LOCUS       sequence-478889-        6034 bp    DNA     circular SYN 17-JUL-2025
DEFINITION  Expresses StayGold and mCherry in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6034)
  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 6034)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 17, 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-StayGold-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..6034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        280..557
                     /label=chicken beta-actin promoter
     primer_bind     836..858
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     920..939
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      978..987
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             984..1634
                     /codon_start=1
                     /product="bright and highly photostable dimeric green 
                     fluorescent protein (Hirano et al., 2022)"
                     /label=StayGold
                     /note="engineered variant of a GFP from Cytaeis uchidae"
                     /translation="MASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKL
                     PMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFM
                     KNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNEVQHIPRDDGVECPVTLLYPLLSDKS
                     KCVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
     CDS             1659..1715
                     /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             1728..2438
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1873..1891)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2141..2160
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2466..3054
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2519..2539)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2937..2948)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3097..3218
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3108..3127)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3162..3181
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3597..4185)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3677..3696)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4356..5147,5148..5216))
                     /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     4979..4998
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5217..5321)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5347..5802)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5484..5505)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5696..5715
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5874..6003
                     /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 gcgcgcgcca ggcggggcgg
      421 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
      481 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
      541 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
      601 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
      661 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
      721 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
      781 gctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc
      841 ggcttctggc gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc
      901 tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa
      961 gaattggatc cggtaccgcc accatggcta gtacaccatt taaatttcaa cttaaaggaa
     1021 ccatcaatgg caaatcgttt accgttgaag gcgaaggtga aggaaattca catgaaggtt
     1081 ctcataaagg aaaatatgtt tgtacaagtg gaaaactacc gatgtcatgg gcagcacttg
     1141 gaacatcctt tggttatgga atgaaatatt ataccaaata tcctagtgga ctgaagaact
     1201 ggtttcatga agtaatgcct gaaggcttta cctacgatcg tcatattcaa tataaaggcg
     1261 atgggagtat ccatgcaaaa caccaacact ttatgaaaaa tgggacttat cacaacattg
     1321 ttgaatttac tggtcaggat tttaaagaaa atagtccagt cttaactgga gatatgaatg
     1381 tctcattacc gaatgaagtt caacatatac ccagagatga tggagtagaa tgcccagtga
     1441 ccttgcttta tcctttatta tcggataaat caaaatgcgt tgaggctcac caaaatacaa
     1501 tctgcaagcc tcttcataat caaccagcac ctgatgtccc atatcactgg attcgtaaac
     1561 aatacacaca aagcaaagat gataccgagg aacgtgatca tatttgtcaa tcagagactc
     1621 tcgaagcaca cttacgaccg gttcacgtgg gaagtggggc tacaaacttc tcactgctga
     1681 aacaggccgg ggatgtggag gaaaacccag gaccacacgt gactagtatg gtgagcaagg
     1741 gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg
     1801 gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg
     1861 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca
     1921 tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc
     1981 ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     2041 aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct
     2101 acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga
     2161 ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg
     2221 agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca
     2281 cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg
     2341 acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc
     2401 gccactccac cggcggcatg gacgagctgt acaagtagta agaattcgat atcaagctta
     2461 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     2521 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     2581 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     2641 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     2701 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     2761 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     2821 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc
     2881 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     2941 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     3001 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat
     3061 accgtcgacc cgggcggccg cttcgagcag acatgataag atacattgat gagtttggac
     3121 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     3181 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     3241 ttatgtttca ggttcagggg gagatgtggg aggtttttta aagcaagtaa aacctctaca
     3301 aatgtggtaa aatcgataag gatcttccta gagcatggct acgtagataa gtagcatggc
     3361 gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     3421 gctcgctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     3481 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3541 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3601 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3661 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3721 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3781 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3841 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3901 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3961 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4021 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4081 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4141 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4201 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4261 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4321 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4381 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4441 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     4501 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4561 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4621 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4681 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4741 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4801 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4861 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4921 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4981 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5041 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5101 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5161 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5221 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5281 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5341 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     5401 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     5461 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     5521 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     5581 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     5641 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     5701 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     5761 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc
     5821 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctgcgcg
     5881 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc
     5941 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     6001 ccttgtagtt aatgattaac ccgccatgct actt
//
