LOCUS       sequence-252561-        6870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  fusion protein of Gaussia luciferase variant slow burn, 
            Channelrhodopsin-2 variant C128S/D156A (stabilized step function 
            opsin), and enhanced yellow fluorescent protein for bioluminescent 
            optogenetics.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6870)
  AUTHORS   Berglund K, Fernandez AM, Gutekunst CN, Hochgeschwender U, Gross RE
  TITLE     Step-function luminopsins for bimodal prolonged neuromodulation.
  JOURNAL   J Neurosci Res. 2019 Apr 7. doi: 10.1002/jnr.24424.
  PUBMED    30957296
REFERENCE   2  (bases 1 to 6870)
  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: "J Neurosci 
            Res. 2019 Apr 7. doi: 10.1002/jnr.24424."
COMMENT     Sequence Label: pAAV-hSyn-sbGLuc-ChR2(CS/DA)-EYFP
FEATURES             Location/Qualifiers
     source          1..6870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   5..134
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        179..626
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             673..1227
                     /codon_start=1
                     /product="enhanced variant of secreted Gaussia luciferase"
                     /label=Gaussia-Dura Luc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     CDS             673..1227
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     regulatory      1267..1276
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1273..2199
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTSPVILIHLSNLTGLSNDYSRRTMGLLVSAIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     CDS             join(2212..2214,2215..2928)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2254..2275)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2515..2534)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2862..2883
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2953..2970
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    3001..3589
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3054..3074)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3472..3483)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3621..4097
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3820..3839)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4137..4277
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4137..4266
                     /label=AAV2 ITR
     rep_origin      4352..4807
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4439..4458)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4649..4670
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4824..4843)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4943..4965
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5003..5021)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5089..5193
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5194..5262,5263..6054)
                     /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(5412..5431)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6225..6813
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6714..6733
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
       61 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
      121 cactaggggt tcctgcggcc gcacgcgtgt gtctagactg cagagggccc tgcgtatgag
      181 tgcaagtggg ttttaggacc aggatgaggc ggggtggggg tgcctacctg acgaccgacc
      241 ccgacccact ggacaagcac ccaaccccca ttccccaaat tgcgcatccc ctatcagaga
      301 gggggagggg aaacaggatg cggcgaggcg cgtgcgcact gccagcttca gcaccgcgga
      361 cagtgccttc gcccccgcct ggcggcgcgc gccaccgccg cctcagcact gaaggcgcgc
      421 tgacgtcact cgccggtccc ccgcaaactc cccttcccgg ccaccttggt cgcgtccgcg
      481 ccgccgccgg cccagccgga ccgcaccacg cgaggcgcga gatagggggg cacgggcgcg
      541 accatctgcg ctgcggcgcc ggcgactcag cgctgcctca gtctgcggtg ggcagcggag
      601 gagtcgtgtc gtgcctgaga gcgcagtcga gaaggtaccg gatcctctag agtcgagctc
      661 aagcttgcca ccatgggagt caaagttctg tttgccctga tctgcatcgc tgtggccgag
      721 gccaagccca ccgagaacaa cgaagacttc aacatcgtgg ccgtggccag caacttcgcg
      781 accacggatc tcgatgctga ccgcgggaag ttgcccggca agaagctgcc gctggaggtg
      841 ctcaaagagc tggaagccaa tgcccggaaa gctggctgca ccaggggctg tctgatctgc
      901 ctgtcccaca tcaagtgcac gcccaagatg aagaagttca tcccaggacg ctgccacacc
      961 tacgaaggcg acaaagagtc cgcacagggc ggcataggcg aggcgatcgt cgacattcct
     1021 gagattcctg ggttcaagga cttggagccc ctggagcagt tcatcgcaca ggtcgatctg
     1081 tgtgtggact gcacaactgg ctgcctcaaa gggcttgcca acgtgcagtg ttctgacctg
     1141 ctcaagaagt ggctgccgca acgctgtgcg acctttgcca gcaagatcca gggccaggtg
     1201 gacaagatca agggggccgg tggtgacggg gatccactag tccagtgtgg tggaattgcc
     1261 cttgctgcca ccatggacta tggcggcgct ttgtctgccg tcggacgcga acttttgttc
     1321 gttactaatc ctgtggtggt gaacgggtcc gtcctggtcc ctgaggatca atgttactgt
     1381 gccggatgga ttgaatctcg cggcacgaac ggcgctcaga ccgcgtcaaa tgtcctgcag
     1441 tggcttgcag caggattcag cattttgctg ctgatgttct atgcctacca aacctggaaa
     1501 tctacatgcg gctgggagga gatctatgtg tgcgccattg aaatggttaa ggtgattctc
     1561 gagttctttt ttgagtttaa gaatccctct atgctctacc ttgccacagg acaccgggtg
     1621 cagtggctgc gctatgcaga gtggctgctc acttctcctg tcatccttat ccacctgagc
     1681 aacctcaccg gcctgagcaa cgactacagc aggagaacca tgggactcct tgtctcagcc
     1741 atcgggacta tcgtgtgggg ggctaccagc gccatggcaa ccggctatgt taaagtcatc
     1801 ttcttttgtc ttggattgtg ctatggcgcg aacacatttt ttcacgccgc caaagcatat
     1861 atcgagggtt atcatactgt gccaaagggt cggtgccgcc aggtcgtgac cggcatggca
     1921 tggctgtttt tcgtgagctg gggtatgttc ccaattctct tcattttggg gcccgaaggt
     1981 tttggcgtcc tgagcgtcta tggctccacc gtaggtcaca cgattattga tctgatgagt
     2041 aaaaattgtt gggggttgtt gggacactac ctgcgcgtcc tgatccacga gcacatattg
     2101 attcacggag atatccgcaa aaccaccaaa ctgaacatcg gcggaacgga gatcgaggtc
     2161 gagactctcg tcgaagacga agccgaggcc ggagccgtgc cagcggccgc cgtgagcaag
     2221 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2281 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2341 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2401 ttcggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     2461 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2521 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2581 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2641 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     2701 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2761 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     2821 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2881 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaca tatgctctag
     2941 ccaattgccg gtcatcatca ccatcaccat tgagtttaat tcgatatcaa gcttatcgat
     3001 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3061 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     3121 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     3181 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     3241 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     3301 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     3361 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     3421 gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3481 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     3541 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca tcgataccga
     3601 gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
     3661 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca
     3721 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
     3781 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga
     3841 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
     3901 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
     3961 tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat
     4021 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
     4081 ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga
     4141 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     4201 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     4261 gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4321 ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg
     4381 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgcctt agcgcccgct
     4441 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     4501 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     4561 cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     4621 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     4681 aactctatct cgggctattc ttttgattta taagggattt tgccgatttc ggtctattgg
     4741 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
     4801 acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc
     4861 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4921 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4981 ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg
     5041 ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     5101 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5161 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5221 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5281 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5341 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5401 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5461 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5521 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5581 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5641 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5701 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5761 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5821 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     5881 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     5941 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6001 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6061 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6121 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6181 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6241 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6301 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6361 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6421 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6481 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6541 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6601 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6661 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6721 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6781 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6841 ttcctggcct tttgctggcc ttttgctcac
//
