LOCUS       sequence-192697-        5850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Minigene reporter for alternative splicing analysis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5850)
  AUTHORS   Stoilov P, Lin CH, Damoiseaux R, Nikolic J, Black DL
  TITLE     A high-throughput screening strategy identifies cardiotonic steroids
            as alternative splicing modulators.
  JOURNAL   Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11218-23. doi: 
            10.1073/pnas.0801661105. Epub 2008 Aug 4.
  PUBMED    18678901
REFERENCE   2  (bases 1 to 5850)
  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: 
            "10.1073/pnas.0801661105"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2008-08-12- 12"; volume: "105"; issue: "32"; pages: 
            "11218-23"
COMMENT     Sequence Label: pFlare9A-Dup34
FEATURES             Location/Qualifiers
     source          1..5850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        143..446
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        447..650
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     601..621
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1014..1023
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1159..1161,1162..1875)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHLKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGTDELYK"
     primer_bind     complement(1201..1222)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1462..1481)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      1882..1891
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1888..2598
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent 
                     protein"
                     /label=mStrawberry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILTPNFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKMRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYIVGIKLDITSHNEDYTIVELYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2033..2051)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2301..2320
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2720..2841
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2757..2776)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2811..2830
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2848..3303)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2985..3006)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3197..3216
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3330..3434
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3436..3793
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3436..3627
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3459..3479)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3644..3779
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3828..4622
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3828..4622
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3882..3901)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4492..4511
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4810..4829)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4854..4901
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5230..5818
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5719..5738
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
       61 taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc
      121 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      181 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      241 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      301 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      361 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      421 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      481 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      541 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      601 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ggtttagtga
      661 accgtcagat ccgctagcgc taccggactc agatctacca ttggtgcacc tgactcctga
      721 ggagaagtct gccgttactg ccctgtgggg caaggtgaac gtggaagagt tggtggtgag
      781 gccctgggcc accagtaagt atcaaggtta caagacaggt ttaaggagac caatagaaac
      841 tgggcatgtg gagacagaga agactcacgc gtttctgaat tcactgactc tctctgccta
      901 ttggtctatt ttcccaccct taggctgctg gtggtgccat ggcaggccct gggcaggttg
      961 gtatcaaggt tacaagacag gtttaaggag accaatggat ccatagtcga ccaccatggt
     1021 ggcttagatc cgggcatgtg gagacagaga agactgttga gtttgtgata agcactgact
     1081 ctctctgcct attggtctat tttccctccc tcagtggcgg tcgaggacaa actctacgcg
     1141 gacttgccag tacgcttcgt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     1201 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1261 ggcgacgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1321 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt tagccgctac
     1381 cccgaccacc tgaagcagca cgacttcttc aagagtgcaa tgcccgaagg ctacgtccag
     1441 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1501 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1561 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcaccgcc
     1621 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     1681 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1741 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     1801 cgcgatcata tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcaccgac
     1861 gagctgtaca agtaaccggt cgccaccatg gtgagcaagg gcgaggagaa taacatggcc
     1921 atcatcaagg agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag
     1981 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     2041 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctaacccc caacttcacc
     2101 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
     2161 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     2221 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     2281 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     2341 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagat gaggctgaag
     2401 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     2461 gtgcagctgc ccggcgccta catcgtcggc atcaagttgg acatcacctc ccacaacgag
     2521 gactacacca tcgtggaact gtacgaacgc gccgagggcc gccactccac cggcggcatg
     2581 gacgagctgt ataagtaagc ggccgcgact ctagatcata atcagccata ccacatttgt
     2641 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     2701 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     2761 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     2821 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     2881 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2941 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     3001 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     3061 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     3121 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     3181 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     3241 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     3301 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3361 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3421 gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     3481 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3541 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3601 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3661 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3721 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3781 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3841 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3901 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3961 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     4021 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4081 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4141 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4201 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4261 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4321 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     4381 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4441 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4501 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4561 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4621 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4681 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4741 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     4801 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     4861 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     4921 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     4981 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     5041 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     5101 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5161 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5221 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5281 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5341 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5401 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5461 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5521 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5581 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5641 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5701 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5761 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5821 cgccagcaac gcggcctttt tacggttcct
//
