LOCUS       sequence-387123-        6267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of mNeptune2.5-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6267)
  AUTHORS   Zhang H, Papadaki S, Sun X, Wang X, Drobizhev M, Yao L, Rehbock M, 
            Koster RW, Wu L, Namikawa K, Piatkevich KD
  TITLE     Quantitative assessment of near-infrared fluorescent proteins.
  JOURNAL   Nat Methods. 2023 Sep 4. doi: 10.1038/s41592-023-01975-z.
  PUBMED    37666982
REFERENCE   2  (bases 1 to 6267)
  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: "Nat 
            Methods. 2023 Sep 4. doi: 10.1038/s41592-023-01975-z."
COMMENT     Sequence Label: pAAV-CAG-mNeptune2.5-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6267
                     /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"
     CDS             985..1716
                     /codon_start=1
                     /product="brighter version of the far-red fluorescent 
                     protein mNeptune2 with slightly blue-shifted spectra"
                     /label=mNeptune2.5
                     /translation="MVSKGEELIKENMHTKLYMEGTVNNHHFKCTHEGEGKPYEGTQTN
                     RIKVVEGGPLPFAFDILATCFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTVTQDTSLQDGCLIYNVKLRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRCD
                     MALKLVGGGHLHCNLKTTYRSKKPAKNLKMPGVYFVDRRLERIKEADNETYVEQHEVAV
                     ARYCDLPSKLGHKLNGMDELYK"
     CDS             1741..1797
                     /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(1810..1812,1813..2526)
                     /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(1810..1812,1813..2523)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1852..1873)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2113..2132)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2460..2481
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2551..3139
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2604..2624)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3022..3033)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3182..3303
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3193..3212)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3247..3266
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3465..3594
                     /label=AAV2 ITR
     rep_origin      complement(3831..4419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3911..3930)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4590..5381,5382..5450))
                     /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     5213..5232
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5451..5555)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5581..6036)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5718..5739)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5930..5949
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6108..6237
                     /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 aggcggggcg
      421 gggcggggcg aggggcgggg cggggcgagg 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 aggagctgat caaggagaac
     1021 atgcacacga agctgtacat ggaaggcacc gtgaacaacc accacttcaa gtgcacccac
     1081 gaaggggagg gcaagcccta cgagggcacc cagaccaaca ggattaaggt ggtggaggga
     1141 ggccccctgc cgttcgcatt cgacatcctg gccacctgct ttatgtacgg gagcaagacc
     1201 ttcatcaacc acacccaggg catccccgat ttctttaagc agtccttccc tgagggcttc
     1261 acatgggaga gagtcaccac atacgaagac gggggcgtgc ttaccgttac ccaggacacc
     1321 agcctccagg acggctgctt gatctacaac gtcaagctca gaggggtgaa cttcccatcc
     1381 aacggccctg tgatgcagaa gaaaacactc ggctgggagg ccagtaccga gaccctgtac
     1441 cccgctgacg gcggcctgga aggcagatgc gacatggccc tgaagctcgt gggcgggggc
     1501 cacctgcact gcaacctgaa gaccacatac agatccaaga aacccgctaa gaacctcaag
     1561 atgcccggcg tctactttgt ggaccgcaga ctggaaagaa tcaaggaggc cgacaatgag
     1621 acctacgtcg agcagcacga ggtggctgtg gccagatact gcgacctccc tagcaaactg
     1681 gggcacaaac ttaatggcat ggacgagctg tacaagcgac cggttcacgt gggaagtggg
     1741 gctacaaact tctcactgct gaaacaggcc ggggatgtgg aggaaaaccc aggaccacac
     1801 gtgactagtg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1861 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1921 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     1981 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     2041 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     2101 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     2161 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     2221 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     2281 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     2341 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     2401 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     2461 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     2521 aagtaagaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     2581 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     2641 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     2701 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     2761 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     2821 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     2881 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     2941 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     3001 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3061 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     3121 ctttgggccg cctccccgca tcgataccgt cgacccgggc ggccgcttcg agcagacatg
     3181 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     3241 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     3301 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat gtgggaggtt
     3361 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcg ataaggatct tcctagagca
     3421 tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat
     3481 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     3541 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcat
     3601 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     3661 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3721 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3781 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3841 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     3901 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     3961 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4021 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4081 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4141 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4201 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4261 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4321 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4381 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4441 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     4501 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     4561 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     4621 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     4681 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     4741 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     4801 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     4861 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     4921 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     4981 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5041 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5101 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5161 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5221 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5281 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5341 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5401 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     5461 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     5521 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     5581 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     5641 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     5701 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     5761 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     5821 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     5881 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     5941 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     6001 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
     6061 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg cgcgctcgct
     6121 cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct
     6181 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttccttgt
     6241 agttaatgat taacccgcca tgctact
//
