LOCUS       sequence-448038-        6621 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 6621)
  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 6621)
  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-WIPI4
FEATURES             Location/Qualifiers
     source          1..6621
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(163..179)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(163..179)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(176..198)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    187..203
                     /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(211..217,218..235,236..241))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    256..277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        336..531
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      382..517
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    537..671
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(574..593)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     628..647
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(739..756)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(910..1498)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(990..1009)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1669..2460,2461..2529))
                     /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     2292..2311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2530..2634)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        2665..3044
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3046..3323
                     /label=chicken beta-actin promoter
     intron          3324..4340
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4264..4286
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4348..4367
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      4414..4423
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4420..5076
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     5034..5056
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             5104..5124
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(6303..6322)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6368..6423
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6422..6441)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
ORIGIN
        1 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
       61 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta
      121 tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt
      181 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
      241 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
      301 tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata
      361 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      421 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      481 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact
      541 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
      601 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
      661 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
      721 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
      781 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
      841 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
      901 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
      961 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1021 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1081 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     1141 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     1201 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1261 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     1321 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     1381 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     1441 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     1501 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     1561 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     1621 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     1681 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     1741 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     1801 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     1861 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     1921 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     1981 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     2041 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     2101 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     2161 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     2221 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     2281 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     2341 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     2401 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     2461 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     2521 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     2581 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     2641 tccccgaaaa gtgccacctg ggtcgacatt gattattgac tagttattaa tagtaatcaa
     2701 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     2761 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     2821 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     2881 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     2941 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     3001 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca
     3061 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta
     3121 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg
     3181 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc
     3241 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata
     3301 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct
     3361 ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga
     3421 gcgggcggga cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg
     3481 tttcttttct gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg
     3541 ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg
     3601 cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg
     3661 tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga
     3721 acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg
     3781 tcgggctgca accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg
     3841 gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc
     3901 aggtgggggt gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc
     3961 gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt
     4021 tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga
     4081 aatctgggag gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg
     4141 gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc
     4201 ctctccagcc tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg
     4261 cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg
     4321 ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt
     4381 ttggcaaaga attcctcgat cgacggtatc gatgccacca tggcatcccc tatactaggt
     4441 tattggaaaa ttaagggcct tgtgcaaccc actcgacttc ttttggaata tcttgaagaa
     4501 aaatatgaag agcatttgta tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt
     4561 gaattgggtt tggagtttcc caatcttcct tattatattg atggtgatgt taaattaaca
     4621 cagtctatgg ccatcatacg ttatatagct gacaagcaca acatgttggg tggttgtcca
     4681 aaagagcgtg cagagatttc aatgcttgaa ggagcggttt tggatattag atacggtgtt
     4741 tcgagaattg catatagtaa agactttgaa actctcaaag ttgattttct tagcaagcta
     4801 cctgaaatgc tgaaaatgtt cgaagatcgt ttatgtcata aaacatattt aaatggtgat
     4861 catgtaaccc atcctgactt catgttgtat gacgctcttg atgttgtttt atacatggac
     4921 ccaatgtgcc tggatgcgtt cccaaaatta gtttgtttta aaaaacgtat tgaagctatc
     4981 ccacaaattg ataagtactt gaaatccagc aagtatatag catggccttt gcagggctgg
     5041 caagccacgt ttggtggtgg cgaccatcct ccaaaatcgg atgattacga tatcccaacg
     5101 accgaaaacc tgtattttca gggtggtacc atgactcaac agccacttcg aggagtgacc
     5161 agcctgcgtt tcaaccaaga ccaaagctgc ttttgctgcg ccatggagac aggtgtgcgc
     5221 atctacaacg tggagccctt gatggagaag gggcatctgg accacgagca ggtgggcagc
     5281 atgggcttgg tggagatgct gcaccgctcc aaccttctgg ccttggtggg cggtggtagt
     5341 agtcccaagt tctcagagat ctcagtgctg atctgggacg atgcccggga gggcaaggac
     5401 tccaaggaga agctggtgct ggagttcacc ttcaccaagc cagtgctttc tgtgcgcatg
     5461 cgccatgaca agatcgtgat cgtgctgaag aaccgcatct atgtgtactc cttccccgac
     5521 aatccccgaa agctgtttga gtttgatacc cgggacaacc ccaaggggct ctgtgacctc
     5581 tgccccagcc tggagaagca actgctagtg ttcccgggac acaagtgtgg gagtctgcaa
     5641 cttgtggacc tggcgagcac aaagcctggc acctcgtctg ctccattcac gatcaatgca
     5701 catcagagtg acatagcctg tgtgtctcta aaccagccag gcactgtagt ggcctcagcc
     5761 tcccagaagg gtacccttat tcgcctcttt gacacacaat ccaaggagaa actggtggag
     5821 ctgcgccgag gcactgaccc tgccaccctc tactgcatta acttcagcca cgactcctcc
     5881 ttcctctgcg cttccagtga taagggtact gtccatatct ttgctctcaa ggatacccgc
     5941 ctcaaccgcc gctccgcgct ggctcgcgtg ggcaaggtgg ggcctatgat tgggcagtac
     6001 gtggactctc agtggagcct ggcgagcttc actgtgcctg ctgagtcagc ttgcatctgc
     6061 gccttcggtc gcaatacttc caagaacgtc aactctgtca ttgccatctg cgtagatggg
     6121 accttccaca aatatgtctt cactcctgat ggaaactgca acagagaggc tttcgacgtg
     6181 taccttgaca tctgtgatga tgatgacttt taactcgagg aattcactcc tcaggtgcag
     6241 gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga
     6301 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     6361 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     6421 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     6481 tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga
     6541 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg
     6601 acttgaggtt agattttttt t
//
