LOCUS       sequence-448249-        7992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HRI 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 7992)
  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 7992)
  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-HRI-GFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..7992
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      982..1437
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1069..1088)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1279..1300
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1463..1567
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1568..1636,1637..2428)
                     /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(1786..1805)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2599..3187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3088..3107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3341..3358
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3475..3496
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3511..3516,3517..3534,3535..3541)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3549..3565
                     /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     3554..3576
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3573..3589
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3573..3589
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3608..3628
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3610..3628
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    3741..3875
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3778..3797)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3832..3851
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(3905..4615)
                     /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     4452..4470
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    4624..5196
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4783..4802)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     5012..5029
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(5204..5923)
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     promoter        complement(7866..7884)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(7867..7884)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(join(7919..7992,1..911))
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 ccatttgcgt cattgcccct ccccattgac gtcaatgggg atgtacttgg cagccatcgc
       61 gggccattta ccgccattga cgtcaatggg agtactgcca atgtaccctg gcgtacttcc
      121 aatagtaatg tacttgccaa gttactatta atagatattg atgtactgcc aagtgggcca
      181 tttaccgtca ttgacgtcaa tagggggcgt gagaacggat atgaatgggc aatgagccat
      241 cccattgacg tcaatggtgg gtggtcctat tgacgtcaat gggcattgag ccaggcgggc
      301 catttaccgt aattgacgtc aatgggggag gcgccatata cgtcaatagg accgcccata
      361 tgacgtcaat aggaaagacc atatatagag accattgacg tcaatggggg agtggctatg
      421 ggcggtatta ggaagcccca tatatggtat atgggaccgc ccattgggag gggctatcta
      481 cgtcaatagg aaaacccata tatggaatac tatatggcat agggccaata catagtattg
      541 aacctggcca atagccatat tggcataggg ccatattgga tattgcctat atattgatcc
      601 tggcatatag ccaatatggc cgccattatt ggcaccatgc caattcaata tggcggactt
      661 ggcacagggc caagacccct cctcaaactt ggcaccgtgc caagtagacc cctccccagt
      721 tggcacagtg ccaggtccgc catattgaat tggcatggtg ccaagtccgc catattgaat
      781 tggctggggt ccagatccac catattgaat tggcacgagt ccatatacgc catattgaat
      841 tggcacaggt ccagatccac catattgaat tggcatgagt ccatatacgc catattgaat
      901 tggcggtcga ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
      961 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1021 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1081 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1141 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1201 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1261 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1321 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1381 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     1441 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1501 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     1561 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     1621 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     1681 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     1741 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     1801 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     1861 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     1921 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     1981 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     2041 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     2101 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     2161 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     2221 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     2281 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     2341 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     2401 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     2461 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     2521 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2581 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2641 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2701 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     2761 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2821 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2881 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2941 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3001 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3061 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3121 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3181 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3241 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3301 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3361 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     3421 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     3481 gagttagctc actcattagg caccccaggc tttacacttt atgctcccgg ctcgtatgtt
     3541 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     3601 caagcgcgca attaaccctc actaaaggga acaaaagctg ggtaccgggc ccaatgcatt
     3661 ggcgccgcgg ccgcgaatta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     3721 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     3781 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     3841 ccaaactcat caatgtatct tatcatgtct ggatctacgt aatacgactc actatagttc
     3901 tagatcactt gtacagctcg tccatgccgc cggtggagtg gcggccctcg gcgcgttcgt
     3961 actgttccac gatggtgtag tcctcgttgt gggaggtgat gtccaacttg atgttgacgt
     4021 tgtaggcgcc gggcagctgc acgggcttct tggccttgta ggtggtcttg acctcagcgt
     4081 cgtagtggcc gccgtccttc agcttcagcc tctgcttgat ctcgcccttc agggcgccgt
     4141 cctcggggta catccgctcg gaggaggcct cccagcccat ggttttcttc tgcattacgg
     4201 ggccgtcgga ggggaagttg gtgccgcgca gcttcacctt gtagatgaac tcgccgtcct
     4261 gcagggagga gtcctgggtc acggtcacca cgccgccgtc ctcgaagttc atcacgcgct
     4321 cccacttgaa gccctcgggg aaggacagct tcaagtagtc ggggatgtcg gcggggtgct
     4381 tcacgtaggc cttggagccg tacatgaact gaggggacag gatgtcccag gcgaagggca
     4441 gggggccacc cttggtcacc ttcagcttgg cggtctgggt gccctcgtag gggcggccct
     4501 cgccctcgcc ctcgatctcg aactcgtggc cgttcacgga gccctccatg tgcaccttga
     4561 agcgcatgaa ctccttgatg atggccatgt tatcctcctc gcccttgctc accattccgg
     4621 aggtattatc gtgtttttca aaggaaaacc acgtccccgt ggttcggggg gcctagacgt
     4681 ttttttaacc tcgactaaac acatgtaaag catgtgcacc gaggccccag atcagatccc
     4741 atacaatggg gtaccttctg ggcatccttc agccccttgt tgaatacgct tgaggagagc
     4801 catttgactc tttccacaac tatccaactc acaacgtggc actggggttg tgccgccttt
     4861 gcaggtgtat cttatacacg tggcttttgg ccgcagaggc acctgtcgcc aggtgggggg
     4921 ttccgctgcc tgcaaagggt cgctacagac gttgtttgtc ttcaagaagc ttccagagga
     4981 actgcttcct tcacgacatt caacagacct tgcattcctt tggcgagagg ggaaagaccc
     5041 ctaggaatgc tcgtcaagaa gacagggcca ggtttccggg ccctcacatt gccaaaagac
     5101 ggcaatatgg tggaaaataa catatagaca aacgcacacc ggccttattc caagcggctt
     5161 cggccagtaa cgttaggggg gggggaggga gaggggctct agatcacttg tacagctcat
     5221 ccatgccgtg ggtgatggcg gcggcggtca cgaagccgaa gtagatcatg tgatcgcgct
     5281 tctcgttggg gtccttggac agggcgctct gggtggacag gtagtggtta tcgggcagca
     5341 gcacagggcc atcgccgatg ggggtattct gctggtagtg gtcggccagc tgcacgctgc
     5401 catcctcgat gttgtggcgg atcttgaagt tcaccttgat gccattcttg gccttgtcgg
     5461 tcatgatgta cacattgtgg gcgttgtagt tgtactccat cttattgccc aggatgttgc
     5521 catcctcctt gaaatcggtg ccggtcagct cgatgcgatt caccagggta tcgccctcga
     5581 acttcacctc ggcgcgcgac ttgtagttgc cgtcatcctc gaagaagatg gtgcgctcct
     5641 ggatgtagcc ctcaggcatg gcgctcttga agaagtcgtg ctgcttcatg tgatcggggt
     5701 agcgtgagaa gcactgcacg ccgtagctca gggtggtcac cagggtgggc cagggcacag
     5761 gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct
     5821 cgccctcgcc gctcacgctg aacttgtggc cattcacatc gccattcagc tcgatcagga
     5881 tgggcacgat gccggtgaac agctcggcgc ccttgctcac catgctgcct ccgcctcctc
     5941 ccacgccccc atcctttccg tcatccctca cccctttgtc ttgagaaagg aggtttagct
     6001 gcttctttag ttctgcaatt tctttttctt gctctattat cttcatctgt agggtgaggt
     6061 taacatttcc agaattttgg aaaagttcac tctgcagcag ctgaatggca gatggtctct
     6121 gcgatgagtt ccttctcgtt aagtgctgga tatacttggc ttgcactgga caccttttac
     6181 ggagggattc cggcaactga ccagttctta aacctgttag aacttctgct cgctccattt
     6241 ctgttccaaa cggctgaaag agctctagca ggaccacacc caagctgtac atatctgact
     6301 tggcatcata ctcagatcct tccaactgtt cgggtgaagc gtacagacaa gtacccactc
     6361 tggacgtatg tgttggtgtt ctcttcccgt ttctgttggt ccagtctgtg ttcttctgta
     6421 ggatgtctgt gcaggccaga ccaaagtctc ctatttttac ttgctgatca gggccatgaa
     6481 gaaaaatatt tcttggcttc agatctcggt gcacaattcc catgttatgt atgtaaaaca
     6541 caccttctac caattcttga aaaatttttg ttgcaacatt ggccataaca taaggacagg
     6601 cagactcgtc cacatactcc cggccccgct tgtttctctc gactatccaa tcccacagcg
     6661 agagctcaca cagctgcatc tggatgtgca gcatcaggtg gtactgtgcc tctgtctgac
     6721 ccaaaaagtt gacattttct tcggaagatt cttcggtgga tgtgaaactc tcctctaggt
     6781 gggaattacg cctgagtggc agctgctgtt ccacaattga actggcagac aaaccagcca
     6841 agccattttc ctggagctcc agggtcgact caagttcacc agattctctt atgactaaat
     6901 tggtggtgta cttcaccgac ttgttattct gattttcagt gtcagattct ccaaagcgtt
     6961 tttctttttc tggggtgggc tcagcaaaga taatggatga gctgctactt tcatcatttt
     7021 taacaccaca ttgctctctg tcctcttcct ggtcggagag cacttccaga gatggcaact
     7081 caatggcagc tctgtctgct cgtggctgaa tcacatgaac atgttctatc cacgcggtgt
     7141 gatagccaac aatattgggg tgctgaagac ctgccagcac cttcacttcc cgtaggacct
     7201 tcatgcaaac tgttttagtt gcacccttaa tcaggatttt ttttattgca taatactgac
     7261 catctaattt attcctgacc ttgtatactc ttccgtatcc accttttcct aagatggcaa
     7321 gttcttcaaa ttcatttaag taacgtgaag tttgtgcttc caaggctact tcccttgatc
     7381 tgattttctg gatacgagaa atatcctcac aaggatcctg acgaactctc tctttagcag
     7441 acctcattaa gtgagtaata gctctgttgt gatgtagtct caatgagcta aactcgtcac
     7501 tacaagtgaa agaagacagc agccccattt tgataaacgt ctggcaaagt agcttaaaca
     7561 cctgtcttga acgaagtggg tttggttcat gcacgtggct caagtgctcc agcaaagaaa
     7621 ccagcaagag ttggtttgca actgcaaaag ggaaggttgg ctgttgtagg ggttctttta
     7681 acacctggat ttctgctgga acatcagatt cgtcatattc ggggtccggg ccctcggcgg
     7741 gaaagtcgat ggccggcggc gcagccacag ccccagcccc gtcgccctcc tcttcgcgct
     7801 tgcggacccc ggagttgccc ccctgcatgg tggcgggatc cgcaaaaaga acaagtagct
     7861 tgtattctat agtgtcacct aaatcaagct tgctccgacg tccccaggca gaatggcggt
     7921 tccctaaacg agcattgctt atatagacct cccattaggc acgcctaccg cccatttacg
     7981 tcaatggaac gc
//
