LOCUS       sequence-190529-        5423 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EGFP-mCherry Met71GAA reporter for methionine misincorporation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5423)
  AUTHORS   Gomes AC, Kordala AJ, Strack R, Wang X, Geslain R, Delaney K, Clark 
            WC, Keenan R, Pan T
  TITLE     A dual fluorescent reporter for the investigation of methionine 
            mistranslation in live cells.
  JOURNAL   RNA. 2016 Mar;22(3):467-76. doi: 10.1261/rna.054163.115. Epub 2016 
            Jan 4.
  PUBMED    26729921
REFERENCE   2  (bases 1 to 5423)
  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: 
            "10.1261/rna.054163.115"; journalName: "RNA"; date: "2016-03"; 
            volume: "22"; issue: "3"; pages: "467-76"
COMMENT     Sequence Label: GAA-Pan
FEATURES             Location/Qualifiers
     source          1..5423
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             98..892
                     /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             98..892
                     /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(152..171)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     762..781
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1080..1099)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1124..1171
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1500..2088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1989..2008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2263..2566
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2567..2770
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2721..2741
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2837..2846
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2843..2845,2846..2848,2849..3559)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2888..2909)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3149..3168)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3496..3517
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      3575..3584
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3581..4291
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFEYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3726..3744)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3994..4013
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    4413..4534
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4450..4469)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4504..4523
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4541..4996)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4678..4699)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4890..4909
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5023..5127
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(5129..5423,1..63)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5129..5320
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5152..5172)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      join(5337..5423,1..49)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
       61 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
      121 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
      181 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
      241 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
      301 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
      361 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
      421 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
      481 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
      541 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
      601 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
      661 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
      721 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
      781 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
      841 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
      901 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
      961 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     1021 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     1081 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     1141 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     1201 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     1261 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     1321 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     1381 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     1441 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     1501 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     1561 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     1621 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     1681 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     1741 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     1801 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     1861 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     1921 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1981 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2041 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2101 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     2161 ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc
     2221 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     2281 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     2341 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     2401 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     2461 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     2521 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     2581 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     2641 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     2701 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     2761 aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggactc agatctcgag
     2821 ctcaagcttc gaattcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt
     2881 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2941 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     3001 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     3061 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     3121 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     3181 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     3241 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     3301 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     3361 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     3421 cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca aagaccccaa
     3481 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3541 catggacgag ctgtacaagt cggatccact agtcgccacc atggtgagca agggcgagga
     3601 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt
     3661 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     3721 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc
     3781 ccctcagttc gaatacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     3841 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     3901 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     3961 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agactatggg
     4021 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     4081 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa
     4141 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac
     4201 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc
     4261 caccggcggc atggacgagc tgtacaagta agcggccgcg actctagatc ataatcagcc
     4321 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     4381 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     4441 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4501 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     4561 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4621 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     4681 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4741 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     4801 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4861 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4921 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4981 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5041 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5101 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     5161 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     5221 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     5281 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     5341 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     5401 tttatgcaga ggccgaggcc gcc
//
