LOCUS       sequence-448231-        5800 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 5800)
  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 5800)
  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: pGEX-4T1_GST-EGFP-ATG13_IDR (206-230aa)
FEATURES             Location/Qualifiers
     source          1..5800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(179..184,185..200,201..207)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    215..231
                     /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             254..910
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     868..890
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             938..958
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(965..967,968..970,971..1681)
                     /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(1010..1031)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1271..1290)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1618..1639
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1846..1868)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2013..2031)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2099..2203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2204..2272,2273..3064)
                     /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(2422..2441)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3235..3823
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3724..3743
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3977..3994
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4067..4144
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4145..5227
                     /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(4164..4183)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5276..5281,5282..5299,5300..5306)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5314..5330
                     /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     5338..5354
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5338..5354
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5350..5800,1..77)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTVRCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5370..5387)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5370..5386)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5379..5401)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tatcgactgt acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat
       61 ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg
      121 gataatgttt tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt
      181 gacaattaat catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca
      241 ggaaacagta ttcatggcat cccctatact aggttattgg aaaattaagg gccttgtgca
      301 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
      361 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
      421 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
      481 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
      541 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
      601 tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga
      661 tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt
      721 gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa
      781 attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc
      841 cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca
      901 tcctccaaaa tcggatgatt acgatatccc aacgaccgaa aacctgtatt ttcagggtgg
      961 taccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1021 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1081 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1141 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     1201 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1261 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1321 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1381 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1441 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1501 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1561 ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat
     1621 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1681 gggtaccggt ggtcctatta tgggaatcat tatcgaccac ttcgtggata gaccataccc
     1741 cagtagctcc cctatgcatc catgcaactg acccgggtga ctcgagcggc cgcatcgtga
     1801 ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca
     1861 gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
     1921 gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga
     1981 tagcggagtg tataattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt
     2041 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     2101 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2161 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     2221 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2281 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2341 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2401 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca
     2461 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2521 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2581 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2641 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2701 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     2761 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2821 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2881 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     2941 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3001 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3061 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     3121 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     3181 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     3241 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     3301 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     3361 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     3421 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     3481 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     3541 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     3601 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     3661 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     3721 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3781 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3841 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3901 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3961 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     4021 ttttctcctt acgcatctgt gcggtatttc acaccgcata aattccgaca ccatcgaatg
     4081 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     4141 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     4201 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     4261 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     4321 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     4381 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     4441 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     4501 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     4561 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     4621 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     4681 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     4741 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     4801 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     4861 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     4921 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     4981 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     5041 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     5101 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     5161 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     5221 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccc caggctttac
     5281 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     5341 gaaacagcta tgaccatgat tacggattca ctggccgtcg ttttacaacg tcgtgactgg
     5401 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     5461 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     5521 gaatggcgct ttgcctggtt tccggcacca gaagcggtgc cggaaagctg gctggagtgc
     5581 gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat
     5641 gcgcccatct acaccaacgt gacctatccc attacggtca atccgccgtt tgttcccacg
     5701 gagaatccga cgggttgtta ctcgctcaca tttaatgttg atgaaagctg gctacaggaa
     5761 ggccagacgc gaattatttt tgatggcgtt ggaattagct
//
