LOCUS       sequence-105669-        2329 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  Expression of the Azure (blue) spectral variant of eGFP in bacteria 
            and in mammalian cells. Could be used as a reporter for quantifying 
            gene targeting and recombineering in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 2329)
  AUTHORS   Valledor M, Hu Q, Schiller P, Myers RS
  TITLE     Fluorescent protein engineering by in vivo site-directed 
            mutagenesis.
  JOURNAL   IUBMB Life. 2012 Aug;64(8):684-9. doi: 10.1002/iub.1041. Epub 2012 
            May 28.
  PUBMED    22639380
REFERENCE   2  (bases 1 to 2329)
  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: "10.1002/iub.1041"; 
            journalName: "IUBMB Life"; date: "2012-08"; volume: "64"; issue: 
            "8"; pages: "684-9"
COMMENT     Sequence Label: pDual-eGFP(H66)
FEATURES             Location/Qualifiers
     source          1..2329
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     intron          35..977
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     934..954
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1038..1057
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1038..1056
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1057..1081
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1096..1118
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1137..1154
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1164..1181
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1185..1217
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     regulatory      1236..1245
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1242..1244,1245..1247,1248..1958)
                     /codon_start=1
                     /product="enhanced blue variant of GFP"
                     /label=EBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1242..1244,1245..1247,1248..1958)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1287..1308)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1548..1567)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1895..1916
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tctttttcgc aacgggtttg ccgccagaac acaggtaagt gccgtgtgtg gttcccgcgg
       61 gcctggcctc tttacgggtt atggcccttg cgtgccttga attacttcca cgcccctggc
      121 tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc
      181 cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg
      241 gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc
      301 tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg
      361 taaatgcggg ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg
      421 gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga
      481 gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc
      541 cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg
      601 aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg
      661 gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg
      721 cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct
      781 tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca
      841 ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat
      901 ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt
      961 tttttcttcc atttcaggtg tcgtgaggaa ttctgcagtc gacggtaccg cgggcccaga
     1021 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     1081 ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc agcagccatc
     1141 atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatggct agcatgactg
     1201 gtggacagca aatgggtcgc ggggatccac cggtcgccac catggtgagc aagggcgagg
     1261 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     1321 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     1381 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgaccc
     1441 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     1501 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     1561 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     1621 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     1681 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     1741 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     1801 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     1861 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     1921 ccgccgggat cactctcggc atggacgagc tgtacaagtc cggactcaga tccgacctcg
     1981 agacctagaa aaacatggag caatcacaag tagcaataca gcagctaaca atgctgcttg
     2041 tgcctggcta gaagcacaag aggaggaaga ggtgggtttt ccagtcacac ctcangtacc
     2101 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg
     2161 gggactggaa gggctaattc actcccaaag aagacaagat atccttgatc tgtggatcta
     2221 ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag gggtcagata
     2281 tccactgacc tttggatggt gctacaagct agtaccagtt gagccagat
//
