LOCUS       sequence-448409-        6210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stability reporter construct of the COX8A mitochondrial targeting 
            sequence (MTS) with mutations that impair recognition by SIFI for 
            transient expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6210)
  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 6210)
  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-MTSmut-cox8A(XXXX)-GFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     332..350
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    504..1076
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(663..682)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     892..909
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(1084..1803)
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     promoter        complement(1964..1982)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1965..1982)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(2017..3001)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     rep_origin      3072..3527
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3159..3178)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3369..3390
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3553..3657
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3658..3726,3727..4518)
                     /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(3876..3895)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4689..5277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5178..5197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5431..5448
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5565..5586
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5601..5606,5607..5624,5625..5631)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5639..5655
                     /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     5644..5666
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5663..5679
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5663..5679
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5698..5718
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5700..5718
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    5831..5965
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5868..5887)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5922..5941
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5995..6210,1..495))
                     /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"
ORIGIN
        1 ctcgcccttc agggcgccgt cctcggggta catccgctcg gaggaggcct cccagcccat
       61 ggttttcttc tgcattacgg ggccgtcgga ggggaagttg gtgccgcgca gcttcacctt
      121 gtagatgaac tcgccgtcct gcagggagga gtcctgggtc acggtcacca cgccgccgtc
      181 ctcgaagttc atcacgcgct cccacttgaa gccctcgggg aaggacagct tcaagtagtc
      241 ggggatgtcg gcggggtgct tcacgtaggc cttggagccg tacatgaact gaggggacag
      301 gatgtcccag gcgaagggca gggggccacc cttggtcacc ttcagcttgg cggtctgggt
      361 gccctcgtag gggcggccct cgccctcgcc ctcgatctcg aactcgtggc cgttcacgga
      421 gccctccatg tgcaccttga agcgcatgaa ctccttgatg atggccatgt tatcctcctc
      481 gcccttgctc accattccgg aggtattatc gtgtttttca aaggaaaacc acgtccccgt
      541 ggttcggggg gcctagacgt ttttttaacc tcgactaaac acatgtaaag catgtgcacc
      601 gaggccccag atcagatccc atacaatggg gtaccttctg ggcatccttc agccccttgt
      661 tgaatacgct tgaggagagc catttgactc tttccacaac tatccaactc acaacgtggc
      721 actggggttg tgccgccttt gcaggtgtat cttatacacg tggcttttgg ccgcagaggc
      781 acctgtcgcc aggtgggggg ttccgctgcc tgcaaagggt cgctacagac gttgtttgtc
      841 ttcaagaagc ttccagagga actgcttcct tcacgacatt caacagacct tgcattcctt
      901 tggcgagagg ggaaagaccc ctaggaatgc tcgtcaagaa gacagggcca ggtttccggg
      961 ccctcacatt gccaaaagac ggcaatatgg tggaaaataa catatagaca aacgcacacc
     1021 ggccttattc caagcggctt cggccagtaa cgttaggggg gggggaggga gaggggctct
     1081 agatcacttg tacagctcat ccatgccgtg ggtgatggcg gcggcggtca cgaagccgaa
     1141 gtagatcatg tgatcgcgct tctcgttggg gtccttggac agggcgctct gggtggacag
     1201 gtagtggtta tcgggcagca gcacagggcc atcgccgatg ggggtattct gctggtagtg
     1261 gtcggccagc tgcacgctgc catcctcgat gttgtggcgg atcttgaagt tcaccttgat
     1321 gccattcttg gccttgtcgg tcatgatgta cacattgtgg gcgttgtagt tgtactccat
     1381 cttattgccc aggatgttgc catcctcctt gaaatcggtg ccggtcagct cgatgcgatt
     1441 caccagggta tcgccctcga acttcacctc ggcgcgcgac ttgtagttgc cgtcatcctc
     1501 gaagaagatg gtgcgctcct ggatgtagcc ctcaggcatg gcgctcttga agaagtcgtg
     1561 ctgcttcatg tgatcggggt agcgtgagaa gcactgcacg ccgtagctca gggtggtcac
     1621 cagggtgggc cagggcacag gcagcttgcc ggtggtgcag atgaacttca gggtcagctt
     1681 gccgtaggtg gcatcgccct cgccctcgcc gctcacgctg aacttgtggc cattcacatc
     1741 gccattcagc tcgatcagga tgggcacgat gccggtgaac agctcggcgc ccttgctcac
     1801 catgctgcct ccgcctcccc caagcttccc ctccggcggc aacgaatgga tcttggcgcg
     1861 cggcactggg agccgccggg ccgagcctgt ctcgccccgc tcctccagcg gcgtctcgac
     1921 ggacatggtg gcgggatccg caaaaagaac aagtagcttg tattctatag tgtcacctaa
     1981 atcaagcttg ctccgacgtc cccaggcaga atggcggttc cctaaacgag cattgcttat
     2041 atagacctcc cattaggcac gcctaccgcc catttacgtc aatggaacgc ccatttgcgt
     2101 cattgcccct ccccattgac gtcaatgggg atgtacttgg cagccatcgc gggccattta
     2161 ccgccattga cgtcaatggg agtactgcca atgtaccctg gcgtacttcc aatagtaatg
     2221 tacttgccaa gttactatta atagatattg atgtactgcc aagtgggcca tttaccgtca
     2281 ttgacgtcaa tagggggcgt gagaacggat atgaatgggc aatgagccat cccattgacg
     2341 tcaatggtgg gtggtcctat tgacgtcaat gggcattgag ccaggcgggc catttaccgt
     2401 aattgacgtc aatgggggag gcgccatata cgtcaatagg accgcccata tgacgtcaat
     2461 aggaaagacc atatatagag accattgacg tcaatggggg agtggctatg ggcggtatta
     2521 ggaagcccca tatatggtat atgggaccgc ccattgggag gggctatcta cgtcaatagg
     2581 aaaacccata tatggaatac tatatggcat agggccaata catagtattg aacctggcca
     2641 atagccatat tggcataggg ccatattgga tattgcctat atattgatcc tggcatatag
     2701 ccaatatggc cgccattatt ggcaccatgc caattcaata tggcggactt ggcacagggc
     2761 caagacccct cctcaaactt ggcaccgtgc caagtagacc cctccccagt tggcacagtg
     2821 ccaggtccgc catattgaat tggcatggtg ccaagtccgc catattgaat tggctggggt
     2881 ccagatccac catattgaat tggcacgagt ccatatacgc catattgaat tggcacaggt
     2941 ccagatccac catattgaat tggcatgagt ccatatacgc catattgaat tggcggtcga
     3001 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     3061 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3121 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3181 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3241 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3301 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3361 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3421 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3481 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     3541 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3601 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     3661 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     3721 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     3781 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     3841 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     3901 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     3961 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4021 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4081 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4141 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4201 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4261 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4321 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4381 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4441 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4501 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4561 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4621 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4681 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4741 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4801 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4861 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4921 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4981 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5041 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5101 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5161 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5221 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5281 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5341 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5401 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5461 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     5521 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     5581 actcattagg caccccaggc tttacacttt atgctcccgg ctcgtatgtt gtgtggaatt
     5641 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
     5701 attaaccctc actaaaggga acaaaagctg ggtaccgggc ccaatgcatt ggcgccgcgg
     5761 ccgcgaatta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     5821 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5881 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5941 caatgtatct tatcatgtct ggatctacgt aatacgactc actatagttc tagatcactt
     6001 gtacagctcg tccatgccgc cggtggagtg gcggccctcg gcgcgttcgt actgttccac
     6061 gatggtgtag tcctcgttgt gggaggtgat gtccaacttg atgttgacgt tgtaggcgcc
     6121 gggcagctgc acgggcttct tggccttgta ggtggtcttg acctcagcgt cgtagtggcc
     6181 gccgtccttc agcttcagcc tctgcttgat
//
