LOCUS       sequence-469842-        5328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the mutant T6B peptide fused with YFP under the human 
            synapsin promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5328)
  AUTHORS   Zolboot N, Xiao Y, Du JX, Ghanem MM, Choi SY, Junn MJ, Zampa F, 
            Huang Z, MacRae IJ, Lippi G
  TITLE     MicroRNA mechanisms instructing Purkinje cell specification.
  JOURNAL   Neuron. 2025 Apr 1:S0896-6273(25)00181-3. doi: 
            10.1016/j.neuron.2025.03.009.
  PUBMED    40179877
REFERENCE   2  (bases 1 to 5328)
  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"; journalName: "Neuron. 
            2025 Apr 1:S0896-6273(25)00181-3. doi: 
            10.1016/j.neuron.2025.03.009."
COMMENT     Sequence Label: pAAV-hSyn-FLAG-HA-T6B-MUT-YFP
FEATURES             Location/Qualifiers
     source          1..5328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        175..622
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      653..662
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             662..685
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             686..712
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(992..994,995..997,998..1711)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1037..1058)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1298..1317)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1645..1666
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1718..1742)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    1752..2340
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1805..1825)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2223..2234)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2370..2387)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2376..2583
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2591..2731
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2591..2720
                     /label=AAV2 ITR
     rep_origin      2806..3261
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2893..2912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3103..3124
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3278..3297)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3397..3419
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3457..3475)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3543..3647
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3648..3716,3717..4508)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3866..3885)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4679..5267
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5168..5187
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttgac tagtagtgca
      181 agtgggtttt aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga
      241 cccactggac aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg
      301 gaggggaaac aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt
      361 gccttcgccc ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac
      421 gtcactcgcc ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc
      481 cgccggccca gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca
      541 tctgcgctgc ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt
      601 cgtgtcgtgc ctgagagcgc agtcgatcta gacgataagg gcgaatctcg aggccaccat
      661 ggactacaag gacgacgatg acaagtaccc ttatgacgtg cccgattacg ctagcggccg
      721 cgattgtcag gctgtcttgc agactctttt gagccgaact gatttggacc ccagggtgct
      781 ctcaaacact ggcgccggcc aaactcaaat taagcaggac acagtggccg acattgaaga
      841 ggtgccaagg cctgagggga aatctgacaa aggaactgag ggggccgaga gcgctgccac
      901 acagaccaag aactcagggg gcgccggaga tgcacccagc caaagcaatc aaatgaagtc
      961 tggagccggg gagctcgtcg ccaccggatc catggtgagc aagggcgagg agctgttcac
     1021 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1081 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1141 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgca
     1201 gtgcttcgcc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1261 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1321 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1381 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1441 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1501 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1561 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc taccagtccg ccctgagcaa
     1621 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     1681 cactctcggc atggacgagc tgtacaagta agtcgacacc cagctttctt gtacaaagtg
     1741 ggaattccga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     1801 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     1861 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     1921 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     1981 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     2041 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     2101 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc
     2161 catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     2221 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     2281 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     2341 atcgggaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2401 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2461 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2521 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg
     2581 ggagggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     2641 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     2701 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     2761 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     2821 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2881 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2941 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3001 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     3061 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3121 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     3181 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3241 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     3301 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     3361 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     3421 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     3481 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     3541 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3601 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     3661 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     3721 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     3781 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     3841 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     3901 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     3961 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4021 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4081 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4141 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4201 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4261 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4321 ctggctggtt tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg
     4381 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     4441 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     4501 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     4561 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     4621 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4681 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4741 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4801 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     4861 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4921 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4981 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5041 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5101 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5161 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5221 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5281 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgt
//
