LOCUS       sequence-239593-        6230 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses botulinum toxin B (aa1-146) fused to AsLOV2-ssrA 
            (iLID)-V416I (in AsLOV).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6230)
  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 6230)
  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"; journalName: "Neuron. 
            2019 Jan 15. pii: S0896-6273(19)30003-0. doi: 
            10.1016/j.neuron.2019.01.002."
COMMENT     Sequence Label: pQL193 BoNT(1-146)-iLID(V416I)
FEATURES             Location/Qualifiers
     source          1..6230
                     /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      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             613..1320
                     /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(758..776)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1026..1045
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(1922..1938)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1923..1939)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     CDS             2438..2875
                     /codon_start=1
                     /product="improved light-inducible dimer when used with 
                     SspB nano or SspB micro (Guntas et al., 2015)"
                     /label=iLID
                     /translation="LATTLERIEKNFIITDPRLPDNPIIFASDSFLQLTEYSREEILGR
                     NCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNVFHLQPMRDYKGDVQ
                     YFIGVQLDGTERLHGAAEREAVCLIKKTAFQIAEAANDENYF"
     polyA_signal    3018..3139
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3055..3074)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3109..3128
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3146..3601)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3283..3304)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3495..3514
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3628..3732
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3734..4091
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3734..3925
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3757..3777)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3942..4077
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4126..4920
                     /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             4126..4920
                     /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(4180..4199)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4790..4809
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5108..5127)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5152..5199
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5528..6116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6017..6036
                     /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 ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
      661 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
      721 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
      781 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
      841 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
      901 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
      961 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     1021 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     1081 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     1141 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     1201 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     1261 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     1321 tgtggcggct agtactccgg tattgcggta cccttgtacg cctgttttat actcccttcc
     1381 cgtaacttag acgcacaaaa ccaagttcaa tagaaggggg tacaaaccag taccaccacg
     1441 aacaagcact tctgtttccc cggtgatgtc gtatagactg cttgcgtggt tgaaagcgac
     1501 ggatccgtta tccgcttatg tacttcgaga agcccagtac cacctcggaa tcttcgatgc
     1561 gttgcgctca gcactcaacc ccagagtgta gcttaggctg atgagtctgg acatccctca
     1621 ccggtgacgg tggtccaggc tgcgttggcg gcctacctat ggctaacgcc atgggacgct
     1681 agttgtgaac aaggtgtgaa gagcctattg agctacataa gaatcctccg gcccctgaat
     1741 gcggctaatc ccaacctcgg agcaggtggt cacaaaccag tgattggcct gtcgtaacgc
     1801 gcaagtccgt ggcggaaccg actactttgg gtgtccgtgt ttccttttat tttattgtgg
     1861 ctgcttatgg tgacaatcac agattgttat cataaagcga attggatagg atcaagctta
     1921 tcgataccgt cgacctcgag ccaccatgcc agttacaata aataatttta attataatga
     1981 tcctattgat aataataata ttattatgat ggagcctcca tttgcgagag gtacggggag
     2041 atattataaa gcttttaaaa tcacagatcg tatttggata ataccggaaa gatatacttt
     2101 tggatataaa cctgaggatt ttaataaaag ttccggtatt tttaatagag atgtttgtga
     2161 atattatgat ccagattact taaatactaa tgataaaaag aatatatttt tacaaacaat
     2221 gatcaagtta tttaatagaa tcaaatcaaa accattgggt gaaaagttat tagagatgat
     2281 tataaatggt ataccttatc ttggagatag acgtgttcca ctcgaagagt ttaacacaaa
     2341 cattgctagt gtaactgtta ataaattaat cagtaatcca ggaggcggcc gcagcggtgg
     2401 cggtggctct ggaggtgggt ccgagctcgg ggagtttctg gcaaccacac tggaacggat
     2461 cgagaaaaat ttcatcatta ctgatccgag actgcctgac aacccaatca tttttgcgag
     2521 cgattccttc ctgcagctga cagaatattc tcgggaagag atcctggggc gcaattgccg
     2581 ttttctgcag ggacccgaga cagaccgtgc cactgttcgg aaaatcagag atgctattga
     2641 caaccagact gaagtgaccg ttcagctgat caattatacc aagagcggca agaagttctg
     2701 gaacgtgttc cacctgcagc cgatgcgcga ttataagggc gacgtccagt acttcattgg
     2761 cgtgcagctg gatggcaccg aacgtcttca tggcgccgct gagcgtgagg cggtctgcct
     2821 gatcaaaaag acagcctttc agattgctga ggcagcgaac gacgaaaatt acttttgact
     2881 taagcccggg atccaccgga tctagataac tgatcataat cagccatacc acatttgtag
     2941 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     3001 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3061 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3121 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     3181 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     3241 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     3301 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     3361 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     3421 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     3481 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     3541 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     3601 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3661 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3721 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     3781 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3841 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3901 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3961 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     4021 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     4081 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     4141 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     4201 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4261 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     4321 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4381 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4441 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4501 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4561 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4621 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     4681 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4741 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4801 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4861 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4921 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4981 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     5041 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     5101 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     5161 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     5221 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     5281 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     5341 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     5401 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     5461 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     5521 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     5581 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5641 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5701 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5761 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5821 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5881 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5941 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6001 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     6061 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     6121 ccggcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     6181 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
