LOCUS       sequence-371193-        6234 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses blue fluorescent protein mTagBFP2 and EGFP in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6234)
  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 6234)
  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-mTagBFP2-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..6234
                     /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"
     CDS             985..1683
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             1708..1764
                     /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(1777..1779,1780..2493)
                     /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(1777..1779,1780..2490)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1819..1840)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2080..2099)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2427..2448
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2518..3106
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2571..2591)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2989..3000)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3149..3270
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3160..3179)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3214..3233
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3432..3561
                     /label=AAV2 ITR
     rep_origin      complement(3798..4386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3878..3897)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4557..5348,5349..5417))
                     /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     5180..5199
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5418..5522)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5548..6003)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5685..5706)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5897..5916
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6075..6204
                     /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 gggggsgggg 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 ccagtaccgc caccatgagc gagctgatta aggagaacat gcacatgaag
     1021 ctgtacatgg agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc
     1081 aagccctacg agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc
     1141 ttcgccttcg acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac
     1201 acccagggca tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga
     1261 gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac
     1321 ggctgcctca tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg
     1381 atgcagaaga aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc
     1441 ggcctggaag gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca
     1501 aacgccaaga ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc
     1561 tactatgtgg actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag
     1621 cagcacgagg tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt
     1681 aatcgaccgg ttcacgtggg aagtggggct acaaacttct cactgctgaa acaggccggg
     1741 gatgtggagg aaaacccagg accacacgtg actagtgtga gcaagggcga ggagctgttc
     1801 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1861 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1921 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1981 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2041 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2101 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2161 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2221 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2281 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2341 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     2401 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2461 atcactctcg gcatggacga gctgtacaag taagaattcg atatcaagct tatcgataat
     2521 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     2581 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     2641 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     2701 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     2761 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     2821 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     2881 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     2941 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3001 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3061 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     3121 cccgggcggc cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     3181 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     3241 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     3301 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     3361 aaaatcgata aggatcttcc tagagcatgg ctacgtagat aagtagcatg gcgggttaat
     3421 cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3481 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     3541 agtgagcgag cgagcgcgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3601 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3661 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3721 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3781 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3841 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3901 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3961 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4021 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4081 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4141 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4201 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4261 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4321 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4381 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4441 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4501 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4561 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4621 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4681 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4741 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4801 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4861 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4921 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4981 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5041 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5101 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5161 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5221 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5281 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5341 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5401 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5461 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5521 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     5581 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     5641 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     5701 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     5761 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     5821 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     5881 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     5941 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     6001 cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     6061 cgctattacg ccagctgcgc gctcgctcgc tcactgaggc cgcccgggca aagcccgggc
     6121 gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg
     6181 ccaactccat cactaggggt tccttgtagt taatgattaa cccgccatgc tact
//
