LOCUS       sequence-238429-        7161 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV plasmid with human synapsin promoter driving mCh and 
            SspB(milli)-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(milli)-BoNT/B(147-441,...
FEATURES             Location/Qualifiers
     source          1..7161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   8..137
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        182..629
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      655..664
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             661..1368
                     /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(806..824)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1074..1093
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1970..1986)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1971..1987)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     regulatory      1989..1998
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2004..2210,2211..2213,2214..2333)
                     /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
                     IVLNLSASVTGNLQLTNDFIQFNAQFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     misc_feature    3295..3883
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3348..3368)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3766..3777)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3915..4391
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4114..4133)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4431..4571
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4431..4560
                     /label=AAV2 ITR
     rep_origin      4646..5101
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4733..4752)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4943..4964
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5118..5137)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5237..5259
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5297..5315)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5383..5487
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5488..5556,5557..6348)
                     /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(5706..5725)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6519..7107
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7008..7027
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cacatgtcct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc
       61 gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc
      121 catcactagg ggttcctgcg gccgcacgcg tgtgtctaga ctgcagaggg ccctgcgtat
      181 gagtgcaagt gggttttagg accaggatga ggcggggtgg gggtgcctac ctgacgaccg
      241 accccgaccc actggacaag cacccaaccc ccattcccca aattgcgcat cccctatcag
      301 agagggggag gggaaacagg atgcggcgag gcgcgtgcgc actgccagct tcagcaccgc
      361 ggacagtgcc ttcgcccccg cctggcggcg cgcgccaccg ccgcctcagc actgaaggcg
      421 cgctgacgtc actcgccggt cccccgcaaa ctccccttcc cggccacctt ggtcgcgtcc
      481 gcgccgccgc cggcccagcc ggaccgcacc acgcgaggcg cgagataggg gggcacgggc
      541 gcgaccatct gcgctgcggc gccggcgact cagcgctgcc tcagtctgcg gtgggcagcg
      601 gaggagtcgt gtcgtgcctg agagcgcagt cgagaaggta ccggatctgc tagcgccacc
      661 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
      721 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
      781 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
      841 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
      901 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
      961 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
     1021 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     1081 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
     1141 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
     1201 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
     1261 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
     1321 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtg tggcggctag
     1381 tactccggta ttgcggtacc cttgtacgcc tgttttatac tcccttcccg taacttagac
     1441 gcacaaaacc aagttcaata gaagggggta caaaccagta ccaccacgaa caagcacttc
     1501 tgtttccccg gtgatgtcgt atagactgct tgcgtggttg aaagcgacgg atccgttatc
     1561 cgcttatgta cttcgagaag cccagtacca cctcggaatc ttcgatgcgt tgcgctcagc
     1621 actcaacccc agagtgtagc ttaggctgat gagtctggac atccctcacc ggtgacggtg
     1681 gtccaggctg cgttggcgac ctacctatgg ctaacgccat gggacgctag ttgtgaacaa
     1741 ggtgtgaaga gcctattgag ctacataaga atcctccggc ccctgaatgc ggctaatccc
     1801 aacctcggag caggtggtca caaaccagtg attggcctgt cgtaacgcgc aagtccgtgg
     1861 cggaaccgac tactttgggt gtccgtgttt ccttttattt tattgtggct gcttatggtg
     1921 acaatcacag attgttatca taaagcgaat tggataggat caagcttatc gataccgtcg
     1981 acctcgaggc caccatggaa tacagctccc cgaaacgccc taagctgctg cgtgaatatt
     2041 acgattggct ggttgataac agctttaccc catatctggt ggtggatgcc acatacctgg
     2101 gcgtgaacgt gcccgtggag tatgtgaaag acggtcagat cgtgctgaat ctgtctgcaa
     2161 gtgtgaccgg caacctgcaa ctgacaaatg attttatcca gttcaacgcc cagtttaagg
     2221 gcgtgtctcg tgaactgtat atcccgatgg gtgccgctct ggccatttac gctcgcgaga
     2281 acggcgatgg tgtgatgttc gaaccagaag aaatctatga cgagctgaat attgagctcg
     2341 gtggcggtgg ctctggaggt ggtgggtccg gaggaggcgg ccgcgaagtg gagcgaaaaa
     2401 aaggtatttt cgcaaattta ataatatttg gacctgggcc agttttaaat gaaaatgaga
     2461 ctatagatat aggtatacaa aatcattttg catcaaggga aggcttcggg ggtataatgc
     2521 aaatgaagtt ttgcccagaa tatgtaagcg tatttaataa tgttcaagaa aacaaaggcg
     2581 caagtatatt taatagacgt ggatattttt cagatccagc cttgatatta atgcatgaac
     2641 ttatacatgt tttacatgga ttatatggca ttaaagtaga tgatttacca attgtaccaa
     2701 atgaaaaaaa attttttatg caatctacag atgctataca ggcagaagaa ctatatacat
     2761 ttggaggaca agatcccagc atcataactc cttctacgga taaaagtatc tatgataaag
     2821 ttttgcaaaa ttttagaggg atagttgata gacttaacaa ggttttagtt tgcatatcag
     2881 atcctaacat taatattaat atatataaaa ataaatttaa agataaatat aaattcgttg
     2941 aagattctga gggaaaatat agtatagatg tagaaagttt tgataaatta tataaaagct
     3001 taatgtttgg ttttacagaa actaatatag cagaaaatgc caaaataaaa actagagctt
     3061 cttattttag tgattcctta ccaccagtaa aaataaaaaa tttattagat aatgaaatct
     3121 atactataga ggaagggttt aatatatctg ataaagatat ggaaaaagaa tatagaggtc
     3181 agaataaagc tataaataaa caagcttatg aagaaattag caaggagcat ttggctgtat
     3241 ataagataca aatgtgtaaa agtgttaaat gaattcgata tcaagcttat cgataatcaa
     3301 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3361 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3421 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3481 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3541 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3601 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     3661 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat
     3721 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     3781 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     3841 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgagcgctg
     3901 ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg
     3961 aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt
     4021 ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc
     4081 aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag
     4141 tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc
     4201 agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt
     4261 tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg
     4321 tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct
     4381 tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc aggaacccct
     4441 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4501 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4561 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4621 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4681 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4741 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4801 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4861 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4921 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     4981 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     5041 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5101 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5161 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5221 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5281 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5341 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5401 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5461 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5521 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5581 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5641 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5701 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5761 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     5821 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     5881 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5941 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6001 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6061 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6121 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6181 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6241 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6301 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6361 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6421 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6481 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6541 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6601 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6661 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6721 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6781 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6841 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6901 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6961 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7021 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7081 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7141 gccttttgct ggccttttgc t
//
