LOCUS       sequence-418290-        6024 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double fluorescent stalling reporter K(AAA)20.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6024)
  AUTHORS   Juszkiewicz S, Hegde RS
  TITLE     Initiation of Quality Control during Poly(A) Translation Requires 
            Site-Specific Ribosome Ubiquitination.
  JOURNAL   Mol Cell. 2017 Feb 16;65(4):743-750.e4. doi: 
            10.1016/j.molcel.2016.11.039. Epub 2017 Jan 5.
  PUBMED    28065601
REFERENCE   2  (bases 1 to 6024)
  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.molcel.2016.11.039"; journalName: "Mol Cell"; date: 
            "2017-02-16- 16"; volume: "65"; issue: "4"; pages: "743-750.e4"
COMMENT     Sequence Label: pmGFP-P2A-K(AAA)20-P2A-RFP
FEATURES             Location/Qualifiers
     source          1..6024
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      547..556
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(553..555,556..558,559..1269)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(598..619)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(859..878)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1285..1341
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1348..1413
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1849..1905
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1912..2619
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2054..2072)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2322..2341
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2752..2873
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2789..2808)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2843..2862
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2880..3335)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3017..3038)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3229..3248
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3362..3466
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3468..3825
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3468..3659
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3491..3511)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3676..3811
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3860..4654
                     /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             3860..4654
                     /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(3914..3933)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4524..4543
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4842..4861)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4886..4933
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5262..5850
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5751..5770
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      601 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      661 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      721 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      781 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      841 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      901 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
      961 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1021 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1081 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1141 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1201 gatcacatgg tccttaagga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1261 ctgtacaagt ccggaggcag cggcgccacc aacttttccc tgctcaagca ggccggcgac
     1321 gtggaagaga atcccggccc cggtactgac tacaaagacc atgacggtga ttataaagat
     1381 catgacatcg attacaagga tgacgatgac aaggccggat ctcctggtcc gacccccagt
     1441 ggcactaacg tgggatcctc actctctgac gaggacttca aggctgtttt tggcatgacc
     1501 cgctctgcct ttgccaactt gcccttgtgg aaacagcaga acctcaagaa ggagaaagga
     1561 ctcttcggat cagggcgctc tcccagcaaa gcagtggccg cccgggcggc gggatccact
     1621 gtccggcaga ggaaaaatgc cagctgtggg acaaggagtg caggccgcac aacctcggca
     1681 ggcaccgggg ggatgtggcg attctacaca gaagattcac ctgggctcaa agttggccct
     1741 aggtcgaccg ccatggcgac caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     1801 aaaaaaaaaa aaaaaaaaaa acccggggga tccggtaccg gaagcggagc tactaacttc
     1861 agcctgctga agcaggctgg agacgtggag gagaaccctg gacctctcga ggtgagcaag
     1921 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     1981 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     2041 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     2101 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     2161 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     2221 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     2281 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     2341 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     2401 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     2461 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     2521 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     2581 cgccactcca ccggcggcat ggacgagctg tacaagtaaa ctggatccac cggatctaga
     2641 taactgatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     2701 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     2761 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     2821 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta
     2881 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     2941 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     3001 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     3061 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     3121 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     3181 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     3241 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     3301 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     3361 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     3421 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag
     3481 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3541 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3601 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3661 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3721 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     3781 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
     3841 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     3901 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     3961 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4021 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
     4081 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4141 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4201 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4261 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4321 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     4381 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     4441 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     4501 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     4561 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     4621 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     4681 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     4741 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     4801 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     4861 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     4921 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     4981 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     5041 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     5101 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5161 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5221 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5281 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5341 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5401 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5461 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5521 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5581 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5641 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5701 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5761 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5821 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5881 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5941 gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt
     6001 tcatagccca tatatggagt tccg
//
