LOCUS       sequence-485698-        7823 bp    DNA     circular SYN 10-SEP-2025
DEFINITION  Optogenetic tool for blue-light inhibition and red-light excitation 
            of neurons fused to mRuby3 in a viral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7823)
  AUTHORS   Adesnik H
  TITLE     Miscellaneous Optogenetic Tools
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7823)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 10, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-hSyn-DIO-BiPoles-mRuby3
FEATURES             Location/Qualifiers
     source          1..7823
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   826..955
                     /label=AAV2 ITR
     polyA_signal    1117..1238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1154..1173)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1208..1227
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1267..1283
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1268..1284
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1287..1875
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1393..1404
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1802..1822
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(1900..1933)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1984..2017)
                     /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)."
     CDS             2987..3694
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTDI
                     ALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAVA
                     KYSNLGGGMDELYK"
     CDS             4055..5098
                     /codon_start=1
                     /product="red light-drivable channelrhodopsin from 
                     Chlamydomonas noctigama (Klapoetke et al., 2014)"
                     /label=Chrimson
                     /translation="ELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWW
                     CDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAI
                     ALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFE
                     WLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSC
                     IYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLS
                     PYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEFV
                     EEEDEDTV"
     protein_bind    5314..5347
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    5398..5431
                     /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)."
     promoter        complement(5477..5924)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   6006..6135
                     /label=AAV2 ITR
     rep_origin      6207..6662
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6294..6313)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6504..6525
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6688..6792
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6793..6861,6862..7653)
                     /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(7011..7030)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctgcg cgctcgctcg
      841 ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca
      901 gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag
      961 ttaatgatta acccgccatg ctacttatct acgtagccat gctctaggaa gatccttatc
     1021 gattttacca catttgtaga ggttttactt gctttaaaaa acctcccaca tctccccctg
     1081 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     1141 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     1201 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgctc gaagcggccg
     1261 cccgggtcga ggtcgacggt atcgatgcgg ggaggcggcc caaagggaga tccgactcgt
     1321 ctgagggcga aggcgaagac gcggaagagg ccgcagagcc ggcagcaggc cgcgggaagg
     1381 aaggtccgct ggattgaggg ccgaagggac gtagcagaag gacgtcccgc gcagaatcca
     1441 ggtggcaaca caggcgagca gccaaggaaa ggacgatgat ttccccgaca acaccacgga
     1501 attgtcagtg cccaacagcc gagcccctgt ccagcagcgg gcaaggcagg cggcgatgag
     1561 ttccgccgtg gcaataggga gggggaaagc gaaagtcccg gaaaggagct gacaggtggt
     1621 ggcaatgccc caaccagtgg gggttgcgtc agcaaacaca gtgcacacca cgccacgttg
     1681 cctgacaacg ggccacaact cctcataaag agacagcaac caggatttat acaaggagga
     1741 gaaaatgaaa gccatacggg aagcaatagc atgatacaaa ggcattaaag cagcgtatcc
     1801 acatagcgta aaaggagcaa catagttaag aataccagtc aatctttcac aaattttgta
     1861 atccagaggt tgattatcga taagcttgat atcgaattca taacttcgta tagcatacat
     1921 tatacgaagt tattctttgc accattctaa agaataacag tgataatttc tgggttaagg
     1981 caaataactt cgtataggat actttatacg aagttatgct agcggaacca tggcatcaca
     2041 ggtcgtctat ggggaatggg catcaactca caccgagtgc tacaatatgt ccagaatcga
     2101 ctccaccttc gtctcactgc tgcagctggt gtgggccgtg gtctcaggct gccagacaat
     2161 cttcatgatt agcagggctc ccaaggtccc ttgggagtcc gtgtacctgc cctttgtcga
     2221 atctatcact tatgcactgg ccagtactgg caacggaacc ctgcagatga gggacggcag
     2281 attctttcct tggagccgaa tggcctcctg gctgtgcacc tgtccaatca tgctgggaca
     2341 gatttctaac atggctctgg tgaagtacaa aagtatccca ctgaatccca ttgcacaggc
     2401 cgcttccatc attagagtgg tcatgggaat caccgctaca atttctcccg cagagtatat
     2461 gaagtggctg ttctttttct ttggggccac ctgcctggtg ttcgagtaca gtgtggtctt
     2521 cacaatcttt caagtggggc tgtatggctt tgaatctgtc gggacacctc tggcacagaa
     2581 ggtggtggtg aggatcaaga tgctgcgcct gatcttcttt attgcttgga ctatgttccc
     2641 aatcgtgtgg ctgatttcac ccaccggagt ctgcgtgatc cacgagaacg tgagcgccat
     2701 tctgtacctg ctggctgacg gcctgtgcaa aaatacatat ggagtgatcc tgtggagcac
     2761 tgcctgggga gtgctggaag ggaagtggga ccccgcatgt ctgccagggc aggagaaacc
     2821 cgaagccgac gatccttttg gcctgaatca cgagaagaat gcccctccta acgacgaagt
     2881 caatatcaga atgtttggaa ggggacgtac ggcggccaag agcaggatca ccagcgaggg
     2941 cgagtacatc cccctggacc agatcgacat caacgtggcg gtacccgtgt ctaagggcga
     3001 agagctgatc aaggaaaata tgcgtatgaa ggtggtcatg gaaggttcgg tcaacggcca
     3061 ccaattcaaa tgcacaggtg aaggagaagg cagaccgtac gagggagtgc aaaccatgag
     3121 gatcaaagtc atcgagggag gacccctgcc atttgccttt gacattcttg ccacgtcgtt
     3181 catgtatggc agccgtacct ttatcaagta cccggccgac atccctgatt tctttaaaca
     3241 gtcctttcct gagggtttta cttgggaaag agttacgaga tacgaagatg gtggagtcgt
     3301 caccgtcacg caggacacca gccttgagga tggcgagctc gtctacaacg tcaaggtcag
     3361 aggggtaaac tttccctcca atggtcccgt gatgcagaag aagaccaagg gttgggagcc
     3421 taatacagag atgatgtatc cagcagatgg tggtctgaga ggatacactg acatcgcact
     3481 gaaagttgat ggtggtggcc atctgcactg caacttcgtg acaacttaca ggtcaaaaaa
     3541 gaccgtcggg aacatcaaga tgcccggtgt ccatgccgtt gatcaccgcc tggaaaggat
     3601 cgaggagagt gacaatgaaa cctacgtagt gcaaagagaa gtggcagttg ccaaatacag
     3661 caaccttggt ggtggcatgg acgagctgta caagaccggt gcagccctgc aggagaagaa
     3721 gtcatgcagc cagcgcatgg ccgaattccg gcaatactgt tggaacccgg acactgggca
     3781 gatgctgggc cgcaccccag cccggtgggt gtggatcagc ctgtactatg cagctttcta
     3841 cgtggtcatg actgggctct ttgccttgtg catctatgtg ctgatgcaga ccattgatcc
     3901 ctacaccccc gactaccagg accagttaaa gtcaccgggg gtaaccttga gaccggatgt
     3961 gtatggggaa agagggctgc agatttccta caacatctct gaaaacagct ctagacaggc
     4021 ccagatcacc ggacgtccgg agggagctgt acaagagctg atcagcagcg ccaccagatc
     4081 tctgtttgcc gccggaggca tcaacccttg gcctaacccc taccaccacg aggacatggg
     4141 ctgtggagga atgacaccta caggcgagtg cttcagcacc gagtggtggt gtgacccttc
     4201 ttacggactg agcgacgccg gatacggata ttgcttcgtg gaggccacag gcggctacct
     4261 ggtcgtggga gtggagaaga agcaggcttg gctgcacagc agaggcacac caggagaaaa
     4321 gatcggcgcc caggtctgcc agtggattgc tttcagcatc gccatcgccc tgctgacatt
     4381 ctacggcttc agcgcttgga aggccacttg cggttgggag gaggtctacg tctgttgcgt
     4441 cgaggtgctg ttcgtgaccc tggagatctt caaggagttc agcagccccg ccacagtgta
     4501 cctgtctacc ggcaaccacg cctattgcct gcgctacttc gagtggctgc tgtcttgccc
     4561 cgtgatcctg atcaagctga gcaacctgag cggcctgaag aacgactaca gcaagcggac
     4621 catgggcctg atcgtgtctt gcgtgggaat gatcgtgttc ggcatggccg caggactggc
     4681 taccgattgg ctcaagtggc tgctgtatat cgtgtcttgc atctacggcg gctacatgta
     4741 cttccaggcc gccaagtgct acgtggaagc caaccacagc gtgcctaaag gccattgccg
     4801 catggtcgtg aagctgatgg cctacgctta cttcgcctct tggggcagct acccaatcct
     4861 ctgggcagtg ggaccagaag gactgctgaa gctgagccct tacgccaaca gcatcggcca
     4921 cagcatctgc gacatcatcg ccaaggagtt ttggaccttc ctggcccacc acctgaggat
     4981 caagatccac gagcacatcc tgatccacgg cgacatccgg aagaccacca agatggagat
     5041 cggaggcgag gaggtggaag tggaagagtt cgtggaggag gaggacgagg acacagtgct
     5101 cgagcagagc cagcccatcc tgaacaccaa ggagatggcc ccccagagca agccccccga
     5161 ggagctggag atgagcagca tgcccagccc cgtggccccc ctgcccgccc gcaccgaggg
     5221 cgtgatcgac atgcgcagca tgagcagcat cgacagcttc atcagctgcg ccaccgactt
     5281 ccccgaggcc acccgcttct gatctggcgc gccataactt cgtataatgt atgctatacg
     5341 aagttattaa gaggtttcat attgctaata gcagctacaa tccagctacc attctgcata
     5401 acttcgtata aagtatccta tacgaagtta ttccggagtc gactctagag gatccttgct
     5461 atcagcttga attcgactgc gctctcaggc acgacacgac tcctccgctg cccaccgcag
     5521 actgaggcag cgctgagtcg ccggcgccgc agcgcagatg gtcgcgcccg tgccccccta
     5581 tctcgcgcct cgcgtggtgc ggtccggctg ggccggcggc ggcgcggacg cgaccaaggt
     5641 ggccgggaag gggagtttgc gggggaccgg cgagtgacgt cagcgcgcct tcagtgctga
     5701 ggcggcggtg gcgcgcgccg ccaggcgggg gcgaaggcac tgtccgcggt gctgaagctg
     5761 gcagtgcgca cgcgcctcgc cgcatcctgt ttcccctccc cctctctgat aggggatgcg
     5821 caatttgggg aatgggggtt gggtgcttgt ccagtgggtc ggggtcggtc gtcaggtagg
     5881 cacccccacc ccgcctcatc ctggtcctaa aacccacttg cactcatacg cagggccctc
     5941 tgcagagatc ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa
     6001 ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
     6061 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
     6121 cgagcgagcg cgcagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca
     6181 gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     6241 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     6301 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     6361 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     6421 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     6481 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     6541 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     6601 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     6661 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     6721 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     6781 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     6841 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     6901 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     6961 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     7021 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     7081 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     7141 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     7201 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     7261 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     7321 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     7381 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     7441 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     7501 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     7561 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     7621 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     7681 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     7741 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     7801 gtagaaaaga tcaaaggatc ttc
//
