LOCUS       sequence-374455-        6296 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the expression and purification of GST-TEV-NDP52 in E. 
            coli Rosetta pLysS. Internal reference: SMC1586.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6296)
  AUTHORS   Nguyen TN, Sawa-Makarska J, Khuu G, Lam WK, Adriaenssens E, 
            Fracchiolla D, Shoebridge S, Bernklau D, Padman BS, Skulsuppaisarn 
            M, Lindblom RSJ, Martens S, Lazarou M
  TITLE     Unconventional initiation of PINK1/Parkin mitophagy by Optineurin.
  JOURNAL   Mol Cell. 2023 May 18;83(10):1693-1709.e9. doi: 
            10.1016/j.molcel.2023.04.021.
  PUBMED    37207627
REFERENCE   2  (bases 1 to 6296)
  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"; doi: 
            "10.1016/j.molcel.2023.04"; journalName: "Mol Cell"; date: 
            "2023-05-18- 18"; volume: "83"; issue: "10"; pages: "1693-1709.e9"
COMMENT     Sequence Label: pGST2-GST-TEV-NDP52
FEATURES             Location/Qualifiers
     source          1..6296
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..938
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(2346..2368)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2513..2531)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2599..2703
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2704..2772,2773..3564)
                     /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(2922..2941)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3735..4323
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4224..4243
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4477..4494
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4567..4644
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4645..5727
                     /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="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVKAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTXLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4664..4683)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5776..5781,5782..5799,5800..5806)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5814..5830
                     /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     5838..5854
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5838..5854
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5850..6296,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5870..5887)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5870..5886)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5879..5901)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg 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 aatcgatgaa aacctgtatt ttcagggatc aggcatggag gagaccatca
      961 aagatccccc cacatcagct gtcttgctgg atcactgtca tttctctcag gtcatcttta
     1021 acagtgtgga gaagttctac atccctggag gggacgtcac atgtcattat accttcaccc
     1081 agcatttcat ccctcgtcga aaggattgga ttggcatctt tagagtgggg tggaagacaa
     1141 cccgtgagta ttacaccttc atgtgggtta ctttgcccat tgacctaaac aacaaatcag
     1201 ctaaacagca ggaagtccaa ttcaaagctt actacctgcc caaggatgat gagtattacc
     1261 agttctgcta tgtggatgag gatggtgtgg tccggggagc aagtattcct ttccaattcc
     1321 gtccagaaaa tgaggaagac atcctggttg ttaccactca gggagaggtg gaagagattg
     1381 agcagcacaa caaggagctt tgcaaagaaa accaggagct gaaggacagc tgtatcagcc
     1441 tccagaagca gaactcagac atgcaggctg agctccaaaa gaagcaggag gagctagaaa
     1501 ccctacagag catcaataag aagttggaac tgaaagtgaa agaacagaag gactattggg
     1561 agacagagct gcttcaactg aaagaacaaa accagaagat gtcctcagaa aatgagaaga
     1621 tgggaatcag agtggatcag cttcaggccc agctgtcaac tcaagagaaa gaaatggaga
     1681 agcttgttca gggagatcaa gataagacag agcagttaga gcagctgaaa aaggaaaatg
     1741 accacctctt tctcagttta actgaacaga ggaaggacca gaagaagctc gagcagacag
     1801 tggagcaaat gaagcagaat gaaactactg caatgaagaa acaacaggaa ttaatggatg
     1861 aaaactttga cctgtcaaaa agactgagtg agaacgaaat tatatgtaat gctctgcaga
     1921 gacagaaaga gagattggaa ggagaaaatg atcttttgaa gagggagaac agcagattgc
     1981 tcagttacat gggtctggat tttaattctt tgccgtatca agtacctact tcagatgaag
     2041 gaggcgcaag acaaaatcca ggacttgcct atggaaaccc atattctggt atccaagaaa
     2101 gttcttcccc cagcccgctc tccatcaaga aatgccctat ctgcaaagca gatgatattt
     2161 gtgatcacac cttggagcaa cagcagatgc agcccctttg tttcaattgt ccaatttgtg
     2221 acaagatctt cccagctaca gagaagcaga tctttgaaga ccacgtgttc tgccactctc
     2281 tttaagcggc cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt
     2341 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     2401 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     2461 atgacccagt cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg
     2521 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     2581 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2641 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2701 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2761 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2821 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2881 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2941 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     3001 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     3061 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     3121 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     3181 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     3241 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     3301 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     3361 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     3421 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     3481 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     3541 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     3601 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3661 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3721 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3781 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3841 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3901 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3961 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4021 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4081 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4141 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     4201 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     4261 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     4321 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     4381 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     4441 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     4501 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     4561 aattccgaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     4621 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     4681 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     4741 aaaacgcggg aaaaagtgaa agcggcgatg gcggagctga attacattcc caaccgcgtg
     4801 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     4861 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     4921 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     4981 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     5041 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     5101 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     5161 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     5221 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     5281 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     5341 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     5401 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     5461 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     5521 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     5581 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccmc cctggcgccc
     5641 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     5701 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     5761 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     5821 cggataacaa tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg
     5881 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     5941 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     6001 agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc
     6061 cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact
     6121 ggcagatgca cggttacgat gcgcccatct acaccaacgt gacctatccc attacggtca
     6181 atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg
     6241 atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaatt
//
