attcaccctc taaatgaagg tttaattgtt
     2221 gatgcaggtt tcttaaaacc attagtaaga ttgttggact ataaggattc agaagaaatt
     2281 caatgtcacg ccgtatctac tttgagaaat ttggctgcat catctgaaaa aaacagaaaa
     2341 gagttcttcg aaagtggtgc tgtcgaaaaa tgtaaggaac tggctctaga ttcacctgtt
     2401 agtgttcaaa gtgaaatttc cgcatgtttt gctattttag ccctagcaga tgtctccaaa
     2461 ttggatctac tggaagcaaa tatcttagat gctttaatcc ctatgacttt ttcccagaac
     2521 caagaagttt ctggtaatgc tgctgctgct ttggcaaatc tatgttcaag agtgaataat
     2581 tatacaaaaa ttattgaagc atgggatcgt ccgaatgaag gcattcgtgg atttctgatt
     2641 agatttttga aaagtgacta tgctaccttt gaacacattg cgctatggac aattttacaa
     2701 ttgctagaaa gtcataatga taaagtggaa gatttggtta aaaatgatga cgacatcatt
     2761 aacggagtga gaaaaatggc agatgtgacc tttgagcgtt tacaaagatc aggaattgat
     2821 gttaaaaatc cggggagcaa caacaatcca agctcgaatg ataacaacag taataacaat
     2881 gacacaggat ctgaacatca acctgtagag gatgcaagtt tggaattgta taatattact
     2941 caacagattt tacaattttt acatcggatc cccgggttaa ttaacagatc cgcctcctcc
     3001 gaggacgtca tcaaggagtt catgcgcttc aaggtgcgca tggagggctc cgtgaacggc
     3061 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     3121 aagctgaagg tgaccaaggg cggccccctg cccttcgcct gggacatcct gtcccctcag
     3181 ttccagtacg gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag
     3241 ctgtccttcc ccgagggctt caagtggggg cgcgtgatga acttcgagga cggcggcgtg
     3301 gtgaccgtga cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg
     3361 cgcggcacca acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag
     3421 gcctccaccg agcggatgta ccccgaggac ggcgccctga agggcgagat caagatgagg
     3481 ctgaagctga aggacggcgg ccactacgac gccgaggtca agaccaccta catggccaag
     3541 aagcccgtgc agctgcccgg cgcctacaag accgacatca agctggacat cacctcccac
     3601 aacgaggact acaccatcgt ggaacagtac gagcgcgccg agggccgcca ctccaccggc
     3661 gccggatctt agggcgcgcc acttctaaat aagcgaattt cttatgattt atgattttta
     3721 ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt
     3781 taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcccc
     3841 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3901 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3961 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     4021 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4081 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4141 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4201 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4261 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4321 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4381 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4441 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4501 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4561 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4621 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4681 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4741 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4801 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4861 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4921 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4981 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5041 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5101 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5161 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5221 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5281 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5341 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5401 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5461 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5521 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5581 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5641 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5701 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5761 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5821 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5881 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5941 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6001 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     6061 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     6121 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     6181 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     6241 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatcgac tacgtcgtta
     6301 aggccgtttc tgacagagta aaattcttga gggaactttc accattatgg gaaatggttc
     6361 aagaaggtat tgacttaaac tccatcaaat ggtcaggtca ttgagtgttt tttatttgtt
     6421 gtattttttt ttttttagag aaaatcctcc aatatataaa ttaggaatca tagtttcatg
     6481 attttctgtt acacctaact ttttgtgtgg tgccctcctc cttgtcaata ttaatgttaa
     6541 agtgcaattc tttttcctta tcacgttgag ccattagtat caatttgctt acctgtattc
     6601 ctttacatcc tcctttttct ccttcttgat aaatgtatgt agattgcgta tatagtttcg
     6661 tctaccctat gaacatattc cattttgtaa tttcgtgtcg tttctattat gaatttcatt
     6721 tataaagttt atgtacaaat atcataaaaa aagagaatct ttttaagcaa ggattttctt
     6781 aacttcttcg gcgacagcat caccgacttc ggtggtactg ttggaaccac ctaaatcacc
     6841 agttctgata cctgcatcca aaaccttttt aactgcatct tcaatggcct taccttcttc
     6901 aggcaagttc aatgacaatt tcaacatcat tgcagcagac aagatagtgg cgatagggtt
     6961 gaccttattc tttggcaaat ctggagcaga accgtggcat ggttcgtaca aaccaaatgc
     7021 ggtgttcttg tctggcaaag aggccaagga cgcagatggc aacaaaccca aggaacctgg
     7081 gataacggag gcttcatcgg agatgatatc accaaacatg ttgctggtga ttataatacc
     7141 atttaggtgg gttgggttct taactaggat catggcggca gaatcaatca attgatgttg
     7201 aaccttcaat gtagggaatt cgttcttgat ggtttcctcc acagtttttc tccataatct
     7261 tgaagaggcc aaaacattag ctttatccaa ggaccaaata ggcaatggtg gctcatgttg
     7321 tagggccatg aaagcggcca ttcttgtgat tctttgcact tctggaacgg tgtattgttc
     7381 actatcccaa gcgacaccat caccatcgtc ttcctttctc ttaccaaagt aaatacctcc
     7441 cactaattct ctgacaacaa cgaagtcagt acctttagca aattgtggct tgattggaga
     7501 taagtctaaa agagagtcgg atgcaaagtt acatggtctt aagttggcgt acaattgaag
     7561 ttctttacgg atttttagta aaccttgttc aggtctaaca ctaccggtac cccatttagg
     7621 accacccaca gcacctaaca aaacggcatc agccttcttg gaggcttcca gcgcctcatc
     7681 tggaagtgga acacctgtag catcgatagc agcaccacca attaaatgat tttcgaaatc
     7741 gaacttgaca ttggaacgaa catcagaaat agctttaaga accttaatgg cttcggctgt
     7801 gatttcttga ccaacgtggt cacctggcaa aacgacgatc ttcttagggg cagacattag
     7861 aatggtatat ccttgaaata tatatatata tattgctgaa atgtaaaagg taagaaaagt
     7921 tagaaattaa gacgattgct aaccacctat tggaaaaaac aataggtcct taaataatat
     7981 tgtcaacttc aagtattgtg atgcaagcat ttagtcatga acgcttctct attctatatg
     8041 aaaagccggt tccggcgctc tcacctttcc tttttctccc aatttttcag ttgaaaaagg
     8101 tatatgcgtc aggcgacctc tgaaattaac aaaaaatttc cagtcatcga atttgattct
     8161 gtgcgatagc gcccctgtgt gttctcgtta tgttgaggaa aaaaataatg gttgctaaga
     8221 gattcgaact cttgcatctt acgatacctg agtattccca cagttaactg cggtcaagat
     8281 atttcttgaa tcaggcgcct tagaccgctc ggccaaacaa ccaattactt gttgagaaat
     8341 agagtataat tatcctataa atataacgtt tttgaacaca catgaacaag gaagtacagg
     8401 acaattgatt ttgaagagaa tgtggatttt gatgtaattg ttgggattcc atttttaata
     8461 aggcaataat attaggtatg t
//
