LOCUS       sequence-194275-        5729 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 5729)
  AUTHORS   Zheng S, Damoiseaux R, Chen L, Black DL
  TITLE     A broadly applicable high-throughput screening strategy identifies 
            new regulators of Dlg4 (Psd-95) alternative splicing.
  JOURNAL   Genome Res. 2013 Jun;23(6):998-1007. doi: 10.1101/gr.147546.112. 
            Epub 2013 May 1.
  PUBMED    23636947
REFERENCE   2  (bases 1 to 5729)
  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: 
            "10.1101/gr.147546.112"; journalName: "Genome Res"; date: "2013-06";
            volume: "23"; issue: "6"; pages: "998-1007"
COMMENT     Sequence Label: pFlare5GdP
FEATURES             Location/Qualifiers
     source          1..5729
                     /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      811..820
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(956..958,959..1672)
                     /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(998..1019)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1259..1278)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      1679..1688
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1685..2395
                     /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(1830..1848)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2098..2117
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2517..2638
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2554..2573)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2608..2627
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2645..3100)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2782..2803)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2994..3013
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3127..3231
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3233..3590
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3233..3424
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3256..3276)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3441..3576
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3625..4419
                     /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             3625..4419
                     /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(3679..3698)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4289..4308
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4607..4626)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4651..4698
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5027..5615
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5516..5535
                     /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 aggtaagtat
      721 caaggttaca agacaggttt aaggagacca atagaaactg ggcatgtgga gacagagaag
      781 actcttgggt ttctgaattc attggatcca ccatggtggc ttagatccgg gcatgtggag
      841 acagagaaga ctgttgagtt tgtgataagc actgactctc tctgcctatt ggtctatttt
      901 ccctccctca gtggcggtcg aggacaaact ctacgcggac ttgccagtac gcttcgtgag
      961 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1021 aaacggccac aagttcagcg tgtccggcga gggcgagggc gacgccacct acggcaagct
     1081 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     1141 caccctgacc tacggcgtgc agtgctttag ccgctacccc gaccacctga agcagcacga
     1201 cttcttcaag agtgcaatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     1261 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1321 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1381 gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga acggcatcaa
     1441 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1501 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1561 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcatatgg tcctgctgga
     1621 gttcgtgacc gccgccggga tcactctcgg caccgacgag ctgtacaagt aaccggtcgc
     1681 caccatggtg agcaagggcg aggagaataa catggccatc atcaaggagt tcatgcgctt
     1741 caaggtgcgc atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     1801 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     1861 gcccttcgcc tgggacatcc taacccccaa cttcacctac ggctccaagg cctacgtgaa
     1921 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     1981 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
     2041 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     2101 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     2161 cggcgccctg aagggcgaga tcaagatgag gctgaagctg aaggacggcg gccactacga
     2221 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacat
     2281 cgtcggcatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaactgta
     2341 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtata agtaagcggc
     2401 cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     2461 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     2521 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     2581 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag
     2641 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     2701 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     2761 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     2821 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     2881 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     2941 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     3001 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3061 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     3121 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3181 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg
     3241 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     3301 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     3361 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3421 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3481 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     3541 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa
     3601 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3661 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3721 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3781 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     3841 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3901 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3961 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4021 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4081 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4141 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4201 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4261 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4321 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4381 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4441 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4501 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4561 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg
     4621 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
     4681 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     4741 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     4801 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     4861 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     4921 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     4981 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5041 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5101 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5161 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     5221 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     5281 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     5341 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     5401 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     5461 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     5521 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     5581 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     5641 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     5701 ctgtggataa ccgtattacc gccatgcat
//
