LOCUS       sequence-348954-        3518 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Site-specific mutagenesis of mOrange for the purposes of creating a 
            site-specific G:G mispair for the purposes of measuring MMR activity
            via Host Cell Reactivation (HCR)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3518)
  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 3518)
  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_mOrange_G299C
FEATURES             Location/Qualifiers
     source          1..3518
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          296..428
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      445..454
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             451..1161
                     /codon_start=1
                     /product="orange monomeric derivative of DsRed fluorescent 
                     protein (Shanet et al., 2004)"
                     /label=mOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTAKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYFKLSFPEGFKWEPVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKMRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVGIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     864..883
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1174..1193)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    1203..1284
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1228..1247
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(1482..2070)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1562..1581)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2151..2945)
                     /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(2151..2945)
                     /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(2262..2281)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2872..2891
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     enhancer        3139..3518
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta caccaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag aggtcgttta gtgaaccgtc agatcactag tagctttatt
      241 gcggtagttt atcacagtta aattgctaac gcagtcagtg ctcgactgat cacaggtaag
      301 tatcaaggtt acaagacagg tttaaggagg ccaatagaaa ctgggcttgt cgagacagag
      361 aagattcttg cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct
      421 ctccacaggg gtaccgctag cgctgccacc atggtgagca agggcgagga gaataacatg
      481 gccatcatca aggagttcat gcgcttcaag gtgcgcatgg agggctccgt gaacggccac
      541 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggctttca gaccgctaag
      601 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
      661 acctacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttcaagctg
      721 tccttccccg agggcttcaa gtgggagccc gtgatgaact tcgaggacgg cggcgtggtg
      781 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
      841 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
      901 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gatgaggctg
      961 aagctgaagg acggcggcca ctacacctcc gaggtcaaga ccacctacaa ggccaagaag
     1021 cccgtgcagc tgcccggcgc ctacatcgtc ggcatcaagt tggacatcac ctcccacaac
     1081 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     1141 atggacgagc tgtacaagta gaagcttgag ctcgatgagt ttggacaaac cacaactaga
     1201 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
     1261 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
     1321 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtacttaa
     1381 gagggggaga ccaaagggcg agacgttaag gcctcacgtg acatgtgagc aaaaggccag
     1441 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     1501 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1561 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1621 ccgcttacgg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1681 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1741 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1801 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1861 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     1921 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1981 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     2041 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     2101 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgccgtc tcagaagaac
     2161 tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc
     2221 acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac
     2281 gctatgtcct gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag
     2341 cggccatttt ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc
     2401 tcgccgtcgg gcatgctcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga
     2461 tgctcttcgt ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctctc
     2521 tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc
     2581 cgccgcattg catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg
     2641 agatcctgcc ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg
     2701 tcgagtacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg
     2761 tcttgcagtt cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc
     2821 tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca
     2881 tagccgaata gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca
     2941 atcataatat tattgaagca tttatcaggg ttcgtctcgt cccggtctcc tcccatgcat
     3001 gtcaatattg gccattagcc atattattca ttggttatat agcataaatc aatattggct
     3061 attggccatt gcatacgttg tatctatatc ataatatgta catttatatt ggctcatgtc
     3121 caatatgacc gccatgttgg cattgattat tgactagtta ttaatagtaa tcaattacgg
     3181 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     3241 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     3301 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     3361 cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg
     3421 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt
     3481 ggcagtacat ctacgtatta gtcatcgcta ttaccatg
//
