LOCUS       sequence-478965-        6089 bp    DNA     circular SYN 17-JUL-2025
DEFINITION  Expresses EGFP and FusionRed in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6089)
  AUTHORS   Zhang H, Lesnov GD, Subach OM, Zhang W, Kuzmicheva TP, Vlaskina AV, 
            Samygina VR, Chen L, Ye X, Nikolaeva AY, Gabdulkhakov A, Papadaki S,
            Qin W, Borshchevskiy V, Perfilov MM, Gavrikov AS, Drobizhev M, 
            Mishin AS, Piatkevich KD, Subach FV
  TITLE     Bright and stable monomeric green fluorescent protein derived from 
            StayGold.
  JOURNAL   Nat Methods. 2024 Apr;21(4):657-665. doi: 
            10.1038/s41592-024-02203-y. Epub 2024 Feb 26.
  PUBMED    38409224
REFERENCE   2  (bases 1 to 6089)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-024-02203-y"; journalName: "Nat Methods"; date: 
            "2024-04"; volume: "21"; issue: "4"; pages: "657-665"
COMMENT     Sequence Label: pAAV-CAG-GFP-P2A-FusionRed
FEATURES             Location/Qualifiers
     source          1..6089
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        280..557
                     /label=chicken beta-actin promoter
     primer_bind     836..858
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     920..939
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      982..991
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(988..990,991..993,994..1704)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(988..990,991..993,994..1704)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1033..1054)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1294..1313)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1641..1662
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1729..1785
                     /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             1798..2496
                     /codon_start=1
                     /product="monomeric red fluorescent protein optimized for 
                     use as a fusion tag (Shemiakina et al., 2012)"
                     /label=FusionRed
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMPMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFMYGSRTFIKHPPGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKVRGVNFPANGPVMQKKTLGWEASTETMYPADGGLEGACDMAL
                     KLVGGGHLICNLETTYRSKKPATNLKMPGVYNVDHRLERIKEADDETYVEQHEVAVARY
                     STGGAGDGGK"
     misc_feature    2521..3109
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2574..2594)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2992..3003)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3152..3273
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3163..3182)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3217..3236
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3652..4240)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3732..3751)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4411..5202,5203..5271))
                     /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     5034..5053
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5272..5376)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5402..5857)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5539..5560)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5751..5770
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5929..6058
                     /label=AAV2 ITR
ORIGIN
        1 atctacgtag ccatgctcta ggaagagtac cattgacgtc aataatgacg tatgttccca
       61 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      121 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
      181 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
      241 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct
      301 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
      361 aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg
      421 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
      481 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
      541 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
      601 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
      661 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
      721 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
      781 gctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc
      841 ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc
      901 tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa
      961 gaattggatc cggtacccta agccaccatg gtgagcaagg gcgaggagct gttcaccggg
     1021 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     1081 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     1141 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     1201 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     1261 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     1321 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     1381 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     1441 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     1501 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     1561 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     1621 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     1681 ctcggcatgg acgagctgta caagcgaccg gttcacgtgg gaagtggggc tacaaacttc
     1741 tcactgctga aacaggccgg ggatgtggag gaaaacccag gaccacacgt gactagtatg
     1801 gtgagcgagc tgattaagga gaacatgccc atgaagctgt acatggaggg caccgtgaac
     1861 aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
     1921 atgagaatca aggtcgtcga gggcggccct ctccccttcg ccttcgacat cctggctacc
     1981 agcttcatgt acggcagcag aaccttcatc aagcaccctc cgggcatccc cgacttcttt
     2041 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
     2101 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
     2161 gttagagggg tgaacttccc agccaacggc cctgtgatgc agaagaaaac actcggctgg
     2221 gaggcctcca ccgagacgat gtaccctgct gacggaggac tggaaggcgc atgtgacatg
     2281 gctctgaagc tcgtgggagg aggacacctg atctgcaacc ttgagacaac atacagatcc
     2341 aagaaacccg ctacgaacct caagatgccc ggcgtctaca acgtggacca cagactggaa
     2401 agaatcaagg aggccgacga tgagacatac gtcgagcagc acgaggtggc tgtggccaga
     2461 tactctactg gtggcgctgg tgatggaggt aaataagaat tcgatatcaa gcttatcgat
     2521 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     2581 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     2641 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     2701 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     2761 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     2821 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     2881 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     2941 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3001 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     3061 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt
     3121 cgacccgggc ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc
     3181 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     3241 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     3301 tttcaggttc agggggagat gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     3361 ggtaaaatcg ataaggatct tcctagagca tggctacgta gataagtagc atggcgggtt
     3421 aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     3481 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3541 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3601 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3661 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3721 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3781 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3841 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3901 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3961 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4021 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4081 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4141 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4201 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4261 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4321 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4381 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4441 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4501 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4561 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4621 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4681 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4741 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4801 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4861 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4921 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4981 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5041 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5101 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5161 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5221 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5281 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5341 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5401 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     5461 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     5521 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     5581 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     5641 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     5701 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     5761 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     5821 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
     5881 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct gcgcgctcgc
     5941 tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc
     6001 tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttccttg
     6061 tagttaatga ttaacccgcc atgctactt
//
