LOCUS       sequence-447443-        7421 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 7421)
  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 7421)
  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: pETDuet1_6xHis-TEV-mCherry-WIPI2d
FEATURES             Location/Qualifiers
     source          1..7421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     RBS             42..64
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             83..100
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             113..133
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             134..838
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(276..294)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     544..563
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        2218..2236
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2237..2261
                     /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)."
     CDS             2370..2414
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2452..2470)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2466..2513
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2550..3005
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2637..2656)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2847..2868
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3120..3911,3912..3980))
                     /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     3743..3762
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3981..4073)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4154..4742
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4643..4662
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4896..4913
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4928..5067
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4993..5012)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5153..5175
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5169..5360)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5932..7014)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6976..6995
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7015..7092)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7298..7317
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7405..7421,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
       61 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cggaaaacct
      121 gtattttcag ggagtgagca agggcgagga ggataacatg gccatcatca aggagttcat
      181 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
      241 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
      301 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
      361 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
      421 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
      481 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
      541 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
      601 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
      661 ctacgacgct gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
      721 ctacaacgtc aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga
      781 acagtacgaa cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc
      841 cggactcaga tctcgagctc aagcttcgaa ttcgatgaac ctggcgagcc agagcgggga
      901 ggccggcgcc ggccagctgc tcttcgccaa cttcaaccag gacaacacgt ccctagctgt
      961 tggtagtaag tccggttata aatttttctc cctttcttct gtggataagc tggaacagat
     1021 ctatgaatgc accgatacgg aagatgtgtg cattgtagag agattgttct ccagcagcct
     1081 agtggccatc gtgagcctta aagcaccaag gaagctaaag gtttgccact ttaagaaggg
     1141 aactgagatc tgcaactaca gctactccaa cacgattctg gctgtgaagc tcaacaggca
     1201 gaggctgata gtatgcctgg aggagtccct gtacatccac aacattcggg acatgaaggt
     1261 gctgcatacg atcagggaga cgcctccaaa ccctgcaggc ctgtgtgcgc tgtcaatcaa
     1321 caacgacaac tgctacttgg cgtacccagg gagcgcgacc atcggagagg tgcaggtctt
     1381 cgataccatt aatttgagag ctgcaaacat gattccggct cacgacagtc ctttagcggc
     1441 actggccttt gacgcaagtg gaactaaact tgccacggct tcggagaagg ggaccgtgat
     1501 tagggtattt tccattccag aaggacaaaa actctttgag tttcggagag gagtaaagag
     1561 gtgcgtgagc atctgctccc tggccttcag catggacggc atgttcctct ccgcctccag
     1621 caacactgag accgtgcaca tcttcaaact cgagactgtg aaagaaaaac ccccagagga
     1681 gcccaccacc tggaccgggt acttcgggaa agtgctcatg gcctccacca gctacctgcc
     1741 ttcccaagtg acagaaatgt tcaaccaggg cagagccttc gccacggtcc gcctgccatt
     1801 ctgcggccac aaaaacatct gctcgctagc cacaattcag aagatcccgc ggttgttggt
     1861 gggtgccgcc gacgggtacc tgtacatgta caacctggac ccccaggagg gcggcgagtg
     1921 tgccctgatg aagcagcacc ggctggacgg cagtctggaa acgaccaatg agatcttgga
     1981 ctctgcctct cacgactgcc ccttagtcac tcagacatac ggcgcagctg caggaaaagc
     2041 ctacacagac gacctgggtg ctgtgggtgg cgcctgcctg gaggacgagg ccagcgccct
     2101 gcgcctggat gaggacagcg agcacccgcc catgattctt cggactgact gagcggccgc
     2161 ataatgctta agtcgaacag aaagtaatcg tattgtacac ggccgcataa tcgaaattaa
     2221 tacgactcac tataggggaa ttgtgagcgg ataacaattc cccatcttag tatattagtt
     2281 aagtataaga aggagatata catatggcag atctcaattg gatatcggcc ggccacgcga
     2341 tcgctgacgt cggtaccctc gagtctggta aagaaaccgc tgctgcgaaa tttgaacgcc
     2401 agcacatgga ctcgtctact agcgcagctt aattaaccta ggctgctgcc accgctgagc
     2461 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     2521 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     2581 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2641 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2701 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2761 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2821 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2881 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2941 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     3001 aatttctggc ggcacgatgg catgagatta tcaaaaagga tcttcaccta gatcctttta
     3061 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3121 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3181 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3241 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     3301 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3361 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3421 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3481 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     3541 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     3601 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     3661 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     3721 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     3781 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     3841 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     3901 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     3961 cggaaatgtt gaatactcat actcttcctt tttcaatcat gattgaagca tttatcaggg
     4021 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggtca
     4081 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4141 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4201 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4261 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4321 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4381 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4441 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4501 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4561 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4621 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4681 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4741 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     4801 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4861 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4921 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     4981 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     5041 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     5101 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5161 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     5221 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     5281 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     5341 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     5401 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     5461 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     5521 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     5581 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     5641 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     5701 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     5761 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcta
     5821 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     5881 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     5941 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6001 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     6061 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     6121 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     6181 gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc
     6241 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     6301 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     6361 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     6421 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     6481 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     6541 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     6601 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     6661 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     6721 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     6781 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     6841 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     6901 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     6961 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     7021 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     7081 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     7141 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     7201 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     7261 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     7321 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     7381 agatcgatct cgatcccgcg aaattaatac gactcactat a
//
