LOCUS       sequence-369134-        6243 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses blue fluorescent protein mBlueberry2 and EGFP in mammalian
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6243)
  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 6243)
  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-mBlueberry2-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..6243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        281..558
                     /label=chicken beta-actin promoter
     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"
     primer_bind     complement(1130..1148)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             1717..1773
                     /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(1786..1788,1789..2502)
                     /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(1786..1788,1789..2499)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1828..1849)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2089..2108)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2436..2457
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2527..3115
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2580..2600)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2998..3009)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3158..3279
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3169..3188)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3223..3242
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3441..3570
                     /label=AAV2 ITR
     rep_origin      complement(3807..4395)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3887..3906)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4566..5357,5358..5426))
                     /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     5189..5208
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5427..5531)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5557..6012)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5694..5715)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5906..5925
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6084..6213
                     /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 rggggggggg
      421 gggggggggg gggggggggg sggggsgagg cggagaggtg 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 aggagaacaa cgtggccatc
     1021 atcaaggagt tcatgagatt caaggtgcac atggagggca gcgtgaacgg ccacgagttc
     1081 gagatcgagg gcgagggcga gggcagaccc tacgagggca cccagaccgc caagctgaag
     1141 gtgaccaagg gcggccccct gcccttcgcc tgggacatcc tgagccccca gttcctgttc
     1201 ggcagcaagg tgtacatcaa gcaccccgcc gacatccccg actacttcaa gctgagcttc
     1261 cccgagggct tcaagtggga gagagtgatg aacttcgagg acggcggcgt ggtgaccgtg
     1321 acccaggaca gcagcctgca ggacggcgtg ttcatctaca aggtgaagct gagaggcacc
     1381 aacttcccca gcgacggccc cgtgatgcag aagaagacca tgggctggga ggccttcagc
     1441 gagagaatgt accccgagga cggcgccctg aagagcgaga tcaagaccag actgaagctg
     1501 aaggacggcg gccactacga cgccgaggtg aagaccacct acaaggccaa gaagcccgtg
     1561 cagctgcccg gcgcctacaa cgtgaacatc aagctggaca tcgtgagcca caacgaggac
     1621 tacaccatcg tggagcagta cgagagagcc gagggcagac acagcaccgg cggcatggac
     1681 gagctgtaca agcgaccggt tcacgtggga agtggggcta caaacttctc actgctgaaa
     1741 caggccgggg atgtggagga aaacccagga ccacacgtga ctagtgtgag caagggcgag
     1801 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     1861 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     1921 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     1981 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     2041 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     2101 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     2161 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     2221 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     2281 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     2341 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     2401 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     2461 gccgccggga tcactctcgg catggacgag ctgtacaagt aagaattcga tatcaagctt
     2521 atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     2581 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     2641 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     2701 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     2761 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     2821 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     2881 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg
     2941 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     3001 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     3061 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga
     3121 taccgtcgac ccgggcggcc gcttcgagca gacatgataa gatacattga tgagtttgga
     3181 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     3241 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     3301 tttatgtttc aggttcaggg ggagatgtgg gaggtttttt aaagcaagta aaacctctac
     3361 aaatgtggta aaatcgataa ggatcttcct agagcatggc tacgtagata agtagcatgg
     3421 cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg
     3481 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg
     3541 ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat gaatcggcca acgcgcgggg
     3601 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3661 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3721 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3781 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3841 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3901 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3961 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4021 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4081 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4141 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4201 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4261 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4321 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4381 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4441 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4501 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4561 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4621 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4681 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4741 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4801 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4861 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     4921 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     4981 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5041 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5101 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5161 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5221 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5281 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5341 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5401 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5461 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5521 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     5581 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     5641 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     5701 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     5761 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     5821 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     5881 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     5941 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     6001 tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     6061 gggcctcttc gctattacgc cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa
     6121 agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
     6181 agggagtggc caactccatc actaggggtt ccttgtagtt aatgattaac ccgccatgct
     6241 act
//
