LOCUS       sequence-448250-        6516 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HRI N-terminal region (residues 1-138) stability reporter construct 
            for transient expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6516)
  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 6516)
  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-N-HRI(1-138)-GFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6516
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     179..197
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    351..923
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(510..529)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     739..756
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(931..1650)
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     promoter        complement(2117..2135)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2118..2135)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(2170..3154)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     rep_origin      3225..3680
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3312..3331)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3522..3543
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3706..3810
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3811..3879,3880..4671)
                     /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(4029..4048)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4842..5430
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5331..5350
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5584..5601
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5718..5739
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5754..5759,5760..5777,5778..5784)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5792..5808
                     /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     5797..5819
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5816..5832
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5816..5832
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5851..5871
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5853..5871
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    5984..6118
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6021..6040)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6075..6094
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6148..6516,1..342))
                     /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 ctgggtcacg gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc acttgaagcc
       61 ctcggggaag gacagcttca agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt
      121 ggagccgtac atgaactgag gggacaggat gtcccaggcg aagggcaggg ggccaccctt
      181 ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc
      241 gatctcgaac tcgtggccgt tcacggagcc ctccatgtgc accttgaagc gcatgaactc
      301 cttgatgatg gccatgttat cctcctcgcc cttgctcacc attccggagg tattatcgtg
      361 tttttcaaag gaaaaccacg tccccgtggt tcggggggcc tagacgtttt tttaacctcg
      421 actaaacaca tgtaaagcat gtgcaccgag gccccagatc agatcccata caatggggta
      481 ccttctgggc atccttcagc cccttgttga atacgcttga ggagagccat ttgactcttt
      541 ccacaactat ccaactcaca acgtggcact ggggttgtgc cgcctttgca ggtgtatctt
      601 atacacgtgg cttttggccg cagaggcacc tgtcgccagg tggggggttc cgctgcctgc
      661 aaagggtcgc tacagacgtt gtttgtcttc aagaagcttc cagaggaact gcttccttca
      721 cgacattcaa cagaccttgc attcctttgg cgagagggga aagaccccta ggaatgctcg
      781 tcaagaagac agggccaggt ttccgggccc tcacattgcc aaaagacggc aatatggtgg
      841 aaaataacat atagacaaac gcacaccggc cttattccaa gcggcttcgg ccagtaacgt
      901 tagggggggg ggagggagag gggctctaga tcacttgtac agctcatcca tgccgtgggt
      961 gatggcggcg gcggtcacga agccgaagta gatcatgtga tcgcgcttct cgttggggtc
     1021 cttggacagg gcgctctggg tggacaggta gtggttatcg ggcagcagca cagggccatc
     1081 gccgatgggg gtattctgct ggtagtggtc ggccagctgc acgctgccat cctcgatgtt
     1141 gtggcggatc ttgaagttca ccttgatgcc attcttggcc ttgtcggtca tgatgtacac
     1201 attgtgggcg ttgtagttgt actccatctt attgcccagg atgttgccat cctccttgaa
     1261 atcggtgccg gtcagctcga tgcgattcac cagggtatcg ccctcgaact tcacctcggc
     1321 gcgcgacttg tagttgccgt catcctcgaa gaagatggtg cgctcctgga tgtagccctc
     1381 aggcatggcg ctcttgaaga agtcgtgctg cttcatgtga tcggggtagc gtgagaagca
     1441 ctgcacgccg tagctcaggg tggtcaccag ggtgggccag ggcacaggca gcttgccggt
     1501 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgct
     1561 cacgctgaac ttgtggccat tcacatcgcc attcagctcg atcaggatgg gcacgatgcc
     1621 ggtgaacagc tcggcgccct tgctcaccat gctgcctccg cctccatcct gacgaactct
     1681 ctctttagca gacctcatta agtgagtaat agctctgttg tgatgtagtc tcaatgagct
     1741 aaactcgtca ctacaagtga aagaagacag cagccccatt ttgataaacg tctggcaaag
     1801 tagcttaaac acctgtcttg aacgaagtgg gtttggttca tgcacgtggc tcaagtgctc
     1861 cagcaaagaa accagcaaga gttggtttgc aactgcaaaa gggaaggttg gctgttgtag
     1921 gggttctttt aacacctgga tttctgctgg aacatcagat tcgtcatatt cggggtccgg
     1981 gccctcggcg ggaaagtcga tggccggcgg cgcagccaca gccccagccc cgtcgccctc
     2041 ctcttcgcgc ttgcggaccc cggagttgcc cccctgcatg gtggcgggat ccgcaaaaag
     2101 aacaagtagc ttgtattcta tagtgtcacc taaatcaagc ttgctccgac gtccccaggc
     2161 agaatggcgg ttccctaaac gagcattgct tatatagacc tcccattagg cacgcctacc
     2221 gcccatttac gtcaatggaa cgcccatttg cgtcattgcc cctccccatt gacgtcaatg
     2281 gggatgtact tggcagccat cgcgggccat ttaccgccat tgacgtcaat gggagtactg
     2341 ccaatgtacc ctggcgtact tccaatagta atgtacttgc caagttacta ttaatagata
     2401 ttgatgtact gccaagtggg ccatttaccg tcattgacgt caataggggg cgtgagaacg
     2461 gatatgaatg ggcaatgagc catcccattg acgtcaatgg tgggtggtcc tattgacgtc
     2521 aatgggcatt gagccaggcg ggccatttac cgtaattgac gtcaatgggg gaggcgccat
     2581 atacgtcaat aggaccgccc atatgacgtc aataggaaag accatatata gagaccattg
     2641 acgtcaatgg gggagtggct atgggcggta ttaggaagcc ccatatatgg tatatgggac
     2701 cgcccattgg gaggggctat ctacgtcaat aggaaaaccc atatatggaa tactatatgg
     2761 catagggcca atacatagta ttgaacctgg ccaatagcca tattggcata gggccatatt
     2821 ggatattgcc tatatattga tcctggcata tagccaatat ggccgccatt attggcacca
     2881 tgccaattca atatggcgga cttggcacag ggccaagacc cctcctcaaa cttggcaccg
     2941 tgccaagtag acccctcccc agttggcaca gtgccaggtc cgccatattg aattggcatg
     3001 gtgccaagtc cgccatattg aattggctgg ggtccagatc caccatattg aattggcacg
     3061 agtccatata cgccatattg aattggcaca ggtccagatc caccatattg aattggcatg
     3121 agtccatata cgccatattg aattggcggt cgactggcgt aatagcgaag aggcccgcac
     3181 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     3241 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3301 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3361 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3421 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3481 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3541 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3601 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     3661 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3721 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3781 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3841 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3901 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3961 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4021 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4081 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4141 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4201 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4261 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4321 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4381 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4441 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4501 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4561 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4621 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4681 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4741 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4801 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4861 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4921 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4981 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5041 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5101 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5161 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5221 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5281 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5341 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5401 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5461 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5521 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     5581 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     5641 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     5701 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     5761 tttatgctcc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     5821 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     5881 ctgggtaccg ggcccaatgc attggcgccg cggccgcgaa ttaaaaaacc tcccacacct
     5941 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     6001 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     6061 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcta
     6121 cgtaatacga ctcactatag ttctagatca cttgtacagc tcgtccatgc cgccggtgga
     6181 gtggcggccc tcggcgcgtt cgtactgttc cacgatggtg tagtcctcgt tgtgggaggt
     6241 gatgtccaac ttgatgttga cgttgtaggc gccgggcagc tgcacgggct tcttggcctt
     6301 gtaggtggtc ttgacctcag cgtcgtagtg gccgccgtcc ttcagcttca gcctctgctt
     6361 gatctcgccc ttcagggcgc cgtcctcggg gtacatccgc tcggaggagg cctcccagcc
     6421 catggttttc ttctgcatta cggggccgtc ggaggggaag ttggtgccgc gcagcttcac
     6481 cttgtagatg aactcgccgt cctgcaggga ggagtc
//
