LOCUS       sequence-368139-        5986 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for a Y66H mutation to convert a GFP to BFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5986)
  AUTHORS   Schene IF, Joore IP, Baijens JHL, Stevelink R, Kok G, Shehata S, 
            Ilcken EF, Nieuwenhuis ECM, Bolhuis DP, van Rees RCM, Spelier SA, 
            van der Doef HPJ, Beekman JM, Houwen RHJ, Nieuwenhuis EES, Fuchs SA
  TITLE     Mutation-specific reporter for optimization and enrichment of prime 
            editing.
  JOURNAL   Nat Commun. 2022 Mar 1;13(1):1028. doi: 10.1038/s41467-022-28656-3.
  PUBMED    35232966
REFERENCE   2  (bases 1 to 5986)
  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: "Nat 
            Commun."; date: "2022-03-1"; volume: "13(1):1028. doi"; pages: " 
            10.1038/s41467-022-28656-3"
COMMENT     Sequence Label: fluoPEER-GFP>BFP
FEATURES             Location/Qualifiers
     source          1..5986
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      110..698
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     599..618
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        873..1176
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1177..1380
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1331..1351
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1419..1428
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1425..1427,1428..1430,1431..2141)
                     /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(1470..1491)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1731..1750)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2157..2213
                     /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             2220..2285
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2683..2739
                     /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             2746..3453
                     /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(2888..2906)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3156..3175
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3586..3707
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3623..3642)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3677..3696
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3714..4169)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3851..3872)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4063..4082
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4196..4300
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4302..4659
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4302..4493
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4325..4345)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4510..4645
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4694..5488
                     /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             4694..5488
                     /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(4748..4767)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5358..5377
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5676..5695)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5720..5767
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
       61 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
      121 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
      181 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
      241 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
      301 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
      361 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
      421 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
      481 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
      541 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
      601 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
      661 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
      721 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
      781 attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg
      841 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
      901 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
      961 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1021 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     1081 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     1141 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     1201 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     1261 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     1321 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     1381 ggtttagtga accgtcagat ccgctagcgc taccggtcgc caccatggtg agcaagggcg
     1441 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1501 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     1561 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     1621 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1681 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     1741 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1801 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1861 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     1921 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1981 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     2041 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtccttaag gagttcgtga
     2101 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtccggaggc agcggcgcca
     2161 ccaacttttc cctgctcaag caggccggcg acgtggaaga gaatcccggc cccggtactg
     2221 actacaaaga ccatgacggt gattataaag atcatgacat cgattacaag gatgacgatg
     2281 acaaggccgg atctcctggt ccgaccccca gtggcactaa cgtgggatcc tcactctctg
     2341 acgaggactt caaggctgtt tttggcatga cccgctctgc ctttgccaac ttgcccttgt
     2401 ggaaacagca gaacctcaag aaggagaaag gactcttcgg atcagggcgc tctcccagca
     2461 aagcagtggc cgcccgggcg gcgggatcca ctgtccggca gaggaaaaat gccagctgtg
     2521 ggacaaggag tgcaggccgc acaacctcgg caggcaccgg ggggatgtgg cgattctaca
     2581 cagaagattc acctgggctc aaagttggcc ctaggtcgac cgtgaccacc ctgaacctac
     2641 ggcgtgcagt gcttcagccg ctaccccggt accggaagcg gagctactaa cttcagcctg
     2701 ctgaagcagg ctggagacgt ggaggagaac cctggacctc tcgaggtgag caagggcgag
     2761 gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc
     2821 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc
     2881 cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg
     2941 tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac
     3001 tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac
     3061 ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag
     3121 gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg
     3181 ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc
     3241 aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac
     3301 aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc
     3361 acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac
     3421 tccaccggcg gcatggacga gctgtacaag taaactggat ccaccggatc tagataactg
     3481 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
     3541 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
     3601 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3661 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgtaaattgt
     3721 aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa
     3781 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt
     3841 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa
     3901 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag
     3961 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt
     4021 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg
     4081 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc
     4141 cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg
     4201 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4261 accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag
     4321 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     4381 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     4441 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     4501 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4561 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4621 tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg
     4681 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     4741 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     4801 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     4861 cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     4921 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     4981 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     5041 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     5101 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     5161 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     5221 gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     5281 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     5341 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     5401 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     5461 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     5521 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     5581 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     5641 ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg
     5701 gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg
     5761 tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga
     5821 ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg
     5881 ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt
     5941 tactcatata tactttagat tgatttaaaa cttcattttt aattta
//
