LOCUS       sequence-424741-        6627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  minigene construct for Clip2 E9 alternative splicing, potential 
            PTBP2 binding sites are deleted.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6627)
  AUTHORS   Zhang M, Ergin V, Lin L, Stork C, Chen L, Zheng S
  TITLE     Axonogenesis Is Coordinated by Neuron-Specific Alternative Splicing 
            Programming and Splicing Regulator PTBP2.
  JOURNAL   Neuron. 2019 Feb 20;101(4):690-706.e10. doi: 
            10.1016/j.neuron.2019.01.022. Epub 2019 Feb 4.
  PUBMED    30733148
REFERENCE   2  (bases 1 to 6627)
  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: 
            "10.1016/j.neuron.2019.01.022"; journalName: "Neuron"; date: 
            "2019-02-20- 20"; volume: "101"; issue: "4"; pages: "690-706.e10"
COMMENT     Sequence Label: pFlare9A-Clip2E9 Mutant
FEATURES             Location/Qualifiers
     source          1..6627
                     /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      1709..1718
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1854..1856,1857..2570)
                     /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(1896..1917)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2157..2176)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      2577..2586
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2583..3293
                     /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(2728..2746)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2996..3015
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3415..3536
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3452..3471)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3506..3525
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3543..3998)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3680..3701)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3892..3911
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4025..4129
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4131..4488
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4131..4322
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4154..4174)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4339..4474
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4523..5317
                     /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             4523..5317
                     /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(4577..4596)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5187..5206
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5505..5524)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5549..5596
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5925..6513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6414..6433
                     /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 ttctaactgc cctcaaatgc acggtggcat gtgcacaggc
      841 acacatacta aatgaataaa taaaatgcac ctaacaaaca aagaaagact gactctttaa
      901 atcccacaaa ggaagaaact ggggtaccca tagccactgc gttatcagag gctggcaagg
      961 ggtggttccc agagcctctt ctgccttgaa cacacgccca gctagcccca gtgatgggag
     1021 ggcacccagg agaaagcaat tccggctcat tgaagtcttg ttcacggaag atgacgcacg
     1081 gaatattcct ttccccacgt ccctgccttc cctgtccacc ctgccatccc tcccccaccc
     1141 ccgccctggg tggagcagac ccagacccag ctggagcacg cgcgcattgg ggagctggag
     1201 cagagcctgc tattggagaa ggcgcaggcc gagcggctgc tcagagaatt agcggataac
     1261 agggtaaacc gcgcctgcca ctgtcaccca cccggccagc gccttggccc ctagatgccc
     1321 cctttgcttt ttaaatcatt tcctctgcca tcacaatggc tcctcttact attgcttttt
     1381 ggagaccctt ctgaactccc ctctgggagg gaagctgaga tttagagtca agctcactgt
     1441 ggccaagtag tgctcttagg gaaggggggt ttggtcctct gggatttctt ccaagttcat
     1501 tgctccttga gcagaggtgt ctctgtgaga ccttccctgt gcctccctcc tgtcccttgc
     1561 cagaaagaag ccttcttgga gtggcctgcc ccttacttgg atagacatgg gcttccttgg
     1621 caggaatgca gacccagagt gttctagcct agagaatctt aagatggcag cagagagcca
     1681 acctggagga cggatccata gtcgaccacc atggtggctt agatccgggc atgtggagac
     1741 agagaagact gttgagtttg tgataagcac tgactctctc tgcctattgg tctattttcc
     1801 ctccctcagt ggcggtcgag gacaaactct acgcggactt gccagtacgc ttcgtgagca
     1861 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1921 acggccacaa gttcagcgtg tccggcgagg gcgagggcga cgccacctac ggcaagctga
     1981 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2041 ccctgaccta cggcgtgcag tgctttagcc gctaccccga ccacctgaag cagcacgact
     2101 tcttcaagag tgcaatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2161 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2221 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2281 acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg
     2341 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2401 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2461 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcatatggtc ctgctggagt
     2521 tcgtgaccgc cgccgggatc actctcggca ccgacgagct gtacaagtaa ccggtcgcca
     2581 ccatggtgag caagggcgag gagaataaca tggccatcat caaggagttc atgcgcttca
     2641 aggtgcgcat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     2701 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     2761 ccttcgcctg ggacatccta acccccaact tcacctacgg ctccaaggcc tacgtgaagc
     2821 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc
     2881 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg
     2941 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     3001 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg
     3061 gcgccctgaa gggcgagatc aagatgaggc tgaagctgaa ggacggcggc cactacgacg
     3121 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacatcg
     3181 tcggcatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaactgtacg
     3241 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtataag taagcggccg
     3301 cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3361 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3421 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3481 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     3541 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3601 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     3661 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     3721 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     3781 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     3841 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     3901 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     3961 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     4021 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4081 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     4141 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4201 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4261 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4321 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4381 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4441 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4501 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4561 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4621 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     4681 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     4741 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     4801 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     4861 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     4921 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     4981 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5041 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5101 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5161 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5221 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5281 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5341 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5401 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5461 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     5521 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     5581 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     5641 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     5701 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     5761 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     5821 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     5881 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     5941 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6001 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6061 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6121 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6181 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6241 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6301 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6361 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6421 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6481 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6541 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6601 gtggataacc gtattaccgc catgcat
//
