LOCUS       sequence-306853-        7102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For packaging and expression of ChrimsonR-LSSmOrange featuring a 
            neuritin (NRN) 3' UTR in neurons via AAV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7102)
  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 7102)
  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-NRN
FEATURES             Location/Qualifiers
     source          1..7102
                     /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        225..672
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      685..694
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             691..1740
                     /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             1759..2463
                     /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     2169..2188
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2498..3086
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2551..2571)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2969..2980)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(4112..4129)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4118..4325
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4365..4505
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4365..4494
                     /label=AAV2 ITR
     rep_origin      4580..5035
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4667..4686)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4877..4898
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5052..5071)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5171..5193
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5231..5249)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5317..5421
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5422..5490,5491..6282)
                     /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(5640..5659)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6453..7041
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6942..6961
                     /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 gcgtttaaca ttatggcctt aggtcacttc atctccatgg
      181 ggttcttctt ctgattttct agactgcaga gggccctgcg tatgagtgca agtgggtttt
      241 aggaccagga tgaggcgggg tgggggtgcc tacctgacga ccgaccccga cccactggac
      301 aagcacccaa cccccattcc ccaaattgcg catcccctat cagagagggg gaggggaaac
      361 aggatgcggc gaggcgcgtg cgcactgcca gcttcagcac cgcggacagt gccttcgccc
      421 ccgcctggcg gcgcgcgcca ccgccgcctc agcactgaag gcgcgctgac gtcactcgcc
      481 ggtcccccgc aaactcccct tcccggccac cttggtcgcg tccgcgccgc cgccggccca
      541 gccggaccgc accacgcgag gcgcgagata ggggggcacg ggcgcgacca tctgcgctgc
      601 ggcgccggcg actcagcgct gcctcagtct gcggtgggca gcggaggagt cgtgtcgtgc
      661 ctgagagcgc agtcgagagg atccgccacc atggctgagc tgatcagcag cgccaccaga
      721 tctctgtttg ccgccggagg catcaaccct tggcctaacc cctaccacca cgaggacatg
      781 ggctgtggag gaatgacacc tacaggcgag tgcttcagca ccgagtggtg gtgtgaccct
      841 tcttacggac tgagcgacgc cggatacgga tattgcttcg tggaggccac aggcggctac
      901 ctggtcgtgg gagtggagaa gaagcaggct tggctgcaca gcagaggcac accaggagaa
      961 aagatcggcg cccaggtctg ccagtggatt gctttcagca tcgccatcgc cctgctgaca
     1021 ttctacggct tcagcgcctg gaaggccact tgcggttggg aggaggtcta cgtctgttgc
     1081 gtcgaggtgc tgttcgtgac cctggagatc ttcaaggagt tcagcagccc cgccacagtg
     1141 tacctgtcta ccggcaacca cgcctattgc ctgcgctact tcgagtggct gctgtcttgc
     1201 cccgtgatcc tgatcagact gagcaacctg agcggcctga agaacgacta cagcaagcgg
     1261 accatgggcc tgatcgtgtc ttgcgtggga atgatcgtgt tcggcatggc cgcaggactg
     1321 gctaccgatt ggctcaagtg gctgctgtat atcgtgtctt gcatctacgg cggctacatg
     1381 tacttccagg ccgccaagtg ctacgtggaa gccaaccaca gcgtgcctaa aggccattgc
     1441 cgcatggtcg tgaagctgat ggcctacgct tacttcgcct cttggggcag ctacccaatc
     1501 ctctgggcag tgggaccaga aggactgctg aagctgagcc cttacgccaa cagcatcggc
     1561 cacagcatct gcgacatcat cgccaaggag ttttggacct tcctggccca ccacctgagg
     1621 atcaagatcc acgagcacat cctgatccac ggcgacatcc ggaagaccac caagatggag
     1681 atcggaggcg aggaggtgga agtggaagag ttcgtggagg aggaggacga ggacacagtg
     1741 gcggcaccgg tagtagcagt gagcaagggc gaggagaata acatggccat catcaaggag
     1801 ttcatgcgct tcaaggtgcg catggagggc tccgtgaacg gccacgagtt cgagatcgag
     1861 ggcgagggcg agggccgccc ctacgagggc tttcagaccg ttaagctgaa ggtgaccaag
     1921 ggtggccccc tgcccttcgc ctgggacatc ttgtcccctc agttcaccta cggctccaag
     1981 gcctacgtga agcaccccgc cgacatcccc gactacctca agctgtcctt ccccgagggc
     2041 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gactcaggac
     2101 tcctccctgc aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc
     2161 tccgacggcc ccgtaatgca gaagaagacc atgggcatgg aggcctcctc cgagcggatg
     2221 taccccgagg acggcgccct gaagggcgag gacaagctca ggctgaagct gaaggacggc
     2281 ggccactaca cctccgaggt caagaccacc tacaaggcca agaagcccgt gcagttgccc
     2341 ggcgcctaca tcgtcgacat caagttggac atcacctccc acaacgagga ctacaccatc
     2401 gtggaacagt acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     2461 aagtccggac tcagatctcg agctcaagct tatcgataat caacctctgg attacaaaat
     2521 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     2581 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     2641 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     2701 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     2761 tcagctcctt tccggaactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     2821 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     2881 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct
     2941 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     3001 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     3061 gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatctgcgtg
     3121 gggccagctc cccccgcgcg cccacccaca ctcactccat gctcccggaa atcgagagga
     3181 agatccatta gttctttggg gacgttgtga ttctctgtga tgctgaaaac actcatatag
     3241 gattgtggga aatcctgatt ctctttttta tttcgtttga tttcttgtgt tttatttgcc
     3301 aaatgttacc aatcagtgag caagcaagca cagccaaaat cggacctcag ctttagtccg
     3361 tcttcacaca caaataagaa aacggcaaac ccaccccatt ttttaatttt attattatta
     3421 attttttttg ttggcaaaag aatctcagga acggccctgg gccacctact atattaatca
     3481 tgctagtaac atgaaaaatg atgggctcct cctaatagga aggcgaggag aggagaaggc
     3541 caggggaatg aattcaagag agatgtccac ggacgaaaca tacggtgaat aattcacgct
     3601 cacgtcgttc ttccacagta tcttgttttg atcatttcca ctgcacattt ctcctcaaga
     3661 aaagcgaaag gacagactgt tggctttgtg tttggaggat aggagggaga gagggaaggg
     3721 gctgaggaaa tctctggggt aagagtaaag gcttccagaa gacatgctgc tatggtcact
     3781 gaggggttag ctttatctgc tgttgttgat gcatccgtcc aagttcactg cctttatttt
     3841 ccctcctccc tcttgtttta gctgttacac acacagtaat acctgaatat ccaacggtat
     3901 agatcacaag ggggggatgt taaatgttaa tctaaaatat agctaaaaaa agattttgac
     3961 ataaaagagc cttgatttta aaaaaaaaag agagagagat gtaatttaaa aagtttatta
     4021 taaattaaat tcagcaaaaa aagatttgct acaaagtata gagaagtata aaataaaagt
     4081 tattgtttga aaaaaaaaaa aaaaaaataa gcctcgactg tgccttctag ttgccagcca
     4141 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4201 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4261 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4321 ggggactgat tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat
     4381 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     4441 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     4501 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     4561 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     4621 gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc
     4681 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     4741 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     4801 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     4861 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg
     4921 ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag
     4981 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     5041 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     5101 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     5161 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     5221 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     5281 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     5341 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     5401 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     5461 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     5521 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     5581 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     5641 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     5701 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     5761 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     5821 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     5881 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     5941 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     6001 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     6061 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     6121 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     6181 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     6241 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     6301 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     6361 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     6421 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     6481 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     6541 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     6601 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     6661 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     6721 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     6781 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     6841 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     6901 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     6961 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     7021 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     7081 tgctggcctt ttgctcacat gt
//
