LOCUS       sequence-374453-        7493 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for the expression and purification of 6xHis-TEV-mCh-NDP52 
            in E. coli Rosetta pLysS. Internal Reference: SMC1148.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7493)
  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 7493)
  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: pETDuet-1_6xHis-TEV-mCh-NDP52
FEATURES             Location/Qualifiers
     source          1..7493
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             35..57
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             76..93
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             106..126
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             127..831
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(269..287)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     537..556
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        2283..2301
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2302..2326
                     /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             2435..2479
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(2517..2535)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2531..2578
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2615..3070
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2702..2721)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2912..2933
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3185..3976,3977..4045))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3808..3827
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4046..4138)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4219..4807
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4708..4727
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4961..4978
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4993..5132
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5058..5077)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5218..5240
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5234..5425)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5997..7079)
                     /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     7041..7060
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7080..7157)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7363..7382
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7470..7488
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    join(7489..7493,1..20)
                     /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)."
ORIGIN
        1 tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat
       61 accatgggca gcagccatca ccatcatcac cacagccagg atccggaaaa cctgtatttt
      121 cagggagtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc
      181 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
      241 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg
      301 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag
      361 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
      421 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
      481 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
      541 gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta ccccgaggac
      601 ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac
      661 gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac
      721 gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
      781 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gtccggactc
      841 agatctcgag ctcaagcttc gaattctgca gtcgacatgg aggagaccat caaagatccc
      901 cccacatcag ctgtcttgct ggatcactgt catttctctc aggtcatctt taacagtgtg
      961 gagaagttct acatccctgg aggggacgtc acatgtcatt ataccttcac ccagcatttc
     1021 atccctcgtc gaaaggattg gattggcatc tttagagtgg ggtggaagac aacccgtgag
     1081 tattacacct tcatgtgggt tactttgccc attgacctaa acaacaaatc agctaaacag
     1141 caggaagtcc aattcaaagc ttactacctg cccaaggatg atgagtatta ccagttctgc
     1201 tatgtggatg aggatggtgt ggtccgggga gcaagtattc ctttccaatt ccgtccagaa
     1261 aatgaggaag acatcctggt tgttaccact cagggagagg tggaagagat tgagcagcac
     1321 aacaaggagc tttgcaaaga aaaccaggag ctgaaggaca gctgtatcag cctccagaag
     1381 cagaactcag acatgcaggc tgagctccaa aagaagcagg aggagctaga aaccctacag
     1441 agcatcaata agaagttgga actgaaagtg aaagaacaga aggactattg ggagacagag
     1501 ctgcttcaac tgaaagaaca aaaccagaag atgtcctcag aaaatgagaa gatgggaatc
     1561 agagtggatc agcttcaggc ccagctgtca actcaagaga aagaaatgga gaagcttgtt
     1621 cagggagatc aagataagac agagcagtta gagcagctga aaaaggaaaa tgaccacctc
     1681 tttctcagtt taactgaaca gaggaaggac cagaagaagc tcgagcagac agtggagcaa
     1741 atgaagcaga atgaaactac tgcaatgaag aaacaacagg aattaatgga tgaaaacttt
     1801 gacctgtcaa aaagactgag tgagaacgaa attatatgta atgctctgca gagacagaaa
     1861 gagagattgg aaggagaaaa tgatcttttg aagagggaga acagcagatt gctcagttac
     1921 atgggtctgg attttaattc tttgccgtat caagtaccta cttcagatga aggaggcgca
     1981 agacaaaatc caggacttgc ctatggaaac ccatattctg gtatccaaga aagttcttcc
     2041 gccagcccgc tctccatcaa gaaatgccct atctgcaaag cagatgatat ttgtgatcac
     2101 accttggagc aacagcagat gcagcccctt tgtttcaatt gtccaatttg tgacaagatc
     2161 ttcccagcta cagagaagca gatctttgaa gaccacgtgt tctgccactc tctctgagcg
     2221 gccgcataat gcttaagtcg aacagaaagt aatcgtattg tacacggccg cataatcgaa
     2281 attaatacga ctcactatag gggaattgtg agcggataac aattccccat cttagtatat
     2341 tagttaagta taagaaggag atatacatat ggcagatctc aattggatat cggccggcca
     2401 cgcgatcgct gacgtcggta ccctcgagtc tggtaaagaa accgctgctg cgaaatttga
     2461 acgccagcac atggactcgt ctactagcgc agcttaatta acctaggctg ctgccaccgc
     2521 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     2581 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     2641 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2701 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2761 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2821 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2881 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2941 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     3001 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     3061 tttacaattt ctggcggcac gatggcatga gattatcaaa aaggatcttc acctagatcc
     3121 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3181 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3241 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3301 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3361 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3421 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3481 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3541 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3601 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3661 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3721 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3781 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3841 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3901 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3961 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4021 cgacacggaa atgttgaata ctcatactct tcctttttca atcatgattg aagcatttat
     4081 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4141 ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     4201 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     4261 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4321 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     4381 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4441 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4501 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4561 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4621 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4681 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4741 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4801 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4861 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4921 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4981 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     5041 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     5101 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     5161 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     5221 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     5281 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     5341 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     5401 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     5461 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     5521 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     5581 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     5641 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     5701 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     5761 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     5821 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     5881 tgctagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5941 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     6001 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6061 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     6121 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     6181 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     6241 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     6301 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     6361 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     6421 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     6481 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     6541 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     6601 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     6661 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     6721 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     6781 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     6841 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     6901 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6961 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     7021 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     7081 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     7141 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     7201 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     7261 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     7321 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     7381 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     7441 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actatagggg aat
//
