LOCUS       sequence-448251-        7836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HRI stability reporter construct with deletion of SIFI degron 
            helices 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7836)
  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 7836)
  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-Delta-hel1/Delta-hel2HRI-GFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..7836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        188..206
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     188..205
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             1993..2712
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     misc_feature    2720..3292
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2887..2904)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3114..3133
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3301..4011
                     /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     complement(3446..3464)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    4041..4175
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4065..4084)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4119..4138
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4288..4308)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4288..4306)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4327..4343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4327..4343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4340..4362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4351..4367
                     /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(4375..4381,4382..4399,4400..4405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4420..4441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4558..4575)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4729..5317)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4809..4828)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5488..6279,6280..6348))
                     /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     6111..6130
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6349..6453)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6479..6934)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6616..6637)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6828..6847
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(7005..7836,1..153)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 caatggcggt aaatggcccg cgatggctgc caagtacatc cccattgacg tcaatgggga
       61 ggggcaatga cgcaaatggg cgttccattg acgtaaatgg gcggtaggcg tgcctaatgg
      121 gaggtctata taagcaatgc tcgtttaggg aaccgccatt ctgcctgggg acgtcggagc
      181 aagcttgatt taggtgacac tatagaatac aagctacttg ttctttttgc ggatcccgcc
      241 accatgcagg ggggcaactc cggggtccgc aagcgcgaag aggagggcga cggggctggg
      301 gctgtggctg cgccgccggc catcgacttt cccgccgagg gcccggaccc cgaatatgac
      361 gaatctgatg ttccagcaga aatccaggtg ttaaaagaac ccctacaaca gccaaccttc
      421 ccttttagct cattgagact acatcacaac agagctatta ctcacttaat gaggtctgct
      481 aaagagagag ttcgtcagga tccttgtgag gatatttctc gtatccagaa aatcagatca
      541 agggaagtag ccttggaagc acaaacttca cgttacttaa atgaatttga agaacttgcc
      601 atcttaggaa aaggtggata cggaagagta tacaaggtca ggaataaatt agatggtcag
      661 tattatgcaa taaaaaaaat cctgattaag ggtgcaacta aaacagtttg catgaaggtc
      721 ctacgggaag tgaaggtgct ggcaggtctt cagcacccca atattgttgg ctatcacacc
      781 gcgtggatag aacatgttca tgtgattcag ccacgagcag acagagctgc cattgagttg
      841 ccatctctgg aagtgctctc cgaccaggaa gaggacagag agcaatgtgg tgttaaaaat
      901 gatgaaagta gcagctcatc cattatcttt gctgagccca ccccagaaaa agaaaaacgc
      961 tttggagaat ctgacactga aaatcagaat aacaagtcgg tgaagtacac caccaattta
     1021 gtcataagag aatctggtga acttgagtcg accctggagc tccaggaaaa tggcttggct
     1081 ggtttgtctg ccagttcaat tgtggaacag cagctgccac tcaggcgtaa ttcccaccta
     1141 gaggagagtt tcacatccac cgaagaatct tccgaagaaa atgtcaactt tttgggtcag
     1201 acagaggcac agtaccacct gatgctgcac atccagatgc agctgtgtga gctctcgctg
     1261 tgggattgga tagtcgagag aaacaagcgg ggccgggagt atgtggacga gtctgcctgt
     1321 ccttatgtta tggccaatgt tgcaacaaaa atttttcaag aattggtaga aggtgtgttt
     1381 tacatacata acatgggaat tgtgcaccga gatctgaagc caagaaatat ttttcttcat
     1441 ggccctgatc agcaagtaaa aataggagac tttggtctgg cctgcacaga catcctacag
     1501 aagaacacag actggaccaa cagaaacggg aagagaacac caacacatac gtccagagtg
     1561 ggtacttgtc tgtacgcttc acccgaacag ttggaaggat ctgagtatga tgccaagtca
     1621 gatatgtaca gcttgggtgt ggtcctgcta gagctctttc agccgtttgg aacagaaatg
     1681 gagcgagcag aagttctaac aggtttaaga actggtcagt tgccggaatc cctccgtaaa
     1741 aggtgtccag tgcaagccaa gtatatccag cacttaacga gaaggaactc atcgcagaga
     1801 ccatctgcca ttcagctgct gcagagtgaa cttttccaaa attctggaaa tgttaacctc
     1861 accctacaga tgaagataat agagcaagaa aaagaaattg cagaactaaa gaagcagcta
     1921 aacctccttt ctcaagacaa aggggtgagg gatgacggaa aggatggggg cgtgggagga
     1981 ggcggaggca gcatggtgag caagggcgcc gagctgttca ccggcatcgt gcccatcctg
     2041 atcgagctga atggcgatgt gaatggccac aagttcagcg tgagcggcga gggcgagggc
     2101 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcctgtg
     2161 ccctggccca ccctggtgac caccctgagc tacggcgtgc agtgcttctc acgctacccc
     2221 gatcacatga agcagcacga cttcttcaag agcgccatgc ctgagggcta catccaggag
     2281 cgcaccatct tcttcgagga tgacggcaac tacaagtcgc gcgccgaggt gaagttcgag
     2341 ggcgataccc tggtgaatcg catcgagctg accggcaccg atttcaagga ggatggcaac
     2401 atcctgggca ataagatgga gtacaactac aacgcccaca atgtgtacat catgaccgac
     2461 aaggccaaga atggcatcaa ggtgaacttc aagatccgcc acaacatcga ggatggcagc
     2521 gtgcagctgg ccgaccacta ccagcagaat acccccatcg gcgatggccc tgtgctgctg
     2581 cccgataacc actacctgtc cacccagagc gccctgtcca aggaccccaa cgagaagcgc
     2641 gatcacatga tctacttcgg cttcgtgacc gccgccgcca tcacccacgg catggatgag
     2701 ctgtacaagt gatctagagc ccctctccct cccccccccc taacgttact ggccgaagcc
     2761 gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt
     2821 ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc
     2881 tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc
     2941 tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc
     3001 cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg
     3061 cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct
     3121 cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat
     3181 ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc
     3241 taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgataa tacctccgga
     3301 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
     3361 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     3421 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     3481 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
     3541 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
     3601 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     3661 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     3721 atgcagaaga aaaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     3781 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     3841 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     3901 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     3961 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtg atctagaact
     4021 atagtgagtc gtattacgta gatccagaca tgataagata cattgatgag tttggacaaa
     4081 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     4141 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     4201 tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cgcggccgcg gcgccaatgc
     4261 attgggcccg gtacccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     4321 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4381 acgagccggg agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4441 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4501 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4561 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4621 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4681 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4741 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4801 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4861 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4921 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4981 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5041 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5101 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5161 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5221 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5281 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5341 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5401 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5461 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5521 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5581 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5641 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5701 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5761 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5821 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5881 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5941 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6001 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6061 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6121 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6181 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6241 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6301 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6361 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6421 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     6481 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     6541 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     6601 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     6661 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     6721 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     6781 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     6841 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     6901 cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac
     6961 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagtcgac cgccaattca
     7021 atatggcgta tatggactca tgccaattca atatggtgga tctggacctg tgccaattca
     7081 atatggcgta tatggactcg tgccaattca atatggtgga tctggacccc agccaattca
     7141 atatggcgga cttggcacca tgccaattca atatggcgga cctggcactg tgccaactgg
     7201 ggaggggtct acttggcacg gtgccaagtt tgaggagggg tcttggccct gtgccaagtc
     7261 cgccatattg aattggcatg gtgccaataa tggcggccat attggctata tgccaggatc
     7321 aatatatagg caatatccaa tatggcccta tgccaatatg gctattggcc aggttcaata
     7381 ctatgtattg gccctatgcc atatagtatt ccatatatgg gttttcctat tgacgtagat
     7441 agcccctccc aatgggcggt cccatatacc atatatgggg cttcctaata ccgcccatag
     7501 ccactccccc attgacgtca atggtctcta tatatggtct ttcctattga cgtcatatgg
     7561 gcggtcctat tgacgtatat ggcgcctccc ccattgacgt caattacggt aaatggcccg
     7621 cctggctcaa tgcccattga cgtcaatagg accacccacc attgacgtca atgggatggc
     7681 tcattgccca ttcatatccg ttctcacgcc ccctattgac gtcaatgacg gtaaatggcc
     7741 cacttggcag tacatcaata tctattaata gtaacttggc aagtacatta ctattggaag
     7801 tacgccaggg tacattggca gtactcccat tgacgt
//
