LOCUS       sequence-348947-        3527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Site-specific mutagenesis of GFP for the purposes of creating a 
            site-specific hypoxanthine lesion for the purposes of measuring MPG 
            activity via Host Cell Reactivation (HCR)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3527)
  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 3527)
  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_GFP_C289T
FEATURES             Location/Qualifiers
     source          1..3527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     178..197
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     437..458
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      500..509
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          526..658
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     promoter        complement(750..953)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(779..799)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        954..1333
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     CDS             1527..2321
                     /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             1527..2321
                     /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(1581..1600)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2191..2210
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      2402..2990
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2891..2910
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3188..3269
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3225..3244)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3279..3298
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(3311..3527,1..497,498..500,501..503))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQECTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3356..3377)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 atcttgaagt tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg
       61 ctgttgtagt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
      121 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
      181 ttgtagttgc cgtcgtcctt gaagaagatg gtgcactcct ggacgtagcc ttcgggcatg
      241 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
      301 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
      361 atgaacttca gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg
      421 aacttgtggc cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac
      481 agctcctcgc ccttgctcac catggtggca gcgctagcgg tacccctgtg gagagaaagg
      541 caaagtggat gtcagtaaga ccaataggtg cctatcagaa acgcaagaat cttctctgtc
      601 tcgacaagcc cagtttctat tggcctcctt aaacctgtct tgtaaccttg atacttacct
      661 gtgatcagtc gagcactgac tgcgttagca atttaactgt gataaactac cgcaataaag
      721 ctactagtga tctgacggtt cactaaacga cctctgctta tatagacctc ccaccgtaca
      781 cgcctaccgc ccatttgcgt caacggggcg gggttattac gacattttgg aaagtcccgt
      841 tgattttggt gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc
      901 cgtgagtcaa accgctatcc acgcccattg gtgtactgcc aaaaccgcat caccatggta
      961 atagcgatga ctaatacgta gatgtactgc caagtaggaa agtcccgtaa ggtcatgtac
     1021 tgggcataat gccaggcggg ccatttaccg tcattgacgt caataggggg cggacttggc
     1081 atatgataca cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga
     1141 cgtcaatgga aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat
     1201 gggcgggggt cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg
     1261 aactccatat atgggctatg aactaatgac cccgtaattg attactatta ataactagtc
     1321 aataatcaat gccaacatgg cggtcatatt ggacatgagc caatataaat gtacatatta
     1381 tgatatagat acaacgtatg caatggccaa tagccaatat tgatttatgc tatataacca
     1441 atgaataata tggctaatgg ccaatattga catgcatggg aggagaccgg gacgagacga
     1501 accctgataa atgcttcaat aatattatga ttgaacaaga tggattgcac gcaggttctc
     1561 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     1621 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     1681 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     1741 cgacgggcgt tccttgcgca gctgtactcg acgttgtcac tgaagcggga agggactggc
     1801 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     1861 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     1921 cattcgacca ccaagcgaaa catcgcatcg agagagcacg tactcggatg gaagccggtc
     1981 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     2041 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     2101 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     2161 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     2221 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     2281 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agacggcatg accaaaatcc
     2341 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     2401 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     2461 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     2521 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     2581 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     2641 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     2701 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     2761 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     2821 ggagaaaggc ggacaggtat cccgtaagcg gcagggtcgg aacaggagag cgcacgaggg
     2881 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     2941 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3001 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg tcacgtgagg
     3061 ccttaacgtc tcgccctttg gtctccccct cttaagtacc acatttgtag aggttttact
     3121 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     3181 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     3241 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcga
     3301 gctcaagctt tcacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga
     3361 actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg
     3421 tgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct
     3481 ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggcgg
//
