LOCUS       sequence-369644-        6218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human ATG101 HORMA W110D/F112D mutant in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6218)
  AUTHORS   Ren X, Nguyen TN, Lam WK, Buffalo CZ, Lazarou M, Yokom AL, Hurley JH
  TITLE     Structural basis for ATG9A recruitment to the ULK1 complex in 
            mitophagy initiation.
  JOURNAL   Sci Adv. 2023 Feb 15;9(7):eadg2997. doi: 10.1126/sciadv.adg2997. 
            Epub 2023 Feb 15.
  PUBMED    36791199
REFERENCE   2  (bases 1 to 6218)
  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: "Sci Adv."; 
            date: "2023-02-15"; volume: "9(7):eadg2997. doi"; pages: " 
            10.1126/sciadv.adg2997. Epub 2023 Feb 15"
COMMENT     Sequence Label: pCAG-GST-ATG101-W110D/F112D
FEATURES             Location/Qualifiers
     source          1..6218
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          661..1677
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1601..1623
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1685..1704
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1751..1760
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1757..2413
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2371..2393
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2441..2461
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3239..3258)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3304..3359
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3358..3377)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3720..3736)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3720..3736)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3733..3755)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3744..3760
                     /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(3768..3774,3775..3792,3793..3798))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3813..3834
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3893..4088
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3939..4074
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4094..4228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4131..4150)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4185..4204
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4296..4313)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4467..5055)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4547..4566)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5226..6017,6018..6086))
                     /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     5849..5868
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6087..6191)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggggcgcg ccrggsgggg sggggsgggg sgrggggsgg
      541 ggsggggsgr ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg
      721 ccccggctct gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct
      781 ccgggctgta attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa
      841 agccttgagg ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc
      901 gtgtgtgtgt gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc
      961 tgcgggcgcg gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg
     1021 ggcggtgccc cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
     1081 tgcgtggggg ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac
     1141 ccccctcccc gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt
     1201 ggcgcggggc tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg
     1261 gggccgcctc gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc
     1321 ggctgtcgag gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg
     1381 cagggacttc ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc
     1441 ccctctagcg ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag
     1501 ggccttcgtg cgtcgccgcg ccgccgtccc cttctccctc tccagcctcg gggctgtccg
     1561 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     1621 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     1681 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt cctcgatcga
     1741 cggtatcgat gccaccatgg catcccctat actaggttat tggaaaatta agggccttgt
     1801 gcaacccact cgacttcttt tggaatatct tgaagaaaaa tatgaagagc atttgtatga
     1861 gcgcgatgaa ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa
     1921 tcttccttat tatattgatg gtgatgttaa attaacacag tctatggcca tcatacgtta
     1981 tatagctgac aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat
     2041 gcttgaagga gcggttttgg atattagata cggtgtttcg agaattgcat atagtaaaga
     2101 ctttgaaact ctcaaagttg attttcttag caagctacct gaaatgctga aaatgttcga
     2161 agatcgttta tgtcataaaa catatttaaa tggtgatcat gtaacccatc ctgacttcat
     2221 gttgtatgac gctcttgatg ttgttttata catggaccca atgtgcctgg atgcgttccc
     2281 aaaattagtt tgttttaaaa aacgtattga agctatccca caaattgata agtacttgaa
     2341 atccagcaag tatatagcat ggcctttgca gggctggcaa gccacgtttg gtggtggcga
     2401 ccatcctcca aaatcggatg attacgatat cccaacgacc gaaaacctgt attttcaggg
     2461 tggtaccatg aattgccggt cagaggtcct ggaagtgagc gtggaaggta gacaggtgga
     2521 agaggctatg ctggccgtgc tgcatacagt gctgctgcac agatcaactg gcaagttcca
     2581 ttacaagaaa gaaggaacat attccatcgg aaccgtgggt actcaggacg tcgattgcga
     2641 cttcattgat tttacttacg tgcgcgtcag ctccgaggaa ctggacagag cactccgtaa
     2701 ggtggtcgga gaattcaaag atgccctgag gaacagtggc ggagacggtc tgggccagat
     2761 gagcctcgag ttctatcaga agaaaaagtc tagagaccca gattcagatg aatgtatccc
     2821 ctgggaagtg tggaccgtga aggtccacgt ggtcgccctg gctactgagc aggaacggca
     2881 gatctgccgc gagaaagtgg gcgaaaagct ctgtgagaag atcatcaaca tcgtggaagt
     2941 catgaaccgt catgagtacc tgcccaagat gcctacacag agcgaggtgg ataatgtctt
     3001 cgacaccggt ctgcgggatg tgcagcccta cctctataag atctcctttc agattacaga
     3061 cgctctgggc acctctgtca ccactacaat gcgcaggctc atcaaagaca ccctggcact
     3121 ctaacccggg tgatatctta attaattaac tcgaggaatt cactcctcag gtgcaggctg
     3181 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
     3241 tttttccctc tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg
     3301 gctaataaag gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac
     3361 tcggaaggac atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga
     3421 gtttggcaac atatgcccat atgctggctg ccatgaacaa aggttggcta taaagaggtc
     3481 atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa gccttgactt
     3541 gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca tccctaaaat
     3601 tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc ccagtcatag
     3661 ctgtccctct tctcttatgg agatccctcg acctgcagcc caagcttggc gtaatcatgg
     3721 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     3781 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     3841 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat ccgcatctca
     3901 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3961 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     4021 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     4081 tttgcaaaaa gctaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4141 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4201 catcaatgta tcttatcatg tctggatccg ctgcattaat gaatcggcca acgcgcgggg
     4261 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4321 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4381 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4441 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4501 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4561 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4621 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4681 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4741 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4801 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4861 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4921 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4981 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5041 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5101 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5161 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5221 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5281 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5341 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5401 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5461 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5521 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5581 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5641 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5701 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5761 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5821 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5881 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5941 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6001 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6061 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6121 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6181 ggggttccgc gcacatttcc ccgaaaagtg ccacctgg
//
