LOCUS       sequence-338755-        7243 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Cation channelrhodopsin ChroME targeted to the neuronal soma and 
            proximal dendrites and separated from nuclear mRuby3 by P2A in a 
            viral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7243)
  AUTHORS   Sridharan S, Gajowa MA, Ogando MB, Jagadisan UK, Abdeladim L, 
            Sadahiro M, Bounds HA, Hendricks WD, Turney TS, Tayler I, Gopakumar 
            K, Oldenburg IA, Brohawn SG, Adesnik H
  TITLE     High-performance microbial opsins for spatially and temporally 
            precise perturbations of large neuronal networks.
  JOURNAL   Neuron. 2022 Jan 27. pii: S0896-6273(22)00008-3. doi: 
            10.1016/j.neuron.2022.01.008.
  PUBMED    35120626
REFERENCE   2  (bases 1 to 7243)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Neuron. 
            2022 Jan 27. pii: S0896-6273(22)00008-3. doi: 
            10.1016/j.neuron.2022.01.008."
COMMENT     Sequence Label: pAAV-hSyn-DIO-ChroME-ST-P2A-H2B-mRuby3
FEATURES             Location/Qualifiers
     source          1..7243
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   382..511
                     /label=AAV2 ITR
     promoter        593..1040
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     protein_bind    complement(1086..1119)
                     /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(1170..1203)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1215..1925)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     CDS             complement(1935..2312)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             complement(2319..2375)
                     /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(2586..2609)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    3609..3642
                     /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    3693..3726
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3751..4339
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3804..3824)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4222..4233)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4342..4358)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4343..4359)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     polyA_signal    4388..4509
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4399..4418)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4453..4472
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4978..5433
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5065..5084)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5275..5296
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5459..5563
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5564..5632,5633..6424)
                     /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(5782..5801)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6595..7183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7084..7103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
       61 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      121 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgc
      181 gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcggcctcan nngtgagyka
      241 gckmrykyak yrgkcacsck sgsskaaccn accacacccg ccgcgcttaa tgcgccgcta
      301 cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg
      361 gcctcttcgc tattacgcca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag
      421 cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag
      481 ggagtggcca actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac
      541 ttatctacgt agccatgctc taggaagatc tctgcagagg gccctgcgta tgagtgcaag
      601 tgggttttag gaccaggatg aggcggggtg ggggtgccta cctgacgacc gaccccgacc
      661 cactggacaa gcacccaacc cccattcccc aaattgcgca tcccctatca gagaggggga
      721 ggggaaacag gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc
      781 cttcgccccc gcctggcggc gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt
      841 cactcgccgg tcccccgcaa actccccttc ccggccacct tggtcgcgtc cgcgccgccg
      901 ccggcccagc cggaccgcac cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc
      961 tgcgctgcgg cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg
     1021 tgtcgtgcct gagagcgcag tcgaattcaa gctgatagca aggatcctct agagtcgact
     1081 ccggaataac ttcgtatagg atactttata cgaagttatg cagaatggta gctggattgt
     1141 agctgctatt agcaatatga aacctcttaa taacttcgta tagcatacat tatacgaagt
     1201 tatggcgcgc ctcacttgta cagctcgtcc atgccaccac caaggttgct gtatttggca
     1261 actgccactt ctctttgcac tacgtaggtt tcattgtcac tctcctcgat cctttccagg
     1321 cggtgatcaa cggcatggac accgggcatc ttgatgttcc cgacggtctt ttttgacctg
     1381 taagttgtca cgaagttgca gtgcagatgg ccaccaccat caactttcag tgcgatgtca
     1441 gtgtatcctc tcagaccacc atctgctgga tacatcatct ctgtattagg ctcccaaccc
     1501 ttggtcttct tctgcatcac gggaccattg gagggaaagt ttacccctct gaccttgacg
     1561 ttgtagacga gctcgccatc ctcaaggctg gtgtcctgcg tgacggtgac gactccacca
     1621 tcttcgtatc tcgtaactct ttcccaagta aaaccctcag gaaaggactg tttaaagaaa
     1681 tcagggatgt cggccgggta cttgataaag gtacggctgc catacatgaa cgacgtggca
     1741 agaatgtcaa aggcaaatgg caggggtcct ccctcgatga ctttgatcct catggtttgc
     1801 actccctcgt acggtctgcc ttctccttca cctgtgcatt tgaattggtg gccgttgacc
     1861 gaaccttcca tgaccacctt catacgcata ttttccttga tcagctcttc gcccttagac
     1921 accatctcga gtgccttagc gctggtgtac ttggtgatgg ccttagtacc ctcggacacg
     1981 gcgtgcttgg ccaactcccc aggcagcagc aggcgcacgg ccgtctggat ctccctggag
     2041 gtgatggtcg agcgcttgtt gtaatgcgcc aggcgggaag cctcacctgc gatgcgctcg
     2101 aaaatgtcgt tcacaaacga attcatgatg cccatggcct tggacgaaat gccggtgtca
     2161 gggtggacct gcttcagaac cttgtacaca tagatggaat agctctcctt gcggctgcgc
     2221 ttgcgcttct tgccgccttt cttctgcgcc ttagtcaccg ccttcttgga gccctttttc
     2281 ggggcgggag cagacttcgc tggctctggc atactagtag gtccagggtt ctcctccacg
     2341 tctccagcct gcttcagcag gctgaagtta gtagctccgc ttcctgccgc gaatctggtg
     2401 gcttcaggga agtccgtggc acagctgatg aagctgtcaa tgctggacat gctccgcatg
     2461 tcgatgacgc cctccgtgcg tgcgggcaga ggggccacgg ggctgggcat gctgctcatc
     2521 tccagctcct ctggaggctt gctctgcggg gccatctcct tggtgttgag gatgggctgg
     2581 gactgcttat cgtcatcgtc tttgtaatct accggtgcgg ccgccactcc tccctcctcc
     2641 tcctcgtcga cgaaggtctc gatctccatg ttctcgccgg ccacgttgat ggtggttgtc
     2701 ttccggatgt cgccgtggat caggatgtgc tcgtggatct tcactctcag gtggtggccc
     2761 aggaagcccc aagtgttctt gctgatcaga tccaggatca ggtggccgat tccgctggtg
     2821 taaggtgtga tcaggcccag tccctcgtgt ccggcgatga acatcacggg gaacatcagc
     2881 caagagcaga agaagacgta ggccatgatc ttgcaaatct tccggcagac gcctttgggc
     2941 agggtgtggt agctctcgat atacaccttg gcgatctgga agaagcaagt cacgccgtag
     3001 aacaggccga tcatgaagaa caggatcttc agggggccct ttgtaaaggc ggctgtgatc
     3061 ccccacacga tgttgccgat gtcggtcacc aggatggtca tggtccgctt gctgtactct
     3121 tcgtgcagtc cggtcaggtt gctcaggtgg atcaggatca cggggcaagt caggagccac
     3181 tcgctgtacc gcagccaaat cacggctcct ccgttggtct ggtacactgt ggctgggctg
     3241 tcgacctcgt ggaacagctc gatgaagcac ttgacgagct cgatcacgca gacgtagacc
     3301 tcctcccagc cggttgtggc cttgaagctg tgccaaccgt agaagatgag ctgcacgata
     3361 gccacgatca cggtgaacca ctggagtccc actgcgatct tgtgctgcag ctcggttccg
     3421 tggttgatgt gggggaagca accgtgatcg gctccggctg ttgtggcgtt agaggtttct
     3481 ccgtgagcgt ctgctgctgg tgtcaccact gctgcggcgc tcagcaggcc tctaattgtg
     3541 gcttcggcgc taggcacggc tgagataaag gcgtgggtca ttgtggcggc tgtttccata
     3601 ctgctagcat aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat
     3661 tatcactgtt attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga
     3721 agttatgaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     3781 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     3841 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3901 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3961 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     4021 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     4081 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     4141 gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg
     4201 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     4261 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     4321 ctttgggccg cctccccgca tcgataccgt cgacctcgac ccgggcggcc gcttcgagca
     4381 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     4441 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     4501 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg
     4561 gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatcttcct
     4621 agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct
     4681 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc
     4741 tttgcccggg cggcskcrgk srgyrwcmgc tcrckcarmk gcrktaatrc grscsyryag
     4801 yctccctctc tgcgcgctcg ctckcgctca ctgaggccgg gcgaccaaag gtcgcccgac
     4861 gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgg cgtaatagcg
     4921 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg
     4981 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     5041 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     5101 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     5161 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     5221 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     5281 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     5341 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     5401 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     5461 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     5521 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     5581 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     5641 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     5701 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     5761 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     5821 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     5881 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     5941 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6001 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6061 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6121 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6181 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6241 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6301 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6361 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6421 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6481 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     6541 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     6601 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     6661 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     6721 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     6781 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     6841 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     6901 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     6961 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7021 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7081 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7141 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7201 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atg
//
