LOCUS       sequence-450904-        4601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses EGFP without a stop codon that produces a GFP-tagged 
            readthrough RQC substrate..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4601)
  AUTHORS   Xu S, Gierisch ME, Barchi E, Poser I, Alberti S, Salomons FA, 
            Dantuma NP
  TITLE     Chemical inhibition of the integrated stress response impairs the 
            ubiquitin-proteasome system.
  JOURNAL   Commun Biol. 2024 Oct 8;7(1):1282. doi: 10.1038/s42003-024-06974-0.
  PUBMED    39379572
REFERENCE   2  (bases 1 to 4601)
  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"; journalName: "Commun 
            Biol."; date: "2024-10-8"; volume: "7(1):1282. doi"; pages: " 
            10.1038/s42003-024-06974-0"
COMMENT     Sequence Label: pCMV-EGFP-nonstop
FEATURES             Location/Qualifiers
     source          1..4601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    591..671
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      673..682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(679..681,682..684,685..1395)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(679..681,682..684,685..1395)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(724..745)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(985..1004)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1332..1353
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1426..1445)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1480..1499
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1517..1972)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1654..1675)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1866..1885
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1999..2103
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2105..2462
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2105..2296
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2128..2148)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2313..2448
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2497..3291
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2497..3291
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2551..2570)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3161..3180
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3479..3498)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3523..3570
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3899..4487
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4388..4407
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      721 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      781 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      841 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
      901 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      961 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1021 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1081 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
     1141 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
     1201 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1261 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1321 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1381 gacgagctgt acaagcgatc gactagtacg cgtgttacaa ataaagcaat agcatcacaa
     1441 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     1501 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     1561 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     1621 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     1681 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     1741 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     1801 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     1861 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     1921 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc
     1981 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2041 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2101 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     2161 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     2221 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2281 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     2341 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     2401 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     2461 aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     2521 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     2581 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     2641 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     2701 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     2761 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     2821 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2881 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2941 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3001 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     3061 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3121 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3181 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3241 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     3301 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     3361 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     3421 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg
     3481 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     3541 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc
     3601 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt
     3661 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg
     3721 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt
     3781 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     3841 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3901 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3961 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4021 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4081 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4141 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4201 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4261 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     4321 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     4381 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     4441 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     4501 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     4561 tatcccctga ttctgtggat aaccgtatta ccgccatgca t
//
