LOCUS       sequence-448028-        6794 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 6794)
  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 6794)
  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_WIPI2d-TEV-GST
FEATURES             Location/Qualifiers
     source          1..6794
                     /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..3322
                     /label=chicken beta-actin promoter
     intron          3323..4339
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4263..4285
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4347..4366
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             5700..5720
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             5724..6377
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     6335..6357
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     primer_bind     complement(6476..6495)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    6541..6596
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(6595..6614)
                     /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 ggggggcgcg cgccaggcgg
     3181 ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca
     3241 gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa
     3301 aaagcgaagc gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc
     3361 cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag
     3421 cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt
     3481 ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg
     3541 gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc
     3601 gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt
     3661 gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa
     3721 caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt
     3781 cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg
     3841 tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca
     3901 ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg
     3961 cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt
     4021 atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa
     4081 atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg
     4141 caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc
     4201 tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc
     4261 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc
     4321 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt
     4381 tggcaaagaa ttcctcgatc gacggtatcg atgccaccat gaacctggcg agccagagcg
     4441 gggaggccgg cgccggccag ctgctcttcg ccaacttcaa ccaggacaac acgtccctag
     4501 ctgttggtag taagtccggt tataaatttt tctccctttc ttctgtggat aagctggaac
     4561 agatctatga atgcaccgat acggaagatg tgtgcattgt agagagattg ttctccagca
     4621 gcctagtggc catcgtgagc cttaaagcac caaggaagct aaaggtttgc cactttaaga
     4681 agggaactga gatctgcaac tacagctact ccaacacgat tctggctgtg aagctcaaca
     4741 ggcagaggct gatagtatgc ctggaggagt ccctgtacat ccacaacatt cgggacatga
     4801 aggtgctgca tacgatcagg gagacgcctc caaaccctgc aggcctgtgt gcgctgtcaa
     4861 tcaacaacga caactgctac ttggcgtacc cagggagcgc gaccatcgga gaggtgcagg
     4921 tcttcgatac cattaatttg agagctgcaa acatgattcc ggctcacgac agtcctttag
     4981 cggcactggc ctttgacgca agtggaacta aacttgccac ggcttcggag aaggggaccg
     5041 tgattagggt attttccatt ccagaaggac aaaaactctt tgagtttcgg agaggagtaa
     5101 agaggtgcgt gagcatctgc tccctggcct tcagcatgga cggcatgttc ctctccgcct
     5161 ccagcaacac tgagaccgtg cacatcttca aactcgagac tgtgaaagaa aaacccccag
     5221 aggagcccac cacctggacc gggtacttcg ggaaagtgct catggcctcc accagctacc
     5281 tgccttccca agtgacagaa atgttcaacc agggcagagc cttcgccacg gtccgcctgc
     5341 cattctgcgg ccacaaaaac atctgctcgc tagccacaat tcagaagatc ccgcggttgt
     5401 tggtgggtgc cgccgacggg tacctgtaca tgtacaacct ggacccccag gagggcggcg
     5461 agtgtgccct gatgaagcag caccggctgg acggcagtct ggaaacgacc aatgagatct
     5521 tggactctgc ctctcacgac tgccccttag tcactcagac atacggcgca gctgcaggaa
     5581 aagcctacac agacgacctg ggtgctgtgg gtggcgcctg cctggaggac gaggccagcg
     5641 ccctgcgcct ggatgaggac agcgagcacc cgcccatgat tcttcggact gacggtaccg
     5701 aaaacctgta cttccaatcc ggtatgtccc ctatactagg ttattggaaa attaagggcc
     5761 ttgtgcaacc cactcgactt cttttggaat atcttgaaga aaaatatgaa gagcatttgt
     5821 atgagcgcga tgaaggtgat aaatggcgaa acaaaaagtt tgaattgggt ttggagtttc
     5881 ccaatcttcc ttattatatt gatggtgatg ttaaattaac acagtctatg gccatcatac
     5941 gttatatagc tgacaagcac aacatgttgg gtggttgtcc aaaagagcgt gcagagattt
     6001 caatgcttga aggagcggtt ttggatatta gatacggtgt ttcgagaatt gcatatagta
     6061 aagactttga aactctcaaa gttgattttc ttagcaagct acctgaaatg ctgaaaatgt
     6121 tcgaagatcg tttatgtcat aaaacatatt taaatggtga tcatgtaacc catcctgact
     6181 tcatgttgta tgacgctctt gatgttgttt tatacatgga cccaatgtgc ctggatgcgt
     6241 tcccaaaatt agtttgtttt aaaaaacgta ttgaagctat cccacaaatt gataagtact
     6301 tgaaatccag caagtatata gcatggcctt tgcagggctg gcaagccacg tttggtggtg
     6361 gcgaccatcc tccaaaaatt gactagctcg aggaattcac tcctcaggtg caggctgcct
     6421 atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcttt
     6481 ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg acttctggct
     6541 aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt ctctcactcg
     6601 gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg gtttagagtt
     6661 tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa agaggtcatc
     6721 agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc ttgacttgag
     6781 gttagatttt tttt
//
