LOCUS       sequence-458883-        6174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein stability reporter construct for E4F1 consisting of aa 
            220-242 for transient overexpression in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6174)
  AUTHORS   Manford AG, Mena EL, Shih KY, Gee CL, McMinimy R, Martinez-Gonzalez 
            B, Sherriff R, Lew B, Zoltek M, Rodriguez-Perez F, Woldesenbet M, 
            Kuriyan J, Rape M
  TITLE     Structural basis and regulation of the reductive stress response.
  JOURNAL   Cell. 2021 Oct 14;184(21):5375-5390.e16. doi: 
            10.1016/j.cell.2021.09.002. Epub 2021 Sep 24.
  PUBMED    34562363
REFERENCE   2  (bases 1 to 6174)
  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: 
            "10.1016/j.cell.2021.09.002"; journalName: "Cell"; date: 
            "2021-10-14- 14"; volume: "184"; issue: "21"; pages: "5375-5390.e16"
COMMENT     Sequence Label: pCS2+ E4F1 degron(220-242)-GFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             178..897
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     misc_feature    905..1477
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1072..1089)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1299..1318
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1486..2196
                     /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(1631..1649)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    2226..2360
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2250..2269)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2304..2323
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2473..2493)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2473..2491)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2512..2528)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2512..2528)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2525..2547)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2536..2552
                     /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(2560..2566,2567..2584,2585..2590))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2605..2626
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2743..2760)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2914..3502)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2994..3013)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3673..4464,4465..4533))
                     /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     4296..4315
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4534..4638)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4664..5119)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4801..4822)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5013..5032
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5190..6174
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcgga tcccgccacc atgcacgagt gcaagctctg tggggcctcc
      121 ttccgcacca agggctcact catccggcac caccggcggc acggaggcgg aggcagcatg
      181 gtgagcaagg gcgccgagct gttcaccggc atcgtgccca tcctgatcga gctgaatggc
      241 gatgtgaatg gccacaagtt cagcgtgagc ggcgagggcg agggcgatgc cacctacggc
      301 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ctgtgccctg gcccaccctg
      361 gtgaccaccc tgagctacgg cgtgcagtgc ttctcacgct accccgatca catgaagcag
      421 cacgacttct tcaagagcgc catgcctgag ggctacatcc aggagcgcac catcttcttc
      481 gaggatgacg gcaactacaa gtcgcgcgcc gaggtgaagt tcgagggcga taccctggtg
      541 aatcgcatcg agctgaccgg caccgatttc aaggaggatg gcaacatcct gggcaataag
      601 atggagtaca actacaacgc ccacaatgtg tacatcatga ccgacaaggc caagaatggc
      661 atcaaggtga acttcaagat ccgccacaac atcgaggatg gcagcgtgca gctggccgac
      721 cactaccagc agaatacccc catcggcgat ggccctgtgc tgctgcccga taaccactac
      781 ctgtccaccc agagcgccct gtccaaggac cccaacgaga agcgcgatca catgatctac
      841 ttcggcttcg tgaccgccgc cgccatcacc cacggcatgg atgagctgta caagtgatct
      901 agagcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
      961 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     1021 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     1081 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     1141 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     1201 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     1261 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     1321 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     1381 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc
     1441 acggggacgt ggttttcctt tgaaaaacac gataatacct ccggaatggt gagcaagggc
     1501 gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca catggagggc
     1561 tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc
     1621 acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc
     1681 ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc
     1741 gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag
     1801 gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac
     1861 aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaaaacc
     1921 atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag
     1981 atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc
     2041 tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac
     2101 atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc
     2161 cactccaccg gcggcatgga cgagctgtac aagtgatcta gaactatagt gagtcgtatt
     2221 acgtagatcc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     2281 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     2341 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
     2401 gggaggtgtg ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc
     2461 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     2521 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccgggagcat
     2581 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     2641 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     2701 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     2761 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     2821 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     2881 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     2941 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3001 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3061 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3121 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3181 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3241 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3301 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     3361 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     3421 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     3481 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     3541 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     3601 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     3661 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     3721 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     3781 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     3841 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     3901 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     3961 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4021 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4081 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4141 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4201 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4261 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4321 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     4381 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     4441 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     4501 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     4561 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     4621 acaaataggg gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat
     4681 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     4741 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     4801 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     4861 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     4921 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     4981 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     5041 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     5101 gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     5161 tcggtgcggg cctcttcgct attacgccag tcgaccgcca attcaatatg gcgtatatgg
     5221 actcatgcca attcaatatg gtggatctgg acctgtgcca attcaatatg gcgtatatgg
     5281 actcgtgcca attcaatatg gtggatctgg accccagcca attcaatatg gcggacttgg
     5341 caccatgcca attcaatatg gcggacctgg cactgtgcca actggggagg ggtctacttg
     5401 gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc aagtccgcca tattgaattg
     5461 gcatggtgcc aataatggcg gccatattgg ctatatgcca ggatcaatat ataggcaata
     5521 tccaatatgg ccctatgcca atatggctat tggccaggtt caatactatg tattggccct
     5581 atgccatata gtattccata tatgggtttt cctattgacg tagatagccc ctcccaatgg
     5641 gcggtcccat ataccatata tggggcttcc taataccgcc catagccact cccccattga
     5701 cgtcaatggt ctctatatat ggtctttcct attgacgtca tatgggcggt cctattgacg
     5761 tatatggcgc ctcccccatt gacgtcaatt acggtaaatg gcccgcctgg ctcaatgccc
     5821 attgacgtca ataggaccac ccaccattga cgtcaatggg atggctcatt gcccattcat
     5881 atccgttctc acgcccccta ttgacgtcaa tgacggtaaa tggcccactt ggcagtacat
     5941 caatatctat taatagtaac ttggcaagta cattactatt ggaagtacgc cagggtacat
     6001 tggcagtact cccattgacg tcaatggcgg taaatggccc gcgatggctg ccaagtacat
     6061 ccccattgac gtcaatgggg aggggcaatg acgcaaatgg gcgttccatt gacgtaaatg
     6121 ggcggtaggc gtgcctaatg ggaggtctat ataagcaatg ctcgtttagg gaac
//
