LOCUS       sequence-460267-        7522 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expressing membrane-localized Voltron2 and membrane-localized 
            CheRiff..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7522)
  AUTHORS   Wong-Campos JD, Park P, Davis H, Qi Y, Tian H, Itkis DG, Kim D, 
            Grimm JB, Plutkis SE, Lavis L, Cohen AE
  TITLE     Voltage dynamics of dendritic integration and back-propagation in 
            vivo.
  JOURNAL   bioRxiv [Preprint]. 2023 May 26:2023.05.25.542363. doi: 
            10.1101/2023.05.25.542363.
  PUBMED    37292691
REFERENCE   2  (bases 1 to 7522)
  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: "bioRxiv 
            [Preprint]. 2023 May 26:2023.05.25.542363. doi: 
            10.1101/2023.05.25.542363."
COMMENT     Sequence Label: PP_072_pAAV_hSyn_DIO-LR-Voltron2-P2A-LR-CheRif...
FEATURES             Location/Qualifiers
     source          1..7522
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   24..164
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   35..164
                     /label=AAV2 ITR
     promoter        209..656
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(694..727)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(778..811)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(850..876)
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             complement(976..1920)
                     /codon_start=1
                     /product="blue light-activated optogenetic actuator derived
                     from Scherffelia dubia channelrhodopsin (Hochbaum et al., 
                     2014)"
                     /label=CheRiff
                     /translation="GGAPAPDAHSAPPGNDSAGGSEYHAPAGYQVNPPYHPVHGYEEQC
                     SSIYIYYGALWEQETARGFQWFAVFLSALFLAFYGWHAYKASVGWEEVYVCSVELIKVI
                     LEIYFEFTSPAMLFLYGGNITPWLRYAEWLLTCPVILIHLSNITGLSEAYNKRTMALLV
                     SDLGTICMGVTAALATGWVKWLFYCIGLVYGTQTFYNAGIIYVESYYIMPAGGCKKLVL
                     AMTAVYYSSWLMFPGLFIFGPEGMHTLSVAGSTIGHTIADLLSKNIWGLLGHFLRIKIH
                     EHIIMYGDIRRPVSSQFLGRKVDVLAFVTEEDKV"
     CDS             complement(1939..1977)
                     /codon_start=1
                     /product="segment of bovine rhodopsin containing a signal 
                     for N-linked glycosylation (Favaloro et al., 2008)"
                     /label=opsin tag
                     /note="The asparagine in the NKT tripeptide will be 
                     glycosylated if this sequence is exposed to the ER lumen."
                     /translation="GPNFYVPFSNKTG"
     CDS             complement(2038..2094)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(3775..3813)
                     /codon_start=1
                     /product="segment of bovine rhodopsin containing a signal 
                     for N-linked glycosylation (Favaloro et al., 2008)"
                     /label=opsin tag
                     /note="The asparagine in the NKT tripeptide will be 
                     glycosylated if this sequence is exposed to the ER lumen."
                     /translation="GPNFYVPFSNKTG"
     regulatory      3876..3885
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3892..3925
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3976..4009
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    4034..4622
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4087..4107)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4505..4516)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4676..4695)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4730..4749
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4808..4948
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4808..4937
                     /label=AAV2 ITR
     rep_origin      5023..5478
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5110..5129)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5320..5341
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5495..5514)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5614..5636
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5674..5692)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5760..5864
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5865..5933,5934..6725)
                     /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(6083..6102)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6896..7484
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7385..7404
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc
       61 cgcccgggca aagcccgggc 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 gatcctctag agtcgactcc ggaataactt cgtataggat actttatacg
      721 aagttatgca gaatggtagc tggattgtag ctgctattag caatatgaaa cctcttaata
      781 acttcgtata gcatacatta tacgaagtta tggcgcgcct taaacttcat tttcatagca
      841 aaatctagaa gcgtaatctg gaacatcgta tgggtatcta gaactagtgt cgacaacatt
      901 gatgtctatt tgatccagag ggatatactc cccttcagaa gtaattctgg atttctcgac
      961 gctcttggcg gccgccactt tatcttcctc tgtcacgaag gccagcacgt ccaccttgcg
     1021 tcccagaaac tgggagctca ctggtctcct gatatcgccg tacataatga tatgctcgtg
     1081 aattttgatc cgcaggaagt gccccagcag tccccaaata ttcttggaca gcaggtcggc
     1141 gatggtatgg ccaatagtgg acccagccac gctcagggtg tgcatgcctt caggcccaaa
     1201 gatgaacagg ccgggaaaca tcagccaact agaatagtac acggcagtca tggccagcac
     1261 cagtttctta cagccgccgg caggcatgat atagtaagac tccacgtaga tgattccagc
     1321 gttgtagaat gtctgggttc catacaccag gccgatacag taaaacagcc acttcaccca
     1381 cccagtggcc agagcggctg tcactcccat gcaaatagtt cccaggtcgg acaccagcag
     1441 agccattgtc cgcttattgt atgcctcact caggccggtg atgttagaca gatgaatcag
     1501 gatcacggga catgtcagca gccattcggc atatctcagc catggggtaa tgttccctcc
     1561 gtacaggaac agcatagcag gactggtgaa ctcgaaatag atctccagaa tcactttgat
     1621 cagctccacg gagcacacgt acacttcctc ccatcccacg ctggccttat aggcgtgcca
     1681 gccgtagaaa gccagaaaca gggcagacag gaacacggca aaccactgga agcccctagc
     1741 tgtttcctgc tcccacaggg ccccatagta gatgtagatg gagctgcact gttcctcata
     1801 cccatgcacg gggtggtagg gtggattcac ttgatatcca gctggggcat ggtactcact
     1861 gcctccggca gaatcgtttc caggtggggc gctgtgagcg tctggagcag gagctccgcc
     1921 actaccggat ctgactacgc ctgtcttatt gctgaaagga acgtagaagt tgggaccttc
     1981 tgttccgtta gccagtccaa gggtcaacaa agccagcaag aggagtattt ggggtcgggg
     2041 accggggttt tcttccacgt ctcctgcttg ctttaacaga gagaagttcg tggctccgga
     2101 gcccacctcg ttctcgtagc agaacacgtt gatgtcgatc tggtccaggg ggatgtactc
     2161 gccctcgctg gtgatcctgc tcttagtggt tggctcgccg gaaatctcga gcgtcgacag
     2221 ccagcgcgcg atctcgctgc cgatcaggtc cgggttgtct tcttgcagca gattcagacc
     2281 cgggccgatg tccacagcct tgcagttagg caggcttttg gccaggcgag cggcttcggc
     2341 cggtgggatc agaacgcctg gggtgcccca gaacagcagc ttcgggacag gggactggtg
     2401 cagccagtcc atgtattctt cgaccagcgc gacgatgttc gctggctcac cggcgattgg
     2461 cagctcgttt gggaagcgcc acagtggctc gcggtcaaca ggattcagga acggctcgcg
     2521 gtaatggtcc atctcgactt cagtcagcgg gcggacgaca cccatcggca gcgtaccctc
     2581 gataaaaacg ttctgatcga tgatcagctt gcggccgacg tcggtggtgc ggaaggcctg
     2641 gaaggtctcg cgggcaaatt ctggccattc gtcccaggtc gggatagggc ggatgaactc
     2701 cataaatgca atacctttga cgcgctctgg attgcgcttg gcccagtgga aacccagagc
     2761 ggagccccag tcgtgaatga ccaggacgac ctcttccaga cccagggctt cgatgaaggc
     2821 atccatgaag cggacgtggt cgtcgaagaa ataacccagg tctggtttgt cggatttgcc
     2881 cataccgatc aggtctggag caatgcagcg atgggtcggt gcaacatgcg ggatgatgtt
     2941 gcgccacacg taggaggagg tcgggttacc gtgcaggaac agcacagggg tgccatcgcg
     3001 cggaccaaca tcgacgtagt gcatgcgctc gcccaggact tccacataat gggggtcgaa
     3061 tggaaagcca gtaccgatct tgaagatagt ctcatgggca atgaggcacc ccattccaaa
     3121 cagtggctta gcgagcacat ccaggattcc catgagaatg gcctcattgt cgacggacag
     3181 cacgccacta ccagacccga aagcccacac gacaggataa aacacccaga ggattgacag
     3241 gaagacgagc agactgacat aggagctatg gtactcgggg cttttcttca gatcgtcttt
     3301 ccaagtggcg ttatacatcc cgtatgtcag gactgcctgc atcacacatc cagcgataaa
     3361 gtacacccat ttgaacttgt gttcatcctc gaagtcgccc agaataccaa acacgatcat
     3421 gaaaatgtct gcgccgatga cccaagaaat catcactccg cccatcttgg tcatgacgat
     3481 gagatcgagc aggagcagtg gtgtggtcag cacccagtta atatatcttg cgtagaccac
     3541 ctgcctttca ccattcattg tcaggtcctg gtagttcacg gccattgcat agtaagcggt
     3601 agcggcgata gtgcagatgg caatgttgat atagtatgcg ctcttcagct ctgaaggccg
     3661 aaagtgcaca gccactccaa aagcaatagc agcagcagcc ataatagcaa agacaatcca
     3721 ctgtgcaatc atgccggtct cggtttccac gtcagcacta ccggatctga ctacgcctgt
     3781 cttattgctg aaaggaacgt agaagttggg accttctgtt ccgttagcca gtccaagggt
     3841 caacaaagcc agcaagagga gtatttgggg tcggcccatg gtggcgctag cataacttcg
     3901 tataaagtat cctatacgaa gttatttgcc ttaacccaga aattatcact gttattcttt
     3961 agaatggtgc aaagaataac ttcgtataat gtatgctata cgaagttatg aattcgatat
     4021 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     4081 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     4141 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     4201 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     4261 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     4321 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     4381 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     4441 tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     4501 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     4561 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     4621 gcatcgatac cgtcgacccg ggcggccgct tcgagcagac atgataagat acattgatga
     4681 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     4741 tgctattgct ttatttgtaa ccattataag ctgcaataaa ctaaccacgt gcggaccgag
     4801 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4861 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     4921 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     4981 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     5041 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     5101 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     5161 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     5221 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     5281 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     5341 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     5401 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5461 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     5521 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     5581 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     5641 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     5701 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     5761 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5821 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5881 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5941 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6001 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6061 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6121 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6181 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6241 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6301 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6361 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6421 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6481 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6541 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6601 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6661 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6721 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6781 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6841 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6901 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6961 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7021 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     7081 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7141 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7201 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7261 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7321 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7381 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7441 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7501 cctttttacg gttcctggcc tt
//
