LOCUS       sequence-469833-        5718 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the mutant T6B peptide fused with YFP under the Purkinje 
            cell-specific L7-6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5718)
  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 5718)
  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-L7-6-FLAG-HA-T6B-MUT-YFP
FEATURES             Location/Qualifiers
     source          1..5718
                     /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"
     regulatory      1043..1052
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1052..1075
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1076..1102
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             join(1382..1384,1385..1387,1388..2101)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1427..1448)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1688..1707)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2035..2056
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2108..2132)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2142..2730
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2195..2215)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2613..2624)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2760..2777)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2766..2973
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2981..3121
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2981..3110
                     /label=AAV2 ITR
     rep_origin      3196..3651
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3283..3302)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3493..3514
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3668..3687)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3787..3809
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3847..3865)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3933..4037
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4038..4106,4107..4898)
                     /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(4256..4275)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5069..5657
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5558..5577
                     /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 tagtggttcc
      181 accctcatgt tggttgatct tcaacattgt cctgacttct tcccacttga cattcctcag
      241 ggtcctgtga tcatggctgg gtctagtgag gttcaaacct gcactgccct acccacaccc
      301 acacccagct cagcgtcagt caggatcaac agttacctag agatcatctt tctggggctt
      361 aagcattggt gggagcagat gggatatgag ttggggattt ggggatgggg gaagatatct
      421 gctccccctc cccctacacc ctagcctttt aaaaggcctt ctcaggtcag agaccaggag
      481 aaaagtatag gagagataca caatggacca ggaagaagaa aagggagagg gaggctcaga
      541 ccttctagac aaggtaagag ggctctggct gactccacca tccgcttctt gaggtctcgg
      601 cacctgtaat tgacaagatt aattcattta tagggcatct aattagcaag taagtctctg
      661 gagtcccctg acccagttac tataacacac agggggtata ggtaggggag tataagagcc
      721 cctcctcagg gcaaatgaat ggattcttag tactgtcccc caagagatag taggtactag
      781 gatttagggg cacttctgag ccccatttcc ctggtaagtg tcccaacccc ccaaatcaac
      841 ccaagcctgg tctcaatcta ggacagtggt agaatgctgt ccctagagtc agtaccatgt
      901 gaaattgtgc tgcaggcagg ggccccaggc tgggaggtgg gggttggggg agtcaggggc
      961 aggtcaggga aggagactca ggtttcattt agagaaattc tgcagacccg tgaggactca
     1021 agtttgtaca aaaaagctcg aggccaccat ggactacaag gacgacgatg acaagtaccc
     1081 ttatgacgtg cccgattacg ctagcggccg cgattgtcag gctgtcttgc agactctttt
     1141 gagccgaact gatttggacc ccagggtgct ctcaaacact ggcgccggcc aaactcaaat
     1201 taagcaggac acagtggccg acattgaaga ggtgccaagg cctgagggga aatctgacaa
     1261 aggaactgag ggggccgaga gcgctgccac acagaccaag aactcagggg gcgccggaga
     1321 tgcacccagc caaagcaatc aaatgaagtc tggagccggg gagctcgtcg ccaccggatc
     1381 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1441 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     1501 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1561 cctcgtgacc accttcggct acggcctgca gtgcttcgcc cgctaccccg accacatgaa
     1621 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     1681 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1741 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1801 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     1861 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     1921 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1981 ctacctgagc taccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     2041 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     2101 agtcgacacc cagctttctt gtacaaagtg ggaattccga taatcaacct ctggattaca
     2161 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
     2221 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
     2281 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
     2341 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
     2401 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
     2461 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
     2521 tggtgttgtc ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga
     2581 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
     2641 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
     2701 gtcggatctc cctttgggcc gcctccccgc atcgggaatt cctagagctc gctgatcagc
     2761 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     2821 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     2881 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     2941 ggattgggaa gagaatagca ggcatgctgg ggagggccgc aggaacccct agtgatggag
     3001 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3061 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     3121 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     3181 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3241 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     3301 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3361 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     3421 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3481 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     3541 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     3601 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     3661 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     3721 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3781 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     3841 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3901 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3961 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4021 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4081 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4141 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4201 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4261 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4321 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4381 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4441 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4501 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4561 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4621 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4681 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4741 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4801 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4861 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4921 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4981 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5041 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5101 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5161 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5221 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5281 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5341 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5401 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5461 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5521 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5581 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5641 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5701 ggccttttgc tcacatgt
//
