LOCUS       sequence-371196-        6252 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses blue fluorescent protein EBFP2 and EGFP in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6252)
  AUTHORS   Papadaki S, Wang X, Wang Y, Zhang H, Jia S, Liu S, Yang M, Zhang D, 
            Jia JM, Koster RW, Namikawa K, Piatkevich KD
  TITLE     Dual-expression system for blue fluorescent protein optimization.
  JOURNAL   Sci Rep. 2022 Jun 17;12(1):10190. doi: 10.1038/s41598-022-13214-0.
  PUBMED    35715437
REFERENCE   2  (bases 1 to 6252)
  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"; journalName: "Sci Rep."; 
            date: "2022-06-17"; volume: "12(1):10190. doi"; pages: " 
            10.1038/s41598-022-13214-0"
COMMENT     Sequence Label: pAAV-CAG-EBFP2-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..6252
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     837..859
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     921..940
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      979..988
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(985..987,988..990,991..1701)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     CDS             1726..1782
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(1795..1797,1798..2511)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1795..1797,1798..2508)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    2536..3124
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2589..2609)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3007..3018)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3167..3288
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3178..3197)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3232..3251
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3450..3579
                     /label=AAV2 ITR
     rep_origin      complement(3816..4404)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3896..3915)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4575..5366,5367..5435))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5198..5217
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5436..5540)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5566..6021)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5703..5724)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5915..5934
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6093..6222
                     /label=AAV2 ITR
ORIGIN
        1 tatctacgta gccatgctct aggaagagta ccattgacgt caataatgac gtatgttccc
       61 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      121 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      181 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      241 tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag ccccacgttc
      301 tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt atttattttt
      361 taattatttt gtgcagcgat gggggcgggg gggggggggg ggcgcgcgcc rggsggggsg
      421 gggsggggsg gggggggggg sggggsgrgg sggrgaggkg cggcggcagc caatcagagc
      481 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
      541 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
      601 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
      661 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct
      721 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc
      781 ggctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca gggcggggtt
      841 cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc atgccttctt
      901 ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc attttggcaa
      961 agaattggat ccggtaccgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     1021 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgaggggc
     1081 gagggcgagg gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc
     1141 aagctgcccg tgccctggcc caccctcgtg accaccctga gccacggcgt gcagtgcttc
     1201 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1261 tacgtccagg agcgcaccat cttcttcaag gacgacggca cctacaagac ccgcgccgag
     1321 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcgt cgacttcaag
     1381 gaggacggca acatcctggg gcacaagctg gagtacaact tcaacagcca caacatctat
     1441 atcatggccg tcaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacgtg
     1501 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1561 cccgtgctgc tgcccgacag ccactacctg agcacccagt ccgtgctgag caaagacccc
     1621 aacgagaagc gcgatcacat ggtcctgctg gagttccgca ccgccgccgg gatcactctc
     1681 ggcatggacg agctgtacaa gcgaccggtt cacgtgggaa gtggggctac aaacttctca
     1741 ctgctgaaac aggccgggga tgtggaggaa aacccaggac cacacgtgac tagtgtgagc
     1801 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1861 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1921 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1981 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2041 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2101 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2161 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2221 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2281 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2341 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2401 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2461 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta agaattcgat
     2521 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     2581 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     2641 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     2701 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     2761 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     2821 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     2881 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     2941 tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3001 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     3061 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     3121 ccgcatcgat accgtcgacc cgggcggccg cttcgagcag acatgataag atacattgat
     3181 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     3241 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     3301 tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa
     3361 aacctctaca aatgtggtaa aatcgataag gatcttccta gagcatggct acgtagataa
     3421 gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc
     3481 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     3541 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcattaatg aatcggccaa
     3601 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     3661 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     3721 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     3781 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     3841 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     3901 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     3961 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4021 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4081 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4141 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4201 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4261 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     4321 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     4381 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     4441 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     4501 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     4561 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     4621 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     4681 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     4741 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     4801 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     4861 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     4921 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     4981 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5041 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5101 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5161 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5221 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     5281 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     5341 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     5401 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     5461 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     5521 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta
     5581 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     5641 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     5701 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     5761 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     5821 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     5881 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     5941 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     6001 atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc
     6061 gatcggtgcg ggcctcttcg ctattacgcc agctgcgcgc tcgctcgctc actgaggccg
     6121 cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
     6181 cgcgcagaga gggagtggcc aactccatca ctaggggttc cttgtagtta atgattaacc
     6241 cgccatgcta ct
//
