LOCUS       sequence-368141-        5999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter for a 5 basepair deletion at the HEK3 locus..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5999)
  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 5999)
  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-HEK3_5bp-del
FEATURES             Location/Qualifiers
     source          1..5999
                     /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             2696..2752
                     /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             2759..3466
                     /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(2901..2919)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3169..3188
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3599..3720
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3636..3655)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3690..3709
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3727..4182)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3864..3885)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4076..4095
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4209..4313
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4315..4672
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4315..4506
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4338..4358)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4523..4658
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4707..5501
                     /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             4707..5501
                     /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(4761..4780)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5371..5390
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5689..5708)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5733..5780
                     /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 cttggggccc agactgagca
     2641 cgtgatggca gaggaaagga agccctgctt cctccagagg ggtaccggaa gcggagctac
     2701 taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac ctctcgaggt
     2761 gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct tcaaggtgca
     2821 catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc
     2881 ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc
     2941 ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga agcaccccgc
     3001 cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat
     3061 gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc aggacggcga
     3121 gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc ccgtaatgca
     3181 gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg acggcgccct
     3241 gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg acgctgaggt
     3301 caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca acgtcaacat
     3361 caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt acgaacgcgc
     3421 cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaaactg gatccaccgg
     3481 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     3541 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     3601 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     3661 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     3721 acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     3781 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     3841 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     3901 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     3961 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     4021 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     4081 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     4141 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     4201 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4261 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     4321 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     4381 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     4441 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     4501 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4561 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     4621 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     4681 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     4741 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     4801 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     4861 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     4921 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     4981 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     5041 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     5101 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     5161 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     5221 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     5281 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     5341 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     5401 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     5461 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     5521 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     5581 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     5641 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     5701 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     5761 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     5821 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     5881 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     5941 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaattta
//
