LOCUS       sequence-194018-        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        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      932..941
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1077..1079,1080..1793)
                     /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(1119..1140)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1380..1399)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      1800..1809
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1806..2516
                     /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(1951..1969)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2219..2238
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2638..2759
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2675..2694)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2729..2748
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2766..3221)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2903..2924)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3115..3134
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3248..3352
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3354..3711
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3354..3545
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3377..3397)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3562..3697
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3746..4540
                     /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             3746..4540
                     /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(3800..3819)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4410..4429
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4728..4747)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4772..4819
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5148..5736
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5637..5656
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctaccatt ggtgcacctg actcctgagg agaagtctgc cgttactgcc
      661 ctgtggggca aggtgaacgt ggaagagttg gtggtgaggc cctgggccac cagtaagtat
      721 caaggttaca agacaggttt aaggagacca atagaaactg ggcatgtgga gacagagaag
      781 actcacgcgt ttctgaattc actgactctc tctgcctatt ggtctatttt cccaccctta
      841 ggctgctggt ggtgccatgg caggccctgg gcaggttggt atcaaggtta caagacaggt
      901 ttaaggagac caatggatcc atagtcgacc accatggtgg cttagatccg ggcatgtgga
      961 gacagagaag actgttgagt ttgtgataag cactgactct ctctgcctat tggtctattt
     1021 tccctccctc agtggcggtc gaggacaaac tctacgcgga cttgccagta cgcttcgtga
     1081 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1141 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgacgccacc tacggcaagc
     1201 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1261 ccaccctgac ctacggcgtg cagtgcttta gccgctaccc cgaccacctg aagcagcacg
     1321 acttcttcaa gagtgcaatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1381 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1441 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1501 agtacaacta caacagccac aacgtctata tcaccgccga caagcagaag aacggcatca
     1561 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     1621 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1681 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcatatg gtcctgctgg
     1741 agttcgtgac cgccgccggg atcactctcg gcaccgacga gctgtacaag taaccggtcg
     1801 ccaccatggt gagcaagggc gaggagaata acatggccat catcaaggag ttcatgcgct
     1861 tcaaggtgcg catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     1921 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     1981 tgcccttcgc ctgggacatc ctaaccccca acttcaccta cggctccaag gcctacgtga
     2041 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     2101 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     2161 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     2221 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
     2281 acggcgccct gaagggcgag atcaagatga ggctgaagct gaaggacggc ggccactacg
     2341 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     2401 tcgtcggcat caagttggac atcacctccc acaacgagga ctacaccatc gtggaactgt
     2461 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtat aagtaagcgg
     2521 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     2581 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     2641 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2701 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     2761 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     2821 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     2881 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     2941 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     3001 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     3061 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     3121 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     3181 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     3241 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     3301 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     3361 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3421 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3481 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3541 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3601 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     3661 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     3721 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     3781 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     3841 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     3901 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     3961 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4021 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4081 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4141 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4201 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4261 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4321 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4381 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4441 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4501 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4561 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4621 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4681 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     4741 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     4801 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     4861 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     4921 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     4981 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5041 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5101 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     5161 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     5221 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     5281 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     5341 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     5401 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     5461 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     5521 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     5581 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     5641 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     5701 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5761 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5821 tctgtggata accgtattac cgccatgcat
//
