LOCUS Exported 6855 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION fusion protein of Gaussia luciferase variant slow burn, Volvox channelrhodopsin-1, and enhanced yellow fluorescent protein for bioluminescent optogenetics. ACCESSION . VERSION . KEYWORDS pAAV-Hb9-LMO3 (sbGLuc-VChR1-EYFP) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6855) AUTHORS Hochgeschwender, U TITLE luciferase opsin fusion proteins for bioluminescent optogenetics JOURNAL unpublished REFERENCE 2 (bases 1 to 6855) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6855 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 5..134 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" regulatory 626..635 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 632..1186 /codon_start=1 /product="enhanced variant of secreted Gaussia luciferase" /label=Gaussia-Dura Luc /note="human codon-optimized" /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG KLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA QGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ RCATFASKIQGQVDKIKGAGGD" CDS 632..1186 /codon_start=1 /product="secreted Gaussia luciferase" /label=hGLuc /note="human codon-optimized" /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG KLPGKKLPLEVLKELEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA QGGIGEAIVDIPEIPGFKDLEPLEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ RCATFASKIQGQVDKIKGAGGD" regulatory 1226..1235 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2141..2857 /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(2183..2204) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(2444..2463) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 2791..2812 /label=EGFP-C /note="EGFP, forward primer" CDS 2887..2928 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 2938..2955 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" misc_feature 2986..3574 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3039..3059) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3457..3468) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3606..4082 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3805..3824) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4122..4262 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4122..4251 /label=AAV2 ITR rep_origin 4337..4792 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4424..4443) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4634..4655 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4809..4828) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4928..4950 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(4988..5006) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5074..5178 /gene="bla" /label=AmpR promoter CDS 5179..6039 /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(5397..5416) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6210..6798 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6699..6718 /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 gcacgcgttg aataaattta agcaggctaa ttaatatata 181 aactagccca atttgtcaag ttgatttgta ttttagttaa ttgtgaaagt aattaccaca 241 tggtcaaatt aacagctttc tggaaatgac caagcctgag gttttatttc cttcctgggt 301 gaagaaaatt catttttcca agctcttgat gtgatgaata aaagtcataa atctgggtga 361 ttggtgcagg cagagtctaa atggcttcat atttcatttt aggtttaata gaaatattca 421 tgctctgttt taatgaaatt aaattgaagg gggatggggc cagagtagtt agctgatgaa 481 ttgacaaaaa ctaatcagct ttattgggaa acaggtttaa gggcacgggc gtgtcaataa 541 cgctcagcct gaccccctct tccattagct aggcaggctg attagagaat tcggtaccgg 601 atcctctaga gtcgagctca agcttgccac catgggagtc aaagttctgt ttgccctgat 661 ctgcatcgct gtggccgagg ccaagcccac cgagaacaac gaagacttca acatcgtggc 721 cgtggccagc aacttcgcga ccacggatct cgatgctgac cgcgggaagt tgcccggcaa 781 gaagctgccg ctggaggtgc tcaaagagct ggaagccaat gcccggaaag ctggctgcac 841 caggggctgt ctgatctgcc tgtcccacat caagtgcacg cccaagatga agaagttcat 901 cccaggacgc tgccacacct acgaaggcga caaagagtcc gcacagggcg gcataggcga 961 ggcgatcgtc gacattcctg agattcctgg gttcaaggac ttggagcccc tggagcagtt 1021 catcgcacag gtcgatctgt gtgtggactg cacaactggc tgcctcaaag ggcttgccaa 1081 cgtgcagtgt tctgacctgc tcaagaagtg gctgccgcaa cgctgtgcga cctttgccag 1141 caagatccag ggccaggtgg acaagatcaa gggggccggt ggtgacggcg acccactagt 1201 ccagtgtggt ggaattgccg gatccgccac catggattac cctgttgcta gaagcctcat 1261 agttcgctac ccaaccgacc tcggaaacgg caccgtctgc atgccaagag gacagtgtta 1321 ctgtgaaggt tggcttcgga gtcgcggcac ttccattgaa aagacaatag caattactct 1381 tcagtgggta gtctttgctt tgtcagtggc ttgcctgggg tggtatgcgt atcaagcgtg 1441 gcgagctacc tgcggatggg aggaggttta cgtagccttg atagaaatga tgaaaagcat 1501 catcgaggcc ttccacgagt tcgacagccc tgcaacactg tggctgtctt cagggaacgg 1561 cgtagtttgg atgcggtatg gcgaatggct cctcacctgc ccggtccttc tgatccatct 1621 gagcaacctc acaggcctga aggacgatta tagcaaaagg actatgggcc tgttggtttc 1681 tgatgtggga tgcatcgtgt ggggcgcaac cagcgccatg tgtacggggt ggacgaagat 1741 cctgttcttc ctcatctcat tgagctatgg tatgtatacc tattttcatg ctgctaaagt 1801 ttatatcgaa gcattccaca cagttccaaa agggatttgt cgagaactgg tccgagtgat 1861 ggcctggaca ttctttgtgg cttggggaat gtttccagtc ctgtttctgc tgggcacgga 1921 aggattcggt catatcagcc cttatggatc tgccattggg cactccatcc tcgacctgat 1981 tgcaaagaac atgtggggtg tgctggggaa ttacctgcgc gtcaaaatcc acgagcacat 2041 cctgttgtat ggcgacatca gaaagaagca gaaaattacg atcgccggcc aagagatgga 2101 ggttgagaca ctggtggctg aagaggagga cgcggccgcc gtgagcaagg gcgaggagct 2161 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 2221 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 2281 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccacct tcggctacgg 2341 cctgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc 2401 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 2461 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 2521 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 2581 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 2641 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 2701 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccgccct 2761 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 2821 cgggatcact ctcggcatgg acgagctgta caagtaacat atgctctaga gggcccgcgg 2881 ttcgaaggta agcctatccc taaccctctc ctcggtctcg attctacgcg taccggtcat 2941 catcaccatc accattgagt ttaattcgat atcaagctta tcgataatca acctctggat 3001 tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 3061 ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 3121 tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 3181 caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 3241 accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 3301 ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 3361 tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc 3421 tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 3481 ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 3541 acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga 3601 tctacgggtg gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca 3661 ctccagtgcc caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg 3721 tgtccttcta taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga 3781 agacaacctg tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat 3841 cttggctcac tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg 3901 agttgttggg attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga 3961 gacggggttt caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc 4021 caccttggcc tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc 4081 ttctgatttt gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga 4141 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 4201 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca 4261 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg 4321 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 4381 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc 4441 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 4501 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat 4561 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 4621 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc 4681 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg 4741 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc 4801 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 4861 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 4921 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 4981 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 5041 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 5101 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 5161 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 5221 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 5281 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 5341 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 5401 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 5461 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 5521 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 5581 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 5641 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 5701 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 5761 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 5821 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 5881 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 5941 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 6001 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 6061 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 6121 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 6181 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 6241 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 6301 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt 6361 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 6421 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 6481 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 6541 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 6601 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 6661 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 6721 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 6781 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 6841 tggccttttg ctcac //