LOCUS       sequence-448199-        6631 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 6631)
  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 6631)
  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_delta206-230
            (191-2...
FEATURES             Location/Qualifiers
     source          1..6631
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(180..185,186..201,202..208)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    216..232
                     /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             255..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MASPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
                     KFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIR
                     YGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVV
                     LYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             939..959
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(966..968,969..971,972..1682)
                     /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(1011..1032)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1272..1291)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1619..1640
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2678..2700)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2845..2863)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2931..3035
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3036..3104,3105..3896)
                     /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(3254..3273)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4067..4655
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4556..4575
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4809..4826
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4899..4976
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4977..6059
                     /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(4996..5015)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6108..6113,6114..6131,6132..6138)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6146..6162
                     /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     6170..6186
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6170..6186
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6182..6631,1..78)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTVRCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6202..6219)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6202..6218)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6211..6233)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttatcgactg tacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta
       61 tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct
      121 ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt
      181 tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac
      241 aggaaacagt attcatggca tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatgat tacgatatcc caacgaccga aaacctgtat tttcagggtg
      961 gtaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1021 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1081 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1141 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     1201 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1261 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1321 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1381 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1441 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1501 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1561 accactacct gagcacccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     1621 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1681 agggtaccgg tggtatcaat ctggccttca tgtcaaccag acagtttgag cgtactccct
     1741 atcgtacagc cggtgaagac accggcgtga tctaccccag tgtcgaggat agccaggaag
     1801 tgtgtaccac ttccttctct acctcaccac cctcacagct ctcttcaagt cgcctgagtt
     1861 atcagcctgc cgctctcgga gtcggtagcg cagacctggc ctacccagtg gtcttcgcag
     1921 caggcctcaa cgcaacccac ccacatcagc tgatggtgcc aggaaaggag ggtggagtcc
     1981 cactggctcc taatcagcca gtgcacggta ctcaggctga ccaggaaagg ctggcaactt
     2041 gcacaccatc cgatagaact cattgtgctg caacacccag ctcctctgag gacaccgaaa
     2101 ctgtgagcaa ctcaagtgag ggtcgcgcct ccccacacga tgtcctggaa accatcttcg
     2161 tgagaaaagt cggcgctttt gtgaacaagc ccattaatca ggtcacactc acctctctgg
     2221 acatcccttt cgctatgttt gcaccaaaga acctcgagct ggaagacact gatcccatgg
     2281 tgaatcctcc agactcaccc gagacagaaa gtcctctcca gggcagtctg cacagcgatg
     2341 gaagctccgg tggctcttca ggcaacacac atgacgattt cgtcatgatt gacttcaaac
     2401 ccgctttttc caaggacgat atcctcccta tggatctggg aaccttctac agggagtttc
     2461 agaatccccc tcagctcagt agcctgagca ttgacatcgg tgcccagtcc atggctgagg
     2521 acctcgattc tctgcctgaa aaactggctg tgcacgagaa gaacgtcaga gaattcgacg
     2581 catttgtgga gaccctgcag tgacccgggt gactcgagcg gccgcatcgt gactgactga
     2641 cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     2701 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     2761 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     2821 tgtataattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     2881 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     2941 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3001 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3061 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3121 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3181 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     3241 cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac
     3301 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     3361 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     3421 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     3481 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     3541 aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc
     3601 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     3661 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     3721 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     3781 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     3841 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     3901 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3961 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4021 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4081 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4141 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4201 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4261 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4321 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4381 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4441 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4501 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4561 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4621 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4681 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4741 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4801 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4861 ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa
     4921 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     4981 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     5041 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     5101 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     5161 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     5221 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     5281 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     5341 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     5401 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     5461 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     5521 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     5581 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     5641 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     5701 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     5761 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     5821 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     5881 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     5941 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     6001 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     6061 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cccaggcttt acactttatg
     6121 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     6181 tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     6241 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     6301 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     6361 ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc
     6421 tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat
     6481 ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc
     6541 gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac
     6601 gcgaattatt tttgatggcg ttggaattag c
//
