LOCUS       sequence-448414-        6385 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ATF4uORF1-2 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 6385)
  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 6385)
  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-ATF4uORF1-2-GFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6385
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     112..130
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     misc_feature    284..856
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(443..462)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     672..689
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(864..1583)
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     regulatory      1794..1803
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1919..1937)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1920..1937)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     promoter        complement(1972..2956)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     rep_origin      3027..3482
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3114..3133)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3324..3345
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3508..3612
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3613..3681,3682..4473)
                     /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(3831..3850)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4644..5232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5133..5152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5386..5403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5520..5541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5556..5561,5562..5579,5580..5586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5594..5610
                     /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     5599..5621
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5618..5634
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5618..5634
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5653..5673
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5655..5673
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    5786..5920
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5823..5842)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5877..5896
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5950..6385,1..275))
                     /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 aaggacagct tcaagtagtc ggggatgtcg gcggggtgct tcacgtaggc cttggagccg
       61 tacatgaact gaggggacag gatgtcccag gcgaagggca gggggccacc cttggtcacc
      121 ttcagcttgg cggtctgggt gccctcgtag gggcggccct cgccctcgcc ctcgatctcg
      181 aactcgtggc cgttcacgga gccctccatg tgcaccttga agcgcatgaa ctccttgatg
      241 atggccatgt tatcctcctc gcccttgctc accattccgg aggtattatc gtgtttttca
      301 aaggaaaacc acgtccccgt ggttcggggg gcctagacgt ttttttaacc tcgactaaac
      361 acatgtaaag catgtgcacc gaggccccag atcagatccc atacaatggg gtaccttctg
      421 ggcatccttc agccccttgt tgaatacgct tgaggagagc catttgactc tttccacaac
      481 tatccaactc acaacgtggc actggggttg tgccgccttt gcaggtgtat cttatacacg
      541 tggcttttgg ccgcagaggc acctgtcgcc aggtgggggg ttccgctgcc tgcaaagggt
      601 cgctacagac gttgtttgtc ttcaagaagc ttccagagga actgcttcct tcacgacatt
      661 caacagacct tgcattcctt tggcgagagg ggaaagaccc ctaggaatgc tcgtcaagaa
      721 gacagggcca ggtttccggg ccctcacatt gccaaaagac ggcaatatgg tggaaaataa
      781 catatagaca aacgcacacc ggccttattc caagcggctt cggccagtaa cgttaggggg
      841 gggggaggga gaggggctct agatcacttg tacagctcat ccatgccgtg ggtgatggcg
      901 gcggcggtca cgaagccgaa gtagatcatg tgatcgcgct tctcgttggg gtccttggac
      961 agggcgctct gggtggacag gtagtggtta tcgggcagca gcacagggcc atcgccgatg
     1021 ggggtattct gctggtagtg gtcggccagc tgcacgctgc catcctcgat gttgtggcgg
     1081 atcttgaagt tcaccttgat gccattcttg gccttgtcgg tcatgatgta cacattgtgg
     1141 gcgttgtagt tgtactccat cttattgccc aggatgttgc catcctcctt gaaatcggtg
     1201 ccggtcagct cgatgcgatt caccagggta tcgccctcga acttcacctc ggcgcgcgac
     1261 ttgtagttgc cgtcatcctc gaagaagatg gtgcgctcct ggatgtagcc ctcaggcatg
     1321 gcgctcttga agaagtcgtg ctgcttcatg tgatcggggt agcgtgagaa gcactgcacg
     1381 ccgtagctca gggtggtcac cagggtgggc cagggcacag gcagcttgcc ggtggtgcag
     1441 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc gctcacgctg
     1501 aacttgtggc cattcacatc gccattcagc tcgatcagga tgggcacgat gccggtgaac
     1561 agctcggcgc ccttgctcac catgctgcct ccgcctccca tgttgcggtg ctttgctgga
     1621 atcgaggaat gtgcttaatt cgaaggtgtc tttgtcggtt acagcaacgc tgctgctgaa
     1681 tgccgtgaga agcgccatgg cttaagccgc tgggggttgc cgctgcagag cctggtgctg
     1741 ctgccgccgc tgcaaaggcc aatgctgccg caggcactgc tgcccctaat acgccatggt
     1801 ggccgtggac cctgagggcg gggaggaggg aaaacgcaca cgcgcagaga aaactacatc
     1861 tgtgggcggg caaagtagaa aggtggcggg atccgcaaaa agaacaagta gcttgtattc
     1921 tatagtgtca cctaaatcaa gcttgctccg acgtccccag gcagaatggc ggttccctaa
     1981 acgagcattg cttatataga cctcccatta ggcacgccta ccgcccattt acgtcaatgg
     2041 aacgcccatt tgcgtcattg cccctcccca ttgacgtcaa tggggatgta cttggcagcc
     2101 atcgcgggcc atttaccgcc attgacgtca atgggagtac tgccaatgta ccctggcgta
     2161 cttccaatag taatgtactt gccaagttac tattaataga tattgatgta ctgccaagtg
     2221 ggccatttac cgtcattgac gtcaataggg ggcgtgagaa cggatatgaa tgggcaatga
     2281 gccatcccat tgacgtcaat ggtgggtggt cctattgacg tcaatgggca ttgagccagg
     2341 cgggccattt accgtaattg acgtcaatgg gggaggcgcc atatacgtca ataggaccgc
     2401 ccatatgacg tcaataggaa agaccatata tagagaccat tgacgtcaat gggggagtgg
     2461 ctatgggcgg tattaggaag ccccatatat ggtatatggg accgcccatt gggaggggct
     2521 atctacgtca ataggaaaac ccatatatgg aatactatat ggcatagggc caatacatag
     2581 tattgaacct ggccaatagc catattggca tagggccata ttggatattg cctatatatt
     2641 gatcctggca tatagccaat atggccgcca ttattggcac catgccaatt caatatggcg
     2701 gacttggcac agggccaaga cccctcctca aacttggcac cgtgccaagt agacccctcc
     2761 ccagttggca cagtgccagg tccgccatat tgaattggca tggtgccaag tccgccatat
     2821 tgaattggct ggggtccaga tccaccatat tgaattggca cgagtccata tacgccatat
     2881 tgaattggca caggtccaga tccaccatat tgaattggca tgagtccata tacgccatat
     2941 tgaattggcg gtcgactggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     3001 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     3061 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3121 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3181 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3241 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3301 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3361 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3421 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     3481 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3541 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3601 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     3661 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     3721 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     3781 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     3841 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     3901 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     3961 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     4021 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     4081 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     4141 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     4201 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     4261 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     4321 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     4381 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     4441 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     4501 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4561 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4621 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4681 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4741 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4801 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4861 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4921 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4981 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     5041 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5101 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5161 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5221 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5281 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5341 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     5401 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     5461 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     5521 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct cccggctcgt
     5581 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat
     5641 tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctgggtac cgggcccaat
     5701 gcattggcgc cgcggccgcg aattaaaaaa cctcccacac ctccccctga acctgaaaca
     5761 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     5821 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     5881 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc tacgtaatac gactcactat
     5941 agttctagat cacttgtaca gctcgtccat gccgccggtg gagtggcggc cctcggcgcg
     6001 ttcgtactgt tccacgatgg tgtagtcctc gttgtgggag gtgatgtcca acttgatgtt
     6061 gacgttgtag gcgccgggca gctgcacggg cttcttggcc ttgtaggtgg tcttgacctc
     6121 agcgtcgtag tggccgccgt ccttcagctt cagcctctgc ttgatctcgc ccttcagggc
     6181 gccgtcctcg gggtacatcc gctcggagga ggcctcccag cccatggttt tcttctgcat
     6241 tacggggccg tcggagggga agttggtgcc gcgcagcttc accttgtaga tgaactcgcc
     6301 gtcctgcagg gaggagtcct gggtcacggt caccacgccg ccgtcctcga agttcatcac
     6361 gcgctcccac ttgaagccct cgggg
//
