LOCUS Exported 8553 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION fusion protein of NanoLuc, Volvox channelrhodopsin-1, and enhanced yellow fluorescent protein for bioluminescent optogenetics. ACCESSION . VERSION . KEYWORDS pcDNA3.1-CAG-NanoLuc-VChR1-EYFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8553) AUTHORS Berglund K, Clissold K, Li HE, Wen L, Park SY, Gleixner J, Klein ME, Lu D, Barter JW, Rossi MA, Augustine GJ, Yin HH, Hochgeschwender U TITLE Luminopsins integrate opto- and chemogenetics by using physical and biological light sources for opsin activation. JOURNAL Proc Natl Acad Sci U S A. 2016 Jan 19;113(3):E358-67. doi: 10.1073/pnas.1510899113. Epub 2016 Jan 5. PUBMED 26733686 REFERENCE 2 (bases 1 to 8553) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8553 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 98..401 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 403..678 /label=chicken beta-actin promoter intron 679..1695 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1619..1641 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" primer_bind 1703..1722 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, forward primer" regulatory 1784..1793 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1841..2350 /codon_start=1 /product="NanoLuc(R) luciferase" /label=Nluc /note="human codon-optimized" /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERILA" regulatory 2390..2399 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3305..4021 /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(3347..3368) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(3608..3627) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 3955..3976 /label=EGFP-C /note="EGFP, forward primer" CDS 4051..4092 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" CDS 4102..4119 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(4142..4159) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4148..4372 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 4418..4846 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4505..4524) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4715..4736 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(4855..4875) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 4860..5189 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 5040..5175 /label=SV40 ori /note="SV40 origin of replication" primer_bind 5102..5121 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 5256..6050 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(5310..5329) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 5920..5939 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 6226..6347 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6263..6282) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6317..6336 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(6396..6412) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6396..6412) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(6409..6431) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 6420..6436 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." promoter complement(6444..6474) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6489..6510 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6627..6644) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(6798..7386) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6878..6897) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7557..8417) /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 8180..8199 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8418..8522) /gene="bla" /label=AmpR promoter ORIGIN 1 gacggatcgg gagatctggc gcgccggtac catgcattag ttattaatag taatcaatta 61 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 121 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 181 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 241 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 301 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 361 cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt 421 tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt 481 tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggcgcgcgcc aggcggggcg 541 gggcggggcg aggggcgggg cggggcgagg cggagaggtg cggcggcagc caatcagagc 601 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag 661 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc 721 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg 781 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct 841 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc 901 ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg 961 cccggcggct gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc 1021 gaggggagcg cggccggggg cggtgccccg cggtgcgggg ggggctgcga ggggaacaaa 1081 ggctgcgtgc ggggtgtgtg cgtggggggg tgagcagggg gtgtgggcgc gtcggtcggg 1141 ctgcaacccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg 1201 gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg 1261 ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc 1321 ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg 1381 taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct 1441 gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg 1501 aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc 1561 cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg 1621 ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc 1681 ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc 1741 aaagaagcta gcgctaccgg actcagatct cgagctcaag cttgccacca tgggagtcaa 1801 agttctgttt gccctgatct gcatcgctgt ggccgaggcc gtcttcacac tcgaagattt 1861 cgttggggac tggcgacaga cagccggcta caacctggac caagtccttg aacagggagg 1921 tgtgtccagt ttgtttcaga atctcggggt gtccgtaact ccgatccaaa ggattgtcct 1981 gagcggtgaa aatgggctga agatcgacat ccatgtcatc atcccgtatg aaggtctgag 2041 cggcgaccaa atgggccaga tcgaaaaaat ttttaaggtg gtgtaccctg tggatgatca 2101 tcactttaag gtgatcctgc actatggcac actggtaatc gacggggtta cgccgaacat 2161 gatcgactat ttcggacggc cgtatgaagg catcgccgtg ttcgacggca aaaagatcac 2221 tgtaacaggg accctgtgga acggcaacaa aattatcgac gagcgcctga tcaaccccga 2281 cggctccctg ctgttccgag taaccatcaa cggagtgacc ggctggcggc tgtgcgaacg 2341 cattctggcg ggcgacccac tagtccagtg tggtggaatt gccggatccg ccaccatgga 2401 ttaccctgtt gctagaagcc tcatagttcg ctacccaacc gacctcggaa acggcaccgt 2461 ctgcatgcca agaggacagt gttactgtga aggttggctt cggagtcgcg gcacttccat 2521 tgaaaagaca atagcaatta ctcttcagtg ggtagtcttt gctttgtcag tggcttgcct 2581 ggggtggtat gcgtatcaag cgtggcgagc tacctgcgga tgggaggagg tttacgtagc 2641 cttgatagaa atgatgaaaa gcatcatcga ggccttccac gagttcgaca gccctgcaac 2701 actgtggctg tcttcaggga acggcgtagt ttggatgcgg tatggcgaat ggctcctcac 2761 ctgcccggtc cttctgatcc atctgagcaa cctcacaggc ctgaaggacg attatagcaa 2821 aaggactatg ggcctgttgg tttctgatgt gggatgcatc gtgtggggcg caaccagcgc 2881 catgtgtacg gggtggacga agatcctgtt cttcctcatc tcattgagct atggtatgta 2941 tacctatttt catgctgcta aagtttatat cgaagcattc cacacagttc caaaagggat 3001 ttgtcgagaa ctggtccgag tgatggcctg gacattcttt gtggcttggg gaatgtttcc 3061 agtcctgttt ctgctgggca cggaaggatt cggtcatatc agcccttatg gatctgccat 3121 tgggcactcc atcctcgacc tgattgcaaa gaacatgtgg ggtgtgctgg ggaattacct 3181 gcgcgtcaaa atccacgagc acatcctgtt gtatggcgac atcagaaaga agcagaaaat 3241 tacgatcgcc ggccaagaga tggaggttga gacactggtg gctgaagagg aggacgcggc 3301 cgccgtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga 3361 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta 3421 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac 3481 cctcgtgacc accttcggct acggcctgca gtgcttcgcc cgctaccccg accacatgaa 3541 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt 3601 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct 3661 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca 3721 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa 3781 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc 3841 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca 3901 ctacctgagc taccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt 3961 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta 4021 acatatgctc tagagggccc gcggttcgaa ggtaagccta tccctaaccc tctcctcggt 4081 ctcgattcta cgcgtaccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca 4141 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 4201 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 4261 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 4321 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 4381 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta 4441 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 4501 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 4561 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 4621 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 4681 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 4741 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 4801 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 4861 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 4921 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 4981 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 5041 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 5101 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 5161 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg 5221 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 5281 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 5341 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 5401 tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 5461 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 5521 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 5581 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 5641 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 5701 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 5761 aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg 5821 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 5881 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 5941 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 6001 ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 6061 ggggttcgcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc 6121 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg 6181 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca 6241 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 6301 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata 6361 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 6421 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 6481 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 6541 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 6601 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 6661 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 6721 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 6781 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 6841 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 6901 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 6961 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 7021 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 7081 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 7141 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 7201 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 7261 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 7321 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 7381 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 7441 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 7501 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 7561 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 7621 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 7681 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 7741 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 7801 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 7861 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 7921 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 7981 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 8041 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 8101 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 8161 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 8221 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 8281 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 8341 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 8401 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 8461 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 8521 cgcacatttc cccgaaaagt gccacctgac gtc //