LOCUS       sequence-448329-        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-TEV-EGFP-ATG13_IDR_191-517aa_(M196A_S197A_R199A)
FEATURES             Location/Qualifiers
     source          1..6706
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..528
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(21..38)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(21..37)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(30..52)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(630..635,636..651,652..658)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    666..682
                     /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             705..1361
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1319..1341
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             1389..1409
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1416..1418,1419..1421,1422..2132)
                     /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(1461..1482)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1722..1741)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2069..2090
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3203..3225)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3370..3388)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3456..3560
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3561..3629,3630..4421)
                     /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(3779..3798)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4592..5180
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5081..5100
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5334..5351
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5424..5501
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5502..6584
                     /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(5521..5540)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6633..6638,6639..6656,6657..6663)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6671..6687
                     /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     join(6695..6706,1..5)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(6695..6706,1..5)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
       61 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
      121 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
      181 tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct
      241 gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga tgcgcccatc
      301 tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg
      361 acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg
      421 cgaattattt ttgatggcgt tggaattagc ttatcgactg cacggtgcac caatgcttct
      481 ggcgtcaggc agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa
      541 ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac
      601 ggttctggca aatattctga aatgagctgt tgacaattaa tcatcggctc gtataatgtg
      661 tggaattgtg agcggataac aatttcacac aggaaacagt attcatggca tcccctatac
      721 taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg gaatatcttg
      781 aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg cgaaacaaaa
      841 agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt gatgttaaat
      901 taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg ttgggtggtt
      961 gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat attagatacg
     1021 gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat tttcttagca
     1081 agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca tatttaaatg
     1141 gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt gttttataca
     1201 tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa cgtattgaag
     1261 ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg cctttgcagg
     1321 gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatgat tacgatatcc
     1381 caacgaccga aaacctgtat tttcagggtg gtaccatggt gagcaagggc gaggagctgt
     1441 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1501 gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct
     1561 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
     1621 tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     1681 tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
     1741 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     1801 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc
     1861 acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc
     1921 gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     1981 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga
     2041 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2101 ggatcactct cggcatggac gagctgtaca agggtaccgg tggtatcaat ctggccttcg
     2161 cggcaaccgc acagtttgag cgtactcccc ctattatggg aatcattatc gaccacttcg
     2221 tggatagacc ataccccagt agctccccta tgcatccatg caactatcgt acagccggtg
     2281 aagacaccgg cgtgatctac cccagtgtcg aggatagcca ggaagtgtgt accacttcct
     2341 tctctacctc accaccctca cagctctctt caagtcgcct gagttatcag cctgccgctc
     2401 tcggagtcgg tagcgcagac ctggcctacc cagtggtctt cgcagcaggc ctcaacgcaa
     2461 cccacccaca tcagctgatg gtgccaggaa aggagggtgg agtcccactg gctcctaatc
     2521 agccagtgca cggtactcag gctgaccagg aaaggctggc aacttgcaca ccatccgata
     2581 gaactcattg tgctgcaaca cccagctcct ctgaggacac cgaaactgtg agcaactcaa
     2641 gtgagggtcg cgcctcccca cacgatgtcc tggaaaccat cttcgtgaga aaagtcggcg
     2701 cttttgtgaa caagcccatt aatcaggtca cactcacctc tctggacatc cctttcgcta
     2761 tgtttgcacc aaagaacctc gagctggaag acactgatcc catggtgaat cctccagact
     2821 cacccgagac agaaagtcct ctccagggca gtctgcacag cgatggaagc tccggtggct
     2881 cttcaggcaa cacacatgac gatttcgtca tgattgactt caaacccgct ttttccaagg
     2941 acgatatcct ccctatggat ctgggaacct tctacaggga gtttcagaat ccccctcagc
     3001 tcagtagcct gagcattgac atcggtgccc agtccatggc tgaggacctc gattctctgc
     3061 ctgaaaaact ggctgtgcac gagaagaacg tcagagaatt cgacgcattt gtggagaccc
     3121 tgcagtgacc cgggtgactc gagcggccgc atcgtgactg actgacgatc tgcctcgcgc
     3181 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     3241 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     3301 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat aattcttgaa
     3361 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     3421 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3481 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3541 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     3601 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     3661 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     3721 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     3781 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     3841 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     3901 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     3961 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     4021 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     4081 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     4141 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     4201 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     4261 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     4321 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     4381 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     4441 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     4501 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     4561 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     4621 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     4681 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     4741 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     4801 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     4861 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     4921 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     4981 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     5041 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     5101 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     5161 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     5221 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     5281 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     5341 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     5401 gtatttcaca ccgcataaat tccgacacca tcgaatggcg caaaaccttt cgcggtatgg
     5461 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     5521 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     5581 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     5641 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     5701 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     5761 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     5821 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     5881 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     5941 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     6001 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     6061 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     6121 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     6181 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     6241 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     6301 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     6361 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     6421 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     6481 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     6541 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     6601 gtaagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     6661 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagct
//
