LOCUS       sequence-348950-        3509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Site-specific mutagenesis of tagBFP for the purposes of creating a 
            clean double strand break for the purposes of measuring NHEJ 
            activity via Host Cell Reactivation (HCR)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3509)
  AUTHORS   Nagel ZD, Margulies CM, Chaim IA, McRee SK, Mazzucato P, Ahmad A, 
            Abo RP, Butty VL, Forget AL, Samson LD
  TITLE     Multiplexed DNA repair assays for multiple lesions and multiple 
            doses via transcription inhibition and transcriptional mutagenesis.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 May 6;111(18):E1823-32. doi: 
            10.1073/pnas.1401182111. Epub 2014 Apr 22.
  PUBMED    24757057
REFERENCE   2  (bases 1 to 3509)
  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: 
            "10.1073/pnas.1401182111"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2014-05-6- 6"; volume: "111"; issue: "18"; pages: "E1823-32"
COMMENT     Sequence Label: pMax_ScaI_BFP
FEATURES             Location/Qualifiers
     source          1..3509
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        77..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          676..808
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     CDS             837..1538
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(1545..1564)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    1574..1655
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1599..1618
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(1853..2441)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1933..1952)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2522..3316)
                     /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             complement(2522..3316)
                     /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(2633..2652)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3243..3262
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 ggcattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag aggtcgttta gtgaaccgtc
      601 agatcactag tagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg
      661 ctcgactgat cacaggtaag tatcaaggtt acaagacagg tttaaggagg ccaatagaaa
      721 ctgggcttgt cgagacagag aagattcttg cgtttctgat aggcacctat tggtcttact
      781 gacatccact ttgcctttct ctccacaggg gtaccgctag cagtactgct gccaccatga
      841 gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc
      901 atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga
      961 gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct
     1021 tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc
     1081 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc
     1141 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca
     1201 gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg
     1261 ccttcaccga gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc
     1321 tgaagctcgt gggcgggagc catctgatcg caaacatcaa gaccacatat agatccaaga
     1381 aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa
     1441 tcaaggaggc caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact
     1501 gcgacctccc tagcaaactg gggcacaagc ttaattgaga gctcgatgag tttggacaaa
     1561 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     1621 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     1681 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     1741 gtggtactta agagggggag accaaagggc gagacgttaa ggcctcacgt gacatgtgag
     1801 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     1861 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     1921 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     1981 ttccgaccct gccgcttacg ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2041 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2101 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2161 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2221 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2281 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2341 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2401 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2461 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgccgt
     2521 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     2581 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     2641 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     2701 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     2761 cgacgagatc ctcgccgtcg ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg
     2821 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     2881 tacgtgctct ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     2941 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     3001 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     3061 cagtgacaac gtcgagtaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     3121 gcgctgcctc gtcttgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     3181 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     3241 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     3301 catcttgttc aatcataata ttattgaagc atttatcagg gttcgtctcg tcccggtctc
     3361 ctcccatgca tgtcaatatt ggccattagc catattattc attggttata tagcataaat
     3421 caatattggc tattggccat tgcatacgtt gtatctatat cataatatgt acatttatat
     3481 tggctcatgt ccaatatgac cgccatgtt
//
