LOCUS       sequence-424740-        6736 bp    DNA     circular SYN 15-APR-2025
DEFINITION  minigene construct for Clip2 E9 alternative splicing .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6736)
  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 6736)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 WT
FEATURES             Location/Qualifiers
     source          1..6736
                     /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      1818..1827
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1963..1965,1966..2679)
                     /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(2005..2026)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2266..2285)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      2686..2695
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2692..3402
                     /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(2837..2855)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3105..3124
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3524..3645
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3561..3580)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3615..3634
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3652..4107)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3789..3810)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4001..4020
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4134..4238
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4240..4597
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4240..4431
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4263..4283)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4448..4583
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4632..5426
                     /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             4632..5426
                     /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(4686..4705)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5296..5315
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5614..5633)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5658..5705
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6034..6622
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6523..6542
                     /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 ggcacccagc cctctcacgc accccacttc tgagacctct ctctcttccc caagcaattc
     1081 cggctcattg atctgttttc agtcttgttc acggaagatg acgcctcctc tctctctctc
     1141 tctctttttc cttctcacgc cctttctttc tttctccgac ttcctgttcc tatattcctt
     1201 tccccacgtc cctgccttcc ctgtccaccc tgccatccct cccccacccc cgccctgggt
     1261 ggagcagacc cagacccagc tggagcacgc gcgcattggg gagctggagc agagcctgct
     1321 attggagaag gcgcaggccg agcggctgct cagagaatta gcggataaca gggtaaaccg
     1381 cgcctgccac tgtcacccac ccggccagcg ccttggcccc tagatgcccc ctttgctttt
     1441 taaatcattt cctctgccat cacaatggct cctcttacta ttgctttttg gagacccttc
     1501 tgaactcccc tctgggaggg aagctgagat ttagagtcaa gctcactgtg gccaagtagt
     1561 gctcttaggg aaggggggtt tggtcctctg ggatttcttc caagttcatt gctccttgag
     1621 cagaggtgtc tctgtgagac cttccctgtg cctccctcct gtcccttgcc agaaagaagc
     1681 cttcttggag tggcctgccc cttacttgga tagacatggg cttccttggc aggaatgcag
     1741 acccagagtg ttctagccta gagaatctta agatggcagc agagagccaa cctggaggac
     1801 ggatccatag tcgaccacca tggtggctta gatccgggca tgtggagaca gagaagactg
     1861 ttgagtttgt gataagcact gactctctct gcctattggt ctattttccc tccctcagtg
     1921 gcggtcgagg acaaactcta cgcggacttg ccagtacgct tcgtgagcaa gggcgaggag
     1981 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     2041 ttcagcgtgt ccggcgaggg cgagggcgac gccacctacg gcaagctgac cctgaagttc
     2101 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     2161 ggcgtgcagt gctttagccg ctaccccgac cacctgaagc agcacgactt cttcaagagt
     2221 gcaatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2281 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2341 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2401 agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggt gaacttcaag
     2461 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2521 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     2581 ctgagcaaag accccaacga gaagcgcgat catatggtcc tgctggagtt cgtgaccgcc
     2641 gccgggatca ctctcggcac cgacgagctg tacaagtaac cggtcgccac catggtgagc
     2701 aagggcgagg agaataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcgcatg
     2761 gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac
     2821 gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc cttcgcctgg
     2881 gacatcctaa cccccaactt cacctacggc tccaaggcct acgtgaagca ccccgccgac
     2941 atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac
     3001 ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga cggcgagttc
     3061 atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt aatgcagaag
     3121 aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg cgccctgaag
     3181 ggcgagatca agatgaggct gaagctgaag gacggcggcc actacgacgc tgaggtcaag
     3241 accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacatcgt cggcatcaag
     3301 ttggacatca cctcccacaa cgaggactac accatcgtgg aactgtacga acgcgccgag
     3361 ggccgccact ccaccggcgg catggacgag ctgtataagt aagcggccgc gactctagat
     3421 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     3481 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     3541 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3601 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa
     3661 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     3721 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     3781 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     3841 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     3901 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     3961 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4021 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4081 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4141 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4201 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     4261 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4321 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     4381 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     4441 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4501 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4561 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag
     4621 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     4681 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     4741 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     4801 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     4861 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     4921 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     4981 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     5041 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     5101 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     5161 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     5221 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     5281 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     5341 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     5401 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     5461 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5521 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5581 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     5641 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     5701 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     5761 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     5821 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
     5881 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5941 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6001 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6061 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6121 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6181 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6241 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6301 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6361 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6421 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6481 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6541 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6601 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6661 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     6721 tattaccgcc atgcat
//
