LOCUS       sequence-472022-        6230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GFP splicing reporter with CORO1B intron.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6230)
  AUTHORS   Bhat P, Chow A, Emert B, Ettlin O, Quinodoz SA, Strehle M, Takei Y, 
            Burr A, Goronzy IN, Chen AW, Huang W, Ferrer JLM, Soehalim E, Goh 
            ST, Chari T, Sullivan DK, Blanco MR, Guttman M
  TITLE     Genome organization around nuclear speckles drives mRNA splicing 
            efficiency.
  JOURNAL   Nature. 2024 May;629(8014):1165-1173. doi: 
            10.1038/s41586-024-07429-6. Epub 2024 May 8.
  PUBMED    38720076
REFERENCE   2  (bases 1 to 6230)
  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/s41586-024-07429-6"; journalName: "Nature"; date: 
            "2024-05"; volume: "629"; issue: "8014"; pages: "1165-1173"
COMMENT     Sequence Label: BFP-GFP-bidirectional MS2 splicing reporter CO...
FEATURES             Location/Qualifiers
     source          1..6230
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      81..90
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(111..322)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        367..670
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     CDS             1426..1491
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(1492..1494,1495..1497,1498..2211)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1492..1494,1495..1497,1498..2208)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1537..1558)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1798..1817)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2145..2166
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2241..2362
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2278..2297)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2332..2351
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   2388..2528
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2388..2517
                     /label=AAV2 ITR
     rep_origin      2603..3058
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2690..2709)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2900..2921
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3075..3094)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3194..3216
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3254..3272)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3340..3444
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3445..3513,3514..4305)
                     /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     complement(3663..3682)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4476..5064
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4965..4984
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   5126..5255
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     5270..5289
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5270..5288
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(join(5592..6230,1..60))
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="ELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVE
                     GGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQ
                     DTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLV
                     GGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDL
                     PSKLGHKLNS"
     CDS             complement(join(5598..6230,1..75))
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDM
                     ALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVA
                     RYCDLPSKLGHKLN"
ORIGIN
        1 atggttgtcc acggtgccct ccatgtacag cttcatgtgc atgttctcct taatcagctc
       61 ttcgccctta gacacggatc ccatggtggc gaagcgtcga ctcacgacac ctgtgttctg
      121 gcggcaaacc cgttgcgaaa aagaacgttc acggcgacta ctgcacttat atacggttct
      181 cccccaccct cgggaaaaag gcggagccag tacacgacat cactttccca gtttaccccg
      241 cgccaccttc tctaggcacc ggttcaattg ccgacccctc cccccaactt ctcggggact
      301 gtgggcgatg tgcgctctgc ccactgacgg gcaccggagc cctatgactt gcatagctgc
      361 gtatggcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      421 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      481 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      541 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      601 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      661 tattaccatg aattgagggc gtcaccgcta aggctccgcc ccagcctggg ctccacaacc
      721 aatgaagggt aatctcgaca aagagcaagg ggtggggcgc gggcgcgcag gtgcagcagc
      781 acacaggctg gtcgggaggg cggggcgcga cgtctgccgt gcggggtccc ggcatcggtt
      841 gcgcgcgcgc tccctcctct cggagagagg gctgtggtaa aacccgtccg gaaaccactt
      901 aagctagagc tagcgaattc gattcgcagt ctagcttaag tggtttccgg aaaccactta
      961 agctagagct agcgaattcg aatttaaatc ggatctggct agcaccatga cgggccgcat
     1021 tgacaaggcc tacccgacgg tgtgtgggca cacgggacct gtcctggaca tcgactggtg
     1081 tcctcacaac gacgaagtca tagccagcgg ctcggaggac tgcacggtca tggtgaggca
     1141 caggactggc tggtagctgc gtcctggtgg cgcaggccag ggtcgtgggt gtggccgagg
     1201 gggccagcaa taggaccagc agaggactga gctcggtggc cctgtcccta ccacaggtgt
     1261 ggcagatccc agagaacggg ctgacctccc cgctgacaga gccggtggtg gtactggagg
     1321 ggcacaccaa gcgagtgggc atcatcgcct ggcaccccac ggcccgaaac gtgctgctca
     1381 gtgcagggcg cgcccagggc aaaacgttcg ggttcgggtg cgccagtaaa gcagacatta
     1441 aactttgatt tgctgaaact tgcaggtgat gtagagtcaa atccaggtcc aatggtgagc
     1501 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1561 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1621 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1681 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     1741 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1801 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1861 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1921 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1981 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2041 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2101 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2161 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aatcgatacc
     2221 gagcgctgct cgaggctagc aacttgttta ttgcagctta taatggttac aaataaagca
     2281 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     2341 ccaaactcat caatgtatct tatcatgtct gcggaccgag cggccgcagg aacccctagt
     2401 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     2461 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     2521 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     2581 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2641 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     2701 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2761 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     2821 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2881 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     2941 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     3001 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     3061 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     3121 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3181 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3241 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     3301 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3361 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3421 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     3481 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     3541 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     3601 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     3661 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     3721 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     3781 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     3841 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     3901 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     3961 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     4021 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4081 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4141 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     4201 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4261 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     4321 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     4381 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     4441 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     4501 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     4561 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     4621 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     4681 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     4741 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     4801 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     4861 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     4921 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     4981 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5041 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5101 ttttgctggc cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag
     5161 gccgcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
     5221 agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgtt aatacgactc
     5281 actatagggc tgaaacaaaa gaagctgttc caatccacga acccgaacct gaatcacttt
     5341 ctttgaattt tttgcgactg gtgttcaagc tgaacgacat ttccgccttg taccttattc
     5401 tgggttgttt cacaagtggg gttattacac catgtgatat gccaacgtca attcttgctg
     5461 tgtacctgca tggtaatgtg atggtggtaa ccgtattcat tggatttatg tgtatttgcc
     5521 agtgatggca aattgccaag gctgctacgg acgatgtgga agatggccca agagcatctg
     5581 gagagacaag cctagctatt aagcttgtgc cccagtttgc tagggaggtc gcagtatctg
     5641 gccactgcca cctcgtgctg ctcgacgtag gtttcgttgt tggcctcctt gattctttcc
     5701 agtctgtagt ccacatagta gacgccaggc atcttgaggt tcttagcggg tttcttggat
     5761 ctatatgtgg tcttggcgtt tgcgatcaga tggctcccgc ccacgagctt cagggccatg
     5821 tcgtttctgc cttccaggcc gccgtcagcg gggtacagcg tttcggtgaa ggcctcccag
     5881 ccgagtgttt tcttctgcat cacagggccg ttggatgtga agttcacccc tctgatcttg
     5941 acgttgtaga tgaggcagcc gtcctggagg ctggtgtcct gggtagcggt cagcacgccc
     6001 ccgtcctcgt atgtggtgac tctctcccat gtgaagccct cagggaagga ctgcttgaag
     6061 aagtcgggga tgccctgggt gtggttgatg aaggtcttgc tgccgtagag gaagctagta
     6121 gccaggatgt cgaaggcgaa ggggagaggg ccgccctcga ccaccttgat tctcatggtc
     6181 tgggtgccct cgtagggctt gccttcgccc tcggatgtgc acttgaagtg
//
