LOCUS       sequence-478876-        6104 bp    DNA     circular SYN 17-JUL-2025
DEFINITION  Expresses EGFP and mCherry in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6104)
  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 6104)
  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-EGFP-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..6104
                     /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..2508
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1943..1961)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2211..2230
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     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"
     rep_origin      complement(3667..4255)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3747..3766)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4426..5217,5218..5286))
                     /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     5049..5068
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5287..5391)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5417..5872)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5554..5575)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5766..5785
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5944..6073
                     /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 gcgcgggccg gggggggggg
      421 gggggggggg gggggggggg 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 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg
     1861 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     1921 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc
     1981 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc
     2041 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     2101 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc
     2161 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg
     2221 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc
     2281 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag
     2341 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac
     2401 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc
     2461 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtagta 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 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3541 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3601 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3661 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3721 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3781 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3841 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3901 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3961 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4021 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4081 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4141 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4201 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4261 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4321 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4381 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4441 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4501 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4561 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4621 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4681 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4741 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4801 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4861 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4921 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4981 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5041 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5101 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5161 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5221 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5281 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5341 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5401 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     5461 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     5521 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     5581 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     5641 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     5701 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     5761 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     5821 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     5881 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     5941 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
     6001 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
     6061 actaggggtt ccttgtagtt aatgattaac ccgccatgct actt
//
