LOCUS       sequence-489855-        6018 bp    DNA     circular SYN 21-OCT-2025
DEFINITION  Expresses StayGold and FusionRed in the cytoplasm of mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6018)
  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 6018)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 21, 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-FusionRed
FEATURES             Location/Qualifiers
     source          1..6018
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        280..556
                     /label=chicken beta-actin promoter
     primer_bind     835..857
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     919..938
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      977..986
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             983..1633
                     /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             1658..1714
                     /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             1727..2425
                     /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    2450..3038
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2503..2523)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2921..2932)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3081..3202
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3092..3111)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3146..3165
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3581..4169)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3661..3680)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4340..5131,5132..5200))
                     /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     4963..4982
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5201..5305)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5331..5786)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5468..5489)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5680..5699
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5858..5987
                     /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 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 ccatggctag tacaccattt aaatttcaac ttaaaggaac
     1021 catcaatggc aaatcgttta ccgttgaagg cgaaggtgaa ggaaattcac atgaaggttc
     1081 tcataaagga aaatatgttt gtacaagtgg aaaactaccg atgtcatggg cagcacttgg
     1141 aacatccttt ggttatggaa tgaaatatta taccaaatat cctagtggac tgaagaactg
     1201 gtttcatgaa gtaatgcctg aaggctttac ctacgatcgt catattcaat ataaaggcga
     1261 tgggagtatc catgcaaaac accaacactt tatgaaaaat gggacttatc acaacattgt
     1321 tgaatttact ggtcaggatt ttaaagaaaa tagtccagtc ttaactggag atatgaatgt
     1381 ctcattaccg aatgaagttc aacatatacc cagagatgat ggagtagaat gcccagtgac
     1441 cttgctttat cctttattat cggataaatc aaaatgcgtt gaggctcacc aaaatacaat
     1501 ctgcaagcct cttcataatc aaccagcacc tgatgtccca tatcactgga ttcgtaaaca
     1561 atacacacaa agcaaagatg ataccgagga acgtgatcat atttgtcaat cagagactct
     1621 cgaagcacac ttacgaccgg ttcacgtggg aagtggggct acaaacttct cactgctgaa
     1681 acaggccggg gatgtggagg aaaacccagg accacacgtg actagtatgg tgagcgagct
     1741 gattaaggag aacatgccca tgaagctgta catggagggc accgtgaaca accaccactt
     1801 caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca tgagaatcaa
     1861 ggtcgtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacca gcttcatgta
     1921 cggcagcaga accttcatca agcaccctcc gggcatcccc gacttcttta agcagtcctt
     1981 ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg tgctgaccgc
     2041 tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaagg ttagaggggt
     2101 gaacttccca gccaacggcc ctgtgatgca gaagaaaaca ctcggctggg aggcctccac
     2161 cgagacgatg taccctgctg acggaggact ggaaggcgca tgtgacatgg ctctgaagct
     2221 cgtgggagga ggacacctga tctgcaacct tgagacaaca tacagatcca agaaacccgc
     2281 tacgaacctc aagatgcccg gcgtctacaa cgtggaccac agactggaaa gaatcaagga
     2341 ggccgacgat gagacatacg tcgagcagca cgaggtggct gtggccagat actctactgg
     2401 tggcgctggt gatggaggta aataagaatt cgatatcaag cttatcgata atcaacctct
     2461 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     2521 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     2581 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     2641 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     2701 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     2761 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     2821 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc
     2881 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     2941 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     3001 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc gacccgggcg
     3061 gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     3121 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     3181 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     3241 gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga
     3301 taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta atcattaact
     3361 acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg
     3421 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3481 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3541 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3601 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3661 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3721 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3781 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3841 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3901 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     3961 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4021 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4081 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4141 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4201 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4261 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4321 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4381 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4441 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4501 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4561 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4621 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4681 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4741 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4801 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4861 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4921 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     4981 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5041 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5101 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5161 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5221 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5281 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct aaattgtaag
     5341 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5401 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5461 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5521 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5581 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5641 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5701 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5761 gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga
     5821 agggcgatcg gtgcgggcct cttcgctatt acgccagctg cgcgctcgct cgctcactga
     5881 ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga
     5941 gcgagcgcgc agagagggag tggccaactc catcactagg ggttccttgt agttaatgat
     6001 taacccgcca tgctactt
//
