LOCUS       sequence-306851-        6481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For packaging and expression of ChrimsonR-LSSmOrange in neurons via 
            AAV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6481)
  AUTHORS   Christoffel DJ, Walsh JJ, Hoerbelt P, Heifets BD, Llorach P, Lopez 
            RC, Ramakrishnan C, Deisseroth K, Malenka RC
  TITLE     Selective filtering of excitatory inputs to nucleus accumbens by 
            dopamine and serotonin.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Jun 15;118(24). pii: 2106648118. doi:
            10.1073/pnas.2106648118.
  PUBMED    34103400
REFERENCE   2  (bases 1 to 6481)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2021-06-15"; volume: "118(24). pii: 
            2106648118. doi"; pages: " 10.1073/pnas.2106648118"
COMMENT     Sequence Label: pAAV-hSyn-ChrimsonR-LSSmOrange
FEATURES             Location/Qualifiers
     source          1..6481
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        341..788
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      801..810
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             807..1856
                     /codon_start=1
                     /product="red light-drivable channelrhodopsin from 
                     Chlamydomonas noctigama (Klapoetke et al., 2014)"
                     /label=ChrimsonR
                     /note="K176R mutant with accelerated closing kinetics"
                     /translation="MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTE
                     WWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSI
                     AIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRY
                     FEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIV
                     SCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLK
                     LSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEE
                     FVEEEDEDTV"
     CDS             1875..2579
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="VSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGF
                     QTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALKG
                     EDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     2285..2304
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2614..3202
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2667..2687)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3085..3096)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3222..3698
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3421..3440)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3744..3884
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3744..3873
                     /label=AAV2 ITR
     rep_origin      3959..4414
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4046..4065)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4256..4277
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4431..4450)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4550..4572
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4610..4628)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4696..4800
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4801..4869,4870..5661)
                     /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(5019..5038)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5832..6420
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6321..6340
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgttgcaaa gatggataaa gttttaaaca gagaggaatc
      181 tttgcagcta atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc
      241 agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcagca
      301 aatggttaat taatctagac tgcagagggc cctgcgtatg agtgcaagtg ggttttagga
      361 ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc
      421 acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga
      481 tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc
      541 ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc
      601 ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg
      661 gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg
      721 ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga
      781 gagcgcagtc gagaggatcc gccaccatgg ctgagctgat cagcagcgcc accagatctc
      841 tgtttgccgc cggaggcatc aacccttggc ctaaccccta ccaccacgag gacatgggct
      901 gtggaggaat gacacctaca ggcgagtgct tcagcaccga gtggtggtgt gacccttctt
      961 acggactgag cgacgccgga tacggatatt gcttcgtgga ggccacaggc ggctacctgg
     1021 tcgtgggagt ggagaagaag caggcttggc tgcacagcag aggcacacca ggagaaaaga
     1081 tcggcgccca ggtctgccag tggattgctt tcagcatcgc catcgccctg ctgacattct
     1141 acggcttcag cgcctggaag gccacttgcg gttgggagga ggtctacgtc tgttgcgtcg
     1201 aggtgctgtt cgtgaccctg gagatcttca aggagttcag cagccccgcc acagtgtacc
     1261 tgtctaccgg caaccacgcc tattgcctgc gctacttcga gtggctgctg tcttgccccg
     1321 tgatcctgat cagactgagc aacctgagcg gcctgaagaa cgactacagc aagcggacca
     1381 tgggcctgat cgtgtcttgc gtgggaatga tcgtgttcgg catggccgca ggactggcta
     1441 ccgattggct caagtggctg ctgtatatcg tgtcttgcat ctacggcggc tacatgtact
     1501 tccaggccgc caagtgctac gtggaagcca accacagcgt gcctaaaggc cattgccgca
     1561 tggtcgtgaa gctgatggcc tacgcttact tcgcctcttg gggcagctac ccaatcctct
     1621 gggcagtggg accagaagga ctgctgaagc tgagccctta cgccaacagc atcggccaca
     1681 gcatctgcga catcatcgcc aaggagtttt ggaccttcct ggcccaccac ctgaggatca
     1741 agatccacga gcacatcctg atccacggcg acatccggaa gaccaccaag atggagatcg
     1801 gaggcgagga ggtggaagtg gaagagttcg tggaggagga ggacgaggac acagtggcgg
     1861 caccggtagt agcagtgagc aagggcgagg agaataacat ggccatcatc aaggagttca
     1921 tgcgcttcaa ggtgcgcatg gagggctccg tgaacggcca cgagttcgag atcgagggcg
     1981 agggcgaggg ccgcccctac gagggctttc agaccgttaa gctgaaggtg accaagggtg
     2041 gccccctgcc cttcgcctgg gacatcttgt cccctcagtt cacctacggc tccaaggcct
     2101 acgtgaagca ccccgccgac atccccgact acctcaagct gtccttcccc gagggcttca
     2161 agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgact caggactcct
     2221 ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg
     2281 acggccccgt aatgcagaag aagaccatgg gcatggaggc ctcctccgag cggatgtacc
     2341 ccgaggacgg cgccctgaag ggcgaggaca agctcaggct gaagctgaag gacggcggcc
     2401 actacacctc cgaggtcaag accacctaca aggccaagaa gcccgtgcag ttgcccggcg
     2461 cctacatcgt cgacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     2521 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     2581 ccggactcag atctcgagct caagcttatc gataatcaac ctctggatta caaaatttgt
     2641 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     2701 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     2761 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     2821 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     2881 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     2941 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     3001 tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc
     3061 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     3121 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     3181 tccctttggg ccgcctcccc gcatcgatac cggctcgaga cgggtggcat ccctgtgacc
     3241 cctccccagt gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc
     3301 taataaaatt aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt
     3361 ggaggggggt ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg
     3421 tctattggga accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct
     3481 cctgggttca agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca
     3541 tgaccaggct cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca
     3601 ggctggtctc caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg
     3661 gattacaggc gtgaaccact gctcccttcc ctgtccttct cgagctgatt ttgtaggtaa
     3721 ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct
     3781 gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc
     3841 ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta
     3901 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     3961 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     4021 acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4081 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4141 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     4201 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4261 ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa
     4321 gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4381 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     4441 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4501 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4561 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     4621 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     4681 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4741 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4801 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4861 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4921 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4981 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5041 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5101 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5161 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5221 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5281 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5341 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5401 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5461 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5521 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5581 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5641 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5701 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5761 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5821 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5881 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5941 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6001 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     6061 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6121 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6181 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6241 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6301 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6361 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6421 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     6481 t
//
