LOCUS       sequence-448328-        6706 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 6706)
  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 6706)
  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 (191-517aa)
FEATURES             Location/Qualifiers
     source          1..6706
                     /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(2754..2776)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2921..2939)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3007..3111
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3112..3180,3181..3972)
                     /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(3330..3349)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4143..4731
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4632..4651
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4885..4902
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4975..5052
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5053..6135
                     /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(5072..5091)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6184..6189,6190..6207,6208..6214)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6222..6238
                     /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     6246..6262
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6246..6262
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6258..6706,1..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(6278..6295)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6278..6294)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6287..6309)
                     /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 gtggtatcaa tctggccttc atgtcaacca gacagtttga gcgtactccc
     1741 cctattatgg gaatcattat cgaccacttc gtggatagac cataccccag tagctcccct
     1801 atgcatccat gcaactatcg tacagccggt gaagacaccg gcgtgatcta ccccagtgtc
     1861 gaggatagcc aggaagtgtg taccacttcc ttctctacct caccaccctc acagctctct
     1921 tcaagtcgcc tgagttatca gcctgccgct ctcggagtcg gtagcgcaga cctggcctac
     1981 ccagtggtct tcgcagcagg cctcaacgca acccacccac atcagctgat ggtgccagga
     2041 aaggagggtg gagtcccact ggctcctaat cagccagtgc acggtactca ggctgaccag
     2101 gaaaggctgg caacttgcac accatccgat agaactcatt gtgctgcaac acccagctcc
     2161 tctgaggaca ccgaaactgt gagcaactca agtgagggtc gcgcctcccc acacgatgtc
     2221 ctggaaacca tcttcgtgag aaaagtcggc gcttttgtga acaagcccat taatcaggtc
     2281 acactcacct ctctggacat ccctttcgct atgtttgcac caaagaacct cgagctggaa
     2341 gacactgatc ccatggtgaa tcctccagac tcacccgaga cagaaagtcc tctccagggc
     2401 agtctgcaca gcgatggaag ctccggtggc tcttcaggca acacacatga cgatttcgtc
     2461 atgattgact tcaaacccgc tttttccaag gacgatatcc tccctatgga tctgggaacc
     2521 ttctacaggg agtttcagaa tccccctcag ctcagtagcc tgagcattga catcggtgcc
     2581 cagtccatgg ctgaggacct cgattctctg cctgaaaaac tggctgtgca cgagaagaac
     2641 gtcagagaat tcgacgcatt tgtggagacc ctgcagtgac ccgggtgact cgagcggccg
     2701 catcgtgact gactgacgat ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     2761 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     2821 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     2881 cgtagcgata gcggagtgta taattcttga agacgaaagg gcctcgtgat acgcctattt
     2941 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     3001 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3061 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3121 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3181 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3241 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3301 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     3361 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3421 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3481 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3541 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3601 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     3661 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     3721 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     3781 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     3841 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3901 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3961 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4021 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4081 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4141 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4201 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4261 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     4321 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4381 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4441 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4501 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4561 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4621 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4681 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4741 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4801 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4861 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     4921 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataaa ttccgacacc
     4981 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     5041 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     5101 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     5161 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     5221 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     5281 tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg
     5341 tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg
     5401 caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg
     5461 gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc
     5521 aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca
     5581 ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg
     5641 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
     5701 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
     5761 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
     5821 attaccgagt ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc
     5881 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
     5941 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
     6001 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
     6061 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     6121 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacccca
     6181 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     6241 tcacacagga aacagctatg accatgatta cggattcact ggccgtcgtt ttacaacgtc
     6301 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     6361 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     6421 tgaatggcga atggcgcttt gcctggtttc cggcaccaga agcggtgccg gaaagctggc
     6481 tggagtgcga tcttcctgag gccgatactg tcgtcgtccc ctcaaactgg cagatgcacg
     6541 gttacgatgc gcccatctac accaacgtga cctatcccat tacggtcaat ccgccgtttg
     6601 ttcccacgga gaatccgacg ggttgttact cgctcacatt taatgttgat gaaagctggc
     6661 tacaggaagg ccagacgcga attatttttg atggcgttgg aattag
//
