LOCUS       sequence-367992-        6206 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TagRFP658 and GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6206)
  AUTHORS   Babakhanova S, Jung EE, Namikawa K, Zhang H, Wang Y, Subach OM, 
            Korzhenevskiy DA, Rakitina TV, Xiao X, Wang W, Shi J, Drobizhev M, 
            Park D, Eisenhard L, Tang H, Koster RW, Subach FV, Boyden ES, 
            Piatkevich KD
  TITLE     Rapid directed molecular evolution of fluorescent proteins in 
            mammalian cells.
  JOURNAL   Protein Sci. 2022 Mar;31(3):728-751. doi: 10.1002/pro.4261. Epub 
            2021 Dec 30.
  PUBMED    34913537
REFERENCE   2  (bases 1 to 6206)
  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"; doi: "10.1002/pro.4261"; 
            journalName: "Protein Sci"; date: "2022-03"; volume: "31"; issue: 
            "3"; pages: "728-751"
COMMENT     Sequence Label: pAAV-CAG-TagRFP658-P2A-GFP
FEATURES             Location/Qualifiers
     source          1..6206
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     gap             396..407
                     /estimated_length=12
     primer_bind     807..829
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     891..910
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             955..1653
                     /codon_start=1
                     /product="far-red monomeric fluorescent protein with an 
                     emission peak at 657 nm (Morozova et al., 2010)"
                     /label=TagRFP657
                     /note="derived from mKate"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTQRIKV
                     VEGGPLPFAFDILATSFMYGSHTFIDHTQGIPDFWKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLHDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEAHTEMLYPTDGGLEGRTALALK
                     LVGGGHLICNFKTIYRSKKPAKNLKMPGVYYVDYRLERIEEADNETYVEQHEVAVARYC
                     DLPTKLGHKLN"
     CDS             1678..1734
                     /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(1747..1749,1750..2463)
                     /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(1747..1749,1750..2460)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1789..1810)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2050..2069)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2397..2418
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2488..3076
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2541..2561)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2959..2970)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3119..3240
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3130..3149)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3184..3203
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3402..3531
                     /label=AAV2 ITR
     rep_origin      complement(3768..4356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3848..3867)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4527..5318,5319..5387))
                     /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     5150..5169
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5388..5492)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5518..5973)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5655..5676)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5867..5886
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6045..6174
                     /label=AAV2 ITR
ORIGIN
        1 tctacgtagc catgctctag gaagagtacc attgacgtca ataatgacgt atgttcccat
       61 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      121 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      181 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      241 gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg
      301 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
      361 attattttgt gcagcgatgg gggcgggggg gggggnnnnn nnnnnnnggg cggggcgagg
      421 cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg
      481 aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg agtcgctgcg
      541 cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc ccggctctga
      601 ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc gggctgtaat
      661 tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag ccttgagggg
      721 ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg gggacggctg
      781 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
      841 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct
      901 ggttattgtg ctgtctcatc attttggcaa aggattggat ccggtaccgc caccatgagc
      961 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt gaacaatcac
     1021 cacttcaagt gcacatcaga gggcgaaggc aagccctacg agggcaccca gacccagaga
     1081 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc taccagcttc
     1141 atgtacggca gtcacacctt catcgaccac acccagggca tccccgactt ctggaagcag
     1201 tccttccctg agggcttcac gtgggagaga gtcaccacat acgaagacgg gggcgtcctg
     1261 accgctaccc aggacaccag cctccatgac ggctgcctca tctacaacgt caagatcaga
     1321 ggggtgaact tcccatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     1381 cacaccgaga tgttgtaccc cactgacggc ggtctggaag gcagaaccgc gctggccctg
     1441 aagctcgtgg gcgggggcca cttaatctgc aacttcaaga ctatatacag atccaagaaa
     1501 cccgctaaga acctcaagat gcccggcgtc tactatgtgg actacagact ggaaagaatc
     1561 gaggaggccg acaatgagac ctacgtcgag cagcacgagg tggctgtggc cagatactgc
     1621 gacctcccta ccaaactggg gcacaagctt aatcgaccgg ttcacgtggg aagtggggct
     1681 acaaacttct cactgctgaa acaggccggg gatgtggagg aaaacccagg accacacgtg
     1741 actagtgtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1801 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1861 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1921 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1981 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2041 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2101 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2161 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2221 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2281 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2341 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2401 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2461 taagaattcg atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga
     2521 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     2581 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
     2641 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
     2701 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
     2761 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
     2821 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
     2881 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
     2941 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
     3001 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
     3061 tgggccgcct ccccgcatcg ataccgtcga cccgggcggc cgcttcgagc agacatgata
     3121 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     3181 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     3241 aacaacaaca attgcattca ttttatgttt caggttcagg gggagatgtg ggaggttttt
     3301 taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatcttcc tagagcatgg
     3361 ctacgtagat aagtagcatg gcgggttaat cattaactac aaggaacccc tagtgatgga
     3421 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     3481 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcattaa
     3541 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3601 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3661 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3721 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3781 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3841 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3901 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3961 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4021 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4081 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4141 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4201 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4261 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4321 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4381 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4441 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4501 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4561 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4621 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4681 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4741 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4801 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4861 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4921 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4981 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5041 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5101 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5161 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5221 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5281 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5341 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5401 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5461 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     5521 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     5581 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     5641 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     5701 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     5761 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     5821 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     5881 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     5941 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     6001 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctgcgc gctcgctcgc
     6061 tcactgaggc cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag
     6121 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt
     6181 taatgattaa cccgccatgc tactta
//
