LOCUS       sequence-400961-        5469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of TwinStrep-Flag tagged ATG13 (363-517).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5469)
  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 5469)
  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)
FEATURES             Location/Qualifiers
     source          1..5469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        129..508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        510..785
                     /label=chicken beta-actin promoter
     intron          786..1802
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1726..1748
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1810..1829
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1877..1886
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1881..1890
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1892..1915
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1952..1975
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1982..2005
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2012..2032
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2615..2634)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2680..2735
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2734..2753)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3096..3112)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3096..3112)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3109..3131)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3120..3136
                     /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(3144..3150,3151..3168,3169..3174))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3189..3210
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3269..3464
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3315..3450
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3470..3604
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3507..3526)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3561..3580
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3672..3689)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3843..4431)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3923..3942)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4602..5393,5394..5462))
                     /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     5225..5244
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(5463..5469,1..98))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
       61 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
      121 cctgggtcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      181 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      241 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      421 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      481 ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct
      541 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
      601 tgcagcgatg ggggcggggg gggggggggs gcgcgccagg cggggcgggg cggggcgagg
      661 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      721 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      781 gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc
      841 gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt
      901 ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc
      961 gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg
     1021 cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg
     1081 agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg
     1141 ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg
     1201 gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc
     1261 tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg
     1321 ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg
     1381 gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg
     1441 ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga
     1501 gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc
     1561 gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg
     1621 gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct
     1681 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1741 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     1801 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattcctcg
     1861 atcgacggta tcgatagcca ccatggcggc ctggtcacac ccgcagttcg agaaaggtgg
     1921 aggtgcccga ggtggatcgg gaggtggatc gtggagccac ccgcagttcg aaaaaggatt
     1981 cgattacaag gatgacgatg acaagggtac cgaaaacctg tacttccaat cccacgatgt
     2041 cctggaaacc atcttcgtga gaaaagtcgg cgcttttgtg aacaagccca ttaatcaggt
     2101 cacactcacc tctctggaca tccctttcgc tatgtttgca ccaaagaacc tcgagctgga
     2161 agacactgat cccatggtga atcctccaga ctcacccgag acagaaagtc ctctccaggg
     2221 cagtctgcac agcgatggaa gctccggtgg ctcttcaggc aacacacatg acgatttcgt
     2281 catgattgac ttcaaacccg ctttttccaa ggacgatatc ctccctatgg atctgggaac
     2341 cttctacagg gagtttcaga atccccctca gctcagtagc ctgagcattg acatcggtgc
     2401 ccagtccatg gctgaggacc tcgattctct gcctgaaaaa ctggctgtgc acgagaagaa
     2461 cgtcagagaa ttcgacgcat ttgtggagac cctgcagtaa cccgggtgat atcttaatta
     2521 attaactcga ggaattcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg
     2581 tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg
     2641 gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca
     2701 ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa
     2761 atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc
     2821 tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg
     2881 ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt
     2941 gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca
     3001 gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat
     3061 ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3121 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3181 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3241 tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc
     3301 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3361 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     3421 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat
     3481 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3541 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     3601 gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3661 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     3721 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     3781 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     3841 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     3901 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     3961 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     4021 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     4081 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     4141 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     4201 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     4261 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     4321 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     4381 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     4441 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     4501 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     4561 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     4621 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4681 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     4741 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     4801 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4861 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     4921 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4981 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     5041 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     5101 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     5161 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     5221 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     5281 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     5341 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     5401 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     5461 atactcttc
//
