LOCUS       sequence-447749-        6071 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 6071)
  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 6071)
  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-Lambda-Protein-Phosphatase
FEATURES             Location/Qualifiers
     source          1..6071
                     /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        868..886
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    887..911
                     /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             1020..1064
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1102..1120)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1116..1163
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1200..1655
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1287..1306)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1497..1518
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1770..2561,2562..2630))
                     /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     2393..2412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2631..2723)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2804..3392
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3293..3312
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3546..3563
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3578..3717
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3643..3662)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3803..3825
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3819..4010)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4582..5664)
                     /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     5626..5645
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5665..5742)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5948..5967
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(6055..6071,1..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 ctcgatgcgg tactacgaaa aaatcgacgg tagcaaatat cgcaatatct gggttgtggg
      181 cgacctgcat ggctgctata caaatctgat gaacaagctt gacactatcg ggttcgataa
      241 taaaaaagac ctgctgatca gcgtgggaga tctggttgac cgtggggctg aaaacgttga
      301 atgcctggaa ctgatcacct tcccctggtt ccgcgccgtg cgtggcaacc atgaacagat
      361 gatgatcgat ggactgagcg aacgcggtaa tgtgaaccat tggctgttaa atggaggagg
      421 ctggtttttc aatctggact acgacaaaga gatcctggcc aaagccttgg cacataaagc
      481 agatgagctg cctctcatca ttgaactggt tagtaaagat aaaaaatatg tgatttgcca
      541 cgcggattac ccttttgacg agtatgaatt tggaaaaccg gtggatcatc agcaggttat
      601 ttggaaccgc gagcgcatct ctaattcgca gaatggcatc gtgaaggaaa ttaagggcgc
      661 cgataccttc atcttcggtc atacgccagc agtgaaaccc ctgaagtttg cgaaccagat
      721 gtatattgat accggcgcgg ttttctgcgg taatttgacg ctcatccagg ttcagggcga
      781 aggtgcctaa gtcgacaagc ttgcggccgc ataatgctta agtcgaacag aaagtaatcg
      841 tattgtacac ggccgcataa tcgaaattaa tacgactcac tataggggaa ttgtgagcgg
      901 ataacaattc cccatcttag tatattagtt aagtataaga aggagatata catatggcag
      961 atctcaattg gatatcggcc ggccacgcga tcgctgacgt cggtaccctc gagtctggta
     1021 aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt
     1081 aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     1141 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     1201 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1261 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1321 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1381 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1441 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1501 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     1561 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     1621 cgcgaatttt aacaaaatat taacgtttac aatttctggc ggcacgatgg catgagatta
     1681 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     1741 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     1801 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     1861 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     1921 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     1981 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     2041 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     2101 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     2161 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     2221 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     2281 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     2341 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     2401 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     2461 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     2521 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     2581 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     2641 tttcaatcat gattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     2701 atgtatttag aaaaataaac aaataggtca tgaccaaaat cccttaacgt gagttttcgt
     2761 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2821 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2881 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2941 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3001 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3061 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3121 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3181 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3241 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3301 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3361 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3421 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3481 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3541 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3601 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     3661 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     3721 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     3781 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     3841 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     3901 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     3961 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4021 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4081 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4141 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4201 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4261 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     4321 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4381 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4441 gtcctcaacg acaggagcac gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg
     4501 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4561 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4621 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4681 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4741 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4801 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4861 gatgtccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     4921 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     4981 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5041 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5101 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5161 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5221 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5281 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5341 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5401 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5461 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5521 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5581 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5641 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5701 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5761 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     5821 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     5881 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     5941 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6001 cggccacgat gcgtccggcg tagaggatcg agatcgatct cgatcccgcg aaattaatac
     6061 gactcactat a
//
