LOCUS       sequence-448234-        6664 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 6664)
  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 6664)
  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: pGEX4T1_GST-EGFP-ATG13_IDR_delta191-204 (205-5...
FEATURES             Location/Qualifiers
     source          1..6664
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(181..186,187..202,203..209)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    217..233
                     /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             256..912
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     870..892
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             940..960
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(967..969,970..972,973..1683)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1012..1033)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1273..1292)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1620..1641
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2712..2734)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2879..2897)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2965..3069
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3070..3138,3139..3930)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3288..3307)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4101..4689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4590..4609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4843..4860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4933..5010
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5011..6093
                     /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     complement(5030..5049)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6142..6147,6148..6165,6166..6172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6180..6196
                     /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)."
     primer_bind     6204..6220
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6204..6220
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6216..6664,1..79)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTVRCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6236..6253)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6236..6252)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6245..6267)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cttatcgact gtacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt
       61 atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc
      121 tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
      181 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
      241 caggaaacag tattcatggc atcccctata ctaggttatt ggaaaattaa gggccttgtg
      301 caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca tttgtatgag
      361 cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat
      421 cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat catacgttat
      481 atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg
      541 cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata tagtaaagac
      601 tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa aatgttcgaa
      661 gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc tgacttcatg
      721 ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga tgcgttccca
      781 aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa gtacttgaaa
      841 tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg tggtggcgac
      901 catcctccaa aatcggatga ttacgatatc ccaacgaccg aaaacctgta ttttcagggt
      961 ggtaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1021 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1081 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1141 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     1201 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1261 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1321 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1381 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1441 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1501 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1561 aaccactacc tgagcaccca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac
     1621 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1681 aagggtaccg gtggtccccc tattatggga atcattatcg accacttcgt ggatagacca
     1741 taccccagta gctcccctat gcatccatgc aactatcgta cagccggtga agacaccggc
     1801 gtgatctacc ccagtgtcga ggatagccag gaagtgtgta ccacttcctt ctctacctca
     1861 ccaccctcac agctctcttc aagtcgcctg agttatcagc ctgccgctct cggagtcggt
     1921 agcgcagacc tggcctaccc agtggtcttc gcagcaggcc tcaacgcaac ccacccacat
     1981 cagctgatgg tgccaggaaa ggagggtgga gtcccactgg ctcctaatca gccagtgcac
     2041 ggtactcagg ctgaccagga aaggctggca acttgcacac catccgatag aactcattgt
     2101 gctgcaacac ccagctcctc tgaggacacc gaaactgtga gcaactcaag tgagggtcgc
     2161 gcctccccac acgatgtcct ggaaaccatc ttcgtgagaa aagtcggcgc ttttgtgaac
     2221 aagcccatta atcaggtcac actcacctct ctggacatcc ctttcgctat gtttgcacca
     2281 aagaacctcg agctggaaga cactgatccc atggtgaatc ctccagactc acccgagaca
     2341 gaaagtcctc tccagggcag tctgcacagc gatggaagct ccggtggctc ttcaggcaac
     2401 acacatgacg atttcgtcat gattgacttc aaacccgctt tttccaagga cgatatcctc
     2461 cctatggatc tgggaacctt ctacagggag tttcagaatc cccctcagct cagtagcctg
     2521 agcattgaca tcggtgccca gtccatggct gaggacctcg attctctgcc tgaaaaactg
     2581 gctgtgcacg agaagaacgt cagagaattc gacgcatttg tggagaccct gcagtgaccc
     2641 gggtgactcg agcggccgca tcgtgactga ctgacgatct gcctcgcgcg tttcggtgat
     2701 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     2761 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     2821 gcagccatga cccagtcacg tagcgatagc ggagtgtata attcttgaag acgaaagggc
     2881 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     2941 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3001 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3061 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3121 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3181 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3241 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3301 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3361 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3421 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3481 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3541 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3601 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3661 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3721 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3781 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3841 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3901 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3961 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4021 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4081 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4141 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4201 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     4261 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4321 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4381 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4441 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4501 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4561 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4621 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4681 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4741 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4801 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4861 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     4921 cgcataaatt ccgacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     4981 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     5041 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     5101 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     5161 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     5221 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     5281 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     5341 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     5401 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     5461 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     5521 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     5581 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     5641 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     5701 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     5761 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     5821 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     5881 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     5941 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     6001 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     6061 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     6121 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     6181 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg gattcactgg
     6241 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     6301 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     6361 cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag
     6421 cggtgccgga aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct
     6481 caaactggca gatgcacggt tacgatgcgc ccatctacac caacgtgacc tatcccatta
     6541 cggtcaatcc gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta
     6601 atgttgatga aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttggaa
     6661 ttag
//
