pAAV-CAG-mTagBFP2-P2A-GFP

LOCUS       pAAV-CAG-mTagBFP2-P2A-GFP              6232 bp DNA     circular SYN 29-MAY-2023
DEFINITION  Expresses TagRFP658 and GFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6232)
  TITLE     Rapid Directed Molecular Evolution of Fluorescent Proteins in 
            Mammalian Cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6232)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 29, 2026 from SnapGene 6.0.2
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
FEATURES             Location/Qualifiers
     source          1..6232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        279..556
                     /label=chicken beta-actin promoter
     primer_bind     965..1001
                     /label=Primer 1
     CDS             983..1681
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     regulatory      983..986
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1659..1707)
                     /label=Primer 2
     CDS             1706..1762
                     /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             1775..2491
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGRLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    2516..3104
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(2987..2998)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3147..3268
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   3430..3559
                     /label=AAV2 ITR
     rep_origin      complement(3796..4384)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4555..5415)
                     /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"
     promoter        complement(5416..5520)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5546..6001)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     repeat_region   6073..6202
                     /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 gggggggggg cgcgcgccag gcggggcggg
      421 gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
      481 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
      541 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
      601 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
      661 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt
      721 tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg
      781 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
      841 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
      901 ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag
      961 aattggatcc ggtaccgcca ccatgagcga gctgattaag gagaacatgc acatgaagct
     1021 gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa
     1081 gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt
     1141 cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac
     1201 ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt
     1261 caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg
     1321 ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat
     1381 gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg
     1441 cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa
     1501 cgccaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta
     1561 ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca
     1621 gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa
     1681 tcgaccggtt cacgtgggaa gtggggctac aaacttctca ctgctgaaac aggccgggga
     1741 tgtggaggaa aacccaggac cacacgtgac tagtgtgagc aagggcgagg agctgttcac
     1801 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1861 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1921 caccggcagg ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1981 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     2041 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     2101 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     2161 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     2221 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     2281 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2341 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     2401 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2461 cactctcggc atggacgagc tgtacaagta agaattcgat atcaagctta tcgataatca
     2521 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     2581 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     2641 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     2701 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     2761 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     2821 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     2881 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     2941 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     3001 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     3061 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc
     3121 cgggcggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
     3181 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3241 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     3301 ggttcagggg gagatgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa
     3361 aatcgataag gatcttccta gagcatggct acgtagataa gtagcatggc gggttaatca
     3421 ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     3481 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     3541 tgagcgagcg agcgcgcagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3601 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3661 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3721 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3781 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3841 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3901 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3961 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4021 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4081 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4141 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4201 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4261 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4321 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4381 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4441 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4501 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4561 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4621 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4681 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4741 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     4801 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     4861 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     4921 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     4981 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5041 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5101 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5161 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5221 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5281 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5341 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5401 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5461 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5521 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     5581 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     5641 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     5701 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     5761 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     5821 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     5881 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     5941 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     6001 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     6061 ctattacgcc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt
     6121 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
     6181 aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta ct
//