LOCUS       sequence-391294-        6730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for the expression and purification of NAP1 in a mammalian 
            expression system (SMC Internal No. 2064).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6730)
  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 6730)
  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: pCAGG-GST-TEV-NAP1 (WT)
FEATURES             Location/Qualifiers
     source          1..6730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          277..1293
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1217..1239
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1301..1320
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1359..2012
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1970..1992
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2019..2036
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2046..2066
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3365..3384)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3430..3485
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3484..3503)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3846..3862)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3846..3862)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3859..3881)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3870..3886
                     /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)."
     promoter        complement(join(3894..3900,3901..3918,3919..3924))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3939..3960
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4019..4214
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4065..4200
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4220..4354
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4257..4276)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4311..4330
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4422..4439)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4593..5181)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4673..4692)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5352..6143,6144..6212))
                     /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     5975..5994
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6213..6317)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6348..6727
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
       61 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      121 cgcgcgccrg gggggggggg gggggggggg gggggggggg gggsgrggsg grgrggkgcg
      181 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      241 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      301 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      361 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt
      421 taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc
      481 cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc
      541 gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt
      601 gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg
      661 ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt
      721 gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt tgctgagcac
      781 ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc
      841 ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc
      901 tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc
      961 agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc
     1021 tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag
     1081 cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc
     1141 cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg
     1201 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
     1261 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg
     1321 ctgtctcatc attttggcaa agaattcctc gagccaccat gtcccctata ctaggttatt
     1381 ggaaaattaa gggccttgtg caacccactc gacttctttt ggaatatctt gaagaaaaat
     1441 atgaagagca tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat
     1501 tgggtttgga gtttcccaat cttccttatt atattgatgg tgatgttaaa ttaacacagt
     1561 ctatggccat catacgttat atagctgaca agcacaacat gttgggtggt tgtccaaaag
     1621 agcgtgcaga gatttcaatg cttgaaggag cggttttgga tattagatac ggtgtttcga
     1681 gaattgcata tagtaaagac tttgaaactc tcaaagttga ttttcttagc aagctacctg
     1741 aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac atatttaaat ggtgatcatg
     1801 taacccatcc tgacttcatg ttgtatgacg ctcttgatgt tgttttatac atggacccaa
     1861 tgtgcctgga tgcgttccca aaattagttt gttttaaaaa acgtattgaa gctatcccac
     1921 aaattgataa gtacttgaaa tccagcaagt atatagcatg gcctttgcag ggctggcaag
     1981 ccacgtttgg tggtggcgac catcctccaa aatcggatct ggttccgcgt ggatccccgg
     2041 aattcgaaaa cctgtatttt cagggaggag gaatggatgc actggtagaa gatgatatct
     2101 gtattctgaa tcatgaaaaa gcccataaga gagatacagt gactccagtt tcaatatatt
     2161 caggagatga atctgttgct tcccattttg ctcttgtcac tgcatatgaa gacatcaaaa
     2221 aacgacttaa ggattcagag aaagagaact ctttgttaaa gaagagaata agatttttgg
     2281 aagaaaagct aatagctcga tttgaagaag aaacaagttc cgtgggacga gaacaagtaa
     2341 ataaggccta tcatgcatat cgagaggttt gcattgatag agataatttg aagagcaaac
     2401 tggacaaaat gaataaagac aactctgaat ctttgaaagt attgaatgag cagctacaat
     2461 ctaaagaagt agaactcctc cagctgagga cagaggtgga aactcagcag gtgatgagga
     2521 atttaaatcc accttcatca aactgggagg tggaaaagtt gagctgtgac ctgaagatcc
     2581 atggtttgga acaagagctg gaactgatga ggaaagaatg tagcgatctc aaaatagaac
     2641 tacagaaagc caaacaaacg gatccatatc aggaagacaa tctgaagagc agagatctcc
     2701 aaaaactaag catttcaagt gataatatgc agcatgcata ctgggaactg aagagagaaa
     2761 tgtctaattt acatctggtg actcaagtac aagctgaact actaagaaaa ctgaaaacct
     2821 caactgcaat caagaaagcc tgtgcccctg taggatgcag tgaagacctt ggaagagaca
     2881 gcacaaaact gcacttgatg aattttactg caacatacac aagacatccc cctctcttac
     2941 caaatggcaa agctctttgt cataccacat cttccccttt accaggagat gtaaaggttt
     3001 tatcagagaa agcaatcctc caatcatgga cagacaatga gagatccatt cctaatgatg
     3061 gtacatgctt tcaggaacac agttcttatg gcagaaattc tctggaagac aattcctggg
     3121 tatttccaag tcctcctaaa tcaagtgaga cagcatttgg ggaaactaaa actaaaactt
     3181 tgcctttacc caaccttcca ccactgcatt acttggatca acataatcag aactgccttt
     3241 ataagaatta atgacgccat taacctgatg ttctggggca agcttactag tcctcagtgc
     3301 aggctgccta tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact
     3361 gagatctttt tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga
     3421 cttctggcta ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc
     3481 tctcactcgg aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg
     3541 tttagagttt ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa
     3601 gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct
     3661 tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc
     3721 taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag
     3781 tcatagctgt ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa
     3841 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3901 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3961 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc
     4021 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4081 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4141 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4201 taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa
     4261 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4321 caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc
     4381 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     4441 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     4501 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4561 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4621 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4681 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4741 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4801 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4861 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4921 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4981 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5041 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5101 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5161 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5221 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5281 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     5341 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     5401 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     5461 ccatctggcc ccagtgctgc aatgatacct cgagagccac gctcaccggc tccagattta
     5521 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5581 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5641 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5701 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5761 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5821 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5881 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5941 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     6001 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     6061 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     6121 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     6181 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     6241 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     6301 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgggtcgac attgattatt
     6361 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     6421 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     6481 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     6541 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     6601 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     6661 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     6721 taccatggtc
//
