LOCUS       sequence-455264-        7278 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 7278)
  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 7278)
  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: pETDuet_6xHis-FKBP8(1-391aa)-thrombin-GST
FEATURES             Location/Qualifiers
     source          1..7278
                     /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             1292..1309
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1316..1969
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKF
                     ELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG
                     VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY
                     MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1924..1946
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     promoter        2072..2090
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2091..2115
                     /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             2224..2268
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2306..2324)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2320..2367
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2404..2859
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2491..2510)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2701..2722
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(2974..3765,3766..3834))
                     /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     3597..3616
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3835..3927)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4008..4596
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4497..4516
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4750..4767
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4782..4924
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4850..4869)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5010..5032
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5026..5217)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5789..6871)
                     /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     6833..6852
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6872..6949)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7155..7174
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7262..7278,1..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 caatggcatc
      121 gtgtgctgaa ccctctgagc cctctgcccc actgcccgcc ggggtcccac cgctcgagga
      181 cttcgaggta ctggatgggg ttgaggatgc agagggtgag gaggaagagg aggaggaaga
      241 ggaggaagag gatgacctga gtgagctgcc accgctggag gacatgggac aacccccggc
      301 ggaggaggct gagcagcctg gggccctggc ccgagagttc cttgctgcca tggagcccga
      361 gcccgcccca gccccggccc cagaagagtg gctggacatt ctggggaacg ggctgttgag
      421 gaagaagacg ctggtcccag ggccgccagg ttcgagccgc ccggtcaagg gccaggtggt
      481 caccgtacat ctgcagacgt cgctggagaa tggcacacgg gtgcaggagg agccggagct
      541 ggtgttcact ctgggtgact gtgacgtcat ccaggccctg gatctcagtg tcccactcat
      601 ggacgtgggg gagacggcca tggtcactgc tgactccaag tactgctacg gcccccaagg
      661 cagcaggagc ccatacatcc ccccgcacgc ggccctgtgc ctggaggtga ccctgaagac
      721 ggctgtggac gggcctgacc tggagatgct cacggggcag gagcgcgtgg ccctggccaa
      781 ccggaagcgg gagtgcggca acgcccacta ccagcgggcg gacttcgtcc tggccgccaa
      841 ctcctacgac ctcgccatca aggctatcac ctccagcgcc aaagtggaca tgacgttcga
      901 ggaggaggca cagctcctgc agttgaaggt gaagtgtctg aacaacctgg cggcctcgca
      961 gctgaagctc gaccactacc gcgcagccct gcgctcctgc agccttgtgc tggagcacca
     1021 gccagacaac atcaaggctc tcttccgcaa gggcaaggtg ctggcccagc agggggagta
     1081 cagtgaggcc atccccatcc tgagggcagc cctgaagctg gaaccttcca acaagacgat
     1141 ccacgcagag ctctcaaagc tggtgaagaa gcatgcggcg cagcggagca cggagaccgc
     1201 cttgtaccgg aaaatgctgg gcaaccccag ccggctgcct gctaagtgcc ctggcaaggg
     1261 tgcctggtcc atcccatgga agtggggatc actggttccg cgtggatccg gcggatcccc
     1321 tatactaggt tattggaaaa ttaagggcct tgtgcaaccc actcgacttc ttttggaata
     1381 tcttgaagaa aaatatgaag agcatttgta tgagcgcgat gaaggtgata aatggcgaaa
     1441 caaaaagttt gaattgggtt tggagtttcc caatcttcct tattatattg atggtgatgt
     1501 taaattaaca cagtctatgg ccatcatacg ttatatagct gacaagcaca acatgttggg
     1561 tggttgtcca aaagagcgtg cagagatttc aatgcttgaa ggagcggttt tggatattag
     1621 atacggtgtt tcgagaattg catatagtaa agactttgaa actctcaaag ttgattttct
     1681 tagcaagcta cctgaaatgc tgaaaatgtt cgaagatcgt ttatgtcata aaacatattt
     1741 aaatggtgat catgtaaccc atcctgactt catgttgtat gacgctcttg atgttgtttt
     1801 atacatggac ccaatgtgcc tggatgcgtt cccaaaatta gtttgtttta aaaaacgtat
     1861 tgaagctatc ccacaaattg ataagtactt gaaatccagc aagtatatag catggccttt
     1921 gcagggctgg caagccacgt ttggtggtgg cgaccatcct ccaaaatagg aattcgagct
     1981 cggcgcgcct gcaggtcgac aagcttgcgg ccgcataatg cttaagtcga acagaaagta
     2041 atcgtattgt acacggccgc ataatcgaaa ttaatacgac tcactatagg ggaattgtga
     2101 gcggataaca attccccatc ttagtatatt agttaagtat aagaaggaga tatacatatg
     2161 gcagatctca attggatatc ggccggccac gcgatcgctg acgtcggtac cctcgagtct
     2221 ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc tactagcgca
     2281 gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     2341 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     2401 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2461 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2521 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2581 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2641 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2701 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2761 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2821 ttaacgcgaa ttttaacaaa atattaacgt ttacaatttc tggcggcacg atggcatgag
     2881 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2941 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3001 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3061 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     3121 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3181 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3241 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3301 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3361 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3421 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     3481 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     3541 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     3601 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     3661 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     3721 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     3781 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     3841 cctttttcaa tcatgattga agcatttatc agggttattg tctcatgagc ggatacatat
     3901 ttgaatgtat ttagaaaaat aaacaaatag gtcatgacca aaatccctta acgtgagttt
     3961 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4021 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4081 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4141 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4201 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4261 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4321 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4381 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4441 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4501 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4561 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4621 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4681 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4741 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4801 cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct
     4861 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     4921 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     4981 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5041 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5101 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5161 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5221 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5281 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5341 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5401 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5461 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     5521 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     5581 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     5641 tagccgggtc ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa
     5701 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5761 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5821 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5881 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5941 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     6001 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     6061 ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     6121 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6181 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6241 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6301 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6361 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6421 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6481 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6541 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6601 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6661 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6721 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6781 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6841 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6901 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     6961 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     7021 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     7081 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     7141 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     7201 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa
     7261 ttaatacgac tcactata
//
