LOCUS       sequence-447580-        7340 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 7340)
  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 7340)
  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_NIX(1-182aa)-EGFP-TEV-GST (WT)
FEATURES             Location/Qualifiers
     source          1..7340
                     /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"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    233..257
                     /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             join(846..848,849..1559)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(888..909)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1149..1168)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1496..1517
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1572..1592
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1599..1616
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1623..2276
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2231..2253
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2289..2333
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2371..2389)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2385..2432
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2469..2924
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2556..2575)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2766..2787
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3039..3830,3831..3899))
                     /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     3662..3681
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3900..3992)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4073..4661
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4562..4581
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4815..4832
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4847..4986
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4912..4931)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5072..5094
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5088..5279)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5851..6933)
                     /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     6895..6914
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6934..7011)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7217..7236
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7324..7340,1..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 cgaattcgag
      121 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag
      181 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt
      241 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata
      301 tgtcgtccca cctagtcgag ccgccgccgc ccctgcacaa caacaacaac aactgcgagg
      361 aaaatgagca gtctctgccc ccgccggccg gcctcaacag ttcctgggtg gagctaccca
      421 tgaacagcag caatggcaat gataatggca atgggaaaaa tggggggctg gaacacgtac
      481 catcctcatc ctccatccac aatggagaca tggagaagat tcttttggat gcacaacatg
      541 aatcaggaca gagtagttcc agaggcagtt ctcactgtga cagcccttcg ccacaagaag
      601 atgggcagat catgtttgat gtggaaatgc acaccagcag ggaccatagc tctcagtcag
      661 aagaagaagt tgtagaagga gagaaggaag tcgaggcttt gaagaaaagt gcggactggg
      721 tatcagactg gtccagtaga cccgaaaaca ttccacccaa ggagttccac ttcagacacc
      781 ctaaacgttc tgtgtcttta agcatgagga aaagtggagc catgaagaaa gggggtattt
      841 tctccgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      901 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
      961 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1021 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1081 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1141 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1201 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1261 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1321 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1381 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1441 actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
     1501 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     1561 gcggcagtgg cgaaaacctg tattttcagg gcggatcact ggttccgcgt ggatccggcg
     1621 gatcccctat actaggttat tggaaaatta agggccttgt gcaacccact cgacttcttt
     1681 tggaatatct tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa ggtgataaat
     1741 ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa tcttccttat tatattgatg
     1801 gtgatgttaa attaacacag tctatggcca tcatacgtta tatagctgac aagcacaaca
     1861 tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga gcggttttgg
     1921 atattagata cggtgtttcg agaattgcat atagtaaaga ctttgaaact ctcaaagttg
     1981 attttcttag caagctacct gaaatgctga aaatgttcga agatcgttta tgtcataaaa
     2041 catatttaaa tggtgatcat gtaacccatc ctgacttcat gttgtatgac gctcttgatg
     2101 ttgttttata catggaccca atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa
     2161 aacgtattga agctatccca caaattgata agtacttgaa atccagcaag tatatagcat
     2221 ggcctttgca gggctggcaa gccacgtttg gtggtggcga ccatcctcca aaatagctcg
     2281 agtctggtaa agaaaccgct gctgcgaaat ttgaacgcca gcacatggac tcgtctacta
     2341 gcgcagctta attaacctag gctgctgcca ccgctgagca ataactagca taaccccttg
     2401 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     2461 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2521 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2581 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2641 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2701 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2761 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2821 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2881 aaaatttaac gcgaatttta acaaaatatt aacgtttaca atttctggcg gcacgatggc
     2941 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3001 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3061 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3121 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     3181 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3241 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3301 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3361 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3421 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3481 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3541 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3601 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3661 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3721 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3781 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3841 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     3901 ctcttccttt ttcaatcatg attgaagcat ttatcagggt tattgtctca tgagcggata
     3961 catatttgaa tgtatttaga aaaataaaca aataggtcat gaccaaaatc ccttaacgtg
     4021 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4081 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4141 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4201 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4261 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4321 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4381 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4441 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4501 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4561 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4621 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4681 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4741 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4801 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     4861 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     4921 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4981 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     5041 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     5101 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     5161 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     5221 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     5281 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     5341 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     5401 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     5461 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     5521 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     5581 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     5641 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     5701 cctagccggg tcctcaacga caggagcacg atcatgctag tcatgccccg cgcccaccgg
     5761 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     5821 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5881 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5941 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     6001 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     6061 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     6121 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     6181 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     6241 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     6301 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     6361 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     6421 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     6481 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     6541 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     6601 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     6661 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     6721 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     6781 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     6841 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     6901 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     6961 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     7021 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     7081 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     7141 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     7201 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     7261 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga
     7321 aattaatacg actcactata
//
