LOCUS       sequence-401046-        6644 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of ULK1 (836-1050) with MBP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6644)
  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 6644)
  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-ULK1 (836-1050)-MBP
FEATURES             Location/Qualifiers
     source          1..6644
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        130..509
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        511..786
                     /label=chicken beta-actin promoter
     intron          787..1803
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1727..1749
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1811..1830
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1878..1887
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2535..2555
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2559..3659
                     /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     3633..3656
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             3669..3698
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(3791..3810)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3856..3911
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3910..3929)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4272..4288)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4272..4288)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4285..4307)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4296..4312
                     /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(4320..4326,4327..4344,4345..4350))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4365..4386
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4445..4640
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4491..4626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4646..4780
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4683..4702)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4737..4756
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4848..4865)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5019..5607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5099..5118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5778..6569,6570..6638))
                     /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     6401..6420
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6639..6644,1..99))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
       61 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
      121 acctgggtcg acattgatta ttgactagtt attaatagta atcaattacg gggtcattag
      181 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      241 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      301 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      361 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      421 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      481 tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc
      541 tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt
      601 gtgcagcgat gggggcgggg gggggggggg ggcgcgccag gcggggcggg gcggggcgag
      661 gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga
      721 aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg
      781 cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc
      841 cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct
      901 tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg
      961 cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggggt
     1021 gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt
     1081 gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg
     1141 gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg
     1201 ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt cggtcgggct gcaacccccc
     1261 ctgcaccccc ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtacg
     1321 gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc
     1381 ggggcggggc cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg cccccggagc
     1441 gccggcggct gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag
     1501 agggcgcagg gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc
     1561 cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc
     1621 ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc
     1681 tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg
     1741 cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta
     1801 cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattcctc
     1861 gatcgacggt atcgatagcc accatggagc aggaacacac tgagatcctg aggggtctca
     1921 gattcaccct gctctttgtg cagcatgtcc tggaaattgc tgcactcaag ggtagtgcta
     1981 gcgaggcagc tggtggacca gaataccagc tgcaggagag cgtggtcgcc gaccagatct
     2041 ctctgctctc aagggaatgg ggcttcgctg agcagctggt cctctatctg aaggtggccg
     2101 aactgctcag tagcggcctg cagtccgcca tcgatcagat tagagctgga aagctctgcc
     2161 tgtcctctac tgtgaaacag gtggtccggc gcctgaacga gctctacaag gccagtgtgg
     2221 tcagctgtca gggactctct ctgcgtctcc agcggttctt tctggacaaa cagcgcctgc
     2281 tcgataggat ccactcaatt acagctgaac gcctgatctt cagccatgca gtgcagatgg
     2341 tccagtccgc agccctggac gaaatgtttc agcacaggga gggatgcgtc cccagatacc
     2401 ataaggctct gctcctgctc gagggtctgc agcacatgct cagcgaccag gccgatatcg
     2461 aaaatgtgac taagtgcaaa ctgtgtattg agaggagact ctccgctctg ctcacaggca
     2521 tctgtgcagg taccgaaaac ctgtacttcc aatccggtat gaaaatcgaa gaaggtaaac
     2581 tggtaatctg gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat
     2641 tcgagaaaga taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat
     2701 tcccacaggt tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct
     2761 ttggtggcta cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg
     2821 acaagctgta tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc
     2881 cgatcgctgt tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa
     2941 aaacctggga agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc
     3001 tgatgttcaa cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt
     3061 atgcgttcaa gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg
     3121 gcgcgaaagc gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag
     3181 acaccgatta ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca
     3241 acggcccgtg ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac
     3301 tgccgacctt caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta
     3361 acgccgccag tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg
     3421 atgaaggtct ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt
     3481 acgaggaaga gttggcgaaa gatccacgta ttgccgccac tatggaaaac gcccagaaag
     3541 gtgaaatcat gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg
     3601 tgatcaacgc cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagactg
     3661 gcagtagcca ccatcaccat caccatcatc accatcacta gctcgaggaa ttcactcctc
     3721 aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat
     3781 accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc
     3841 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt
     3901 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt
     3961 atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc
     4021 tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa
     4081 aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa
     4141 catccctaaa attttcctta catgttttac tagccagatt tttcctcctc tcctgactac
     4201 tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag cccaagcttg
     4261 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4321 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4381 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg
     4441 atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4501 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4561 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4621 gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata
     4681 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4741 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc
     4801 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4861 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4921 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4981 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5041 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5101 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5161 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5221 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5281 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5341 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5401 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5461 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5521 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5581 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5641 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5701 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5761 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5821 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5881 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5941 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6001 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6061 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6121 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6181 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6241 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6301 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6361 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6421 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6481 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6541 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6601 aagggaataa gggcgacacg gaaatgttga atactcatac tctt
//
