LOCUS       sequence-478890-        6135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mClover3 and FusionRed in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6135)
  AUTHORS   Zhang H, Lesnov GD, Subach OM, Zhang W, Kuzmicheva TP, Vlaskina AV, 
            Samygina VR, Chen L, Ye X, Nikolaeva AY, Gabdulkhakov A, Papadaki S,
            Qin W, Borshchevskiy V, Perfilov MM, Gavrikov AS, Drobizhev M, 
            Mishin AS, Piatkevich KD, Subach FV
  TITLE     Bright and stable monomeric green fluorescent protein derived from 
            StayGold.
  JOURNAL   Nat Methods. 2024 Apr;21(4):657-665. doi: 
            10.1038/s41592-024-02203-y. Epub 2024 Feb 26.
  PUBMED    38409224
REFERENCE   2  (bases 1 to 6135)
  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: 
            "10.1038/s41592-024-02203-y"; journalName: "Nat Methods"; date: 
            "2024-04"; volume: "21"; issue: "4"; pages: "657-665"
COMMENT     Sequence Label: pAAV-CAG-mClover3-P2A-FusionRed
FEATURES             Location/Qualifiers
     source          1..6135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     882..904
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     966..985
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1028..1037
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1034..1036,1037..1039,1040..1750)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1079..1100)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1687..1708
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1775..1831
                     /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             1844..2542
                     /codon_start=1
                     /product="monomeric red fluorescent protein optimized for 
                     use as a fusion tag (Shemiakina et al., 2012)"
                     /label=FusionRed
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMPMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFMYGSRTFIKHPPGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKVRGVNFPANGPVMQKKTLGWEASTETMYPADGGLEGACDMAL
                     KLVGGGHLICNLETTYRSKKPATNLKMPGVYNVDHRLERIKEADDETYVEQHEVAVARY
                     STGGAGDGGK"
     misc_feature    2567..3155
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2620..2640)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3038..3049)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3198..3319
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3209..3228)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3263..3282
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3698..4286)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3778..3797)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4457..5248,5249..5317))
                     /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     5080..5099
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5318..5422)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5448..5903)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5585..5606)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5797..5816
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5975..6104
                     /label=AAV2 ITR
ORIGIN
        1 atctacgtag ccatgctcta ggaagagtac cattgacgtc aataatgacg tatgttccca
       61 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      121 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
      181 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
      241 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct
      301 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
      361 aattattttg tgcagcgatg ggggcggggg gggggggggg cggcgccggg gggggggggg
      421 tggggggggg gggggggggg gggggggcgg gggggggggg gggcggggcg gggcgagggg
      481 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
      541 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
      601 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      661 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      721 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      781 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggctgtcc
      841 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
      901 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
      961 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tggatccggt
     1021 accataagcc accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct
     1081 ggtcgagctg gacggcgacg taaacggcca caagttcagc gtccgcggcg agggcgaggg
     1141 cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt
     1201 gccctggccc accctcgtga ccaccttcgg ctacggcgtg gcctgcttca gccgctaccc
     1261 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga
     1321 gcgcaccatc tctttcaagg acgacggtac ctacaagacc cgcgccgagg tgaagttcga
     1381 gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa
     1441 catcctgggg cacaagctgg agtacaactt caacagccac tacgtctata tcacggccga
     1501 caagcagaag aactgcatca aggctaactt caagatccgc cacaacgttg aggacggcag
     1561 cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct
     1621 gcccgacaac cactacctga gccatcagtc caagctgagc aaagacccca acgagaagcg
     1681 cgatcacatg gtcctgctgg agttcgtgac cgccgccggg attacacatg gcatggacga
     1741 gctgtacaag cgaccggttc acgtgggaag tggggctaca aacttctcac tgctgaaaca
     1801 ggccggggat gtggaggaaa acccaggacc acacgtgact agtatggtga gcgagctgat
     1861 taaggagaac atgcccatga agctgtacat ggagggcacc gtgaacaacc accacttcaa
     1921 gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga gaatcaaggt
     1981 cgtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctaccagct tcatgtacgg
     2041 cagcagaacc ttcatcaagc accctccggg catccccgac ttctttaagc agtccttccc
     2101 tgagggcttc acatgggaga gagtcaccac atacgaagac gggggcgtgc tgaccgctac
     2161 ccaggacacc agcctccagg acggctgcct catctacaac gtcaaggtta gaggggtgaa
     2221 cttcccagcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg cctccaccga
     2281 gacgatgtac cctgctgacg gaggactgga aggcgcatgt gacatggctc tgaagctcgt
     2341 gggaggagga cacctgatct gcaaccttga gacaacatac agatccaaga aacccgctac
     2401 gaacctcaag atgcccggcg tctacaacgt ggaccacaga ctggaaagaa tcaaggaggc
     2461 cgacgatgag acatacgtcg agcagcacga ggtggctgtg gccagatact ctactggtgg
     2521 cgctggtgat ggaggtaaat aagaattcga tatcaagctt atcgataatc aacctctgga
     2581 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     2641 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     2701 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     2761 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     2821 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     2881 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     2941 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     3001 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     3061 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     3121 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac ccgggcggcc
     3181 gcttcgagca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     3241 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     3301 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     3361 ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa
     3421 ggatcttcct agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca
     3481 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgact
     3541 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     3601 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     3661 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     3721 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     3781 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     3841 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     3901 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3961 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     4021 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     4081 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     4141 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     4201 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     4261 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     4321 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     4381 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     4441 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4501 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4561 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4621 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4681 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4741 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4801 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4861 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     4921 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4981 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5041 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5101 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5161 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5221 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5281 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5341 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5401 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt
     5461 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     5521 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     5581 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     5641 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     5701 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     5761 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     5821 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     5881 taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg
     5941 gcgatcggtg cgggcctctt cgctattacg ccagctgcgc gctcgctcgc tcactgaggc
     6001 cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
     6061 agcgcgcaga gagggagtgg ccaactccat cactaggggt tccttgtagt taatgattaa
     6121 cccgccatgc tactt
//
