LOCUS       sequence-448248-        6125 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein stability reporter construct for transient expression in 
            mammalian cells. Stability of GFP-fusion protein can be assed by 
            flow cytometry by normalizing to mCherry expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6125)
  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 6125)
  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-GFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..49)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    626..760
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(650..669)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     704..723
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(873..893)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(873..891)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(912..928)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(912..928)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(925..947)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    936..952
                     /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(960..966,967..984,985..990))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1005..1026
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1143..1160)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1314..1902)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1394..1413)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2073..2864,2865..2933))
                     /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     2696..2715
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2934..3038)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3064..3519)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3201..3222)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3413..3432
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3590..4574
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        4609..4627
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4609..4626
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      4659..4668
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4703..5422
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     misc_feature    5430..6002
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5597..5614)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5824..5843
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(6011..6125,1..596)
                     /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 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
       61 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
      121 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
      181 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
      241 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
      301 ccgtaatgca gaagaaaacc atgggctggg aggcctcctc cgagcggatg taccccgagg
      361 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
      421 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
      481 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
      541 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtgatcta
      601 gaactatagt gagtcgtatt acgtagatcc agacatgata agatacattg atgagtttgg
      661 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
      721 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
      781 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc
      841 aatgcattgg gcccggtacc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
      901 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
      961 aacatacgag ccgggagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     1021 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     1081 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1141 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1201 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1261 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1321 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1381 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1441 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1501 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1561 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1621 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1681 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1741 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1801 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1861 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1921 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1981 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2041 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2101 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2161 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2221 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     2281 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     2341 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     2401 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     2461 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     2521 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     2581 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     2641 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     2701 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     2761 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     2821 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2881 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2941 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3001 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3061 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     3121 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     3181 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3241 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     3301 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     3361 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     3421 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3481 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     3541 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag tcgaccgcca
     3601 attcaatatg gcgtatatgg actcatgcca attcaatatg gtggatctgg acctgtgcca
     3661 attcaatatg gcgtatatgg actcgtgcca attcaatatg gtggatctgg accccagcca
     3721 attcaatatg gcggacttgg caccatgcca attcaatatg gcggacctgg cactgtgcca
     3781 actggggagg ggtctacttg gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc
     3841 aagtccgcca tattgaattg gcatggtgcc aataatggcg gccatattgg ctatatgcca
     3901 ggatcaatat ataggcaata tccaatatgg ccctatgcca atatggctat tggccaggtt
     3961 caatactatg tattggccct atgccatata gtattccata tatgggtttt cctattgacg
     4021 tagatagccc ctcccaatgg gcggtcccat ataccatata tggggcttcc taataccgcc
     4081 catagccact cccccattga cgtcaatggt ctctatatat ggtctttcct attgacgtca
     4141 tatgggcggt cctattgacg tatatggcgc ctcccccatt gacgtcaatt acggtaaatg
     4201 gcccgcctgg ctcaatgccc attgacgtca ataggaccac ccaccattga cgtcaatggg
     4261 atggctcatt gcccattcat atccgttctc acgcccccta ttgacgtcaa tgacggtaaa
     4321 tggcccactt ggcagtacat caatatctat taatagtaac ttggcaagta cattactatt
     4381 ggaagtacgc cagggtacat tggcagtact cccattgacg tcaatggcgg taaatggccc
     4441 gcgatggctg ccaagtacat ccccattgac gtcaatgggg aggggcaatg acgcaaatgg
     4501 gcgttccatt gacgtaaatg ggcggtaggc gtgcctaatg ggaggtctat ataagcaatg
     4561 ctcgtttagg gaaccgccat tctgcctggg gacgtcggag caagcttgat ttaggtgaca
     4621 ctatagaata caagctactt gttctttttg cggatcccgc caccatggga gacggacgaa
     4681 cgtctctgga ggcggaggca gcatggtgag caagggcgcc gagctgttca ccggcatcgt
     4741 gcccatcctg atcgagctga atggcgatgt gaatggccac aagttcagcg tgagcggcga
     4801 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     4861 gctgcctgtg ccctggccca ccctggtgac caccctgagc tacggcgtgc agtgcttctc
     4921 acgctacccc gatcacatga agcagcacga cttcttcaag agcgccatgc ctgagggcta
     4981 catccaggag cgcaccatct tcttcgagga tgacggcaac tacaagtcgc gcgccgaggt
     5041 gaagttcgag ggcgataccc tggtgaatcg catcgagctg accggcaccg atttcaagga
     5101 ggatggcaac atcctgggca ataagatgga gtacaactac aacgcccaca atgtgtacat
     5161 catgaccgac aaggccaaga atggcatcaa ggtgaacttc aagatccgcc acaacatcga
     5221 ggatggcagc gtgcagctgg ccgaccacta ccagcagaat acccccatcg gcgatggccc
     5281 tgtgctgctg cccgataacc actacctgtc cacccagagc gccctgtcca aggaccccaa
     5341 cgagaagcgc gatcacatga tctacttcgg cttcgtgacc gccgccgcca tcacccacgg
     5401 catggatgag ctgtacaagt gatctagagc ccctctccct cccccccccc taacgttact
     5461 ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata
     5521 ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt
     5581 cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa
     5641 gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag
     5701 cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca
     5761 cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc
     5821 aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat
     5881 tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta
     5941 aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgataa
     6001 tacctccgga atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat
     6061 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     6121 gggcg
//
