LOCUS       sequence-339583-        6901 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses an mCherry-2A-eGFP dual reporter that contains a UAG codon
            in position 58 (W58X) of eGFP, in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6901)
  AUTHORS   Montiel-Gonzalez MF, Vallecillo-Viejo IC, Rosenthal JJ
  TITLE     An efficient system for selectively altering genetic information 
            within mRNAs.
  JOURNAL   Nucleic Acids Res. 2016 Dec 1;44(21):e157. doi: 10.1093/nar/gkw738. 
            Epub 2016 Aug 23.
  PUBMED    27557710
REFERENCE   2  (bases 1 to 6901)
  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: "Nucleic 
            Acids Res."; date: "2016-12-1"; volume: "44(21):e157. doi"; pages: "
            10.1093/nar/gkw738. Epub 2016 Aug 23"
COMMENT     Sequence Label: mCherry-2A-eGFP(W58X) pcDNA3.1(-)
FEATURES             Location/Qualifiers
     source          1..6901
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      927..936
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1650..1706
                     /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             1707..1730
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2034..2053)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2492..2509)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2498..2722
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2768..3196
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2855..2874)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3065..3086
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3205..3225)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3210..3539
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3390..3525
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3452..3471
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3606..4400
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3660..3679)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4270..4289
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4574..4695
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4611..4630)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4665..4684
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4744..4760)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4744..4760)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4757..4779)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4768..4784
                     /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(4792..4798,4799..4816,4817..4822))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4837..4858
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4975..4992)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5146..5734)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5226..5245)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5905..6696,6697..6765))
                     /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     6528..6547
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6766..6870)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga ctcgaggcca ccatggtgag caagggcgag gaggacaaca
      961 tggccatcat taaggagttc atgaggttca aggtccacat ggagggaagc gtgaacggcc
     1021 acgagttcga gatcgaggga gagggcgagg gcagacctta cgagggcacc cagaccgcca
     1081 agctgaaggt caccaaggga ggccccctcc cctttgcttg ggacatcctg tccccccagt
     1141 tcatgtacgg cagcaaggcc tatgtcaagc accccgccga tatccccgac tacctgaagc
     1201 tgtccttccc cgagggcttc aagtgggaga gggtgatgaa cttcgaggac ggcggcgtgg
     1261 tcaccgtgac acaggactcc agcctccagg acggcgagtt catctacaag gtgaagctga
     1321 ggggcaccaa cttccctagc gacggccctg tcatgcagaa gaagaccatg ggatgggagg
     1381 ccagctccga gaggatgtac cccgaggacg gcgctctgaa gggcgagatc aagcagaggc
     1441 tcaagctgaa ggacggcggc cactacgacg ccgaggtgaa gaccacctac aaagccaaga
     1501 agcccgtgca actgcccggc gcctacaacg tcaacatcaa gctcgacatc acctcccaca
     1561 acgaggacta caccatcgtg gagcaatacg agagggccga gggaagacac agcacaggcg
     1621 gcatggacga gctgtacaag ggctccggcg ccaccaactt cagcctgctc aaacaagccg
     1681 gcgatgtgga ggagaaccct ggacccgact acaaggacga cgacgacaag gtgagcaaag
     1741 gcgaggagct gttcaccggc gtggtgccta tcctggtcga gctggacggc gatgtgaacg
     1801 gccacaagtt cagcgtgtcc ggcgaaggag agggcgatgc cacctatggc aagctgaccc
     1861 tgaagtttat ttgcactact ggaaaactac ctgttccata gccaacactt gtcactaccc
     1921 tgacatacgg cgtgcaatgc ttcagcaggt accccgacca catgaaacag cacgacttct
     1981 tcaagtccgc catgcccgag ggctacgtgc aggagaggac catctttttc aaagacgacg
     2041 gcaactacaa gaccagggct gaggtgaagt tcgagggcga taccctcgtg aacagaatcg
     2101 agctcaaggg catcgacttc aaggaggacg gcaacatcct cggccacaag ctggaataca
     2161 attacaactc ccacaacgtc tatatcatgg ccgataagca gaagaacggc attaaggtca
     2221 atttcaagat caggcacaac atcgaggacg gcagcgtgca gctggccgat cactaccagc
     2281 agaacacccc catcggagac ggacctgtgc tgctgcctga caaccattat ctgagcaccc
     2341 agtccgccct gtccaaggac cctaacgaga agagggacca catggtcctc ctggagttcg
     2401 tgaccgccgc tggcatcacc ctgggaatgg acgagctcta caaatgagga tccgagctcg
     2461 gtaccaagct taagtttaaa ccgctgatca gcctcgactg tgccttctag ttgccagcca
     2521 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2581 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2641 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2701 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     2761 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2821 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2881 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2941 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     3001 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3061 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3121 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3181 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3241 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3301 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3361 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3421 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     3481 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3541 agctcccggg agcttgtata tccattttcg gatctgatca agagacagga tgaggatcgt
     3601 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3661 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3721 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3781 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3841 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3901 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     3961 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     4021 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     4081 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc
     4141 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     4201 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     4261 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     4321 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4381 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4441 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4501 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4561 cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4621 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4681 catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc
     4741 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4801 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4861 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4921 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4981 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5041 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5101 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5161 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5221 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5281 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5341 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5401 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5461 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5521 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5581 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5641 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5701 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5761 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5821 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5881 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5941 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6001 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6061 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6121 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6181 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6241 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6301 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6361 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6421 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6481 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6541 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6601 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6661 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6721 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6781 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6841 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     6901 c
//
