LOCUS       sequence-478761-        6018 bp    DNA     circular SYN 16-JUL-2025
DEFINITION  Expresses mStayGold(E138D) 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 Jul 16, 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-mStayGold(E138D)-P2A-FusionRed
FEATURES             Location/Qualifiers
     source          1..6018
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(51..62)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    211..332
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(222..241)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     276..295
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(711..1299)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(791..810)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1470..2261,2262..2330))
                     /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     2093..2112
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2331..2435)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2461..2916)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2598..2619)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2810..2829
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2988..3117
                     /label=AAV2 ITR
     promoter        3428..3704
                     /label=chicken beta-actin promoter
     primer_bind     3983..4005
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4067..4086
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      4125..4134
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4131..4781
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ivorra-Molla et al., 2023)"
                     /label=mStayGold (E138D)
                     /note="contains the monomerizing E138D mutation"
                     /translation="MASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKGKYVCTSGKL
                     PMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGSIHAKHQHFM
                     KNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNDVQHIPRDDGVECPVTLLYPLLSDKS
                     KCVEAHQNTICKPLHNQPAPDVPYHWIRKQYTQSKDDTEERDHICQSETLEAHL"
     CDS             4806..4862
                     /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             4875..5573
                     /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    join(5598..6018,1..168)
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5651..5671)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
ORIGIN
        1 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
       61 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
      121 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgtc
      181 gacccgggcg gccgcttcga gcagacatga taagatacat tgatgagttt ggacaaacca
      241 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
      301 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
      361 ttcaggttca gggggagatg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg
      421 gtaaaatcga taaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta
      481 atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
      541 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
      601 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
      661 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
      721 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
      781 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
      841 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
      901 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
      961 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1021 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     1081 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     1141 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     1201 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     1261 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     1321 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     1381 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     1441 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     1501 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     1561 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     1621 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     1681 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     1741 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     1801 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     1861 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     1921 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     1981 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     2041 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     2101 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     2161 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     2221 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     2281 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     2341 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     2401 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     2461 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     2521 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     2581 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     2641 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     2701 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     2761 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     2821 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     2881 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
     2941 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg cgcgctcgct
     3001 cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct
     3061 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttccttgt
     3121 agttaatgat taacccgcca tgctacttat ctacgtagcc atgctctagg aagagtacca
     3181 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     3241 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     3301 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     3361 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     3421 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
     3481 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
     3541 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
     3601 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
     3661 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     3721 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     3781 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     3841 gcttggttta atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc
     3901 gggagggccc tttgtgcggg gggagcggct cggggctgtc cgcgggggga cggctgcctt
     3961 cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc
     4021 tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt
     4081 attgtgctgt ctcatcattt tggcaaagaa ttggatccgg taccgccacc atggctagta
     4141 caccatttaa atttcaactt aaaggaacca tcaatggcaa atcgtttacc gttgaaggcg
     4201 aaggtgaagg aaattcacat gaaggttctc ataaaggaaa atatgtttgt acaagtggaa
     4261 aactaccgat gtcatgggca gcacttggaa catcctttgg ttatggaatg aaatattata
     4321 ccaaatatcc tagtggactg aagaactggt ttcatgaagt aatgcctgaa ggctttacct
     4381 acgatcgtca tattcaatat aaaggcgatg ggagtatcca tgcaaaacac caacacttta
     4441 tgaaaaatgg gacttatcac aacattgttg aatttactgg tcaggatttt aaagaaaata
     4501 gtccagtctt aactggagat atgaatgtct cattaccgaa tgacgttcaa catataccca
     4561 gagatgatgg agtagaatgc ccagtgacct tgctttatcc tttattatcg gataaatcaa
     4621 aatgcgttga ggctcaccaa aatacaatct gcaagcctct tcataatcaa ccagcacctg
     4681 atgtcccata tcactggatt cgtaaacaat acacacaaag caaagatgat accgaggaac
     4741 gtgatcatat ttgtcaatca gagactctcg aagcacactt acgaccggtt cacgtgggaa
     4801 gtggggctac aaacttctca ctgctgaaac aggccgggga tgtggaggaa aacccaggac
     4861 cacacgtgac tagtatggtg agcgagctga ttaaggagaa catgcccatg aagctgtaca
     4921 tggagggcac cgtgaacaac caccacttca agtgcacatc cgagggcgaa ggcaagccct
     4981 acgagggcac ccagaccatg agaatcaagg tcgtcgaggg cggccctctc cccttcgcct
     5041 tcgacatcct ggctaccagc ttcatgtacg gcagcagaac cttcatcaag caccctccgg
     5101 gcatccccga cttctttaag cagtccttcc ctgagggctt cacatgggag agagtcacca
     5161 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
     5221 tcatctacaa cgtcaaggtt agaggggtga acttcccagc caacggccct gtgatgcaga
     5281 agaaaacact cggctgggag gcctccaccg agacgatgta ccctgctgac ggaggactgg
     5341 aaggcgcatg tgacatggct ctgaagctcg tgggaggagg acacctgatc tgcaaccttg
     5401 agacaacata cagatccaag aaacccgcta cgaacctcaa gatgcccggc gtctacaacg
     5461 tggaccacag actggaaaga atcaaggagg ccgacgatga gacatacgtc gagcagcacg
     5521 aggtggctgt ggccagatac tctactggtg gcgctggtga tggaggtaaa taagaattcg
     5581 atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     5641 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     5701 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     5761 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     5821 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     5881 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     5941 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     6001 cctttccttg gctgctcg
//
