LOCUS       sequence-438323-        6886 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of TwinStrep-Flag -ATG13 C-terminal and ULK1 
            MIT domain fusion complex fused with GFP-10His.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6886)
  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 6886)
  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-TSF-ATG13 (363-517)-ULK1 (836-1050)-msfGFP
FEATURES             Location/Qualifiers
     source          1..6886
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..663
                     /label=chicken beta-actin promoter
     intron          664..1680
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1604..1626
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1688..1707
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1755..1764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1759..1768
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1770..1793
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1830..1853
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1860..1883
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1890..1910
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3042..3062
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(3069..3071,3072..3074,3075..3785)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3114..3135)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3722..3743
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3786..3815
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     primer_bind     complement(3908..3927)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3973..4028
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4027..4046)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4389..4405)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4389..4405)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4402..4424)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4413..4429
                     /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(4437..4443,4444..4461,4462..4467))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4482..4503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4562..4757
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4608..4743
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4763..4897
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4800..4819)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4854..4873
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4965..4982)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5136..5724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5216..5235)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5895..6686,6687..6755))
                     /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     6518..6537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6756..6860)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg
      541 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
      601 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
      661 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      721 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      781 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      841 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
      961 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     1021 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     1081 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     1141 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     1201 cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg
     1261 gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc
     1321 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     1381 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     1441 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     1501 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     1561 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1621 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1681 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcctcgat
     1741 cgacggtatc gatagccacc atggcggcct ggtcacaccc gcagttcgag aaaggtggag
     1801 gtgcccgagg tggatcggga ggtggatcgt ggagccaccc gcagttcgaa aaaggattcg
     1861 attacaagga tgacgatgac aagggtaccg aaaacctgta cttccaatcc cacgatgtcc
     1921 tggaaaccat cttcgtgaga aaagtcggcg cttttgtgaa caagcccatt aatcaggtca
     1981 cactcacctc tctggacatc cctttcgcta tgtttgcacc aaagaacctc gagctggaag
     2041 acactgatcc catggtgaat cctccagact cacccgagac agaaagtcct ctccagggca
     2101 gtctgcacag cgatggaagc tccggtggct cttcaggcaa cacacatgac gatttcgtca
     2161 tgattgactt caaacccgct ttttccaagg acgatatcct ccctatggat ctgggaacct
     2221 tctacaggga gtttcagaat ccccctcagc tcagtagcct gagcattgac atcggtgccc
     2281 agtccatggc tgaggacctc gattctctgc ctgaaaaact ggctgtgcac gagaagaacg
     2341 tcagagaatt cgacgcattt gtggagaccc tgcagggtag cgacgaagcg atggagcagg
     2401 aacacactga gatcctgagg ggtctcagat tcaccctgct ctttgtgcag catgtcctgg
     2461 aaattgctgc actcaagggt agtgctagcg aggcagctgg tggaccagaa taccagctgc
     2521 aggagagcgt ggtcgccgac cagatctctc tgctctcaag ggaatggggc ttcgctgagc
     2581 agctggtcct ctatctgaag gtggccgaac tgctcagtag cggcctgcag tccgccatcg
     2641 atcagattag agctggaaag ctctgcctgt cctctactgt gaaacaggtg gtccggcgcc
     2701 tgaacgagct ctacaaggcc agtgtggtca gctgtcaggg actctctctg cgtctccagc
     2761 ggttctttct ggacaaacag cgcctgctcg ataggatcca ctcaattaca gctgaacgcc
     2821 tgatcttcag ccatgcagtg cagatggtcc agtccgcagc cctggacgaa atgtttcagc
     2881 acagggaggg atgcgtcccc agataccata aggctctgct cctgctcgag ggtctgcagc
     2941 acatgctcag cgaccaggcc gatatcgaaa atgtgactaa gtgcaaactg tgtattgaga
     3001 ggagactctc cgctctgctc acaggcatct gtgcaggtac cgaaaacctg tacttccaat
     3061 ccggttctat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     3121 agctggacgg cgacgtaaac ggccacaagt tcagcgtgcg cggcgagggc gagggcgatg
     3181 ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     3241 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     3301 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     3361 ccatctcctt caaggacgac ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg
     3421 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     3481 tggggcacaa gctggagtac aacttcaaca gccacaacgt ctatatcacg gccgacaagc
     3541 agaagaacgg catcaaggcg aacttcaaga tccgccacaa cgtcgaggac ggcagcgtgc
     3601 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     3661 acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc
     3721 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     3781 acaagcatca ccatcaccat caccatcacc atcactagct cgaggaattc actcctcagg
     3841 tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct cacaaatacc
     3901 actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc
     3961 tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt
     4021 gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt
     4081 tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa ggttggctat
     4141 aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc catagaaaag
     4201 ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt tctttaacat
     4261 ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc tgactactcc
     4321 cagtcatagc tgtccctctt ctcttatgga gatccctcga cctgcagccc aagcttggcg
     4381 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4441 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4501 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc
     4561 cgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     4621 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     4681 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     4741 gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct tataatggtt acaaataaag
     4801 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     4861 gtccaaactc atcaatgtat cttatcatgt ctggatccgc tgcattaatg aatcggccaa
     4921 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4981 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5041 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5101 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5161 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5221 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5281 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5341 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5401 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5461 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5521 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5581 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5641 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5701 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5761 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5821 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5881 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5941 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6001 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6061 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6121 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6181 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6241 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6301 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6361 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6421 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6481 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6541 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6601 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6661 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6721 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6781 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6841 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctg
//
