LOCUS       sequence-329328-        8544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ER localized Vac8-GFP 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 8544)
  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 8544)
  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-GFP
FEATURES             Location/Qualifiers
     source          1..8544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     16..35
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        16..34
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2352..2380)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2975..2995
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3120..3144)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     primer_bind     complement(3211..3231)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3211..3229)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3250..3266)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3250..3266)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3263..3285)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3274..3290
                     /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(3298..3304,3305..3322,3323..3328))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3343..3364
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3481..3498)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3652..4240)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3732..3751)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4411..5202,5203..5271))
                     /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     5034..5053
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5272..5376)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5444..5462
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5500..5522)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5622..5641
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(6135..7229)
                     /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(7230..7637)
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     rep_origin      complement(7938..8393)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8075..8096)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     8287..8306
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8519..8541
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(8533..8544,1..6)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     join(8534..8544,1..6)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccga ggactcaaaa
       61 cgaaaaggaa aaaaatacga gtggaaagaa aaaatgaacc ctttccagta attgataata
      121 atacaatcat tcattattag aaatatatta gttctcaatt caaggcagaa tagtgctgtc
      181 attgtgatca aatttttgtg tgagtctgct ttgctttctg tttttttttg ttgactcgct
      241 gttcaatctc ctacttttcc ttttgttgtt aaaataaggt attatatatt tatacaaccc
      301 tataaaaagc aattagccaa tttccaatcc tacgaagaca ggaactgagc aaactataag
      361 ggtgttcttt cttctgtact atatatacat ttgcaactat gtccgaattt aatgaaacaa
      421 aattctccaa caacgggacg ttttttgaaa cggaagagcc aattgtggag acgaaatcaa
      481 tctccgttta taccccactc atatatgtct ttattctggt ggtgtccctt gtgatgtttg
      541 cttcaagcta cagaaagaag ttgaaagatt cttcagacga ggccagtgtc tcaccaattg
      601 ccgataatga aagagaagcg gtgactttat tactagggta tttggaggat aaagaccaat
      661 tagactttta ctctggtggt ccactaaagg ccctaactac tttggtgtat tcagataatc
      721 taaatttgca aaggagcgcc gctctggcat tcgcagaaat tactgaaaaa tacgtccgtc
      781 aggtctctag agaggtactg gaacccatct taatattatt acaaagccaa gatccacaga
      841 ttcaagtggc cgcttgtgca gccttgggta atttggccgt taataatgaa aataaattat
      901 taattgtgga aatgggaggc ttagagccat taataaacca aatgatgggt gacaatgtag
      961 aagttcaatg taatgctgtt ggatgcatta ccaatttagc cacaagggat gacaataaac
     1021 ataagattgc tacttctggt gctttaattc cattgactaa gctagctaaa tccaaacata
     1081 taagagttca gagaaatgct acaggtgcat tattaaatat gacacattct gaggaaaata
     1141 gaaaagaatt ggttaatgca ggtgctgtgc ctgtattggt atctttactg tcgtccaccg
     1201 atcctgatgt tcagtactat tgtactactg ctttatcaaa tatagcagtg gatgaagcca
     1261 atcgcaagaa gttggctcaa actgagccaa gattggtttc caaattggtt agtctgatgg
     1321 attctccatc ttcaagggta aaatgtcaag ctactttggc tttacgaaac ctagcgtccg
     1381 atacaagcta ccaattagaa attgtaaggg ctggtgggtt gcctcatttg gtaaagttga
     1441 ttcaaagtga ttctatacca ctggttttgg ctagtgtggc atgcattaga aacatttcta
     1501 ttcaccctct aaatgaaggt ttaattgttg atgcaggttt cttaaaacca ttagtaagat
     1561 tgttggacta taaggattca gaagaaattc aatgtcacgc cgtatctact ttgagaaatt
     1621 tggctgcatc atctgaaaaa aacagaaaag agttcttcga aagtggtgct gtcgaaaaat
     1681 gtaaggaact ggctctagat tcacctgtta gtgttcaaag tgaaatttcc gcatgttttg
     1741 ctattttagc cctagcagat gtctccaaat tggatctact ggaagcaaat atcttagatg
     1801 ctttaatccc tatgactttt tcccagaacc aagaagtttc tggtaatgct gctgctgctt
     1861 tggcaaatct atgttcaaga gtgaataatt atacaaaaat tattgaagca tgggatcgtc
     1921 cgaatgaagg cattcgtgga tttctgatta gatttttgaa aagtgactat gctacctttg
     1981 aacacattgc gctatggaca attttacaat tgctagaaag tcataatgat aaagtggaag
     2041 atttggttaa aaatgatgac gacatcatta acggagtgag aaaaatggca gatgtgacct
     2101 ttgagcgttt acaaagatca ggaattgatg ttaaaaatcc ggggagcaac aacaatccaa
     2161 gctcgaatga taacaacagt aataacaatg acacaggatc tgaacatcaa cctgtagagg
     2221 atgcaagttt ggaattgtat aatattactc aacagatttt acaattttta catcggatcc
     2281 ccgggttaat taattcgcga attcggggag gcggaggtgg agctagcaaa ggagaagaac
     2341 tcttcactgg agttgtccca attcttgttg aattagatgg tgatgttaac ggccacaagt
     2401 tctctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc ctgaagttca
     2461 tctgcactac tggcaaactg cctgttccat ggccaacact agtcactact ctgtgctatg
     2521 gtgttcaatg cttttcaaga tacccggatc atatgaaacg gcatgacttt ttcaagagtg
     2581 ccatgcccga aggttatgta caggaaagga ccatcttctt caaagatgac ggcaactaca
     2641 agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag
     2701 gtattgactt caaggaagat ggcaacattc tgggacacaa attggaatac aactataact
     2761 cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagtg aacttcaaga
     2821 cccgccacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa caaaatactc
     2881 caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca caatctgccc
     2941 tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg
     3001 ctgggattac acatggcatg gatgaactgt acaacatcga ttgaggcgcg ccacttctaa
     3061 ataagcgaat ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag
     3121 tgtatacaaa ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa
     3181 ctctttcctg taggtcaggt tgctttctcc cctttagtga gggttaattg cgcgcttggc
     3241 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3301 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3361 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3421 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3481 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3541 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3601 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3661 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3721 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3781 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3841 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3901 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3961 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4021 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4081 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4141 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4201 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4261 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4321 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4381 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4441 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4501 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4561 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4621 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4681 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4741 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4801 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4861 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4921 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4981 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5041 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5101 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5161 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5221 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5281 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5341 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5401 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     5461 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     5521 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     5581 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     5641 actgagagtg caccatatcg actacgtcgt taaggccgtt tctgacagag taaaattctt
     5701 gagggaactt tcaccattat gggaaatggt tcaagaaggt attgacttaa actccatcaa
     5761 atggtcaggt cattgagtgt tttttatttg ttgtattttt ttttttttag agaaaatcct
     5821 ccaatatata aattaggaat catagtttca tgattttctg ttacacctaa ctttttgtgt
     5881 ggtgccctcc tccttgtcaa tattaatgtt aaagtgcaat tctttttcct tatcacgttg
     5941 agccattagt atcaatttgc ttacctgtat tcctttacat cctccttttt ctccttcttg
     6001 ataaatgtat gtagattgcg tatatagttt cgtctaccct atgaacatat tccattttgt
     6061 aatttcgtgt cgtttctatt atgaatttca tttataaagt ttatgtacaa atatcataaa
     6121 aaaagagaat ctttttaagc aaggattttc ttaacttctt cggcgacagc atcaccgact
     6181 tcggtggtac tgttggaacc acctaaatca ccagttctga tacctgcatc caaaaccttt
     6241 ttaactgcat cttcaatggc cttaccttct tcaggcaagt tcaatgacaa tttcaacatc
     6301 attgcagcag acaagatagt ggcgatgggg ttgaccttat tctttggcaa atctggagca
     6361 gaaccgtggc atggttcgta caaaccaaat gcggtgttct tgtctggcaa agaggccaag
     6421 gacgcagatg gcaacaaacc caaggaacct gggataacgg aggcttcatc ggagatgata
     6481 tcaccaaaca tgttgctggt gattataata ccatttaggt gggttgggtt cttaactagg
     6541 atcatggcgg cagaatcaat caattgatgt tgaaccttca atgtagggaa ttcgttcttg
     6601 atggtttcct ccacagtttt tctccataat cttgaagagg ccaaaacatt agctttatcc
     6661 aaggaccaaa taggcaatgg tggctcatgt tgtagggcca tgaaagcggc cattcttgtg
     6721 attctttgca cttctggaac ggtgtattgt tcactatccc aagcgacacc atcaccatcg
     6781 tcttcctttc tcttaccaaa gtaaatacct cccactaatt ctctgacaac aacgaagtca
     6841 gtacctttag caaattgtgg cttgattgga gataagtcta aaagagagtc ggatgcaaag
     6901 ttacatggtc ttaagttggc gtacaattga agttctttac ggatttttag taaaccttgt
     6961 tcaggtctaa cactaccggt accccattta ggaccaccca cagcacctaa caaaacggca
     7021 tcagccttct tggaggcttc cagcgcctca tctggaagtg gaacacctgt agcatcgata
     7081 gcagcaccac caattaaatg attttcgaaa tcgaacttga cattggaacg aacatcagaa
     7141 atagctttaa gaaccttaat ggcttcggct gtgatttctt gaccaacgtg gtcacctggc
     7201 aaaacgacga tcttcttagg ggcagacatt agaatggtat atccttgaaa tatatatata
     7261 tatattgctg aaatgtaaaa ggtaagaaaa gttagaaagt aagacgattg ctaaccacct
     7321 attggaaaaa acaataggtc cttaaataat attgtcaact tcaagtattg tgatgcaagc
     7381 atttagtcat gaacgcttct ctattctata tgaaaagccg gttccggcgc tctcaccttt
     7441 cctttttctc ccaatttttc agttgaaaaa ggtatatgcg tcaggcgacc tctgaaatta
     7501 acaaaaaatt tccagtcatc gaatttgatt ctgtgcgata gcgcccctgt gtgttctcgt
     7561 tatgttgagg aaaaaaataa tggttgctaa gagattcgaa ctcttgcatc ttacgatacc
     7621 tgagtattcc cacagttaac tgcggtcaag atatttcttg aatcaggcgc cttagaccgc
     7681 tcggccaaac aaccaattac ttgttgagaa atagagtata attatcctat aaatataacg
     7741 tttttgaaca cacatgaaca aggaagtaca ggacaattga ttttgaagag aatgtggatt
     7801 ttgatgtaat tgttgggatt ccatttttaa taaggcaata atattaggta tgtagatata
     7861 ctagaagttc tcctcgaccg gtcgatatgc ggtgtgaaat accgcacaga tgcgtaagga
     7921 gaaaataccg catcaggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     7981 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     8041 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     8101 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     8161 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     8221 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     8281 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     8341 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtccattcg
     8401 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     8461 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     8521 cagtcacgac gttgtaaaac gacg
//
