LOCUS       sequence-485697-        7392 bp    DNA     circular SYN 10-SEP-2025
DEFINITION  Cation channelrhodopsin ChRmine targeted to the neuronal soma and 
            proximal dendrites in a viral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7392)
  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 7392)
  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: "Neuron. 
            2022 Jan 27. pii: S0896-6273(22)00008-3. doi: 
            10.1016/j.neuron.2022.01.008."
COMMENT     Sequence Label: pAAV-CAG-DIO-ChRmine-ST-P2A-H2B-mRuby3
FEATURES             Location/Qualifiers
     source          1..7392
                     /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"
     polyA_signal    1095..1216
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1132..1151)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1186..1205
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1245..1261
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1246..1262
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1265..1853
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1371..1382
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1780..1800
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(1878..1911)
                     /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(1962..1995)
                     /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             3010..3033
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             3262..3318
                     /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             3325..3702
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             3712..4422
                     /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"
     protein_bind    4434..4467
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    4518..4551
                     /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)."
     primer_bind     complement(4602..4621)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     complement(4683..4705)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(4984..5261)
                     /label=chicken beta-actin promoter
     repeat_region   5575..5704
                     /label=AAV2 ITR
     rep_origin      5776..6231
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5863..5882)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6073..6094
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6257..6361
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6362..6430,6431..7222)
                     /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(6580..6599)
                     /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 ccgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag
      901 agggagtggc caactccatc actaggggtt ccttgtagtt aatgattaac ccgccatgct
      961 acttatctac gtagccatgc tctaggaaga tccttatcga ttttaccaca tttgtagagg
     1021 ttttacttgc tttaaaaaac ctcccacatc tccccctgaa cctgaaacat aaaatgaatg
     1081 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     1141 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     1201 tcatcaatgt atcttatcat gtctgctcga agcggccgcc cgggtcgagg tcgacggtat
     1261 cgatgcgggg aggcggccca aagggagatc cgactcgtct gagggcgaag gcgaagacgc
     1321 ggaagaggcc gcagagccgg cagcaggccg cgggaaggaa ggtccgctgg attgagggcc
     1381 gaagggacgt agcagaagga cgtcccgcgc agaatccagg tggcaacaca ggcgagcagc
     1441 caaggaaagg acgatgattt ccccgacaac accacggaat tgtcagtgcc caacagccga
     1501 gcccctgtcc agcagcgggc aaggcaggcg gcgatgagtt ccgccgtggc aatagggagg
     1561 gggaaagcga aagtcccgga aaggagctga caggtggtgg caatgcccca accagtgggg
     1621 gttgcgtcag caaacacagt gcacaccacg ccacgttgcc tgacaacggg ccacaactcc
     1681 tcataaagag acagcaacca ggatttatac aaggaggaga aaatgaaagc catacgggaa
     1741 gcaatagcat gatacaaagg cattaaagca gcgtatccac atagcgtaaa aggagcaaca
     1801 tagttaagaa taccagtcaa tctttcacaa attttgtaat ccagaggttg attatcgata
     1861 agcttgatat cgaattcata acttcgtata gcatacatta tacgaagtta ttctttgcac
     1921 cattctaaag aataacagtg ataatttctg ggttaaggca aataacttcg tataggatac
     1981 tttatacgaa gttatgctag caccatggca cacgcaccag gcaccgacca gatgttctac
     2041 gtgggcacaa tggacggctg gtatctggat accaagctga actccgtggc catcggcgcc
     2101 cactggtctt gctttatcgt gctgacaatc accacattct acctgggcta tgagtcctgg
     2161 acatctcggg gccctagcaa gagaacctcc ttttacgccg gctatcagga ggagcagaac
     2221 ctggccctgt ttgtgaattt ctttgccatg ctgagctact tcggcaagat cgtggccgac
     2281 acactgggcc acaacttcgg cgatgtgggc cccttcatca tcggcttcgg caattacagg
     2341 tatgccgact acatgctgac ctgcccaatg ctggtgtacg atctgctgta tcagctgagg
     2401 gccccctatc gcgtgtcttg tagcgccatc atctttgcca tcctgatgtc tggcgtgctg
     2461 gccgagttct acgcagaggg cgaccctagg ctgaggaatg gcgcctacgc ctggtatggc
     2521 tttggctgtt tctggtttat cttcgcctac tctatcgtga tgagcatcgt ggccaagcag
     2581 tatagccggc tggcccagct ggcccaggat acaggcgccg agcacagcct gcacgtgctg
     2641 aagttcgccg tgtttacctt ctccatgctg tggattctgt ttcccctggt gtgggccatc
     2701 tgccctagag gcttcggctg gatcgacgat aactggacag aggtggccca ctgcgtgtgc
     2761 gacatcgtgg ccaagtcctg ttacggcttt gccctggccc ggttcagaaa gacctatgat
     2821 gaggagctgt ttcggctgct ggagcagctg ggacacgacg aggatgagtt ccagaagctg
     2881 gagctggata tgaggctgag cagcaatggc gagcgcctgc ggagactgtc tgcggccgcc
     2941 aagagcagga tcaccagcga gggcgagtac atccccctgg accagatcga catcaacgtg
     3001 gtaccggtag attacaaaga cgatgacgat aagcagtccc agcccatcct caacaccaag
     3061 gagatggccc cgcagagcaa gcctccagag gagctggaga tgagcagcat gcccagcccc
     3121 gtggcccctc tgcccgcacg cacggagggc gtcatcgaca tgcggagcat gtccagcatt
     3181 gacagcttca tcagctgtgc cacggacttc cctgaagcca ccagattctt ctgctacgag
     3241 aacgaggtgg caggaagcgg agctactaac ttcagcctgc tgaagcaggc tggagacgtg
     3301 gaggagaacc ctggacctac tagtatgcca gagccagcga agtctgctcc cgccccgaaa
     3361 aagggctcca agaaggcggt gactaaggcg cagaagaaag gcggcaagaa gcgcaagcgc
     3421 agccgcaagg agagctattc catctatgtg tacaaggttc tgaagcaggt ccaccctgac
     3481 accggcattt cgtccaaggc catgggcatc atgaattcgt ttgtgaacga cattttcgag
     3541 cgcatcgcag gtgaggcttc ccgcctggcg cattacaaca agcgctcgac catcacctcc
     3601 agggagatcc agacggccgt gcgcctgctg ctgcctgggg agttggccaa gcacgccgtg
     3661 tccgagggta ctaaggccat caccaagtac accagcgcta aggcactcga gatggtgtct
     3721 aagggcgaag agctgatcaa ggaaaatatg cgtatgaagg tggtcatgga aggttcggtc
     3781 aacggccacc aattcaaatg cacaggtgaa ggagaaggca gaccgtacga gggagtgcaa
     3841 accatgagga tcaaagtcat cgagggagga cccctgccat ttgcctttga cattcttgcc
     3901 acgtcgttca tgtatggcag ccgtaccttt atcaagtacc cggccgacat ccctgatttc
     3961 tttaaacagt cctttcctga gggttttact tgggaaagag ttacgagata cgaagatggt
     4021 ggagtcgtca ccgtcacgca ggacaccagc cttgaggatg gcgagctcgt ctacaacgtc
     4081 aaggtcagag gggtaaactt tccctccaat ggtcccgtga tgcagaagaa gaccaagggt
     4141 tgggagccta atacagagat gatgtatcca gcagatggtg gtctgagagg atacactgac
     4201 atcgcactga aagttgatgg tggtggccat ctgcactgca acttcgtgac aacttacagg
     4261 tcaaaaaaga ccgtcgggaa catcaagatg cccggtgtcc atgccgttga tcaccgcctg
     4321 gaaaggatcg aggagagtga caatgaaacc tacgtagtgc aaagagaagt ggcagttgcc
     4381 aaatacagca accttggtgg tggcatggac gagctgtaca agtgaggcgc gccataactt
     4441 cgtataatgt atgctatacg aagttattaa gaggtttcat attgctaata gcagctacaa
     4501 tccagctacc attctgcata acttcgtata aagtatccta tacgaagtta ttccggagtc
     4561 gactctagag gatccaattc tttgccaaaa tgatgagaca gcacaataac cagcacgttg
     4621 cccaggagct gtaggaaaaa gaagaaggca tgaacatggt tagcagaggc tctagagccg
     4681 ccggtcacac gccagaagcc gaaccccgcc ctgccccgtc ccccccgaag gcagccgtcc
     4741 ccccgcggac agccccgagc cgctcccccc gcacaaaggg ccctcccgga gcccctcaag
     4801 gctttcacgc agccacagaa aagaaacaag ccgtcattaa accaagcgct aattacagcc
     4861 cggaggagaa gggccgtccc gcccgctcac ctgtgggagt aacgcggtca gtcagagccg
     4921 gggcgggcgg cgcgaggcgg cggcggagcg gggcacgggg cgaaggcagc gcgcagcgac
     4981 tcccgcccgc cgcgcgcttc gctttttata gggccgccgc cgccgccgcc tcgccataaa
     5041 aggaaacttt cggagcgcgc cgctctgatt ggctgccgcc gcacctctcc gcctcgcccc
     5101 gccccgcccc tcgccccgcc ccgccccgcc tggcgcgcgc cccccccccc cccccgcccc
     5161 catcgctgca caaaataatt aaaaaataaa taaatacaaa attgggggtg gggagggggg
     5221 ggagatgggg agagtgaagc agaacgtggg gctcacctcg accatggtaa tagcgatgac
     5281 taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg
     5341 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
     5401 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
     5461 agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg gtacagatct
     5521 tcctagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac
     5581 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     5641 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     5701 gcagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     5761 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     5821 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5881 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5941 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     6001 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     6061 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     6121 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     6181 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     6241 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6301 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     6361 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     6421 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     6481 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     6541 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6601 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6661 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6721 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6781 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6841 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6901 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6961 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     7021 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     7081 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     7141 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     7201 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     7261 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     7321 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7381 caaaggatct tc
//
