LOCUS       sequence-472021-        6843 bp    DNA     circular SYN 07-MAY-2025
DEFINITION  GFP splicing reporter with FRG1 intron.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6843)
  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 6843)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 7, 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
            FR...
FEATURES             Location/Qualifiers
     source          1..6843
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        40..144
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(145..213,214..1005)
                     /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(363..382)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1176..1764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1665..1684
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1826..1955
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     primer_bind     1970..1989
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1970..1988
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             complement(2292..2990)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="ELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVVE
                     GGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQ
                     DTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKLV
                     GGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCDL
                     PSKLGHKLNS"
     CDS             complement(2298..3005)
                     /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"
     regulatory      3011..3020
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3041..3252)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        3297..3600
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     CDS             4969..5034
                     /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(5035..5037,5038..5040,5041..5754)
                     /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(5035..5037,5038..5040,5041..5751)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5080..5101)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5341..5360)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5688..5709
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5784..5905
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5821..5840)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5875..5894
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5931..6071
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5931..6060
                     /label=AAV2 ITR
     rep_origin      6146..6601
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6233..6252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6443..6464
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6618..6637)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6737..6759
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6797..6815)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
       61 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
      121 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
      181 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
      241 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
      301 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
      361 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
      421 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
      481 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
      541 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
      601 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
      661 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
      721 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
      781 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
      841 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
      901 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
      961 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     1021 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     1081 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     1141 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     1201 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     1261 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     1321 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     1381 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     1441 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     1501 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     1561 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     1621 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     1681 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     1741 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     1801 ttttgctggc cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag
     1861 gccgcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag
     1921 agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgtt aatacgactc
     1981 actatagggc tgaaacaaaa gaagctgttc caatccacga acccgaacct gaatcacttt
     2041 ctttgaattt tttgcgactg gtgttcaagc tgaacgacat ttccgccttg taccttattc
     2101 tgggttgttt cacaagtggg gttattacac catgtgatat gccaacgtca attcttgctg
     2161 tgtacctgca tggtaatgtg atggtggtaa ccgtattcat tggatttatg tgtatttgcc
     2221 agtgatggca aattgccaag gctgctacgg acgatgtgga agatggccca agagcatctg
     2281 gagagacaag cctagctatt aagcttgtgc cccagtttgc tagggaggtc gcagtatctg
     2341 gccactgcca cctcgtgctg ctcgacgtag gtttcgttgt tggcctcctt gattctttcc
     2401 agtctgtagt ccacatagta gacgccaggc atcttgaggt tcttagcggg tttcttggat
     2461 ctatatgtgg tcttggcgtt tgcgatcaga tggctcccgc ccacgagctt cagggccatg
     2521 tcgtttctgc cttccaggcc gccgtcagcg gggtacagcg tttcggtgaa ggcctcccag
     2581 ccgagtgttt tcttctgcat cacagggccg ttggatgtga agttcacccc tctgatcttg
     2641 acgttgtaga tgaggcagcc gtcctggagg ctggtgtcct gggtagcggt cagcacgccc
     2701 ccgtcctcgt atgtggtgac tctctcccat gtgaagccct cagggaagga ctgcttgaag
     2761 aagtcgggga tgccctgggt gtggttgatg aaggtcttgc tgccgtagag gaagctagta
     2821 gccaggatgt cgaaggcgaa ggggagaggg ccgccctcga ccaccttgat tctcatggtc
     2881 tgggtgccct cgtagggctt gccttcgccc tcggatgtgc acttgaagtg atggttgtcc
     2941 acggtgccct ccatgtacag cttcatgtgc atgttctcct taatcagctc ttcgccctta
     3001 gacacggatc ccatggtggc gaagcgtcga ctcacgacac ctgtgttctg gcggcaaacc
     3061 cgttgcgaaa aagaacgttc acggcgacta ctgcacttat atacggttct cccccaccct
     3121 cgggaaaaag gcggagccag tacacgacat cactttccca gtttaccccg cgccaccttc
     3181 tctaggcacc ggttcaattg ccgacccctc cccccaactt ctcggggact gtgggcgatg
     3241 tgcgctctgc ccactgacgg gcaccggagc cctatgactt gcatagctgc gtatggcgtt
     3301 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     3361 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     3421 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     3481 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     3541 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     3601 aattgagggc gtcaccgcta aggctccgcc ccagcctggg ctccacaacc aatgaagggt
     3661 aatctcgaca aagagcaagg ggtggggcgc gggcgcgcag gtgcagcagc acacaggctg
     3721 gtcgggaggg cggggcgcga cgtctgccgt gcggggtccc ggcatcggtt gcgcgcgcgc
     3781 tccctcctct cggagagagg gctgtggtaa aacccgtccg gaaaccactt aagctagagc
     3841 tagcgaattc gaattcgcta gcttagctta agtggtttcc ggaaaccact taagctagag
     3901 ctagcgaatt cgaatttaaa tcggatctgg ctagcaccat gggaatctgg tggacagtaa
     3961 caaactttgg tgaaatttca ggaaccatag ccattgaaat ggatgaggga acctatatac
     4021 atgcactcaa caatggtctt tttaccctgg gagctccaca caaagaaggt ttgtgtctgg
     4081 aagggaagat agctgcgtcc tggtggcgca gggctgccac aagtgtagat tttaggacat
     4141 tcattcactt aggccaaact ctaactagtc tcaaacattt tccaaaggaa tggaaccgtt
     4201 gcatttgact cttccaattt tttttaattc cttaatattt accagccatt ggcaagtccc
     4261 tctttctaat ataagcattt gaaaacattc cgtgtagttc cagaagagta tctttggaaa
     4321 tcaaaacaat atgaataatt aaaatagtaa gtggaaggaa aaaagaaaac ctatttgagt
     4381 caaacatgtt tgaattactt ttttattcag acctattacc aaaactaacc tgggtgcatt
     4441 taacaatttg aagtttcctc attttcttta gccctttaga ggaagcacta gtgagatacg
     4501 aagtaattag aagataaaaa gcagtatcat ttggtcagca aggccatcca ttgaataaat
     4561 gaaagcattt agctttttta aataagtgct tatttatgac tgtattttaa aacataaaga
     4621 gaagctatct cgaaagttat taaataaaca ttatatcacc ctgtcttact cagtgtataa
     4681 aattagcact gtagttaaaa gaaggtaaaa tagatcttga ttccaaagat acagtattac
     4741 agtgacatca gtatatctga atattcttac atttaattgc agaagaaaat agccacattt
     4801 ttaaagcaaa ataatgtctt tatgtttata gcaaatgtca aatttatttt caaatgtttt
     4861 ttctccaata gttgatgagg gccctagtcc tccagagcag tttacggctg tcaaattatc
     4921 tgattccagg gcgcgcccag ggcaaaacgt tcgggttcgg gtgcgccagt aaagcagaca
     4981 ttaaactttg atttgctgaa acttgcaggt gatgtagagt caaatccagg tccaatggtg
     5041 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     5101 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     5161 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     5221 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     5281 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     5341 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     5401 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     5461 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     5521 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     5581 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     5641 agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     5701 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaatcgat
     5761 accgagcgct gctcgaggct agcaacttgt ttattgcagc ttataatggt tacaaataaa
     5821 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5881 tgtccaaact catcaatgta tcttatcatg tctgcggacc gagcggccgc aggaacccct
     5941 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     6001 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     6061 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     6121 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     6181 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     6241 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     6301 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     6361 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     6421 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     6481 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     6541 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     6601 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     6661 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     6721 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     6781 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     6841 atg
//
