LOCUS       sequence-238430-        7161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV plasmid with human synapsin promoter driving mCh and 
            SspB(micro)-BoNT/B amino acids 147-441 separated with an IRES 
            element. Co-express with BoNT/B(1-146)-iLID construct for PA-BoNT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7161)
  AUTHORS   Liu Q, Sinnen BL, Boxer EE, Schneider MW, Grybko MJ, Buchta WC, 
            Gibson ES, Wysoczynski CL, Ford CP, Gottschalk A, Aoto J, Tucker CL,
            Kennedy MJ
  TITLE     A Photoactivatable Botulinum Neurotoxin for Inducible Control of 
            Neurotransmission.
  JOURNAL   Neuron. 2019 Jan 15. pii: S0896-6273(19)30003-0. doi: 
            10.1016/j.neuron.2019.01.002.
  PUBMED    30704911
REFERENCE   2  (bases 1 to 7161)
  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. 
            2019 Jan 15. pii: S0896-6273(19)30003-0. doi: 
            10.1016/j.neuron.2019.01.002."
COMMENT     Sequence Label: pAAV hSyn mCh-IRES-SspB(micro)-BoNT/B(147-441,...
FEATURES             Location/Qualifiers
     source          1..7161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   35..164
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        209..656
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      682..691
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             688..1395
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(833..851)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1101..1120
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1997..2013)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1998..2014)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     regulatory      2016..2025
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2031..2237,2238..2240,2241..2360)
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein and with the mutation R74Q to reduce the 
                     affinity for iLID"
                     /label=SspB micro
                     /note="dimerizes with iLID at moderate affinity to create 
                     iLID micro (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNAQFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     misc_feature    3322..3910
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3375..3395)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3793..3804)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3942..4418
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4141..4160)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4458..4598
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4458..4587
                     /label=AAV2 ITR
     rep_origin      4673..5128
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4760..4779)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4970..4991
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5145..5164)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5264..5286
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5324..5342)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5410..5514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5515..5583,5584..6375)
                     /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(5733..5752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6546..7134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7035..7054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg
       61 ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg
      121 agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc gcacgcgtgt
      181 gtctagactg cagagggccc tgcgtatgag tgcaagtggg ttttaggacc aggatgaggc
      241 ggggtggggg tgcctacctg acgaccgacc ccgacccact ggacaagcac ccaaccccca
      301 ttccccaaat tgcgcatccc ctatcagaga gggggagggg aaacaggatg cggcgaggcg
      361 cgtgcgcact gccagcttca gcaccgcgga cagtgccttc gcccccgcct ggcggcgcgc
      421 gccaccgccg cctcagcact gaaggcgcgc tgacgtcact cgccggtccc ccgcaaactc
      481 cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg cccagccgga ccgcaccacg
      541 cgaggcgcga gatagggggg cacgggcgcg accatctgcg ctgcggcgcc ggcgactcag
      601 cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc gtgcctgaga gcgcagtcga
      661 gaaggtaccg gatctgctag cgccaccatg gtgagcaagg gcgaggagga taacatggcc
      721 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
      781 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
      841 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
      901 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc
      961 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1021 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     1081 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1141 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     1201 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     1261 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     1321 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     1381 gacgagctgt acaagtgtgg cggctagtac tccggtattg cggtaccctt gtacgcctgt
     1441 tttatactcc cttcccgtaa cttagacgca caaaaccaag ttcaatagaa gggggtacaa
     1501 accagtacca ccacgaacaa gcacttctgt ttccccggtg atgtcgtata gactgcttgc
     1561 gtggttgaaa gcgacggatc cgttatccgc ttatgtactt cgagaagccc agtaccacct
     1621 cggaatcttc gatgcgttgc gctcagcact caaccccaga gtgtagctta ggctgatgag
     1681 tctggacatc cctcaccggt gacggtggtc caggctgcgt tggcgaccta cctatggcta
     1741 acgccatggg acgctagttg tgaacaaggt gtgaagagcc tattgagcta cataagaatc
     1801 ctccggcccc tgaatgcggc taatcccaac ctcggagcag gtggtcacaa accagtgatt
     1861 ggcctgtcgt aacgcgcaag tccgtggcgg aaccgactac tttgggtgtc cgtgtttcct
     1921 tttattttat tgtggctgct tatggtgaca atcacagatt gttatcataa agcgaattgg
     1981 ataggatcaa gcttatcgat accgtcgacc tcgaggccac catggaatac agctccccga
     2041 aacgccctaa gctgctgcgt gaatattacg attggctggt tgataacagc tttaccccat
     2101 atctggtggt ggatgccaca tacctgggcg tgaacgtgcc cgtggagtat gtgaaagacg
     2161 gtcagatcgt gctgaatctg tctgcaagtg cgaccggcaa cctgcaactg acaaatgatt
     2221 ttatccagtt caacgcccag tttaagggcg tgtctcgtga actgtatatc ccgatgggtg
     2281 ccgctctggc catttacgct cgcgagaacg gcgatggtgt gatgttcgaa ccagaagaaa
     2341 tctatgacga gctgaatatt gagctcggtg gcggtggctc tggaggtggt gggtccggag
     2401 gaggcggccg cgaagtggag cgaaaaaaag gtattttcgc aaatttaata atatttggac
     2461 ctgggccagt tttaaatgaa aatgagacta tagatatagg tatacaaaat cattttgcat
     2521 caagggaagg cttcgggggt ataatgcaaa tgaagttttg cccagaatat gtaagcgtat
     2581 ttaataatgt tcaagaaaac aaaggcgcaa gtatatttaa tagacgtgga tatttttcag
     2641 atccagcctt gatattaatg catgaactta tacatgtttt acatggatta tatggcatta
     2701 aagtagatga tttaccaatt gtaccaaatg aaaaaaaatt ttttatgcaa tctacagatg
     2761 ctatacaggc agaagaacta tatacatttg gaggacaaga tcccagcatc ataactcctt
     2821 ctacggataa aagtatctat gataaagttt tgcaaaattt tagagggata gttgatagac
     2881 ttaacaaggt tttagtttgc atatcagatc ctaacattaa tattaatata tataaaaata
     2941 aatttaaaga taaatataaa ttcgttgaag attctgaggg aaaatatagt atagatgtag
     3001 aaagttttga taaattatat aaaagcttaa tgtttggttt tacagaaact aatatagcag
     3061 aaaatgccaa aataaaaact agagcttctt attttagtga ttccttacca ccagtaaaaa
     3121 taaaaaattt attagataat gaaatctata ctatagagga agggtttaat atatctgata
     3181 aagatatgga aaaagaatat agaggtcaga ataaagctat aaataaacaa gcttatgaag
     3241 aaattagcaa ggagcatttg gctgtatata agatacaaat gtgtaaaagt gttaaatgaa
     3301 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     3361 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3421 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     3481 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     3541 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     3601 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     3661 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     3721 tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc
     3781 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     3841 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     3901 gcctccccgc atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc
     3961 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc
     4021 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt
     4081 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
     4141 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct
     4201 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca
     4261 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca
     4321 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg
     4381 gattacaggc gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt
     4441 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     4501 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     4561 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
     4621 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
     4681 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4741 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4801 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4861 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     4921 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     4981 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
     5041 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5101 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
     5161 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     5221 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     5281 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
     5341 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     5401 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5461 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5521 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5581 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5641 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5701 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5761 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5821 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5881 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     5941 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6001 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6061 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6121 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6181 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6241 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6301 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6361 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6421 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6481 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6541 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6601 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6661 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6721 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6781 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6841 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6901 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6961 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7021 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7081 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7141 agcaacgcgg cctttttacg g
//
