LOCUS       sequence-210857-        6698 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ATG8 fused at the N terminus to GFP under the control of 
            the endogenous promoter in the pRS416 vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6698)
  AUTHORS   Guan J, Stromhaug PE, George MD, Habibzadegah-Tari P, Bevan A, Dunn 
            WA Jr, Klionsky DJ
  TITLE     Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, 
            pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia 
            pastoris.
  JOURNAL   Mol Biol Cell. 2001 Dec;12(12):3821-38.
  PUBMED    11739783
REFERENCE   2  (bases 1 to 6698)
  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: "Mol Biol 
            Cell."; date: "2001-12"; volume: "12(12)"; pages: "3821-38"
COMMENT     Sequence Label: GFP-ATG8(416)/GFP-AUT7(416)
FEATURES             Location/Qualifiers
     source          1..6698
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     105..124
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        148..363
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     CDS             364..1167
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     rep_origin      complement(1298..1753)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1435..1456)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1647..1666
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1879..1901
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1893..1910
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1894..1910
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1917..1936
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1917..1935
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2568..3275
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI
                     CTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNY
                     KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF
                     KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFV
                     TAAGITHGMDELYK"
     primer_bind     complement(2592..2620)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3215..3235
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3834..3850)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3835..3851)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     promoter        complement(3879..3897)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3918..3934)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3918..3934)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3931..3953)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3942..3958
                     /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(3966..3972,3973..3990,3991..3996))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4011..4032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4149..4166)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4320..4908)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4400..4419)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5079..5870,5871..5939))
                     /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     5702..5721
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5940..6044)
                     /gene="bla"
                     /label=AmpR promoter
     misc_feature    6081..6584
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     primer_bind     6625..6643
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(join(6681..6698,1..5))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
       61 cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat
      121 tgtactgaga gtgcaccata ccaccttttc aattcatcat ttttttttta ttcttttttt
      181 tgatttcggt ttccttgaaa tttttttgat tcggtaatct ccgaacagaa ggaagaacga
      241 aggaaggagc acagacttag attggtatat atacgcatat gtagtgttga agaaacatga
      301 aattgcccag tattcttaac ccaactgcac agaacaaaaa cctgcaggaa acgaagataa
      361 atcatgtcga aagctacata taaggaacgt gctgctactc atcctagtcc tgttgctgcc
      421 aagctattta atatcatgca cgaaaagcaa acaaacttgt gtgcttcatt ggatgttcgt
      481 accaccaagg aattactgga gttagttgaa gcattaggtc ccaaaatttg tttactaaaa
      541 acacatgtgg atatcttgac tgatttttcc atggagggca cagttaagcc gctaaaggca
      601 ttatccgcca agtacaattt tttactcttc gaagacagaa aatttgctga cattggtaat
      661 acagtcaaat tgcagtactc tgcgggtgta tacagaatag cagaatgggc agacattacg
      721 aatgcacacg gtgtggtggg cccaggtatt gttagcggtt tgaagcaggc ggcagaagaa
      781 gtaacaaagg aacctagagg ccttttgatg ttagcagaat tgtcatgcaa gggctcccta
      841 tctactggag aatatactaa gggtactgtt gacattgcga agagcgacaa agattttgtt
      901 atcggcttta ttgctcaaag agacatgggt ggaagagatg aaggttacga ttggttgatt
      961 atgacacccg gtgtgggttt agatgacaag ggagacgcat tgggtcaaca gtatagaacc
     1021 gtggatgatg tggtctctac aggatctgac attattattg ttggaagagg actatttgca
     1081 aagggaaggg atgctaaggt agagggtgaa cgttacagaa aagcaggctg ggaagcatat
     1141 ttgagaagat gcggccagca aaactaaaaa actgtattat aagtaaatgc atgtatacta
     1201 aactcacaaa ttagagcttc aatttaatta tatcagttat taccctatgc ggtgtgaaat
     1261 accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaagcgt taatattttg
     1321 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     1381 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     1441 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     1501 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     1561 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     1621 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     1681 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     1741 ctacagggcg cgtccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     1801 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     1861 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattgtaat
     1921 acgactcact atagggcgaa ttggagctcc accgcggtgg cggccgctct agaactagta
     1981 ggtctcgcaa gagagctggt caacagaatc caaaaattga gaaagaagtg tggtttggaa
     2041 gccaccgacg atgttttagt ggagtacgaa ttagttaaag atactatcga ctttgaagcc
     2101 attgtcaaag aacattttga tatgttaagc aagacctgta gatccgacat tgccaaatat
     2161 gacggctcaa agacagaccc aattggtgat gaagaacaat ctattaatga caccattttc
     2221 aaattaaaag tgttcaaatt atgaaaacaa ctcatataaa tacgtacaaa tttttctcta
     2281 ctcgaagtga tatagatgta tatgtgtaag tttacgttta agttagagtc atgtaatgct
     2341 aactgtctcc accgataatg ttgtataata cccgtgaaat catagcacat gatatatcat
     2401 cacccggagg ccggttattt tcggcggcgg caaaaatatt tggtataatt atggaaatac
     2461 aaaaagggga accattaaag gttgaggagg ggattgataa gagaatctaa taattgtaaa
     2521 gttgagaaaa tcataataaa ataattacta gagacatggg atccggtaaa ggagaagaac
     2581 ttttcactgg agttgtccca attcttgttg aattagatgg tgatgttaat gggcacaaat
     2641 tttctgtcag tggagagggt gaaggtgatg caacatacgg aaaacttacc cttaaattta
     2701 tttgcactac tggaaaacta cctgttccat ggccaacact tgtcactact ttcacttatg
     2761 gtgttcaatg cttttcaaga tacccagatc atatgaaacg gcatgacttt ttcaagagtg
     2821 ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac gggaactaca
     2881 agacacgtgc tgaagtcaag tttgaaggtg atacccttgt taatagaatc gagttaaaag
     2941 gtattgattt taaagaagat ggaaacattc ttggacacaa attggaatac aactataact
     3001 cacacaatgt atacatcatg gcagacaaac aaaagaatgg aatcaaagtt aacttcaaaa
     3061 ttagacacaa cattgaagat ggaagcgttc aactagcaga ccattatcaa caaaatactc
     3121 caattggcga tggccctgtc cttttaccag acaaccatta cctgtccaca caatctgccc
     3181 tttcgaaaga tcccaacgaa aagagagacc acatggtcct tcttgagttt gtaacagctg
     3241 ctgggattac acatggcatg gatgaactat acaaaggatc caagtctaca tttaagtctg
     3301 aatatccatt tgaaaaaagg aaggcggagt cggagaggat tgctgacagg ttcaagaata
     3361 ggatacctgt gatttgcgaa aaagctgaaa agtcagatat tccagagatt gataagcgta
     3421 aatatctagt tcctgctgac cttaccgtag ggcaatttgt ttatgttata agaaagagaa
     3481 ttatgctacc ccctgagaag gccatcttca tttttgtcaa tgatactttg ccacctactg
     3541 cggcgttgat gtctgccata tatcaagaac acaaggataa ggacgggttt ttgtatgtca
     3601 cttactcagg agaaaataca tttggcaggt agtcttttat atgaaaagaa atgaagcgtt
     3661 aacatctaaa atcgaaatta tagggactca ttagccatct attgttccag tataagaatg
     3721 tttacataaa aaattcgaaa aaaaaagata aaataaggga aaaatatata tatattcatt
     3781 aagaagtttt gtacacaacg gagccaagat ttcgaattcg atatcaagct tatcgatacc
     3841 gtcgacctcg agggggggcc cggtaccagc ttttgttccc tttagtgagg gttaatttcg
     3901 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3961 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4021 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4081 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     4141 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4201 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4261 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     4321 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     4381 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     4441 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     4501 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     4561 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     4621 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     4681 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     4741 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     4801 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     4861 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     4921 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     4981 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5041 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5101 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5161 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5221 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     5281 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     5341 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     5401 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     5461 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     5521 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     5581 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     5641 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     5701 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     5761 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     5821 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     5881 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     5941 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6001 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6061 gtgccacctg ggtccttttc atcacgtgct ataaaaataa ttataattta aattttttaa
     6121 tataaatata taaattaaaa atagaaagta aaaaaagaaa ttaaagaaaa aatagttttt
     6181 gttttccgaa gatgtaaaag actctagggg gatcgccaac aaatactacc ttttatcttg
     6241 ctcttcctgc tctcaggtat taatgccgaa ttgtttcatc ttgtctgtgt agaagaccac
     6301 acacgaaaat cctgtgattt tacattttac ttatcgttaa tcgaatgtat atctatttaa
     6361 tctgcttttc ttgtctaata aatatatatg taaagtacgc tttttgttga aattttttaa
     6421 acctttgttt attttttttt cttcattccg taactcttct accttcttta tttactttct
     6481 aaaatccaaa tacaaaacat aaaaataaat aaacacagag taaattccca aattattcca
     6541 tcattaaaag atacgaggcg cgtgtaagtt acaggcaagc gatccgtcta agaaaccatt
     6601 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     6661 ttcggtgatg acggtgaaaa cctctgacac atgcagct
//
