LOCUS       sequence-478888-        6139 bp    DNA     circular SYN 17-JUL-2025
DEFINITION  Expresses mNeonGreen and mCherry in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6139)
  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 6139)
  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-mNeonGreen-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     880..902
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     964..983
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1026..1035
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1032..1739
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1287..1296
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1764..1820
                     /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             1833..2543
                     /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(1978..1996)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2246..2265
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2571..3159
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2624..2644)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3042..3053)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3202..3323
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3213..3232)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3267..3286
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3702..4290)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3782..3801)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4461..5252,5253..5321))
                     /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     5084..5103
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5322..5426)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5452..5907)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5589..5610)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5801..5820
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5979..6108
                     /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 gggggggggc ggcgcccgtg cgtgcggggg
      421 gggggggggg gggggggggg gggggtgggg gtgggggggg gggggggggg gcgaggggcg
      481 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      541 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      601 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      661 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      721 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      781 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggctgtccgc
      841 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
      901 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
      961 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg gatccggtac
     1021 cctaagccac catggtgagc aagggcgagg aggataacat ggcctctctc ccagcgacac
     1081 atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg ggtcagggca
     1141 ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag ggtgacctcc
     1201 agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag tacctgccct
     1261 accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccggc taccaagtcc
     1321 atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc tacacctacg
     1381 agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct gctgacggtc
     1441 ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag acttacccca
     1501 acgacaaaac catcatcagt acctttaagt ggagttacac cacgggaaat ggcaagcgct
     1561 accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct aactatctga
     1621 agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag accgagctca
     1681 acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag ctgtacaagc
     1741 gaccggttca cgtgggaagt ggggctacaa acttctcact gctgaaacag gccggggatg
     1801 tggaggaaaa cccaggacca cacgtgacta gtatggtgag caagggcgag gaggataaca
     1861 tggccatcat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     1921 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     1981 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     2041 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     2101 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     2161 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     2221 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     2281 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     2341 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     2401 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     2461 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg
     2521 gcatggacga gctgtacaag tagtaagaat tcgatatcaa gcttatcgat aatcaacctc
     2581 tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc
     2641 tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca
     2701 ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg
     2761 tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca
     2821 ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg
     2881 cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg
     2941 acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg
     3001 ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg
     3061 accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc
     3121 ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacccgggc
     3181 ggccgcttcg agcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     3241 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     3301 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     3361 agggggagat gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg
     3421 ataaggatct tcctagagca tggctacgta gataagtagc atggcgggtt aatcattaac
     3481 tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     3541 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3601 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     3661 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3721 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3781 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3841 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3901 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3961 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4021 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4081 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4141 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4201 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4261 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4321 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4381 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4441 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4501 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4561 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4621 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     4681 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     4741 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     4801 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4861 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4921 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4981 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5041 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5101 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5161 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5221 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5281 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5341 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5401 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     5461 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     5521 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     5581 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     5641 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     5701 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     5761 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     5821 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     5881 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     5941 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct gcgcgctcgc tcgctcactg
     6001 aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
     6061 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttccttg tagttaatga
     6121 ttaacccgcc atgctactt
//
