LOCUS       sequence-448406-        6436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stability reporter construct of the COX8A mitochondrial targeting 
            sequence (MTS) for transient expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6436)
  AUTHORS   Haakonsen DL, Heider M, Ingersoll AJ, Vodehnal K, Witus SR, Uenaka 
            T, Wernig M, Rape M
  TITLE     Stress response silencing by an E3 ligase mutated in 
            neurodegeneration.
  JOURNAL   Nature. 2024 Feb;626(8000):874-880. doi: 10.1038/s41586-023-06985-7.
            Epub 2024 Jan 31.
  PUBMED    38297121
REFERENCE   2  (bases 1 to 6436)
  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: 
            "10.1038/s41586-023-06985-7"; journalName: "Nature"; date: 
            "2024-02"; volume: "626"; issue: "8000"; pages: "874-880"
COMMENT     Sequence Label: pcs2-MTS-cox8A-GFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      94..549
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(181..200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     391..412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        575..679
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(680..748,749..1540)
                     /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     complement(898..917)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1711..2299
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2200..2219
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2453..2470
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2587..2608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2623..2628,2629..2646,2647..2653)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2661..2677
                     /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     2666..2688
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2685..2701
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2685..2701
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2720..2740
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2722..2740
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    2853..2987
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2890..2909)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2944..2963
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     gap             3109..3208
                     /estimated_length=100
     primer_bind     3663..3681
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    3835..4407
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3994..4013)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     4223..4240
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(4415..5134)
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     CDS             complement(5183..5257)
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     promoter        complement(5295..5313)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5296..5313)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(5348..6332)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 gccatattga attggcggtc gactggcgta atagcgaaga ggcccgcacc gatcgccctt
       61 cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg
      121 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      181 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      241 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      301 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
      361 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
      421 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
      481 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
      541 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
      601 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
      661 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
      721 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
      781 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
      841 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
      901 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
      961 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     1021 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     1081 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     1141 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     1201 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     1261 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     1321 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     1381 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     1441 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     1501 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     1561 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     1621 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1681 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1741 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1801 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     1861 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1921 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1981 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2041 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2101 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     2161 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2221 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     2281 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2341 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2401 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2461 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     2521 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     2581 cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgctccc
     2641 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
     2701 ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tgggtaccgg
     2761 gcccaatgca ttggcgccgc ggccgcgaat taaaaaacct cccacacctc cccctgaacc
     2821 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     2881 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2941 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctac gtaatacgac
     3001 tcactatagt tctagatcac ttgtacagct cgtccatgcc gccggtggag tggcggccct
     3061 cggcgcgttc gtactgttcc acgatggtgt agtcctcgtt gtgggaggnn nnnnnnnnnn
     3121 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
     3181 nnnnnnnnnn nnnnnnnnnn nnnnnnnnga tgtccaactt gatgttgacg ttgtaggcgc
     3241 cgggcagctg cacgggcttc ttggccttgt aggtggtctt gacctcagcg tcgtagtggc
     3301 cgccgtcctt cagcttcagc ctctgcttga tctcgccctt cagggcgccg tcctcggggt
     3361 acatccgctc ggaggaggcc tcccagccca tggttttctt ctgcattacg gggccgtcgg
     3421 aggggaagtt ggtgccgcgc agcttcacct tgtagatgaa ctcgccgtcc tgcagggagg
     3481 agtcctgggt cacggtcacc acgccgccgt cctcgaagtt catcacgcgc tcccacttga
     3541 agccctcggg gaaggacagc ttcaagtagt cggggatgtc ggcggggtgc ttcacgtagg
     3601 ccttggagcc gtacatgaac tgaggggaca ggatgtccca ggcgaagggc agggggccac
     3661 ccttggtcac cttcagcttg gcggtctggg tgccctcgta ggggcggccc tcgccctcgc
     3721 cctcgatctc gaactcgtgg ccgttcacgg agccctccat gtgcaccttg aagcgcatga
     3781 actccttgat gatggccatg ttatcctcct cgcccttgct caccattccg gaggtattat
     3841 cgtgtttttc aaaggaaaac cacgtccccg tggttcgggg ggcctagacg tttttttaac
     3901 ctcgactaaa cacatgtaaa gcatgtgcac cgaggcccca gatcagatcc catacaatgg
     3961 ggtaccttct gggcatcctt cagccccttg ttgaatacgc ttgaggagag ccatttgact
     4021 ctttccacaa ctatccaact cacaacgtgg cactggggtt gtgccgcctt tgcaggtgta
     4081 tcttatacac gtggcttttg gccgcagagg cacctgtcgc caggtggggg gttccgctgc
     4141 ctgcaaaggg tcgctacaga cgttgtttgt cttcaagaag cttccagagg aactgcttcc
     4201 ttcacgacat tcaacagacc ttgcattcct ttggcgagag gggaaagacc cctaggaatg
     4261 ctcgtcaaga agacagggcc aggtttccgg gccctcacat tgccaaaaga cggcaatatg
     4321 gtggaaaata acatatagac aaacgcacac cggccttatt ccaagcggct tcggccagta
     4381 acgttagggg ggggggaggg agaggggctc tagatcactt gtacagctca tccatgccgt
     4441 gggtgatggc ggcggcggtc acgaagccga agtagatcat gtgatcgcgc ttctcgttgg
     4501 ggtccttgga cagggcgctc tgggtggaca ggtagtggtt atcgggcagc agcacagggc
     4561 catcgccgat gggggtattc tgctggtagt ggtcggccag ctgcacgctg ccatcctcga
     4621 tgttgtggcg gatcttgaag ttcaccttga tgccattctt ggccttgtcg gtcatgatgt
     4681 acacattgtg ggcgttgtag ttgtactcca tcttattgcc caggatgttg ccatcctcct
     4741 tgaaatcggt gccggtcagc tcgatgcgat tcaccagggt atcgccctcg aacttcacct
     4801 cggcgcgcga cttgtagttg ccgtcatcct cgaagaagat ggtgcgctcc tggatgtagc
     4861 cctcaggcat ggcgctcttg aagaagtcgt gctgcttcat gtgatcgggg tagcgtgaga
     4921 agcactgcac gccgtagctc agggtggtca ccagggtggg ccagggcaca ggcagcttgc
     4981 cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc
     5041 cgctcacgct gaacttgtgg ccattcacat cgccattcag ctcgatcagg atgggcacga
     5101 tgccggtgaa cagctcggcg cccttgctca ccatgctgcc tccgcctccc ccaagcttcc
     5161 cctccggcgg caacgaatgg atcttggcgc gcggcactgg gagccgccgg gccgagcctg
     5221 tcaagccccg cagcagcagc ggcgtcagga cggacatggt ggcgggatcc gcaaaaagaa
     5281 caagtagctt gtattctata gtgtcaccta aatcaagctt gctccgacgt ccccaggcag
     5341 aatggcggtt ccctaaacga gcattgctta tatagacctc ccattaggca cgcctaccgc
     5401 ccatttacgt caatggaacg cccatttgcg tcattgcccc tccccattga cgtcaatggg
     5461 gatgtacttg gcagccatcg cgggccattt accgccattg acgtcaatgg gagtactgcc
     5521 aatgtaccct ggcgtacttc caatagtaat gtacttgcca agttactatt aatagatatt
     5581 gatgtactgc caagtgggcc atttaccgtc attgacgtca atagggggcg tgagaacgga
     5641 tatgaatggg caatgagcca tcccattgac gtcaatggtg ggtggtccta ttgacgtcaa
     5701 tgggcattga gccaggcggg ccatttaccg taattgacgt caatggggga ggcgccatat
     5761 acgtcaatag gaccgcccat atgacgtcaa taggaaagac catatataga gaccattgac
     5821 gtcaatgggg gagtggctat gggcggtatt aggaagcccc atatatggta tatgggaccg
     5881 cccattggga ggggctatct acgtcaatag gaaaacccat atatggaata ctatatggca
     5941 tagggccaat acatagtatt gaacctggcc aatagccata ttggcatagg gccatattgg
     6001 atattgccta tatattgatc ctggcatata gccaatatgg ccgccattat tggcaccatg
     6061 ccaattcaat atggcggact tggcacaggg ccaagacccc tcctcaaact tggcaccgtg
     6121 ccaagtagac ccctccccag ttggcacagt gccaggtccg ccatattgaa ttggcatggt
     6181 gccaagtccg ccatattgaa ttggctgggg tccagatcca ccatattgaa ttggcacgag
     6241 tccatatacg ccatattgaa ttggcacagg tccagatcca ccatattgaa ttggcatgag
     6301 tccatatacg ccatattgaa ttggcggtcg actggcgtaa tagcgaagag gcccgcaccg
     6361 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     6421 cattaagcgc ggcggg
//
