LOCUS       sequence-448031-        7834 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of recombinant protein for purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7834)
  AUTHORS   Adriaenssens E, Schaar S, Cook ASI, Stuke JFM, Sawa-Makarska J, 
            Nguyen TN, Ren X, Schuschnig M, Romanov J, Khuu G, Uoselis L, 
            Lazarou M, Hummer G, Hurley JH, Martens S
  TITLE     Reconstitution of BNIP3/NIX-mitophagy initiation reveals 
            hierarchical flexibility of the autophagy machinery.
  JOURNAL   Nat Cell Biol. 2025 Jul 25. doi: 10.1038/s41556-025-01712-y.
  PUBMED    40715440
REFERENCE   2  (bases 1 to 7834)
  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: "Nat Cell 
            Biol. 2025 Jul 25. doi: 10.1038/s41556-025-01712-y."
COMMENT     Sequence Label: pCAG_GST-TEV-EGFP-ATG13
FEATURES             Location/Qualifiers
     source          1..7834
                     /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      1754..1763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1760..2416
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2374..2396
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2444..2464
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2471..2473,2474..2476,2477..3187)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2516..2537)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2777..2796)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3124..3145
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3717..3728
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     primer_bind     complement(4856..4875)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4921..4976
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4975..4994)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5337..5353)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5337..5353)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5350..5372)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5361..5377
                     /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(5385..5391,5392..5409,5410..5415))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5430..5451
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5510..5705
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5556..5691
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5711..5845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5748..5767)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5802..5821
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5913..5930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6084..6672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6164..6183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6843..7634,7635..7703))
                     /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     7466..7485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7704..7808)
                     /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 gatgccacca tggcatcccc tatactaggt tattggaaaa ttaagggcct
     1801 tgtgcaaccc actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta
     1861 tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc
     1921 caatcttcct tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg
     1981 ttatatagct gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc
     2041 aatgcttgaa ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa
     2101 agactttgaa actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt
     2161 cgaagatcgt ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt
     2221 catgttgtat gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt
     2281 cccaaaatta gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt
     2341 gaaatccagc aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg
     2401 cgaccatcct ccaaaatcgg atgattacga tatcccaacg accgaaaacc tgtattttca
     2461 gggtggtacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     2521 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     2581 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     2641 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     2701 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     2761 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     2821 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     2881 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     2941 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     3001 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3061 cgacaaccac tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga
     3121 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3181 gtacaagggt accgagaccg acctcaacag ccaggaccgt aaagacctcg acaagttcat
     3241 taagtttttc gccctcaaga ccgtgcaggt gatcgtgcag gcccggctgg gagagaaaat
     3301 ctgcacacgc agctcctctt cacccaccgg ttctgactgg ttcaacctgg ctattaagga
     3361 tatccctgaa gtgactcacg aagcaaagaa agcactcgct ggacagctgc cagcagtggg
     3421 tcgttcaatg tgtgtcgaga tctccctgaa gacatctgaa ggagactcaa tggagctcga
     3481 aatttggtgc ctggagatga acgaaaagtg tgataaagag atcaaagtga gctacaccgt
     3541 ctataatcgt ctgtccctgc tcctgaaatc tctcctggcc atcactcggg tgacacctgc
     3601 ttaccgcctc agtaggaagc agggtcatga atatgtgatt ctgtacagga tctatttcgg
     3661 cgaggtccag ctctccggac tgggtgaagg ctttcagact gtgcgtgtcg gaaccgtggg
     3721 aactccagtc ggcacaatta ccctctcttg cgcataccgt atcaatctgg ccttcatgtc
     3781 aaccagacag tttgagcgta ctccccctat tatgggaatc attatcgacc acttcgtgga
     3841 tagaccatac cccagtagct cccctatgca tccatgcaac tatcgtacag ccggtgaaga
     3901 caccggcgtg atctacccca gtgtcgagga tagccaggaa gtgtgtacca cttccttctc
     3961 tacctcacca ccctcacagc tctcttcaag tcgcctgagt tatcagcctg ccgctctcgg
     4021 agtcggtagc gcagacctgg cctacccagt ggtcttcgca gcaggcctca acgcaaccca
     4081 cccacatcag ctgatggtgc caggaaagga gggtggagtc ccactggctc ctaatcagcc
     4141 agtgcacggt actcaggctg accaggaaag gctggcaact tgcacaccat ccgatagaac
     4201 tcattgtgct gcaacaccca gctcctctga ggacaccgaa actgtgagca actcaagtga
     4261 gggtcgcgcc tccccacacg atgtcctgga aaccatcttc gtgagaaaag tcggcgcttt
     4321 tgtgaacaag cccattaatc aggtcacact cacctctctg gacatccctt tcgctatgtt
     4381 tgcaccaaag aacctcgagc tggaagacac tgatcccatg gtgaatcctc cagactcacc
     4441 cgagacagaa agtcctctcc agggcagtct gcacagcgat ggaagctccg gtggctcttc
     4501 aggcaacaca catgacgatt tcgtcatgat tgacttcaaa cccgcttttt ccaaggacga
     4561 tatcctccct atggatctgg gaaccttcta cagggagttt cagaatcccc ctcagctcag
     4621 tagcctgagc attgacatcg gtgcccagtc catggctgag gacctcgatt ctctgcctga
     4681 aaaactggct gtgcacgaga agaacgtcag agaattcgac gcatttgtgg agaccctgca
     4741 gcccgggtga tatcttaatt aattaactcg aggaattcac tcctcaggtg caggctgcct
     4801 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     4861 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     4921 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
     4981 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
     5041 tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc
     5101 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag
     5161 gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc ctaaaatttt
     5221 ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca gtcatagctg
     5281 tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta atcatggtca
     5341 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5401 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5461 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg catctcaatt
     5521 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     5581 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     5641 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     5701 gcaaaaagct aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5761 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5821 caatgtatct tatcatgtct ggatccgctg cattaatgaa tcggccaacg cgcggggaga
     5881 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     5941 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     6001 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6061 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6121 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6181 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6241 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6301 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6361 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6421 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6481 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6541 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6601 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6661 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6721 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6781 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6841 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     6901 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     6961 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     7021 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7081 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7141 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7201 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7261 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7321 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7381 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7441 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7501 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7561 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7621 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7681 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7741 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7801 gttccgcgca catttccccg aaaagtgcca cctg
//
