LOCUS       sequence-391292-        7420 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for the expression and purification of mCherry NAP1 in a 
            mammalian expression system (SMC Internal No. 2065).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7420)
  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 7420)
  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-mCherry-NAP1 (WT)
FEATURES             Location/Qualifiers
     source          1..7420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        75..454
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          734..1750
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1674..1696
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1758..1777
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1816..2469
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2427..2449
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2479..2499
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2500..3207
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2645..2663)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2913..2932
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4512..4531)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4577..4632
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4631..4650)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4993..5009)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4993..5009)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5006..5028)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5017..5033
                     /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(5041..5047,5048..5065,5066..5071))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5086..5107
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5166..5361
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5212..5347
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5367..5501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5404..5423)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5458..5477
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5569..5586)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5740..6328)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5820..5839)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6499..7290,7291..7359))
                     /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     7122..7141
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7360..7420,1..44))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
       61 gtgccacctg ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      121 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      181 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      241 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      301 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      361 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      421 gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt
      481 cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt
      541 attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccggggg gggggggggg
      601 gggggggggg gggggggggg sgrggsggrg rggkgcggcg gcagccaatc agagcggcgc
      661 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
      721 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
      781 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
      841 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct
      901 gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc
      961 ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg
     1021 cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg
     1081 gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg
     1141 cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca
     1201 accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct
     1261 ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt
     1321 gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc
     1381 ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc
     1441 gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag
     1501 gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga
     1561 aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc
     1621 tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg
     1681 cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt
     1741 tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga
     1801 attcctcgag ccaccatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
     1861 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
     1921 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
     1981 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
     2041 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
     2101 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
     2161 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
     2221 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
     2281 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
     2341 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
     2401 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
     2461 cctccaaaat cggatctgga aaacctgtat tttcagggca tggtgagcaa gggcgaggag
     2521 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     2581 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     2641 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     2701 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     2761 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     2821 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     2881 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     2941 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     3001 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     3061 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     3121 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     3181 accggcggca tggacgagct gtacaagggc ggcggcggca tggatgcact ggtagaagat
     3241 gatatctgta ttctgaatca tgaaaaagcc cataagagag atacagtgac tccagtttca
     3301 atatattcag gagatgaatc tgttgcttcc cattttgctc ttgtcactgc atatgaagac
     3361 atcaaaaaac gacttaagga ttcagagaaa gagaactctt tgttaaagaa gagaataaga
     3421 tttttggaag aaaagctaat agctcgattt gaagaagaaa caagttccgt gggacgagaa
     3481 caagtaaata aggcctatca tgcatatcga gaggtttgca ttgatagaga taatttgaag
     3541 agcaaactgg acaaaatgaa taaagacaac tctgaatctt tgaaagtatt gaatgagcag
     3601 ctacaatcta aagaagtaga actcctccag ctgaggacag aggtggaaac tcagcaggtg
     3661 atgaggaatt taaatccacc ttcatcaaac tgggaggtgg aaaagttgag ctgtgacctg
     3721 aagatccatg gtttggaaca agagctggaa ctgatgagga aagaatgtag cgatctcaaa
     3781 atagaactac agaaagccaa acaaacggat ccatatcagg aagacaatct gaagagcaga
     3841 gatctccaaa aactaagcat ttcaagtgat aatatgcagc atgcatactg ggaactgaag
     3901 agagaaatgt ctaatttaca tctggtgact caagtacaag ctgaactact aagaaaactg
     3961 aaaacctcaa ctgcaatcaa gaaagcctgt gcccctgtag gatgcagtga agaccttgga
     4021 agagacagca caaaactgca cttgatgaat tttactgcaa catacacaag acatccccct
     4081 ctcttaccaa atggcaaagc tctttgtcat accacatctt cccctttacc aggagatgta
     4141 aaggttttat cagagaaagc aatcctccaa tcatggacag acaatgagag atccattcct
     4201 aatgatggta catgctttca ggaacacagt tcttatggca gaaattctct ggaagacaat
     4261 tcctgggtat ttccaagtcc tcctaaatca agtgagacag catttgggga aactaaaact
     4321 aaaactttgc ctttacccaa ccttccacca ctgcattact tggatcaaca taatcagaac
     4381 tgcctttata agaattaatg acgccattaa cctgatgttc tggggcaagc ttactagtcc
     4441 tcagtgcagg ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa
     4501 taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     4561 catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt
     4621 ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag
     4681 tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg
     4741 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga
     4801 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta
     4861 acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta
     4921 ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt
     4981 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     5041 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     5101 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg
     5161 gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     5221 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     5281 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     5341 ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat
     5401 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5461 gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg
     5521 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5581 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5641 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5701 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5761 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5821 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5881 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5941 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6001 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6061 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6121 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6181 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6241 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6301 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6361 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6421 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6481 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6541 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6601 gggcttacca tctggcccca gtgctgcaat gatacctcga gagccacgct caccggctcc
     6661 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6721 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6781 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6841 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6901 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6961 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7021 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7081 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7141 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7201 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7261 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7321 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7381 ttgaagcatt tatcagggtt attgtctcat gagcggatac
//
