LOCUS       sequence-448300-        6593 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 6593)
  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 6593)
  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)-THROMBIN-GST
            (E72A/L75A/...
FEATURES             Location/Qualifiers
     source          1..6593
                     /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             852..869
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             876..1529
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1484..1506
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1542..1586
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1624..1642)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1638..1685
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1722..2177
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1809..1828)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2019..2040
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2292..3083,3084..3152))
                     /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     2915..2934
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3153..3245)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3326..3914
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3815..3834
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4068..4085
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4100..4239
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4165..4184)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4325..4347
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4341..4532)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5104..6186)
                     /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     6148..6167
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6187..6264)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6470..6489
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6577..6593,1..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 tggcgaagat tgctttggct gcacaacatg
      541 catcaggaca 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 tctccggatc actggttccg cgtggatccg gcggatcccc tatactaggt tattggaaaa
      901 ttaagggcct tgtgcaaccc actcgacttc ttttggaata tcttgaagaa aaatatgaag
      961 agcatttgta tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt gaattgggtt
     1021 tggagtttcc caatcttcct tattatattg atggtgatgt taaattaaca cagtctatgg
     1081 ccatcatacg ttatatagct gacaagcaca acatgttggg tggttgtcca aaagagcgtg
     1141 cagagatttc aatgcttgaa ggagcggttt tggatattag atacggtgtt tcgagaattg
     1201 catatagtaa agactttgaa actctcaaag ttgattttct tagcaagcta cctgaaatgc
     1261 tgaaaatgtt cgaagatcgt ttatgtcata aaacatattt aaatggtgat catgtaaccc
     1321 atcctgactt catgttgtat gacgctcttg atgttgtttt atacatggac ccaatgtgcc
     1381 tggatgcgtt cccaaaatta gtttgtttta aaaaacgtat tgaagctatc ccacaaattg
     1441 ataagtactt gaaatccagc aagtatatag catggccttt gcagggctgg caagccacgt
     1501 ttggtggtgg cgaccatcct ccaaaatagc tcgagtctgg taaagaaacc gctgctgcga
     1561 aatttgaacg ccagcacatg gactcgtcta ctagcgcagc ttaattaacc taggctgctg
     1621 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1681 ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc
     1741 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1801 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1861 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1921 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1981 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     2041 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2101 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     2161 attaacgttt acaatttctg gcggcacgat ggcatgagat tatcaaaaag gatcttcacc
     2221 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     2281 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     2341 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     2401 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     2461 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     2521 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     2581 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     2641 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     2701 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     2761 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     2821 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     2881 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     2941 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3001 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3061 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     3121 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaatc atgattgaag
     3181 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3241 acaaataggt catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3301 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     3361 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     3421 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     3481 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     3541 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     3601 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3661 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3721 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3781 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3841 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3901 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3961 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4021 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4081 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4141 cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     4201 tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc
     4261 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4321 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag
     4381 ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc
     4441 gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc
     4501 atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg
     4561 ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg ggttactgat
     4621 gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt atggatgcgg
     4681 cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac agatgtaggt
     4741 gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat ggtgcagggc
     4801 gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat tcatgttgtt
     4861 gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat
     4921 tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc
     4981 acgatcatgc tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     5041 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     5101 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5161 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     5221 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     5281 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     5341 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg
     5401 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5461 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5521 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5581 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5641 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5701 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5761 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5821 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5881 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5941 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     6001 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     6061 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     6121 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     6181 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     6241 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     6301 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     6361 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     6421 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     6481 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     6541 cgtagaggat cgagatcgat ctcgatcccg cgaaattaat acgactcact ata
//
