LOCUS       sequence-387122-        6452 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of miRFP2-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6452)
  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 6452)
  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-miRFP2-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6452
                     /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             1948..2004
                     /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(2017..2019,2020..2733)
                     /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(2017..2019,2020..2730)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2059..2080)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2320..2339)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2667..2688
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2758..3346
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2811..2831)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3229..3240)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3389..3510
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3400..3419)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3454..3473
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(4016..4604)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4096..4115)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4775..5566,5567..5635))
                     /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     5398..5417
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5636..5740)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5766..6221)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5903..5924)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6115..6134
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6293..6422
                     /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 caccatggtc gcaggacacg caagtggaag ccccgacttt
     1021 ggaaccgcca gtccgagcga ctgtgagcac gaagaaatcc atctggccgg atccatccag
     1081 ccacacggaa cactgctggt ggtctccgag cctgaccata gaataattca ggcatctgcc
     1141 aacgccgctg aattcctgaa tctgggaagt gtgctgggaa tccccctggc tgagatcgac
     1201 ggggatctac tgatcaagat tctgccccac ctggacctta ccgcagaagg aatgccagtg
     1261 gccgtcaggt gccgcattgg aaacccctcc acagagtacg acggactgat gcatcgacca
     1321 cctgaaggag gactgatcat tgagctggaa cgggctgggc catccattga tctgagcgga
     1381 acactggctc ccgcactgga gcgaattcga actgcaggat ccctgagggc tctgtgcgac
     1441 gatactgtac tgctgttcca gcagtgtacc ggctacgacc gagtgatggt ctatcggttc
     1501 gatgaacggg ggcacggaga ggtgtattca gaaattcatg tgactggact ggagagctac
     1561 tttggcaaca ggtatcctag ctccctcgtg ccacagatgg cccggcggct gtacgtgaga
     1621 cagagggtgc gcgtcctggt agatgtcacc tatcagcctg tgccactgga gccaagactg
     1681 agtcccctga ctggcaggga cctggatatg tcagggtgtt ttctgcgctc tatgagtcct
     1741 actcacctgc agttcctgaa gaacatgggc gtgcgggcaa cactggtggt cagcctggtg
     1801 gtcggaggca aactgtgggg gctggtgatc tgccaccatt atctgccacg cttcatccat
     1861 tttgagctgc gagcaatttg tgtgctgctg gccgaagcta tcgcaacccg cattacagcc
     1921 ctgcgaccgg ttcacgtggg aagtggggct acaaacttct cactgctgaa acaggccggg
     1981 gatgtggagg aaaacccagg accacacgtg actagtgtga gcaagggcga ggagctgttc
     2041 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     2101 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     2161 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     2221 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     2281 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     2341 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     2401 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     2461 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     2521 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     2581 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     2641 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2701 atcactctcg gcatggacga gctgtacaag taagaattcg atatcaagct tatcgataat
     2761 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     2821 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     2881 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     2941 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     3001 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     3061 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     3121 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     3181 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     3241 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     3301 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcatcg ataccgtcga
     3361 cccgggcggc cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca
     3421 actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt
     3481 gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt
     3541 caggttcagg gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt
     3601 aaaatcgata aggatcttcc tagagcatgg ctacgtagat aagtagcatg gcgggttaat
     3661 cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     3721 gctcactgag gccgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     3781 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3841 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3901 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3961 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4021 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4081 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4141 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4201 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4261 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4321 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4381 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4441 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4501 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4561 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4621 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4681 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4741 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4801 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4861 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4921 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4981 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5041 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5101 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5161 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5221 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5281 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5341 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5401 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5461 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5521 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5581 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5641 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5701 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5761 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5821 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5881 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5941 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     6001 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     6061 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     6121 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     6181 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
     6241 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctgcgcgc
     6301 tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc
     6361 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc
     6421 cttgtagtta atgattaacc cgccatgcta ct
//
