LOCUS       sequence-348956-        3488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Site-specific mutagenesis of mPlum for the purposes of creating a 
            site-specific O6-MeG lesion for the purposes of measuring MGMT 
            activity via Host Cell Reactivation (HCR)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3488)
  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 3488)
  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_mPlum_C207G/T208C
FEATURES             Location/Qualifiers
     source          1..3488
                     /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..1131
                     /codon_start=1
                     /product="far-red monomeric derivative of DsRed fluorescent
                     protein (Wang et al., 2004)"
                     /label=mPlum
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKEHMEGSVNGHEFEIEGEGEGRPYEGTQTA
                     RLKVTKGGPLPFAWDILSPQIMYGPKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG
                     VVTVTQDSSLQDGEFIYKVKVRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEMK
                     MRLRLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRH
                     STGA"
     primer_bind     852..871
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1046..1069
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1144..1163)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    1173..1254
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1198..1217
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(1452..2040)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1532..1551)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2121..2915)
                     /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(2121..2915)
                     /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(2232..2251)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2842..2861
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     enhancer        3109..3488
                     /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 ggtcatcaag
      481 gagttcatgc gcttcaagga gcacatggag ggctccgtga acggccacga gttcgagatc
      541 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaggct gaaggtgacc
      601 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagatcat gtacgggccc
      661 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
      721 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
      781 gactcctccc tgcaggacgg cgagttcatc tacaaggtga aggtgcgcgg caccaacttc
      841 ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg
      901 atgtaccccg aggacggcgc cctgaagggc gagatgaaga tgaggctgag gctgaaggac
      961 ggcggccact acgacgccga ggtcaagacc acctacatgg ccaagaagcc cgtgcagctg
     1021 cccggcgcct acaagaccga catcaagctg gacatcacct cccacaacga ggactacacc
     1081 atcgtggaac agtacgagcg cgccgagggc cgccactcca ccggcgccta gaagcttgag
     1141 ctcgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     1201 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     1261 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
     1321 aagtaaaacc tctacaaatg tggtacttaa gagggggaga ccaaagggcg agacgttaag
     1381 gcctcacgtg acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     1441 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     1501 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     1561 tccctcgtgc gctctcctgt tccgaccctg ccgcttacgg gatacctgtc cgcctttctc
     1621 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1681 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1741 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1801 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1861 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     1921 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1981 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2041 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2101 gggattttgg tcatgccgtc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
     2161 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
     2221 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
     2281 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
     2341 caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgctcgc cttgagcctg
     2401 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
     2461 agaccggctt ccatccgagt acgtgctctc tcgatgcgat gtttcgcttg gtggtcgaat
     2521 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
     2581 ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc
     2641 agccagtccc ttcccgcttc agtgacaacg tcgagtacag ctgcgcaagg aacgcccgtc
     2701 gtggccagcc acgatagccg cgctgcctcg tcttgcagtt cattcagggc accggacagg
     2761 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
     2821 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
     2881 ggagaacctg cgtgcaatcc atcttgttca atcataatat tattgaagca tttatcaggg
     2941 ttcgtctcgt cccggtctcc tcccatgcat gtcaatattg gccattagcc atattattca
     3001 ttggttatat agcataaatc aatattggct attggccatt gcatacgttg tatctatatc
     3061 ataatatgta catttatatt ggctcatgtc caatatgacc gccatgttgg cattgattat
     3121 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     3181 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     3241 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     3301 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     3361 tgccaagtcc gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     3421 agtacatgac cttacgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     3481 ttaccatg
//
