LOCUS       sequence-480247-        6194 bp    DNA     circular SYN 24-JUL-2025
DEFINITION  Expresses mNeonGreen and FusionRed in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6194)
  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 6194)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 24, 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-mNeonGreen-P2A-FusionRed
FEATURES             Location/Qualifiers
     source          1..6194
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     950..972
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1034..1053
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1096..1105
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1102..1809
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1357..1366
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1834..1890
                     /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             1903..2601
                     /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    2626..3214
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2679..2699)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3097..3108)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3257..3378
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3268..3287)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3322..3341
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3757..4345)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3837..3856)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4516..5307,5308..5376))
                     /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     5139..5158
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5377..5481)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5507..5962)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5644..5665)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5856..5875
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6034..6163
                     /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 ggggggcccg gggggggggg
      421 gggggggggg gggggggggg gggggggggg gggggggggg ggggggggcg cgccaggggg
      481 gggggggggg ggggggtgtt ttttgtgggg ggggggggcg ggggggccct caaaaaaaga
      541 agagcgggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc
      601 tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg
      661 cgcggcgggc gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct
      721 cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg
      781 gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt
      841 ggctgcgtga aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg
      901 ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc
      961 tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc
     1021 ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg
     1081 gatccggtac cctaagccac catggtgagc aagggcgagg aggataacat ggcctctctc
     1141 ccagcgacac atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg
     1201 ggtcagggca ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag
     1261 ggtgacctcc agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag
     1321 tacctgccct accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccggc
     1381 taccaagtcc atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc
     1441 tacacctacg agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct
     1501 gctgacggtc ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaag
     1561 acttacccca acgacaaaac catcatcagt acctttaagt ggagttacac cacgggaaat
     1621 ggcaagcgct accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct
     1681 aactatctga agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag
     1741 accgagctca acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag
     1801 ctgtacaagc gaccggttca cgtgggaagt ggggctacaa acttctcact gctgaaacag
     1861 gccggggatg tggaggaaaa cccaggacca cacgtgacta gtatggtgag cgagctgatt
     1921 aaggagaaca tgcccatgaa gctgtacatg gagggcaccg tgaacaacca ccacttcaag
     1981 tgcacatccg agggcgaagg caagccctac gagggcaccc agaccatgag aatcaaggtc
     2041 gtcgagggcg gccctctccc cttcgccttc gacatcctgg ctaccagctt catgtacggc
     2101 agcagaacct tcatcaagca ccctccgggc atccccgact tctttaagca gtccttccct
     2161 gagggcttca catgggagag agtcaccaca tacgaagacg ggggcgtgct gaccgctacc
     2221 caggacacca gcctccagga cggctgcctc atctacaacg tcaaggttag aggggtgaac
     2281 ttcccagcca acggccctgt gatgcagaag aaaacactcg gctgggaggc ctccaccgag
     2341 acgatgtacc ctgctgacgg aggactggaa ggcgcatgtg acatggctct gaagctcgtg
     2401 ggaggaggac acctgatctg caaccttgag acaacataca gatccaagaa acccgctacg
     2461 aacctcaaga tgcccggcgt ctacaacgtg gaccacagac tggaaagaat caaggaggcc
     2521 gacgatgaga catacgtcga gcagcacgag gtggctgtgg ccagatactc tactggtggc
     2581 gctggtgatg gaggtaaata agaattcgat atcaagctta tcgataatca acctctggat
     2641 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt
     2701 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc
     2761 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg
     2821 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc
     2881 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa
     2941 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat
     3001 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc
     3061 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt
     3121 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag
     3181 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc cgggcggccg
     3241 cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
     3301 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     3361 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     3421 gagatgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa aatcgataag
     3481 gatcttccta gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa
     3541 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgactc
     3601 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3661 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3721 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3781 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3841 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3901 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3961 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ccgggctgtg tgcacgaacc
     4021 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4081 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4141 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     4201 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4261 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4321 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4381 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4441 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     4501 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4561 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4621 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4681 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4741 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4801 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4861 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4921 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4981 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5041 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5101 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5161 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5221 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5281 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5341 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5401 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5461 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     5521 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     5581 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     5641 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     5701 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     5761 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     5821 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     5881 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     5941 aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg
     6001 cgatcggtgc gggcctcttc gctattacgc cagctgcgcg ctcgctcgct cactgaggcc
     6061 gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga
     6121 gcgcgcagag agggagtggc caactccatc actaggggtt ccttgtagtt aatgattaac
     6181 ccgccatgct actt
//
