LOCUS       sequence-403405-        6468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of ATG13 (363-517) with MBP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6468)
  AUTHORS   Chen M, Ren X, Nguyen TN, Khuu G, Cook ASI, Lazarou M, Hurley JH
  TITLE     Structure and activation of the human autophagy-initiating 
            ULK1C:PI3KC3-C1 supercomplex
  JOURNAL   bioRxiv 2023.06.01.543278
REFERENCE   2  (bases 1 to 6468)
  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: "bioRxiv 
            2023.06.01.543278"
COMMENT     Sequence Label: pCAG-ATG13 (363-517)-MBP
FEATURES             Location/Qualifiers
     source          1..6468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..659
                     /label=chicken beta-actin promoter
     intron          660..1676
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1600..1622
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1684..1703
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2230..2250
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2254..3354
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     3328..3351
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3364..3393
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(3486..3505)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3551..3606
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3605..3624)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3967..3983)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3967..3983)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3980..4002)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3991..4007
                     /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(4015..4021,4022..4039,4040..4045))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4060..4081
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4140..4335
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4186..4321
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4341..4475
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4378..4397)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4432..4451
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4543..4560)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4714..5302)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4794..4813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5473..6264,6265..6333))
                     /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     6096..6115
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6334..6438)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg
      541 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
      601 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
      661 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
      721 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
      781 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
      841 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct
      961 gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg
     1021 gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     1081 gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc
     1141 cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg
     1201 gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg
     1261 ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg
     1321 gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc
     1381 agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc
     1441 cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg
     1501 gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc
     1561 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
     1621 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
     1681 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc ctcgatcgac
     1741 ggtatcgatg ccaccatgca cgatgtcctg gaaaccatct tcgtgagaaa agtcggcgct
     1801 tttgtgaaca agcccattaa tcaggtcaca ctcacctctc tggacatccc tttcgctatg
     1861 tttgcaccaa agaacctcga gctggaagac actgatccca tggtgaatcc tccagactca
     1921 cccgagacag aaagtcctct ccagggcagt ctgcacagcg atggaagctc cggtggctct
     1981 tcaggcaaca cacatgacga tttcgtcatg attgacttca aacccgcttt ttccaaggac
     2041 gatatcctcc ctatggatct gggaaccttc tacagggagt ttcagaatcc ccctcagctc
     2101 agtagcctga gcattgacat cggtgcccag tccatggctg aggacctcga ttctctgcct
     2161 gaaaaactgg ctgtgcacga gaagaacgtc agagaattcg acgcatttgt ggagaccctg
     2221 cagggtaccg aaaacctgta cttccaatcc ggtatgaaaa tcgaagaagg taaactggta
     2281 atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag
     2341 aaagataccg gaattaaagt caccgttgag catccggata aactggaaga gaaattccca
     2401 caggttgcgg caactggcga tggccctgac attatcttct gggcacacga ccgctttggt
     2461 ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag
     2521 ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc
     2581 gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc
     2641 tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg
     2701 ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
     2761 ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg
     2821 aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacacc
     2881 gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc
     2941 ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg
     3001 accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc
     3061 gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa
     3121 ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag
     3181 gaagagttgg cgaaagatcc acgtattgcc gccactatgg aaaacgccca gaaaggtgaa
     3241 atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc
     3301 aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactggcagt
     3361 agccaccatc accatcacca tcatcaccat cactagctcg aggaattcac tcctcaggtg
     3421 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     3481 tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     3541 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     3601 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     3661 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     3721 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     3781 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     3841 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     3901 gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta
     3961 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4021 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4081 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg
     4141 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4201 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4261 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4321 ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac aaataaagca
     4381 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4441 ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa tcggccaacg
     4501 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4561 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4621 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4681 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4741 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4801 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4861 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4921 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4981 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5041 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5101 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5161 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5221 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5281 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5341 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5401 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5461 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5521 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5581 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     5641 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     5701 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5761 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5821 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5881 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5941 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6001 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6061 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6121 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6181 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6241 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6301 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6361 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6421 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgggtc
//
