LOCUS       sequence-387181-        6210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the protein of E2-Crimson-P2A-GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6210)
  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 6210)
  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-E2-Crimson-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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..1659
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHM
                     ALKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     1383..1402
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1684..1740
                     /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(1753..1755,1756..2469)
                     /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(1753..1755,1756..2466)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1795..1816)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2056..2075)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2403..2424
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2494..3082
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2547..2567)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2965..2976)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3125..3246
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3136..3155)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3190..3209
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3408..3537
                     /label=AAV2 ITR
     rep_origin      complement(3774..4362)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3854..3873)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4533..5324,5325..5393))
                     /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     5156..5175
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5394..5498)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5524..5979)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5661..5682)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5873..5892
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6051..6180
                     /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 ggggygsgsc rggsggggsg
      421 gggsggggcg rggggcgggg 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 caccatggat agcactgaga acgtcatcaa gcccttcatg
     1021 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgtg
     1081 ggcgagggca agccctacga gggcacccag accgccaagc tgcaagtgac caagggcggc
     1141 cccctgccct tcgcctggga catcctgtcc ccccagttct tctacggctc caaggcgtac
     1201 atcaagcacc ccgccgacat ccccgactac ctcaagcagt ccttccccga gggcttcaag
     1261 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
     1321 ctgcaggacg gcaccctcat ctaccacgtg aagttcatcg gcgtgaactt cccctccgac
     1381 ggccccgtaa tgcagaagaa gactctgggc tgggagccct ccactgagcg caactacccc
     1441 cgcgacggcg tgctgaaggg cgagaaccac atggcgctga agctgaaggg cggcggccac
     1501 tacctgtgtg agttcaagtc catctacatg gccaagaagc ccgtgaagct gcccggctac
     1561 cactacgtgg actacaagct cgacatcacc tcccacaacg aggactacac cgtggtggag
     1621 cagtacgagc gcgccgaggc ccgccaccac ctgttccagc gaccggttca cgtgggaagt
     1681 ggggctacaa acttctcact gctgaaacag gccggggatg tggaggaaaa cccaggacca
     1741 cacgtgacta gtgtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1801 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1861 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1921 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     1981 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     2041 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2101 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2161 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     2221 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     2281 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     2341 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     2401 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2461 tacaagtaag aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt
     2521 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     2581 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     2641 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     2701 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     2761 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     2821 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     2881 tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc
     2941 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     3001 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     3061 tccctttggg ccgcctcccc gcatcgatac cgtcgacccg ggcggccgct tcgagcagac
     3121 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     3181 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     3241 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga gatgtgggag
     3301 gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tcttcctaga
     3361 gcatggctac gtagataagt agcatggcgg gttaatcatt aactacaagg aacccctagt
     3421 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     3481 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     3541 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     3601 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3661 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     3721 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     3781 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     3841 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     3901 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     3961 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4021 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4081 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4141 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4201 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4261 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4321 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4381 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4441 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     4501 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     4561 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     4621 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     4681 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     4741 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     4801 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     4861 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     4921 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     4981 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     5041 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     5101 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     5161 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     5221 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     5281 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     5341 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     5401 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     5461 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     5521 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     5581 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     5641 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     5701 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     5761 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     5821 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     5881 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     5941 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca ttcaggctgc
     6001 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctgcgcgctc
     6061 gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg
     6121 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
     6181 tgtagttaat gattaacccg ccatgctact
//
