LOCUS       sequence-202809-        6000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double fluorescent stalling reporter K(AAA)12.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6000)
  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 6000)
  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)12-P2A-RFP
FEATURES             Location/Qualifiers
     source          1..6000
                     /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             1825..1881
                     /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             1888..2595
                     /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(2030..2048)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2298..2317
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2728..2849
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2765..2784)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2819..2838
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2856..3311)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2993..3014)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3205..3224
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3338..3442
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3444..3801
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3444..3635
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3467..3487)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3652..3787
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3836..4630
                     /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             3836..4630
                     /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(3890..3909)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4500..4519
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4818..4837)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4862..4909
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5238..5826
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5727..5746
                     /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 aaaaaaaccc
     1801 gggggatccg gtaccggaag cggagctact aacttcagcc tgctgaagca ggctggagac
     1861 gtggaggaga accctggacc tctcgaggtg agcaagggcg aggaggataa catggccatc
     1921 atcaaggagt tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc
     1981 gagatcgagg gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag
     2041 gtgaccaagg gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac
     2101 ggctccaagg cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc
     2161 cccgagggct tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg
     2221 acccaggact cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc
     2281 aacttcccct ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc
     2341 gagcggatgt accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg
     2401 aaggacggcg gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg
     2461 cagctgcccg gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac
     2521 tacaccatcg tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac
     2581 gagctgtaca agtaaactgg atccaccgga tctagataac tgatcataat cagccatacc
     2641 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
     2701 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
     2761 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2821 ggtttgtcca aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt
     2881 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2941 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     3001 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     3061 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3121 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3181 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3241 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3301 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3361 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3421 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     3481 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3541 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3601 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3661 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3721 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     3781 ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat
     3841 tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta
     3901 tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca
     3961 ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga
     4021 cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga
     4081 cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct
     4141 cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg
     4201 gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga
     4261 gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca
     4321 tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga
     4381 ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg
     4441 cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc
     4501 gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt
     4561 gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga
     4621 gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca
     4681 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     4741 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4801 cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg
     4861 acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg
     4921 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     4981 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5041 gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag
     5101 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     5161 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     5221 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     5281 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     5341 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     5401 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     5461 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     5521 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     5581 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     5641 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     5701 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     5761 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     5821 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     5881 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
     5941 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
//
