LOCUS       sequence-252563-        6625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  fusion protein of Gaussia luciferase variant slow burn, synthetic 
            channelrhodopsin CheRiff, and enhanced green fluorescent protein for
            bioluminescent optogenetics.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6625)
  AUTHORS   Ikefuama EC, Kendziorski GE, Anderson K, Shafau L, Prakash M, 
            Hochgeschwender U, Petersen ED
  TITLE     Improved Locomotor Recovery in a Rat Model of Spinal Cord Injury by 
            BioLuminescent-OptoGenetic (BL-OG) Stimulation with an Enhanced 
            Luminopsin.
  JOURNAL   Int J Mol Sci. 2022 Oct 27;23(21):12994. doi: 10.3390/ijms232112994.
  PUBMED    36361784
REFERENCE   2  (bases 1 to 6625)
  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: "Int J Mol 
            Sci."; date: "2022-10-27"; volume: "23(21):12994. doi"; pages: " 
            10.3390/ijms232112994"
COMMENT     Sequence Label: pAAV-hSyn-sbGLuc-CheRiff-EGFP
FEATURES             Location/Qualifiers
     source          1..6625
                     /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        181..628
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             658..1212
                     /codon_start=1
                     /product="enhanced variant of secreted Gaussia luciferase"
                     /label=Gaussia-Dura Luc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     CDS             658..1212
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     CDS             1258..2205
                     /codon_start=1
                     /product="blue light-activated optogenetic actuator derived
                     from Scherffelia dubia channelrhodopsin (Hochbaum et al., 
                     2014)"
                     /label=CheRiff
                     /translation="MGGAPAPDAHSAPPGNDSAGGSEYHAPAGYQVNPPYHPVHGYEEQ
                     CSSIYIYYGALWEQETARGFQWFAVFLSALFLAFYGWHAYKASVGWEEVYVCSVELIKV
                     ILEIYFEFTSPAMLFLYGGNITPWLRYAEWLLTCPVILIHLSNITGLSEAYNKRTMALL
                     VSDLGTICMGVTAALATGWVKWLFYCIGLVYGTQTFYNAGIIYVESYYIMPAGGCKKLV
                     LAMTAVYYSSWLMFPGLFIFGPEGMHTLSVAGSTIGHTIADLLSKNIWGLLGHFLRIKI
                     HEHIIMYGDIRRPVSSQFLGRKVDVLAFVTEEDKV"
     CDS             join(2275..2277,2278..2991)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2275..2277,2278..2988)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2317..2338)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2578..2597)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2925..2946
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3016..3604
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3069..3089)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3487..3498)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3646..3663)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3652..3859
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3888..4028
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3888..4017
                     /label=AAV2 ITR
     rep_origin      4103..4558
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4190..4209)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4400..4421
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4575..4594)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4694..4716
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4754..4772)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4840..4944
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4945..5013,5014..5805)
                     /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(5163..5182)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5976..6564
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6465..6484
                     /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 gcgttgtaca gtgtctagac tgcagagggc cctgcgtatg
      181 agtgcaagtg ggttttagga ccaggatgag gcggggtggg ggtgcctacc tgacgaccga
      241 ccccgaccca ctggacaagc acccaacccc cattccccaa attgcgcatc ccctatcaga
      301 gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg
      361 gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc
      421 gctgacgtca ctcgccggtc ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg
      481 cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg
      541 cgaccatctg cgctgcggcg ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg
      601 aggagtcgtg tcgtgcctga gagcgcagtc gagaaggatc cggcgcgccg gtaccacatg
      661 ggagtcaaag ttctgtttgc cctgatctgc atcgctgtgg ccgaggccaa gcccaccgag
      721 aacaacgaag acttcaacat cgtggccgtg gccagcaact tcgcgaccac ggatctcgat
      781 gctgaccgcg ggaagttgcc cggcaagaag ctgccgctgg aggtgctcaa agagctggaa
      841 gccaatgccc ggaaagctgg ctgcaccagg ggctgtctga tctgcctgtc ccacatcaag
      901 tgcacgccca agatgaagaa gttcatccca ggacgctgcc acacctacga aggcgacaaa
      961 gagtccgcac agggcggcat aggcgaggcg atcgtcgaca ttcctgagat tcctgggttc
     1021 aaggacttgg agcccctgga gcagttcatc gcacaggtcg atctgtgtgt ggactgcaca
     1081 actggctgcc tcaaagggct tgccaacgtg cagtgttctg acctgctcaa gaagtggctg
     1141 ccgcaacgct gtgcgacctt tgccagcaag atccagggcc aggtggacaa gatcaagggg
     1201 gccggtggtg acggcgaccc actagtccag tgtggtggaa ttgccggttc cgccactatg
     1261 ggcggagctc ctgctccaga cgctcacagc gccccacctg gaaacgattc tgccggaggc
     1321 agtgagtacc atgccccagc tggatatcaa gtgaatccac cctaccaccc cgtgcatggg
     1381 tatgaggaac agtgcagctc catctacatc tactatgggg ccctgtggga gcaggaaaca
     1441 gctaggggct tccagtggtt tgccgtgttc ctgtctgccc tgtttctggc tttctacggc
     1501 tggcacgcct ataaggccag cgtgggatgg gaggaagtgt acgtgtgctc cgtggagctg
     1561 atcaaagtga ttctggagat ctatttcgag ttcaccagtc ctgctatgct gttcctgtac
     1621 ggagggaaca ttaccccatg gctgagatat gccgaatggc tgctgacatg tcccgtgatc
     1681 ctgattcatc tgtctaacat caccggcctg agtgaggcat acaataagcg gacaatggct
     1741 ctgctggtgt ccgacctggg aactatttgc atgggagtga cagccgctct ggccactggg
     1801 tgggtgaagt ggctgtttta ctgtatcggc ctggtgtatg gaacccagac attctacaac
     1861 gctggaatca tctacgtgga gtcttactat atcatgcctg ccggcggctg taagaaactg
     1921 gtgctggcca tgactgccgt gtactattct agttggctga tgtttcccgg cctgttcatc
     1981 tttgggcctg aaggcatgca caccctgagc gtggctgggt ccactattgg ccataccatc
     2041 gccgacctgc tgtccaagaa tatttgggga ctgctggggc acttcctgcg gatcaaaatt
     2101 cacgagcata tcattatgta cggcgatatc aggagaccag tgagctccca gtttctggga
     2161 cgcaaggtgg acgtgctggc cttcgtgaca gaggaagata aagtggcggc cgccaagagc
     2221 aggatcacca gcgagggcga gtacatcccc ctggaccaga tcgacatcaa cgtggtgagc
     2281 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2341 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2401 accctgaagt tcatttgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2461 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2521 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2581 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2641 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2701 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2761 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2821 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     2881 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     2941 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta agaattcgat
     3001 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3061 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3121 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3181 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3241 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3301 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3361 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     3421 tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac
     3481 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     3541 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     3601 ccgcatcgat accgagcgct gctcgagcta gagctcgctg atcagcctcg actgtgcctt
     3661 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3721 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3781 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3841 atagcaggca tgctggggag gtaaccacgt gcggaccgag cggccgcagg aacccctagt
     3901 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     3961 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     4021 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4081 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     4141 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     4201 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     4261 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     4321 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     4381 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     4441 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     4501 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     4561 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     4621 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4681 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4741 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     4801 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4861 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4921 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4981 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     5041 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     5101 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     5161 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     5221 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     5281 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     5341 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     5401 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     5461 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     5521 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     5581 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     5641 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     5701 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     5761 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5821 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5881 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5941 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6001 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     6061 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     6121 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     6181 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     6241 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     6301 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     6361 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     6421 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     6481 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     6541 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     6601 ttttgctggc cttttgctca catgt
//
